Evodiamine-based NQO1 agonist as well as preparation method and application thereof

Abstract

The invention discloses an evodiamine-based NQO1 agonist as well as a preparation method and application thereof, and particularly relates to an evodiamine (EVO) amido bond-connected NQO1 [NAD (P) H: quinone oxidoreductase 1] agonist which shows relatively good non-small cell lung cancer (NSCLC) resistance activity (IC50 is 2.72 M) through a cytotoxicity experiment. The compound provided by the invention promotes A549 cell apoptosis, blocks a cell cycle to a G2 / M period, causes active oxygen outbreak, and down-regulates the expression of Keap1 and up-regulates the expressions of Nrf2, NQO1 and HO-1 after acting in A549 cells for 24 hours. The results show that the compound can improve the cell antioxidant ability by adjusting the Keap1 / Nrf2 / NQO1 antioxidant pathway. In-vivo anti-tumor experiments show that the compound can inhibit proliferation of tumor tissues (TGI = 39.13%). In a word, the research result shows that the compound has a strong excitation effect on an NQO1 signal channel, and a potential opportunity is provided for improving treatment of non-small cell lung cancer.

Description

Technical Field

[0001] The present invention belongs to biomedical technology, and specifically relates to a preparation method and application of an NQO1 agonist connected by an amide bond of evodiamine. Background Art

[0002] Currently, the treatment of non-small cell lung cancer mainly includes surgery, radiotherapy and chemotherapy, but the long-term survival rate of patients with advanced lung cancer is still low due to the drug resistance of patients and the low sensitivity of tumor cells to traditional chemotherapy drugs. In recent years, with the development of targeted therapy and immunotherapy, the treatment effect has improved, but the toxic side effects, treatment resistance and poor efficacy of traditional drugs still exist. Therefore, it is urgent to develop new anticancer drugs with high efficiency and low toxicity. In tumor treatment research, NQO1, as an antioxidant enzyme, has been found to be closely related to the occurrence and development of various tumors. Studies have shown that NQO1 can reduce the damage of reactive oxygen species (ROS) to cells by regulating the redox balance in cells, thereby inducing apoptosis of cancer cells [DPP9 regulates NQO1 and ROS to promote resistance to chemotherapy inliver cancer cells]. However, most of the existing NQO1 agonists have shortcomings such as poor stability, insufficient targeting or short drug action time, which limits their clinical application. Evodiamine (EVO), as a natural alkaloid, interferes with the proliferation, migration and invasion of cancer cells through multiple pathways, but its mechanism of action has not yet been fully clarified, and the efficacy and anti-cancer potential of Evodiamine still have room for improvement. Summary of the invention

[0003] As a natural alkaloid, Evodiamine (EVO) still has room for improvement in its efficacy and anti-cancer potential. The present invention discloses multiple derivatives of Evodiamine connected by amide bonds, which are combined with indolequinone through amide bonds. As NQO1 agonists, they solve the shortcomings of most existing agonists, such as poor stability, insufficient targeting or short drug action time, and broaden their clinical applications.

[0004] The present invention adopts the following technical solution: An NQO1 agonist based on evodiamine, whose chemical structure is one of the following chemical structures: ;

[0005] In the above chemical structural formula, n is 1-10, preferably 2-8, more preferably 2-5, such as 2-4.

[0006] The present invention discloses a method for preparing the NQO1 agonist based on evodiamine, comprising the following steps: reacting amino-substituted evodiamine with indolequinone to prepare the NQO1 agonist based on evodiamine.

[0007] Among them, the chemical structural formula of amino-substituted evodiamine is as follows:

[0008] The chemical structure of indolequinone is as follows:

[0009] R1 is the following substituent:

[0010] In the above chemical structural formula, n is 1-10, preferably 2-8, more preferably 2-5, such as 2-4.

[0011] In the above technical scheme, in the presence of EDCI / DMAP system, amino-substituted evodiamine reacts with indolequinone to prepare the above-mentioned NQO1 agonist based on evodiamine. In the present invention, amino-substituted evodiamine reacts with indolequinone with different connecting bonds, carboxylic acid and amine condense to form amide bonds, and the reaction has high selectivity and efficiency. The reaction time is 6 to 8 hours.

[0012] In the present invention, the alkyl group of the intermediate is a straight-chain substituent having 2 to 4 carbon atoms and connected by a disulfide bond; preferably, the molar ratio of aminoevodiamine to indolequinone is 1:1.

[0013] The present invention discloses a medicine, which uses the NQO1 agonist based on evodiamine as an active ingredient. Specifically, the medicine comprises an effective therapeutic amount of the NQO1 agonist based on evodiamine and a pharmaceutically acceptable excipient, carrier or diluent.

[0014] The present invention discloses the use of the evodiamine-based NQO1 agonist or a pharmaceutically acceptable salt or a pharmaceutical composition thereof in preparing the NQO1 agonist or as the NQO1 agonist.

[0015] The present invention discloses the use of the evodiamine-based NQO1 agonist or a pharmaceutically acceptable salt or a pharmaceutical composition thereof in the preparation of an anti-tumor drug or an anti-tumor cell drug.

[0016] The present invention uses evodiamine substituted with amino groups to react with indolequinones with different connecting bonds to prepare NQO1 agonists containing evodiamine and indolequinone structural units. Existing NQO1 agonists have problems such as poor drug stability and targeting, and although evodiamine compounds have certain anti-cancer potential, their anti-cancer activity and selectivity when used alone need to be improved. In view of the shortcomings of existing NQO1 agonists and evodiamine compounds, the present invention has developed a series of NQO1 agonists connected by evodiamine amide bonds. These novel compounds not only maintain the natural anti-cancer activity of evodiamine through innovative structural design, but also significantly improve its anti-tumor effect in vivo and in vitro by enhancing the interaction with NQO1. In addition, experimental results show that these compounds can significantly enhance the antioxidant capacity of cells by regulating the Keap1 / Nrf2 / NQO1 antioxidant pathway, thereby inhibiting the proliferation of non-small cell lung cancer cells and inducing their apoptosis. Compared with the prior art, the present invention provides an NQO1 agonist with better stability and stronger activity, and provides a new solution for the treatment of non-small cell lung cancer. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 The bioreductive agonistic effects of compounds 11b and 12d on NQO1; (A) the antiproliferative activity of dicoumarol on A549 cells, (B) the cytotoxicity evaluation of compounds 11b and 12d dependent on NQO1.

[0018] Figure 2 To characterize the apoptotic effects of compounds 11b and 12d, A549 cells were treated with different concentrations of the compounds; A549 cells were incubated with different concentrations of 11b and 12d (0, 2, 4, and 8 µM) for 48 h; at least three independent experiments were performed for each condition.

[0019] Figure 3 To investigate the cell cycle effects of compounds 11b and 12d, A549 cells were treated with different concentrations of compounds; compounds 11b and 12d induced G2 / M arrest, and A549 cells were incubated with different concentrations of 11b and 12d (0, 2, 4, and 8 µM) for 48 h; at least three independent experiments were performed for each condition.

[0020] Figure 4Compound 11b inhibits tumor growth. C57BL / 6 mice were divided into control group, 11b group, 5-Fu group and EVO group (n = 6). Except for the control group, the other groups were given 20 mg / kg of the corresponding drug once a day for 13 consecutive days; (A) Experimental scheme, (B) Changes of tumor volume over time, (C) Changes of mouse body weight over time (D, E) Weight and size of the tumor on the last day, (F) Mouse liver index, (G) Mouse kidney index, (H) Immunohistochemical staining of Bax, Bcl2, and Caspase3, (1) Pathological sections of liver, kidney, and tumor tissues. DETAILED DESCRIPTION

[0021] As an example, the present invention discloses a preparation method and application of an NQO1 agonist connected by an amide bond of evodiamine. The preparation method is as follows: 2-aminoevodiamine (100 mg, 0.314 mmol) is dissolved in 5 mL of anhydrous DCM, followed by the addition of compound 8 (109 mg, 0.314 mmol) and EDCI (300.98 mg, 1.57 mmol), the mixture is cooled to 0°C, DMAP (38.36 mg, 0.314 mmol) is added, stirred at room temperature for 24-48 h, monitored by TCL, after the reaction is completed, the solvent is removed, the mixture is dissolved in DCM, washed with water and saturated saline, dried over anhydrous Na2SO4, concentrated under reduced pressure, separated and purified by silica gel column, and eluted with CH3OH / DCM (2-4%, Rf=0.4). A yellow solid compound 9 is obtained (yield 48-62%). The synthesis methods of compounds 9a-9d, 10a-10d, 11a-11d, and 12a-12d are similar.

[0022] The present invention successfully designed and synthesized 16 new compounds targeting NQO1, and then evaluated their biological activities in vitro. In order to analyze the cytotoxicity of these targeted compounds, the MTT method was used to preliminarily screen the antiproliferative activities of compounds 9a-9d, 10a-10d, 11a-11d and 12a-12d containing indolequinone nuclei on human lung cancer cells A549, H460, PC9 and gefitinib-resistant cells PC9 / GR, as well as human normal liver cell line LO2, for 48 hours. EVO was used as a positive control. Most of the compounds had antitumor effects on these cancer cells, and IC 50 The range of 11b and 12d was between 2.54 and 34.48 µM. 11b and 12d showed good anti-NSCLC activity in A549 cells, IC 50They were 2.72 µM and 3.66 µM, respectively, even higher than EVO's 19.65 µM. Apoptosis is an important target for anti-tumor therapy. Annexin V / PI staining flow cytometry was used to further detect the effects of compounds 11b and 12d on tumor cell apoptosis, cell cycle, reactive oxygen species and mitochondrial membrane potential. Flow cytometry results showed that 11b and 12d could induce apoptosis of A549 cells, block their cell cycle at the G2 / M phase, and induce a significant increase in reactive oxygen species. Western blotting analysis further showed that 11b and 12d could inhibit Keap1 expression and promote the expression of Nrf2, NQO1 and HO-1 after 24 hours of treatment of A549 cells, indicating that these two compounds enhanced the antioxidant defense capacity of cells by regulating the Keap1 / Nrf2 / NQO1 antioxidant pathway. In vivo studies confirmed that compound 11b could effectively inhibit tumor growth, with a tumor growth inhibition rate of 39.13%.

[0023] The chemical structures of the four evodiamine derivatives are as follows:

[0024] The above four evodiamine derivatives (2-NH-EVO, 3-NH-EVO, 2-NH-10-OCH3-EVO, 3-NH-10-OCH3-EVO) are respectively assembled with indolequinone through amide bonds. Specifically, the amino-substituted evodiamine is reacted with indolequinone with different connecting bonds to prepare the evodiamine indolequinone complex; the synthetic route is as follows:

[0025] Reagents and conditions: (i) DMF / POCl3. (ii) DMF / NaH / CH3I. (iii) AcOH / HNO3. (iv) Sn / HCl / EtOH, reflux (v) Fremy's salt / Acetone. (vi) NaBH4 / MeOH / THF. (vii) Anhydride / DMAP / DCM.

[0026]

[0027] Synthesis of compounds 9a, 9b, 9c, 9d, 10a, 10b, 10c, 10d, 11a, 11b, 11c, 11d, 12a, 12b, 12c, 12d, reagents and conditions: (i) EDCI / DMAP / DCM, rt, 6-8h.

[0028] In the experimental part of the present invention, a total of 16 new compounds targeting NQO1 were synthesized, among which 11b and 12d showed good anti-NSCLC activity in cytotoxicity experiments, IC 50 The results of flow cytometry showed that 11b and 12d could induce apoptosis of A549 cells, arrest the cell cycle at the G2 / M phase, and induce a significant increase in reactive oxygen species. Western blotting analysis further showed that 11b and 12d could inhibit Keap1 expression and promote the expression of Nrf2, NQO1 and HO-1 after 24 hours of treatment of A549 cells, indicating that these two compounds enhanced the antioxidant defense capacity of cells by regulating the Keap1 / Nrf2 / NQO1 antioxidant pathway. In vivo studies confirmed that compound 11b could effectively inhibit tumor growth, with a tumor growth inhibition rate of 39.13%. Therefore, the study showed that 11b and 12d showed excellent anti-cancer potential by activating the NQO1 signaling pathway, providing new candidate drugs for the treatment of non-small cell lung cancer.

[0029] The present invention bonds different raw materials by chemical means to obtain NQO1 agonists with reduced toxic side effects and additive or synergistic pharmacological effects. The raw materials and reagents involved in the reaction process, unless otherwise specified, are all analytically pure and purchased from Sigma Aldrich Trading Co. Ltd. The nuclear magnetic resonance spectrometer is a German company Bruker AVANCE400 and Bruker AVANCE500 model, TMS is used as the internal standard, and the solvent is DMSO- d 6 or CDCl3, chemical shift (d) and coupling constant ( J ) in ppm and Hz respectively. ESI mass spectrometry was performed by Aglient 7250 & JEOL-JMS-T100LP AccuTOF mass spectrometer, and TLC analysis was performed by silica gel GF 254 (Qingdao Marine Chemistry, China). Cell lines A549, H460, PC9, PC9 / GR and LO2 were purchased from Wuhan Purcell Life Science Co., Ltd., China. The specific experimental operations and tests (including statistical data analysis) of the present invention are conventional techniques, and the animal experiments comply with the regulations of Shaanxi University of Chinese Medicine.

[0030] The present invention discloses a coupling reaction between aminoevodiamine and indolequinone to obtain an NQO1 agonist containing an evodiamine structural unit. Also disclosed is the use of the NQO1 agonist containing the evodiamine structural unit in the preparation of an anti-tumor drug. The preferred tumor is lung cancer. The synthesis route of the final product is shown above.

[0031] Preparation of NQO1 agonists containing evodiamine structural units

[0032] 3-Formyl-5-methoxy-2-methylindole (2) POCl3 (0.85 mL, 9.28 mmol) was added to 3 mL of anhydrous DMF and stirred at 0 °C for 10 min to prepare Vilsmeier reagent. 5-Methoxy-2-methylindole (1.04 g, 6.45 mmol) was then dissolved in 3 mL of anhydrous DMF, cooled to 0 °C, and the prepared Vilsmeier reagent was added dropwise. After the addition, the reaction mixture was stirred at 0 °C for 30 min, and then the reaction mixture was dropped into an ice 2 M NaOH (50 mL) solution, DCM (100 mL) was added, and the extraction was separated. The aqueous layer was extracted with DCM (50 mL), and the organic layers were combined, washed with brine, dried over anhydrous Na2SO4, and the solvent was removed. The residue was washed with glacial ethyl acetate to obtain 1.05 g (90%) of light brown compound 2. 1 H NMR(400 MHz, Chloroform-d)δ10.04 (s,1H), 7.74 (d, J = 2.2 Hz, 1H), 7.23 (d, J = 8.8 Hz,1H), 6.88 (dd, J = 8.8,2.5 Hz, 1H), 3.88 (s, 3H), 2.72 (s, 3H). 13 CNMR (101 MHz, Chloroform-d) δ184.47,162.71, 156.40, 147.94, 130.22, 126.91, 113.07, 111.82, 102.88,55.87, 36.61. The structural formula of compound 2 is as follows:

[0033] The structural formulas of other compounds are shown in Table 1.

[0034] Table 1 Summary of compound structures ; ; ;

[0035] (Ethoxycarbonylmethyl)-3-formyl-5-methoxy-2-methylindole (3) Compound 2 (1.4 g, 7.40 mmol) and NaH (0.44 g, 60% mineral oil, 11.10 mmol) were dissolved in anhydrous DMF under N2 protection. The mixture was stirred at room temperature for 2 h. Subsequently, iodomethane (0.91 mL, 14.6 mmol) was added dropwise at 0°C and stirred at room temperature for another 2 h. The mixture was dissolved in DCM and extracted with water and NaCl solution in sequence. The mixture was dried over anhydrous Na2SO4 and the solvent was removed. The mixture was purified on a silica gel column and eluted with ethyl acetate / petroleum ether (1:2, Rf =0.3) to obtain 3.68 g (97.0%) of a white solid 3. 1 H NMR (400 MHz,Chloroform-d)δ10.09 (s,1H), 7.79 (d, J = 2.4 Hz, 1H), 7.19 (d, J = 8.8 Hz, 1H), 6.91 (dd,J = 8.8,2.5 Hz, 1H), 3.89 (s,3H), 3.68 (s,3H), 2.66 (s,3H). 13 C NMR (101 MHz, Chloroform-d) δ183.86, 162.56, 156.60, 147.92, 131.88, 126.31, 112.84, 109.97,102.97,55.88, 29.73, 10.42.

[0036] 4-Amino-1-(ethoxycarbonylformic acid methyl ester)-3-formyl-5-methoxy-2-methylindole (5) Compound 2 (1.4 g, 7.40 mmol) and NaH (0.44 g, 60% mineral oil, 11.10 mmol) were dissolved in anhydrous DMF under N2 protection. The mixture was stirred at room temperature for 2 h. Subsequently, iodomethane (0.91 mL, 14.6 mmol) was added dropwise at 0°C and stirred at room temperature for another 2 h. The mixture was dissolved in DCM and extracted with water and NaCl solution in sequence. The mixture was dried over anhydrous Na2SO4 and the solvent was removed. The mixture was purified on a silica gel column and eluted with ethyl acetate / petroleum ether (1:2, Rf =0.3) to obtain 3.68 g (97.0%) of a white solid 3. 1H NMR (400 MHz,Chloroform-d)δ10.09 (s,1H), 7.79 (d, J = 2.4 Hz, 1H), 7.19 (d, J = 8.8 Hz, 1H), 6.91 (dd,J = 8.8,2.5 Hz, 1H), 3.89 (s,3H), 3.68 (s,3H), 2.66 (s,3H). 13 C NMR (101 MHz, Chloroform-d) δ183.86, 162.56, 156.60, 147.92, 131.88, 126.31, 112.84, 109.97,102.97,55.88, 29.73, 10.42.

[0037] 1-(Ethoxycarbonylmethyl)-3-formyl-5-methoxy-2-methylindole-4,7-dione (6) Compound 5 (0.20 g, 0.68 mmol) was dissolved in acetone, and then NaH2PO4 / Na2HPO4 buffer (18.9 mL, 0.3 M, pH 6.0) was added, and Freund's salt (0.59 g, 2.2 mmol) was added under stirring. The reaction was carried out at room temperature for 3 h, monitored by TCL, extracted with DCM (100 mL×3), and the organic layers were combined, extracted with water and NaCl solution in turn, and dried with Na2SO4. The solvent was removed, and 6 (0.134 g, 84%) was obtained as a red solid without purification. 1 H NMR (400MHz, Chloroform-d) δ10.55 (s, 1H), 5.71 (s, 1H), 3.94 (s, 3H), 3.85 (s, 3H), 2.62 (s, 3H). 13 C NMR (10 MHz, Chloroform-d)δ188.34, 179.06, 177.87, 159.84, 142.71,120.00,106.75, 56.77, 32.26, 11.33.

[0038] 1-(Ethoxycarbonylmethyl)-3-(hydroxymethyl)-5-methoxy-2-methylindole-4,7-dione (7) Compound 6 (0.113 g, 0.48 mmol) was dissolved in an equal proportion of anhydrous methanol and anhydrous tetrahydrofuran. NaBH4 (0.91 g, 2.40 mmol) was added in an ice bath and stirred for 8 min. The reaction progress was monitored by TCL. The mixture was then quenched with saturated NH4Cl solution, extracted with DCM, and then washed with water and brine in sequence. The mixture was dried over anhydrous Na2SO4, concentrated, and purified by silica gel column. The mixture was eluted with EA / PE (1:1, Rf = 0.21) to obtain 0.41 g (67.7%) of 7 as an orange-red solid. 1 HNMR (400 MHz, Chloroform-d)δ5.64 (s, 1H), 4.62 (d, J = 7.1 Hz,2H), 3.89 (s,3H), 3.83 (s, 3H), 2.23 (s, 3H). 13 C NMR (101 MHz, Chloroform-d)δ179.69,179.05, 160.11, 135.05, 129.87, 123.20, 122.49, 107.53, 57.00, 56.34, 32.82,9.97.

[0039] Compound 8 In order to improve the rate and yield of compound 8, succinic acid, glutaric acid, adipic acid and 3,3'-dithiodipropionic acid were first prepared into anhydrides using oxalyl chloride: succinic acid (4 g, 11.8 mmol), glutaric acid (4 g, 13.2 mmol), adipic acid (4 g, 14.6 mmol) and 3,3'-dithiodipropionic acid (4 g, 19 mmol) were dissolved in 12 mL of oxalyl chloride, refluxed at 67 °C for 1.5 h, and concentrated under reduced pressure to remove the solvent. The residue was precipitated in cold ether (80 mL) for at least 4 h, and the mixture was filtered to remove the ether and dried at room temperature.

[0040] (1) 4-((5-methoxy-1,2-dimethyl-4,7-dioxo-4,7-dihydro-1H-indol-3-yl)methoxy)-4-oxobutanoic acid (8a) Succinic anhydride (85 mg, 0.85 mmol) and DMAP (51.9 mg, 0.43 mmol) were dissolved in anhydrous DCM, and the mixture was stirred at room temperature for 30 min, followed by the addition of compound 7 (102 mg, 0.43 mmol) and the reaction was continued for 12 h. The solvent was removed, and the residue was dissolved in DCM, extracted with water and NaCl solution in turn, dried with anhydrous Na2SO4, concentrated, purified on a silica gel column, and eluted with PE:EA (1:1, Rf=0.3) to finally obtain an orange-yellow solid compound 8a (68%). 1 H NMR (400MHz, Chloroform-d) δ 5.62 (s, 1H), 5.28 (s, 2H), 3.90 (s, 3H), 3.80 (s, 3H), 2.69~2.65 (m, 2H), 2.63~2.59 (m, 2H), 2.27 (s, 3H); 13 C NMR (101 MHz, Chloroform-d) δ 179.03, 177.74, 177.36, 172.20, 159.83, 138.11, 129.25,124.96,115.75,106.79, 57.06, 56.59, 32.55, 29.01, 28.80, 9.65 ppm.

[0041] (2) 5-((5-methoxy-1,2-dimethyl-4,7-dioxo-4,7-dihydro-1H-indol-3-yl)methoxy)-5-oxopentanyl (8b) Glutaric anhydride (119.7 mg, 1.05 mmol) and DMAP (42.7 mg, 0.35 mmol) were dissolved in anhydrous DCM, and the mixture was stirred at room temperature for 30 min, followed by the addition of compound 7 (84 mg, 0.35 mmol) and the reaction was continued for 12 h. The solvent was removed, and the residue was dissolved in DCM, extracted with water and NaCl solution in turn, dried with anhydrous Na2SO4, concentrated, purified on a silica gel column, and eluted with PE:EA (1:1, Rf=0.3) to finally obtain an orange-yellow solid compound 8b (72%). 1 H NMR(400 MHz, Chloroform-d) δ5.62 (s, 1H), 5.25 (s, 2H), 3.90 (s, 3H), 3.80 (s,3H), 2.42~2.37 (m, 4H), 2.28 (s, 3H), 1.97~1.92 (m, 2H); 13C NMR (101 MHz, Chloroform-d) δ 179.04, 178.31, 177.74, 172.98, 159.85, 137.99, 129.32,121.89, 115.88,106.79, 56.82, 56.59, 33.24, 33.04, 32.56, 20.02, 9.68 ppm.

[0042] (3) 6-((5-methoxy-1,2-dimethyl-4,7-dioxo-4,7-dihydro-1H-indol-3-yl)methoxy)-6-oxohexanoic acid (8c) Adipic anhydride (134.5 mg, 1.05 mmol) and DMAP (42.7 mg, 0.35 mmol) were dissolved in anhydrous DCM, and the mixture was stirred at room temperature for 30 min, followed by the addition of compound 7 (84 mg, 0.35 mmol) and the reaction was continued for 12 h. The solvent was removed, and the residue was dissolved in DCM, extracted with water and NaCl solution in turn, dried with anhydrous Na2SO4, concentrated, purified on a silica gel column, and eluted with PE:EA (1:1, Rf=0.3) to finally obtain an orange-yellow solid compound 8c (80%). 1 H NMR (400 MHz, DMSO-d6) δ 5.73 (s, 1H), 4.57 (s, 2H), 3.83 (s, 3H), 3.75 (s, 3H), 2.74~2.66 (m, 2H), 2.24 (s, 3H), 2.04~1.93 (m, 1H), 1.23 (s, 3H), 1.10 (t, J= 7.5 Hz, 2H); 13 C NMR (101 MHz, DMSO-d6) δ 177.99, 177.37, 159.19, 137.08,127.52, 121.67, 120.42,106.56, 56.37, 53.20, 37.96, 31.87, 30.89, 16.33,13.61, 9.15 ppm.

[0043] (4) 3-((3-((5-methoxy-1,2-dimethyl-4,7-dioxo-4,7-dihydro-1H-indol-3-yl)methoxy)-3-oxopropyl)disulfide propionic acid (8d) 3,3'-dithiodipropionic anhydride (191.4 mg, 1.05 mmol) and DMAP (42.7 mg, 0.35 mmol) were dissolved in anhydrous DCM, and the mixture was stirred at room temperature for 30 min, followed by the addition of compound 7 (84 mg, 0.35 mmol) and the reaction was continued for 12 h. The solvent was removed, and the residue was dissolved in DCM, extracted with water and NaCl solution in turn, dried over anhydrous Na2SO4, concentrated, purified on a silica gel column, and eluted with PE:EA (1:1, Rf=0.3) to finally obtain an orange-yellow viscous solid compound 8d (70%). 1 H NMR (400 MHz, DMSO-d6) δ5.73 (s, 1H), 4.56 (s, 2H), 3.83 (s, 3H), 3.75 (s, 3H), 2.75~2.65 (m, 2H), 2.24 (s, 3H), 1.23 (s, 2H), 1.10 (t, J = 7.5Hz, 2H); 13 C NMR (101 MHz, DMSO-d6) δ 177.98, 177.38, 159.20, 142.28, 137.09,127.53, 121.66, 120.42, 106.55,56.37, 53.21, 31.88, 16.34, 13.61, 9.15 ppm.

[0044] (1) (5-methoxy-1,2-dimethyl-4,7-dioxo-4,7-dihydro-1H-indol-3-yl)methyl 4-(14-methyl-5-oxo-5,7,8,13,13b,14-hexahydroindol[2,3':3,4]pyrido[2,1-b]quinazolin-2-yl)amino)-4-oxobutanoate (9a) 2-Aminoevodiamine (100 mg, 0.314 mmol) was dissolved in 5 mL of anhydrous DCM, followed by the addition of compound 8a (105 mg, 0.314 mmol) and EDCI (300.98 mg, 1.57 mmol), and the mixture was cooled to 0°C and DMAP (38.36 mg, 0.314 mmol) was added, followed by stirring at room temperature for 6-8 h, monitored by TCL, and after the reaction was completed, the solvent was removed, the mixture was dissolved in DCM, washed with water and saturated brine, dried over anhydrous Na2SO4, concentrated under reduced pressure, separated and purified on a silica gel column, and eluted with DCM / CH3OH (6:1-1:1). Yellow solid compound 9a (yield 51%) was obtained. 1H NMR (400MHz, DMSO-d6) δ 10.96 (s, 1H), 10.17 (d, J = 16.5 Hz, 1H), 7.69–7.58 (m, 1H), 7.50–7.33 (m, 3H), 7.12 – 6.91 (m, 3H), 6.13 (s, 1H), 5.73 (s, 1H), 5.15 (s,2H), 3.76 (d, J = 18.3 Hz, 6H), 3.56 (d, J = 39.3 Hz, 2H), 3.18 (s, 2H), 2.96(s, 3H), 2.59 (s,4H), 2.21 (s, 3H); 1 3C NMR (101 MHz, DMSO-d6) δ 178.61,177.54, 172.56, 170.83,164.67, 159.68, 149.54, 144.34, 139.03, 136.82,131.79, 129.23, 128.63, 126.54, 122.23,121.30, 119.33, 118.57, 115.21,113.73, 112.09, 111.79, 110.87, 107.13, 105.84, 70.85,56.95, 56.72, 51.85,36.99, 32.53, 31.63, 29.07, 19.86, 9.54;MS (ESI, positive) found(M+H):636.24, calc (C 35 H 33 N5O7, m / z): 635.23.

[0045] (2) (5-methoxy-1,2-dimethyl-4,7-dioxo-4,7-dihydro-1H-indol-3-yl)methyl 5-(14-methyl-5-oxo-5,7,8,13,13b,14-hexahydroindol[2,3:3,4]pyrido[2,1-b]quinazolin-2-yl)amino)-5-oxopentanoate (9b) 2-Aminoevodiamine (100 mg, 0.314 mmol) was dissolved in 5 mL of anhydrous DCM, followed by the addition of compound 8b (109 mg, 0.314 mmol) and EDCI (300.98 mg, 1.57 mmol), and the mixture was cooled to 0°C and DMAP (38.36 mg, 0.314 mmol) was added, followed by stirring at room temperature for 6-8 h, monitored by TCL, and after the reaction was completed, the solvent was removed, the mixture was dissolved in DCM, washed with water and saturated brine, dried over anhydrous Na2SO4, concentrated under reduced pressure, separated and purified on a silica gel column, and eluted with DCM / CH3OH (6:1-1:1). Yellow solid compound 9b (yield 65%) was obtained. 1 H NMR (400MHz, DMSO-d6) δ 10.98 (s, 1H), 10.10 (d, J = 9.0 Hz, 1H), 7.67 (d, J= 8.5 Hz,1H), 7.52 (s, 1H), 7.44 (d, J = 7.8 Hz, 1H), 7.35 (d, J = 8.1 Hz, 1H), 7.10(t, J =7.5 Hz, 1H), 6.98 (q, J = 9.7, 8.7 Hz, 2H), 6.13 (s, 1H), 5.15 (s,1H), 3.82 (s, 1H), 3.73 (s,1H), 3.60 (s, 3H), 3.35 (s, 3H), 2.96 (s, 5H), 2.50 (s, 2H), 2.42 2.28 (m, 5H), 2.23 (s, 1H), 1.85 (p, J = 7.4 Hz, 3H); 13C NMR(101 MHz, DMSO-d6)δ178.65, 177.54, 173.48,172.91, 171.65, 164.66, 159.70,149.61, 144.39, 138.96, 136.84, 131.73, 129.24, 128.71,126.54, 122.23,121.31, 119.33, 118.58, 115.26, 113.87, 112.09, 111.80, 111.08, 107.16,106.10, 70.82, 56.95, 56.67, 51.73, 33.23, 33.08, 20.78, 20.73, 19.87, 9.54.MS (ESI,positive) found (M+H): 650.26, calc (C 36 H 35 N5O7, m / z): 649.69.

[0046] (3) (5-methoxy-1,2-dimethyl-4,7-dioxo-4,7-dihydro-1H-indol-3-yl)methyl 6-(14-methyl-5-oxo-5,7,8,13,13b,14-hexahydroindol[2',3':3,4]pyrido[2,1-b]quinazolin-2-yl)amino)-6-oxohexanoate (9c) 2-aminoevodiamine (100 mg, 0.314 mmol) was dissolved in 5 mL of anhydrous DCM, followed by the addition of compound 8c (114 mg, 0.314 mmol) and EDCI (300.98 mg, 1.57 mmol), and the mixture was cooled to 0°C and DMAP (38.36 mg, 0.314 mmol) was added, followed by stirring at room temperature for 6-8 h, monitored by TCL, and after the reaction was completed, the solvent was removed, the mixture was dissolved in DCM, washed with water and saturated brine, dried over anhydrous Na2SO4, concentrated under reduced pressure, separated and purified on a silica gel column, and eluted with DCM / CH3OH (6:1-1:1). Yellow solid compound 9c was obtained (yield 60%). 1H NMR (400MHz, DMSO-d6)δ10.96 (s, 1H), 10.07 (d, J = 8.9 Hz, 1H), 7.66 (d, J= 8.4 Hz,1H), 7.52 (s, 1H), 7.44 (d, J = 7.7 Hz, 1H), 7.35 (d, J = 8.0 Hz, 1H), 7.09(t, J =7.4 Hz, 1H), 7.03–6.94 (m, 2H), 6.13 (s, 1H), 5.75 (d, J = 4.2 Hz,1H), 5.13 (s, 2H), 3.82(s, 3H), 3.73 (s, 3H), 3.24–3.13 (m, 2H), 2.96 (s,3H), 2.78–2.64 (m, 2H), 2.30 (d, J =10.6 Hz, 4H), 2.22 (s, 3H), 1.56 (s,4H).; 13 C NMR (101 MHz, DMSO-d6)δ178.65,177.52, 173.12, 172.02, 164.67,159.69, 149.58, 144.44, 138.96, 136.83, 131.76, 129.23,128.70, 126.54,122.23, 121.31, 119.33, 118.58, 115.31, 113.79, 112.08, 111.79, 111.02,107.16, 106.01, 70.83, 56.96, 56.57, 41.65, 37.00, 33.62, 32.57, 24.86,24.55, 19.87, 9.53.MS (ESI, positive) found (M+H): 664.27, calc (C 37 H 37 N5O7, m / z): 663.72.

[0047] (4) (5-methoxy-1,2-dimethyl-4,7-dioxo-4,7-dihydro-1H-indol-3-yl)methyl 3-((3-((14-methyl-5-oxo-5,7,8,13,13b,14-hexahydroindol[2',3':3,4]pyrido[2,1-b]quinazolin-2-yl)amino)-3-oxopropyl)disulfide)propanoate (9d) 2-Aminoevodiamine (100 mg, 0.314 mmol) was dissolved in 5 mL of anhydrous DCM, followed by the addition of compound 8d (134 mg, 0.314 mmol) and EDCI (300.98 mg, 1.57 mmol), and the mixture was cooled to 0°C and DMAP (38.36 mg, 0.314 mmol) was added, followed by stirring at room temperature for 6-8 h, monitored by TCL, and after the reaction was completed, the solvent was removed, the mixture was dissolved in DCM, washed with water and saturated brine, dried over anhydrous Na2SO4, concentrated under reduced pressure, separated and purified on a silica gel column, and eluted with DCM / CH3OH (6:1-1:1). Yellow solid compound 9d was obtained (yield 52%). 1 H NMR (400MHz, DMSO-d6)δ10.96 (s, 1H), 10.21 (s, 1H), 7.66 (d, J = 8.5 Hz,1H), 7.50 (s,1H), 7.44 (d, J = 7.7 Hz, 1H), 7.34 (d, J = 8.1 Hz, 1H), 7.09 (t, J = 7.5 Hz, 1H), 7.01–6.94 (m, 2H), 6.13 (s, 1H), 5.75 (d, J = 1.8 Hz, 2H), 5.17 (s, 2H), 3.82 (s, 3H), 3.74 (s, 3H), 3.19 (td, J = 12.4, 4.6 Hz, 2H), 2.97–2.90 (m,9H), 2.73–2.66 (m, 4H), 2.22(s, 3H); 13 C NMR (101 MHz, DMSO-d6)δ178.66,177.56, 171.53, 170.11, 164.63, 159.70,149.55, 144.20, 139.08, 136.82,131.76, 129.29, 128.70, 126.54, 122.24, 121.32, 119.34,118.58, 115.06,113.91, 112.09, 111.80, 111.01, 107.17, 70.83, 56.98, 36.99, 36.61, 33.89,33.24, 32.59, 19.86, 9.58. MS (ESI, positive) found (M+H): 728.22, calc(C 37 H 37N5O7S2,m / z): 727.85.

[0048] (1) (5-methoxy-1,2-dimethyl-4,7-dioxo-4,7-dihydro-1H-indol-3-yl)methyl 4-(10-methoxy-14-methyl-5-oxo-5,7,8,13,13b,14-hexahydroindol[2',3':3,4]pyrido[2,1-b]quinazolin-2-yl)amino)-4-oxobutanoate (10a) 2-Amino-10-methoxyevodiamine (100 mg, 0.287 mmol) was dissolved in 5 mL of anhydrous DCM, followed by the addition of compound 8a (96 mg, 0.287 mmol) and EDCI (275.1 mg, 1.435 mmol), and the mixture was cooled to 0°C and DMAP (35.06 mg, 0.287 mmol) was added, followed by stirring at room temperature for 6-8 h, monitored by TCL, and after the reaction was completed, the solvent was removed, the mixture was dissolved in DCM, washed with water and saturated brine, dried over anhydrous Na2SO4, concentrated under reduced pressure, separated and purified on a silica gel column, and eluted with DCM / CH3OH (6:1-1:1). A brown solid compound 10a (yield 64%) was obtained. 1 H NMR (400 MHz, DMSO-d6)δ11.00 (s, 1H), 9.96 (s, 1H), 8.01 (s,1H), 7.69 (d, J= 11.1 Hz, 1H), 7.25 (d, J = 8.8 Hz, 1H), 7.05 (d, J = 8.8 Hz,1H), 7.01–6.97 (m, 1H),6.75 (dd, J = 8.7, 2.3 Hz, 1H), 6.01 (s, 1H), 5.75 (s,1H), 5.14 (s, 2H), 3.78 (s, 3H), 3.76 (s,3H), 3.73 (s, 3H), 3.22–3.13 (m, 2H), 2.87–2.78 (m, 3H), 2.63 (s, 2H), 2.55 (s, 4H), 2.20 (s, 3H). 13C NMR (101MHz, DMSO-d6)δ178.60, 177.52, 172.56, 169.93, 164.27, 159.65,153.82, 145.52,139.00, 134.02, 132.18, 130.89, 128.60, 126.59, 124.88, 121.49, 121.27,120.35, 118.66, 115.24, 112.75, 111.84, 107.10, 100.65, 70.26, 69.73, 56.92,56.61, 55.84,55.35, 36.95, 32.52, 31.34, 29.27, 20.21, 9.53. MS (ESI,positive) found (M+H): 666.25,calc (C 36 H 35 N5O8, m / z): 665.69.

[0049] (2) (5-methoxy-1,2-dimethyl-4,7-dioxo-4,7-dihydro-1H-indol-3-yl)methyl 5-((10-methoxy-14-methyl-5-oxo-5,7,8,13,13b,14-hexahydroindol[2',3':3,4]pyrido[2,1-b]quinazolin-2-yl)amino)-5-oxopentanoate (10b) 2-Amino-10-methoxyevodiamine (100 mg, 0.287 mmol) was dissolved in 5 mL of anhydrous DCM, followed by the addition of compound 8b (100 mg, 0.287 mmol) and EDCI (275.1 mg, 1.435 mmol), and the mixture was cooled to 0°C and DMAP (35.06 mg, 0.287 mmol) was added, followed by stirring at room temperature for 6-8 h, monitored by TCL, and after the reaction was completed, the solvent was removed, the mixture was dissolved in DCM, washed with water and saturated brine, dried over anhydrous Na2SO4, concentrated under reduced pressure, separated and purified on a silica gel column, and eluted with DCM / CH3OH (6:1-1:1). A brown solid compound 10b (yield 54%) was obtained. 1H NMR (400 MHz, DMSO-d6)δ10.99 (s, 1H), 9.91 (d, J = 11.2 Hz, 1H), 8.03 (dd, J= 6.0, 2.4 Hz, 1H), 7.78– 7.72 (m, 1H), 7.24 (d, J = 8.8 Hz, 1H),7.08 (d, J = 8.7 Hz, 1H),6.99 (d, J = 2.1 Hz, 1H), 6.76 (d, J = 8.8 Hz, 1H),6.01 (s, 1H), 5.75 (s, 2H), 5.15 (s, 1H),3.85–3.71 (m, 6H), 3.59 (s, 3H),3.21–3.12 (m, 2H), 2.86 2.79 (m, 2H), 2.63 (s, 3H), 2.39–2.28 (m, 5H), 2.23 (s, 1H), 1.83 (s, 2H), 1.23 (s, 1H). 13 C NMR(101 MHz, DMSO-d6)δ178.67, 173.50, 172.93, 170.80, 164.30, 159.71, 153.82,145.57, 139.01,133.99, 132.16, 130.92, 126.59, 125.15, 121.44, 120.33,118.87, 112.75, 112.45, 111.83,107.16, 100.65, 69.76, 56.96, 56.65, 55.84,55.36, 51.72, 36.97, 35.63, 33.11, 32.59, 20.90,20.19, 9.56. MS (ESI,positive) found (M+H): 680.27, calc (C 37 H 37 N5O8, m / z): 679.72.

[0050] (3) (5-methoxy-1,2-dimethyl-4,7-dioxo-4,7-dihydro-1H-indol-3-yl)methyl 6-((10-methoxy-14-methyl-5-oxo-5,7,8,13,13b,14-hexahydroindol[2',3':3,4]pyrido[2,1-b]quinazolin-2-yl)amino)-6-oxohexanoate (10c) 2-Amino-10-methoxyevodiamine (100 mg, 0.287 mmol) was dissolved in 5 mL of anhydrous DCM, followed by the addition of compound 8c (104 mg, 0.287 mmol) and EDCI (275.1 mg, 1.435 mmol), and the mixture was cooled to 0°C and DMAP (35.06 mg, 0.287 mmol) was added, followed by stirring at room temperature for 6-8 h, monitored by TCL, and after the reaction was completed, the solvent was removed, the mixture was dissolved in DCM, washed with water and saturated brine, dried over anhydrous Na2SO4, concentrated under reduced pressure, separated and purified on a silica gel column, and eluted with DCM / CH3OH (6:1-1:1). A brown solid compound 10c (yield 54%) was obtained. 1 H NMR (400 MHz, DMSO-d6)δ10.96 (s, 1H), 10.06 (s, 1H), 7.65 (d, J =8.5 Hz,1H), 7.52 (s, 1H), 7.34 (d, J = 8.1 Hz, 1H), 7.09 (t, J = 7.5 Hz, 1H),6.97 (dd, J = 11.5, 7.8Hz, 2H), 6.13 (s, 1H), 5.75 (s, 1H), 5.14 (s, 2H),3.83 (s, 3H), 3.73 (s, 3H), 3.59 (s, 1H),3.19 (dq, J = 12.2, 4.2, 3.7 Hz,1H), 2.93 (d, J = 20.2 Hz, 5H), 2.76–2.69 (m, 1H), 2.35–2.26 (m, 4H), 2.23 (s, 3H), 1.61–1.51 (m, 4H). 13 C NMR (101 MHz, DMSO-d6)δ178.67,173.13, 172.04,164.67, 159.71, 149.58, 139.00, 136.82, 131.78, 129.23, 126.54, 119.33,118.59, MS (ESI, positive) found (M+H):694.29, calc (C 38 H 39N5O8,m / z): 693.74.

[0051] (4) (5-methoxy-1,2-dimethyl-4,7-dioxo-4,7-dihydro-1H-indol-3-yl)methyl 3-((3-((10-methoxy-14-methyl-5-oxo-5,7,8,13,13b,14-hexahydroindol[2',3':3,4]pyrido[2,1-b]quinazolin-2-yl)amino)-3-oxopropyl)disulfonyl)propanoate (10d) 2-Amino-10-methoxyevodiamine (100 mg, 0.287 mmol) was dissolved in 5 mL of anhydrous DCM, followed by the addition of compound 8d (122 mg, 0.287 mmol) and EDCI (275.1 mg, 1.435 mmol), and the mixture was cooled to 0°C and DMAP (35.06 mg, 0.287 mmol) was added, followed by stirring at room temperature for 6-8 h, monitored by TCL, and after the reaction was completed, the solvent was removed, the mixture was dissolved in DCM, washed with water and saturated brine, dried over anhydrous Na2SO4, concentrated under reduced pressure, separated and purified on a silica gel column, and eluted with DCM / CH3OH (6:1-1:1). A brown solid compound 10d (yield 57%) was obtained. 1 H NMR (400 MHz, DMSO-d6)δ10.99 (s, 1H), 10.03 (s, 1H), 8.03 (d, J =2.2 Hz,1H), 7.75 (dd, J = 8.7, 2.3 Hz, 1H), 7.24 (d, J = 8.7 Hz, 1H), 7.07(d, J = 8.7 Hz, 1H), 6.98(s, 1H), 6.75 (d, J = 10.9 Hz, 1H), 6.01 (s, 1H), 5.74 (d, J = 5.1 Hz, 1H), 5.16 (s, 2H), 3.82(s, 3H), 3.74 (d, J = 7.0 Hz, 6H), 3.21–3.12 (m, 2H), 3.00–2.87 (m, 6H), 2.81 (s, 3H), 2.50 (s, 4H), 2.22 (s, 3H). 13C NMR (101 MHz, DMSO-d6)δ178.64, 177.54,171.53,169.23, 164.27, 159.70, 153.81, 145.64, 139.08, 133.75, 132.16,130.93, 128.67, 126.59,125.13, 121.30, 120.23, 118.89, 115.06, 112.75,112.45, 111.83, 107.15, 100.63, 69.78,56.97, 55.84, 36.95, 36.34, 34.05,33.89, 33.27, 32.58, 20.19, 9.59. MS (ESI, positive)found (M+H): 758.23, calc(C 38 H 39 N5O8S2, m / z): 757.87.

[0052] (1) (5-methoxy-1,2-dimethyl-4,7-dioxo-4,7-dihydro-1H-indol-3-yl)methyl 4-(14-methyl-5-oxo-5,7,8,13,13b,14-hexahydroindol[2',3':3,4]pyrido[2,1-b]quinazolin-3-yl)amino)-4-oxobutanoate (11a) 3-Aminoevodiamine (100 mg, 0.314 mmol) was dissolved in 5 mL of anhydrous DCM, followed by the addition of compound 8a (105 mg, 0.314 mmol) and EDCI (300.98 mg, 1.57 mmol), and the mixture was cooled to 0°C and DMAP (38.36 mg, 0.314 mmol) was added, followed by stirring at room temperature for 6-8 h and monitoring by TCL. After the reaction was completed, the solvent was removed and the mixture was dissolved in DCM, washed with water and saturated brine, dried over anhydrous Na2SO4, concentrated under reduced pressure, separated and purified on a silica gel column, and eluted with DCM / CH3OH (6:1-1:1). Yellow solid compound 11a was obtained (yield 57%). 1H NMR (400 MHz, DMSO-d6) δ11.20 (s, 1H), 9.98 (s, 1H), 8.02 (d, J = 2.6 Hz, 1H), 7.70 (d, J = 8.7 Hz, 1H), 7.50 (d, J = 7.8 Hz, 1H), 7.37 (d, J = 8.1 Hz, 1H),7.14–7.08 (m,2H), 7.03 (d, J = 7.4 Hz, 1H), 6.04 (s, 1H), 5.72 (s, 1H), 5.16(s, 3H), 3.79 (s, 3H), 3.73 (s,3H), 3.21 –3.16 (m, 2H), 2.85 (d, J = 7.2 Hz,2H), 2.62 (s, 3H), 2.55 (s, 4H), 2.21 (s, 3H). 13 C NMR (101 MHz, DMSO-d6)δ178.61, 177.55, 172.56, 170.83, 164.67, 159.68, 149.55,144.34, 139.03,136.83, 131.79, 129.23, 128.63, 126.54, 122.23, 121.30, 119.33, 118.57,115.21, 113.73, 112.09, 111.79, 110.87, 107.13, 105.84, 70.85, 56.95, 56.72,51.85, 36.99,32.53, 31.63, 29.07, 28.84, 19.86, 9.53. MS (ESI, positive)found (M+H): 636.24, calc(C 35 H 33 N5O7, m / z): 635.67.

[0053] (2) (5-methoxy-1,2-dimethyl-4,7-dioxo-4,7-dihydro-1H-indol-3-yl)methyl 5-(14-methyl-5-oxo-5,7,8,13,13b,14-hexahydroindol[2,3:3,4]pyrido[2,1-b]quinazoline-3-amino)-5-oxopentanoate (11b) 3-Aminoevodiamine (100 mg, 0.314 mmol) was dissolved in 5 mL of anhydrous DCM, followed by the addition of compound 8b (109 mg, 0.314 mmol) and EDCI (300.98 mg, 1.57 mmol), and the mixture was cooled to 0°C and DMAP (38.36 mg, 0.314 mmol) was added, followed by stirring at room temperature for 6-8 h, monitored by TCL, and after the reaction was completed, the solvent was removed, the mixture was dissolved in DCM, washed with water and saturated brine, dried over anhydrous Na2SO4, concentrated under reduced pressure, separated and purified on a silica gel column, and eluted with DCM / CH3OH (6:1-1:1). Yellow solid compound 11b was obtained (yield 65%). 1 H NMR (400MHz, Chloroform-d)δ8.58 (s, 1H), 8.22–8.14 (m, 2H), 7.60 (d, J =7.8 Hz, 1H),7.52 (d, J = 8.2 Hz, 1H), 7.32 (t, J = 7.6 Hz, 1H), 7.19 (t, J = 7.5 Hz, 1H),6.40 (s, 1H), 5.64 (s, 1H), 5.20 (s, 2H), 3.92 (s, 3H), 3.79 (s, 3H), 3.31(td, J = 12.8, 12.3,4.3 Hz, 2H), 3.10– 3.00 (m, 2H), 2.66 (s, 3H), 2.50 (t, J= 7.5 Hz, 2H), 2.46–2.40 (m,2H), 2.31 (s, 3H), 2.09–2.04 (m, 2H). 13C NMR (101MHz, DMSO-d6)δ178.24, 177.12,172.49, 170.38, 163.86, 159.26, 146.84, 145.15,138.71 (d, J = 25.0 Hz), 136.66, 133.61,129.83, 128.26, 125.83, 124.68,121.88, 121.08, 120.85, 120.03, 118.87, 118.39, 114.83,111.59, 106.72, 69.23,56.52, 56.20, 36.56, 35.14, 32.80, 32.15, 20.49, 19.65, 9.11. MS(ESI,positive) found (M+H): 649.22, calc (C 36 H 35 N5O7, m / z): 649.69.

[0054] (3) (5-methoxy-1,2-dimethyl-4,7-dioxo-4,7-dihydro-1H-indol-3-yl)methyl 6-(14-methyl-5-oxo-5,7,8,13,13b,14-hexahydroindol[2,3:3,4]pyrido[2,1-b]quinazolin-3-yl)amino)-6-oxohexanoate (11c) 3-Aminoevodiamine (100 mg, 0.314 mmol) was dissolved in 5 mL of anhydrous DCM, followed by the addition of compound 8c (114 mg, 0.314 mmol) and EDCI (300.98 mg, 1.57 mmol), and the mixture was cooled to 0°C and DMAP (38.36 mg, 0.314 mmol) was added, followed by stirring at room temperature for 6-8 h, monitored by TCL, and after the reaction was completed, the solvent was removed, the mixture was dissolved in DCM, washed with water and saturated brine, dried over anhydrous Na2SO4, concentrated under reduced pressure, separated and purified on a silica gel column, and eluted with DCM / CH3OH (6:1-1:1). Yellow solid compound 11c was obtained (yield 70%). 1H NMR (400MHz, DMSO-d6)δ11.19 (s, 1H), 9.90 (s, 1H), 8.04 (s, 1H), 7.76 (dd, J= 8.7,2.3 Hz, 1H), 7.50 (d, J = 7.6 Hz, 1H), 7.36 (d, J = 8.1 Hz, 1H), 7.11 (dd, J= 14.4,8.0 Hz, 3H), 6.04 (s, 1H), 5.75 (d, J = 2.5 Hz, 1H), 5.14 (s, 2H),3.83 (s, 3H), 3.73 (s, 3H),2.87–2.82 (m, 2H), 2.62 (s, 3H), 2.28 (d, J = 6.0Hz, 5H), 2.23 (s, 3H), 1.59–1.51 (m, 4H). 13 C NMR (101 MHz, DMSO-d6)δ178.65,177.52, 173.12, 172.02, 164.67, 159.69,149.58, 144.44, 138.96, 136.83,131.76, 129.23, 128.70, 126.54, 122.23, 121.31, 119.33,118.58, 115.31,113.79, 112.08, 111.79, 111.02, 107.16, 106.01, 70.83, 56.96, 56.57, 37.00,36.61, 33.62, 32.57, 24.86, 24.55, 19.87, 9.53. MS (ESI, positive) found (M+2H): 665.28,calc (C 37 H 37 N5O7, m / z): 663.72.

[0055] (4) (5-methoxy-1,2-dimethyl-4,7-dioxo-4,7-dihydro-1H-indol-3-yl)methyl 3-((3-((14-methyl-5-oxo-5,7,8,13,13b,14-hexahydroindol[2',3':3,4]pyrido[2,1-b]quinazolin-3-yl)amino)-3-oxopropyl)disulfonyl)propanoate (11d) 3-Aminoevodiamine (100 mg, 0.314 mmol) was dissolved in 5 mL of anhydrous DCM, followed by the addition of compound 8d (134 mg, 0.314 mmol) and EDCI (300.98 mg, 1.57 mmol), and the mixture was cooled to 0°C and DMAP (38.36 mg, 0.314 mmol) was added, followed by stirring at room temperature for 6-8 h, monitored by TCL, and after the reaction was completed, the solvent was removed, the mixture was dissolved in DCM, washed with water and saturated brine, dried over anhydrous Na2SO4, concentrated under reduced pressure, separated and purified on a silica gel column, and eluted with DCM / CH3OH (6:1-1:1). Yellow solid compound 11d was obtained (yield 59%). 1 H NMR (400MHz, DMSO-d6)δ11.18 (s, 1H), 10.05 (s, 1H), 8.04 (s, 1H), 7.76 (d, J= 11.1Hz, 1H), 7.49 (d, J = 7.8 Hz, 1H), 7.36 (d, J = 8.1 Hz, 1H), 7.14–7.07 (m,2H),7.01 (t, J = 7.4 Hz, 1H), 6.04 (s, 1H), 5.73 (s, 1H), 5.16 (s, 2H), 3.81(s, 3H), 3.73 (s, 3H),3.36 (s, 6H), 3.23–3.12 (m, 1H), 2.94 (dt, J = 14.7,6.9 Hz, 4H), 2.68 (t, J = 6.5 Hz, 4H), 2.22 (s, 3H). 13 C NMR (101 MHz, DMSO-d6)δ178.62, 177.53, 171.53, 169.25, 164.27,159.68, 145.68, 139.07, 137.11,133.87, 130.27, 128.66, 126.28, 125.10, 122.33, 121.48,121.29, 120.42,119.30, 118.87, 118.74, 115.05, 112.08, 112.03, 107.15, 69.69, 56.96,36.99,36.34, 34.04, 33.88, 33.26, 32.58, 20.11, 9.59. MS (ESI, positive) found (MH):726.20, calc (C 37 H37 N5O7S2, m / z): 727.85.

[0056] (1) (5-methoxy-1,2-dimethyl-4,7-dioxo-4,7-dihydro-1H-indol-3-yl)methyl 4-(10-methoxy-14-methyl-5-oxo-5,7,8,13,13b,14-hexahydroindol[2',3':3,4]pyrido[2,1-b]quinazolin-3-yl)amino)-4-oxobutanoate (12a) 3-Amino-10-methoxyevodiamine (100 mg, 0.287 mmol) was dissolved in 5 mL of anhydrous DCM, followed by the addition of compound 8a (96 mg, 0.287 mmol) and EDCI (275.1 mg, 1.435 mmol), and the mixture was cooled to 0°C and DMAP (35.06 mg, 0.287 mmol) was added, followed by stirring at room temperature for 6-8 h, monitored by TCL, and after the reaction was completed, the solvent was removed, the mixture was dissolved in DCM, washed with water and saturated brine, dried over anhydrous Na2SO4, concentrated under reduced pressure, separated and purified on a silica gel column, and eluted with DCM / CH3OH (6:1-1:1). A brown solid compound 12a (yield 66%) was obtained. 1 H NMR (400 MHz, DMSO-d6)δ11.01 (s, 1H), 9.99 (d, J = 21.4 Hz, 1H), 8.03 (d, J= 16.4 Hz, 1H), 7.75–7.66 (m, 1H), 7.25 (d, J = 9.6 Hz, 1H), 7.07 (t, J= 8.9 Hz, 1H), 6.99(s, 1H), 6.76 (dd, J = 8.8, 2.3 Hz, 1H), 6.58 (d, J = 5.4Hz, 1H), 6.01 (s, 1H), 5.73 (d, J =14.7 Hz, 1H), 5.15 (s, 1H), 3.79 (s, 2H), 3.74 (d, J = 9.8 Hz, 5H), 3.59 (d, J = 6.5 Hz, 2H), 3.18 (q, J = 7.9, 5.7 Hz, 3H), 2.94 (s, 5H), 2.85–2.80 (m, 2H), 2.59 (s, 2H), 2.55 (s, 2H), 2.21 (s, 2H). 13C NMR (101 MHz, DMSO-d6) δ 178.62, 177.55, 173.29, 172.57, 170.01,169.94, 164.28, 159.67, 153.82, 149.57, 145.53, 139.03, 132.17, 130.89,128.62, 126.59,121.49, 121.28, 120.36, 118.74, 118.66, 115.25, 112.75,112.46, 111.84, 107.12, 100.65,69.74, 56.93, 56.62, 55.84, 51.81, 32.54,31.22, 29.27, 29.01, 20.20, 9.54. MS (ESI,positive) found (M+H): 666.25, calc(C 36 H 35 N5O8, m / z): 665.69.

[0057] (2) (5-methoxy-1,2-dimethyl-4,7-dioxo-4,7-dihydro-1H-indol-3-yl)methyl 5-(10-methoxy-14-methyl-5-oxo-5,7,8,13,13b,14-hexahydroindol[2',3':3,4]pyrido[2,1-b]quinazolin-3-yl)amino)-5-oxopentanoate (12b) 3-Amino-10-methoxyevodiamine (100 mg, 0.287 mmol) was dissolved in 5 mL of anhydrous DCM, followed by the addition of compound 8b (100 mg, 0.287 mmol) and EDCI (275.1 mg, 1.435 mmol), and the mixture was cooled to 0°C and DMAP (35.06 mg, 0.287 mmol) was added, followed by stirring at room temperature for 6-8 h, monitored by TCL, and after the reaction was completed, the solvent was removed, the mixture was dissolved in DCM, washed with water and saturated brine, dried over anhydrous Na2SO4, concentrated under reduced pressure, separated and purified on a silica gel column, and eluted with DCM / CH3OH (6:1-1:1). A brown solid compound 12b (yield 59%) was obtained. 1H NMR (400 MHz, DMSO-d6)δ11.00 (s, 1H), 9.90 (d, J = 9.4 Hz, 1H), 8.03(d, J =2.3 Hz, 1H), 7.79–7.73 (m, 1H), 7.24 (d, J = 8.8 Hz, 1H), 7.07 (d, J =8.7 Hz, 1H), 6.99 (d,J = 2.1 Hz, 1H), 6.75 (dd, J = 8.8, 2.3 Hz, 1H), 6.01(s, 1H), 5.74 (d, J = 4.9 Hz, 1H), 5.13(s, 2H), 3.82 (s, 2H), 3.74 (d, J =10.0 Hz, 6H), 3.21–3.12 (m, 2H), 2.85–2.79 (m, 2H), 2.63 (s, 3H), 2.27 (q, J = 7.3 Hz, 4H), 2.22 (s, 3H). 13 C NMR (101MHz, DMSO-d6) δ178.65, 177.53, 173.14, 171.18, 164.30, 159.70, 153.81,145.54, 138.99, 134.08, 132.17,130.91, 128.69, 126.58, 125.09, 121.47,121.30, 120.36, 118.81, 115.32, 112.75, 112.45,111.83, 107.16, 100.64, 69.76,56.96, 56.56, 55.84, 36.97, 36.35, 33.62, 32.58, 25.00,24.56, 20.20, 9.55. MS(ESI, positive) found (M+): 679.51, calc (C 37 H 37 N5O8, m / z):679.72.

[0058] (3) (5-methoxy-1,2-dimethyl-4,7-dioxo-4,7-dihydro-1H-indol-3-yl)methyl 6-(10-methoxy-14-methyl-5-oxo-5,7,8,13,13b,14-hexahydroindol[2',3':3,4]pyrido[2,1-b]quinazolin-3-yl)amino)-6-oxohexanoate (12c) 3-Amino-10-methoxyevodiamine (100 mg, 0.287 mmol) was dissolved in 5 mL of anhydrous DCM, followed by the addition of compound 8c (104 mg, 0.287 mmol) and EDCI (275.1 mg, 1.435 mmol), and the mixture was cooled to 0°C and DMAP (35.06 mg, 0.287 mmol) was added, followed by stirring at room temperature for 6-8 h, monitored by TCL, and after the reaction was completed, the solvent was removed, the mixture was dissolved in DCM, washed with water and saturated brine, dried over anhydrous Na2SO4, concentrated under reduced pressure, separated and purified on a silica gel column, and eluted with DCM / CH3OH (6:1-1:1). A brown solid compound 12c (yield 64%) was obtained. 1 H NMR (400 MHz, DMSO-d6)δ10.99 (s, 1H), 10.03 (s, 1H), 8.03 (d, J= 2.3 Hz,1H), 7.75 (dd, J = 8.7, 2.3 Hz, 1H), 7.24 (d, J = 8.8 Hz, 1H), 7.08(d, J = 8.8 Hz, 1H), 6.98(d, J = 1.9 Hz, 1H), 6.75 (dd, J = 8.8, 2.2 Hz, 1H), 6.02 (s, 1H), 5.75 (d, J = 2.0 Hz, 1H), 5.17 (s, 2H), 3.83 (s, 3H), 3.75 (d, J= 5.9 Hz, 6H), 3.17 (d, J = 5.1 Hz, 2H), 2.94 (dt, J =14.7, 6.9 Hz, 5H), 2.71–2.63 (m, 8H), 2.23 (s, 3H). 13C NMR (101 MHz, DMSO-d6)δ178.66, 177.56,169.23, 164.26, 159.71, 153.81, 145.64, 139.10, 133.76, 132.16, 130.93,128.68, 126.59, 125.13, 121.36, 120.25, 118.89, 115.07, 112.75, 112.45,111.83, 107.17,100.63, 69.77, 56.98, 55.84, 36.95, 36.34, 34.05, 33.88,33.26, 32.60, 20.19, 9.60. MS (ESI,positive) found (M+H): 694.88, calc(C 38 H 39 N5O8, m / z): 693.74.

[0059] (4) (5-methoxy-1,2-dimethyl-4,7-dioxo-4,7-dihydro-1H-indol-3-yl)methyl 3-((3-((10-methoxy-14-methyl-5-oxo-5,7,8,13,13b,14-hexahydroindol[2',3':3,4]pyrido[2,1-b]quinazolin-3-yl)amino)-3-oxopropyl)disulfonyl)propanoate (12d) 3-Amino-10-methoxyevodiamine (100 mg, 0.287 mmol) was dissolved in 5 mL of anhydrous DCM, followed by the addition of compound 8d (122 mg, 0.287 mmol) and EDCI (275.1 mg, 1.435 mmol), and the mixture was cooled to 0°C and DMAP (35.06 mg, 0.287 mmol) was added, followed by stirring at room temperature for 6-8 h, monitored by TCL, and after the reaction was completed, the solvent was removed, the mixture was dissolved in DCM, washed with water and saturated brine, dried over anhydrous Na2SO4, concentrated under reduced pressure, separated and purified on a silica gel column, and eluted with DCM / CH3OH (6:1-1:1). A brown solid compound 12d (yield 67%) was obtained. 1H NMR (400 MHz, DMSO-d6)δ11.00 (s, 1H), 9.89 (s, 1H), 8.03 (d, J = 2.3Hz, 1H), 7.75 (dd, J = 8.7, 2.4 Hz, 1H), 7.24 (d, J = 8.8 Hz, 1H), 7.07 (d, J= 8.8 Hz, 1H), 6.99 (d, J= 2.1 Hz, 1H), 6.75 (dd, J = 8.8, 2.3 Hz, 1H), 6.01(s, 1H), 5.74 (s, 1H), 5.13 (s, 2H), 3.82(s, 3H), 3.74 (d, J = 10.0 Hz, 6H), 3.17 (d, J = 4.9 Hz, 4H), 2.85–2.78 (m, 2H), 2.63 (s, 3H), 2.27 (q, J = 7.3 Hz, 4H), 2.22 (s, 3H). 13 C NMR (101 MHz, DMSO-d6)δ178.65,177.53, 173.14, 171.18, 164.30, 159.70, 153.81, 145.55, 138.99,134.09, 132.17, 130.91,128.69, 126.58, 125.08, 121.48, 121.30, 120.36,118.82, 115.32, 112.75, 112.45, 111.83,107.16, 100.64, 69.76, 56.96, 56.55,55.84, 49.06, 36.97, 36.34, 33.62, 32.58, 25.00, 24.56,20.20, 9.55. MS (ESI, positive) found (M+H): 758.23, calc (C 38 H 39 N5O8, m / z): 757.87.

[0060] Example 2 Cytotoxicity Experiment A549, H460, PC9, PC9 / GR, and LO2 cell lines were purchased from Wuhan Pucell Life Science Co., Ltd., China. A549, H460, PC9, and LO2 cells were maintained in Roswell Park Memorial Institute (RPMI)-1640 medium. PC9 / GR cells were maintained in DMEM medium containing 10% (v / v) fetal bovine serum (FBS), 100 U / mL penicillin, and 100 mg / mL streptomycin. 4000 cells were seeded in each well of a 96-well plate for viability assay. After 12 h of cell adhesion, the upper layer of culture medium was removed, and drugs were prepared according to the set concentration gradient. The adherent cells were exposed to the corresponding drugs for 48 h, incubated with 0.5 mg / mL MTT for 4 h, and the OD 492 Cell viability was determined. All experiments were performed in triplicate.

[0061] To analyze the cytotoxicity of these targeted compounds, the MTT method was used to preliminarily screen the compounds 9a-9d, 10a-10d, 11a-11d and 12a-12d containing indolequinone cores and evaluate their antiproliferative activity against human lung cancer cells A549, H460, PC9 and gefitinib-resistant cells PC9 / GR. In addition, the effects of these compounds on human normal liver cell line LO2 within 48 hours were also evaluated. Evodiamine (EVO) was used as a positive control. As shown in Table 2, most of the compounds had antitumor effects on these cancer cells, with IC 50 Between 2.54 and 34.48 µM. Compared with EVO, IC 50 Among them, compounds 11b and 12d had better effects on A549 cell lines and PC9 cell lines. On A549 cells, IC 50 The values ​​are 2.72µM and 3.66µM respectively, which are even higher than EVO’s 19.65µM. On PC9 cells, IC 50 The selectivity index (SI) of the compound between normal cells and tumor cells was then calculated. The SI is defined as the IC of normal hepatocyte LO2. 50 IC values ​​of A549 cells 50 The SI value of EVO was 0.16, while the SI values ​​of compounds 11b and 12d increased to 3.07 (19.2 times) and 1.80 (11.3 times), respectively. Compounds 11b and 12d showed weak cytotoxicity against LO2 cells, indicating that these compounds selectively killed cancer cells rather than normal cells.

[0062] Table 2 Antiproliferative effects (IC 50 Summary

[0063] In the study of antiproliferative activity of tumor cells, compound 11b showed the lowest IC against A549 cells. 50 value (2.72±0.12 µM), and therefore was considered to be a potential antitumor drug.

[0064] Example 3 Bioreductive agonism of compounds 11b and 12d on NQO1 To verify that compounds 11b and 12d can target NQO1 to trigger anticancer activity, dicoumarol (DIC), a typical competitive inhibitor of NQO1 enzyme, was selected, which inhibits its catalytic efficiency by interacting with the NAD(P)H binding site on the NQO1 enzyme. A549 cells were selected to verify NQO1 targeting.

[0065] First, the toxicity of DIC on A549 cells was verified by exposing A549 cells to DIC for 48 h. Figure 1 A. IC of tumor cells by DIC after 48h of culture 50 The value was 140.5±0.15µM, indicating that pre-incubation of A549 cells with 10µM DIC for 1h had little effect on tumor cell proliferation. Therefore, A549 cells were co-incubated with 10µM DIC, and then compound 11b and compound 12d were added. A549 cells without DIC incubation were used as controls. The inhibitory effect was detected at 24h, 48h and 72h ( Figure 1 B) IC 50 The values ​​are shown in Table 3. Without the addition of DIC, the IC values ​​of compound 11b at 24 h, 48 h and 72 h 50 The IC values ​​of compound 12d at 24h, 48h and 72h were 4.17µM, 1.86µM and 1.25µM respectively. 50 At the same time, after pretreatment with the competitive inhibitor of NQO1, DIC for 1 h, compound 11b was added. The IC values ​​of compound 11b at 24 h, 48 h, and 72 h were 5.52 μM, 3.00 μM, and 1.72 μM, respectively. 50 The antiproliferative activity of compound 11b against A549 decreased by 1.83, 2.12, and 1.47 times, respectively (Table 3). The IC values ​​of compound 12d at 24h, 48h, and 72h were 50 The anti-tumor activity of compound 11b and 12d against A549 cells was 8.77, 4.80, and 2.62 µM, respectively. The anti-proliferative activity of compound 12d against A549 cells decreased by 1.59, 1.60, and 1.52 times, respectively. This indicates that after pretreatment of A549 cells with enzyme inhibitors, the anti-tumor activity of compounds 11b and 12d decreased, suggesting that the anti-tumor proliferation activity of the compounds is closely related to NQO1.

[0066] Table 3 Evaluation of the cytotoxicity of compounds 11b and 12d on NQO1-dependent cells

[0067] Example 4 Effects of Compounds 11b and 12d on Cell Apoptosis In order to better observe whether the inhibitory effect of compounds 11b and 12d on tumor cells in the MTT experiment is related to the ability to induce apoptosis, the Annexin V-FITC / PI kit was used to detect the effect of the compounds on cell apoptosis. Figure 2 As shown. The induction effect of compounds 11b and 12d on A549 cells was concentration-dependent. After compound 11b was used to treat A549 cells at 0, 2, 4 and 8 µM, the total apoptosis rates were 2.18%, 5.00%, 14.30% and 27.86%, respectively. Compound 12d was measured under the same concentration gradient. Compound 12d was used to treat A549 cells at 2, 4 and 8 µM, and the total apoptosis rates were 5.98, 11.49% and 34.54%, respectively, indicating that compounds 11b and 12d can effectively induce apoptosis of A549 cells.

[0068] Example 5 Effects of Compounds 11b and 12d on Cell Cycle Most anti-tumor drugs inhibit cell proliferation by inducing cell cycle arrest. In order to study the effect of compounds on the proliferation cycle of A549 cells, propidium iodide (PI) staining was used to detect the effect of 11b and 12d on the cell cycle. A549 cells were incubated at 0, 2, 4, and 8 µM 11b and 12d concentrations for 48 h. PI was used for staining alone, and flow cytometry was used to detect cell cycle distribution. The results are shown in Figure 3 As shown. Compound 11b increased the percentage of cells in the G2 / M phase from 12.52% to 31.52%. Compound 12d increased the proportion of cells in the G2 / M phase from 12.52% to 24.65%. EVO also increased the proportion of cells in the G2 / M phase to 21.00%, which is consistent with previous literature reports. The above results indicate that these compounds may inhibit cancer cell proliferation in the G2 / M phase through cell cycle arrest. At the same time, the proportion of cells in the G1 phase decreased, and the proportion of cells in the S phase remained basically unchanged.

[0069] Example 6 In vivo antitumor activity of compound 11b To evaluate the in vivo antitumor activity of compound 11b, a mouse Lewis lung cancer (LLC) xenograft model was used in this study. Figure 4 A. Six-week-old male C57BL / 6 mice were selected and injected subcutaneously with 1×10 6LLC cell suspension (100 μL) was inoculated into the right axillary area of ​​the mice to induce tumor formation. After inoculation, the growth of the tumor was observed and measured every day. Usually, after 7 days, the tumor volume reached about 100 mm³, at which time the formal treatment experiment began. The mice were randomly divided into four groups: control group, EVO group, compound 11b group and 5-fluorouracil (5-Fu) positive control group. During the treatment phase, each mouse received an intraperitoneal injection once a day. The EVO group and compound 11b group were both given a dose of 20 mg / kg for 13 days; the 5-Fu group was used as a positive control with the same administration method, dose and time. In order to evaluate the tumor growth inhibition effect, the length and width of the tumor were measured regularly with a caliper, and the tumor volume was calculated using the formula (tumor volume = 0.5×length×width²). At the same time, the weight changes of each mouse were recorded to evaluate the systemic toxicity of the drug and the health status of the mice.

[0070] The experimental results showed that during the treatment, the tumor volume of mice in all groups gradually increased over time, but the tumor growth rate of the compound 11b group and the 5-Fu group was significantly slower than that of the control group and the EVO group ( Figure 4 B). At the end of treatment, the tumor volume of the compound 11b group was significantly smaller than that of the control group, and the indolequinone-modified EVO structure showed a better tumor inhibition effect compared with EVO. In particular, the antitumor activity of compound 11b was not significantly different from that of the 5-Fu group under the same administration route, dose and time, and the tumor growth inhibition rate (TGI) was 39.13% and 27.54%, respectively.

[0071] At the same time, by monitoring the weight changes of mice, the results showed that ( Figure 4 C), the weight of mice in each group increased slightly during the treatment, and the difference was not significant. The calculation results of the liver and kidney index (ratio of liver and kidney weight to body weight) showed that the liver and kidney index of the EVO group was slightly higher than that of the control group, indicating that there may be mild liver and kidney damage; while the liver and kidney index of the compound 11b group was lower, indicating that it had less damage to liver and kidney function and no obvious toxicity.

[0072] After the experiment, the mice were anesthetized by intraperitoneal injection of pentobarbital and the tumors and major organs (liver and kidney) were removed for pathological sectioning and histological analysis. The results of liver sectioning showed that the liver tissues of the 5-FU group and the EVO group were relaxed, while there was no significant difference in the liver tissues of the compound 11b group and the control group ( Figure 4 F). In the kidney section analysis, the glomerular interstitial space of the 5-FU and EVO groups was enlarged and the renal tubular structure was disordered, while the compound 11b group was closer to the normal control group ( Figure 4G). Observation of tumor tissue sections revealed that the nuclei of tumor tissue cells in the control group and EVO group were enlarged, disordered, and stained darker, indicating that the tumor cells were in a highly proliferative state; in contrast, the tumor cells in the compound 11b group were arranged more loosely, with an imbalanced nuclear-cytoplasmic ratio, and some cells were vacuolated, indicating that the tumor cells had undergone apoptosis or necrosis ( Figure 4 I).

[0073] To further explore the anti-tumor mechanism of compound 11b, immunohistochemical analysis of tumor tissues was performed, mainly detecting the expression of pro-apoptotic proteins Bax, Caspase3 and anti-apoptotic protein Bcl2 ( Figure 4 H). The results showed that the staining of Bax and Caspase3 in the compound 11b group was significantly stronger than that in the control group and the EVO group, and stronger than that in the 5-Fu group, indicating that compound 11b promoted the apoptosis of tumor cells by activating the apoptosis pathway. In contrast, the Bcl2 staining in the compound 11b group was weak, further verifying that it inhibited the survival of tumor cells by inhibiting the expression of Bcl2. In summary, compound 11b showed good anti-tumor effect in vivo and had low toxicity, and had good development potential.

[0074] in conclusion The present invention reacts amino-substituted evodiamine with indolequinones with different connecting bonds to prepare the evodiamine indolequinone complexes (9a-12d) with NQO1 agonism. The structures of these complexes are analyzed by hydrogen spectrum, carbon spectrum and mass spectrum.

[0075] The present invention relates to a novel treatment scheme for tumor microenvironment with high expression of NQO1 protein, specifically the design and synthesis of a series of EVO derivatives with NQO1 targeting specificity. In vitro antiproliferative activity tests showed that compounds 11b and 12d exhibited significant antitumor effects on A549 cells, with IC 50 The values ​​were 2.72 and 3.80 µM, respectively, which were significantly better than the parent compound EVO. Flow cytometry analysis further confirmed that compounds 11b and 12d could not only promote apoptosis of A549 cells, but also arrest the cell cycle at the G2 / M phase. The above research results show that compounds 11b and 12d show great potential in the treatment of non-small cell lung cancer. Therefore, the present invention provides a new compound with important research value and clinical application prospects, which is worthy of further research and development.

Claims

1. A NQO1 agonist based on evodiamine, characterized in that: The chemical structural formula of the NQO1 agonist based on evodiamine is one of the following chemical structural formulas: ； ； n is 1 to 10.

2. The NQO1 agonist based on evodiamine according to claim 1, characterized in that: n is 2 to 8.

3. The method for preparing the NQO1 agonist based on evodiamine according to claim 1, characterized in that: The method comprises the following steps: reacting amino-substituted evodiamine with indolequinone to prepare the evodiamine-based NQO1 agonist.

4. The method for preparing the NQO1 agonist based on evodiamine according to claim 3, characterized in that: The amino-substituted evodiamine is one of 2-NH-EVO, 3-NH-EVO, 2-NH-10-OCH3-EVO and 3-NH-10-OCH3-EVO; The chemical structure of indolequinone is as follows: ； R1 is the following substituent: ； n is 1 to 10.

5. The method for preparing the NQO1 agonist based on evodiamine according to claim 3, characterized in that: In the presence of EDCI / DMAP system, amino-substituted evodiamine reacts with indolequinone to prepare the evodiamine-based NQO1 agonist.

6. A medicine, comprising the NQO1 agonist based on evodiamine according to claim 1 as an active ingredient.

7. Use of the evodiamine-based NQO1 agonist or a pharmaceutically acceptable salt or pharmaceutical composition thereof according to claim 1 in the preparation of an NQO1 agonist or as an NQO1 agonist.

8. Use of the evodiamine-based NQO1 agonist or a pharmaceutically acceptable salt or pharmaceutical composition thereof according to claim 1 in the preparation of anti-tumor drugs or anti-tumor cell drugs.

9. Use of the evodiamine-based NQO1 agonist or a pharmaceutically acceptable salt thereof or a pharmaceutical composition according to claim 1 in the preparation of an antiproliferative drug for drug-resistant cells.

10. Use of amino-substituted evodiamine and indolequinone as raw materials in the preparation of the evodiamine-based NQO1 agonist according to claim 1.

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