An nqo1 agonist based on evodiamine and preparation method and application thereof

Abstract

The application discloses an NQO1 agonist based on evodiamine and a preparation method and application thereof, and particularly relates to an NQO1[NAD(P)H:quinone oxidoreductase 1] agonist connected by an evodiamine (EVO) amide bond, which shows good anti-non-small cell lung cancer (NSCLC) activity (IC 50 The compound promotes A549 cell apoptosis, blocks the cell cycle to the G2 / M phase, causes active oxygen burst, down-regulates the expression of Keap1, and up-regulates the expression of Nrf2, NQO1 and HO-1 after acting on A549 cells for 24 hours. This indicates that the compound improves the cell antioxidant capacity by regulating the Keap1 / Nrf2 / NQO1 antioxidant pathway. In vivo anti-tumor experiments show that the compound can inhibit the proliferation of tumor tissues (TGI=39.13%). In summary, the research results of the application show that the compound has a strong agonizing effect on the NQO1 signal pathway, and provides a potential opportunity for improving the treatment of non-small cell lung cancer.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to a method for preparing and applying an NQO1 agonist linked by an amide bond in Evodia rutaecarpa alkaloid. Background Technology

[0002] Currently, the main treatments for non-small cell lung cancer (NSCLC) include surgery, radiotherapy, and chemotherapy. However, due to drug resistance in advanced lung cancer patients and the low sensitivity of tumor cells to traditional chemotherapy drugs, the long-term survival rate remains low. In recent years, with the development of targeted therapy and immunotherapy, treatment effects have improved, but the problems of toxic side effects, treatment resistance, and poor efficacy of traditional drugs still exist. Therefore, there is an urgent need to develop novel anticancer drugs with high efficacy 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 intracellular redox balance, thereby inducing apoptosis in cancer cells [DPP9 regulates NQO1 and ROS to promote resistance to chemotherapy in liver cancer cells]. However, most existing NQO1 agonists have drawbacks such as poor stability, insufficient targeting, or short duration of action, which limits their clinical application. Evodiamine (EVO), a natural alkaloid, interferes with the proliferation, migration, and invasion of cancer cells through multiple pathways, but its mechanism of action is not yet fully understood, and there is still room for improvement in the efficacy and anti-cancer potential of Evodiamine. Summary of the Invention

[0003] Evodiamine (EVO), a natural alkaloid, still has room for improvement in its efficacy and anticancer potential. This invention discloses several amide-linked derivatives of Evodiamine, which are combined with indolequinone via amide bonds. These derivatives serve as NQO1 agonists, overcoming the shortcomings of existing agonists such as poor stability, insufficient targeting, or short duration of action, thus broadening their clinical applications.

[0004] The present invention adopts the following technical solution: An NQO1 agonist based on evodiamine has the following chemical structural formula: ;

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

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

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

[0008] The chemical structural formula of indolequinone is as follows:

[0009] R1 is a substituent for the following:

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

[0011] In the above technical solution, in the presence of an EDCI / DMAP system, amino-substituted evodiamine is reacted with indolequinone to prepare the aforementioned evodiamine-based NQO1 agonist. This invention reacts amino-substituted evodiamine with indolequinones carrying different linkages; the carboxylic acid and amine undergo condensation to form an amide bond, resulting in a reaction with high selectivity and efficiency. The reaction time is 6–8 hours.

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

[0013] This invention discloses a medicament with the aforementioned evodiamine-based NQO1 agonist as its active ingredient. Specifically, the medicament comprises an effective therapeutic amount of the aforementioned evodiamine-based NQO1 agonist and pharmaceutically acceptable excipients, carriers, or diluents.

[0014] This invention discloses the use of the above-mentioned NQO1 agonists based on evodiamine or pharmaceutically acceptable salts or pharmaceutical compositions thereof in the preparation of NQO1 agonists or as NQO1 agonists.

[0015] This invention discloses the use of the above-mentioned NQO1 agonist based on evodiamine or its pharmaceutically acceptable salt or pharmaceutical composition in the preparation of antitumor drugs or antitumor cell drugs.

[0016] This invention relates to the application of amino-substituted evodiamine in the preparation of NQO1 agonists containing evodiamine and indolequinone structural units through the reaction of evodiamine with indolequinones with different linkages. Existing NQO1 agonists suffer from poor drug stability and targeting, while evodiamine compounds, although possessing certain anticancer potential, require improvement in their individual anticancer activity and selectivity. This invention addresses the shortcomings of existing NQO1 agonists and evodiamine compounds by developing a series of evodiamine-amide-linked NQO1 agonists. These novel compounds, through innovative structural design, not only maintain the natural anticancer activity of evodiamine but also significantly enhance their in vitro and in vivo antitumor effects by strengthening their interaction with NQO1. Furthermore, 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 apoptosis. Compared with existing technologies, this invention provides an NQO1 agonist with better stability and stronger activity, offering a new solution for the treatment of non-small cell lung cancer. Attached Figure Description

[0017] Figure 1 (A) The bioreductive agonist effect of compounds 11b and 12d on NQO1; (B) The antiproliferative activity of dicumarol in A549 cells; (C) The NQO1-dependent cytotoxicity evaluation of compounds 11b and 12d.

[0018] Figure 2 To determine the apoptosis results 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 hours; at least three independent experiments were performed for each case.

[0019] Figure 3 To determine the cell cycle results of compounds 11b and 12d, A549 cells were treated with different concentrations of the 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 hours; at least three independent experiments were performed for each case.

[0020] Figure 4Compound 11b was used to inhibit 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 protocol, (B) Tumor volume change over time, (C) Mouse body weight change over time (D, E) Tumor weight and size 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 Implementation

[0021] As an example, this invention discloses a method for preparing and applying an amide-linked NQO1 agonist of evodiamine. The preparation method involves dissolving 100 mg (0.314 mmol) of 2-aminoevodiamine 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). After cooling the mixture to 0°C, DMAP (38.36 mg, 0.314 mmol) is added. The mixture is stirred at room temperature for 24–48 h, monitored by TCL. After the reaction is complete, the solvent is removed, and the mixture is dissolved in DCM. The mixture is washed with water and saturated brine, dried over anhydrous Na₂SO₄, concentrated under reduced pressure, purified by silica gel column chromatography, and eluted with CH₃OH / DCM (2–4%, Rf = 0.4). A yellow solid compound 9 is obtained (yield 48–62%). The synthesis methods for compounds 9a-9d, 10a-10d, 11a-11d, and 12a-12d are similar.

[0022] This invention successfully designed and synthesized 16 novel compounds targeting NQO1, and then evaluated their bioactivity in vitro. To analyze the cytotoxicity of these targeted compounds, the MTT assay was used to preliminarily screen compounds containing an indolequinone nucleus (9a-9d, 10a-10d, 11a-11d, and 12a-12d) for their antiproliferative activity against human lung cancer cells A549, H460, and PC9, as well as gefitinib-resistant PC9 / GR cells, and the human normal hepatocyte cell line LO2, for 48 hours. EVO was used as a positive control. Most compounds showed antitumor activity against these cancer cells, with an IC50 value of [missing value]. 50 The effective concentration ranged from 2.54 to 34.48 µM. 11b and 12d showed good anti-non-small cell lung cancer activity in A549 cells, with an IC50 concentration of [missing value]. 50The concentrations 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. Further investigation using Annexin V / PI staining and flow cytometry was conducted to examine the effects of compounds 11b and 12d on tumor cell apoptosis, cell cycle, reactive oxygen species (ROS), and mitochondrial membrane potential. Flow cytometry results showed that 11b and 12d induced apoptosis in A549 cells, arrested the cell cycle at the G2 / M phase, and significantly increased ROS. Western blotting analysis further showed that after 24 hours of treatment in A549 cells, 11b and 12d inhibited Keap1 expression and promoted the expression of Nrf2, NQO1, and HO-1, indicating that these two compounds enhanced the cellular antioxidant defense capacity by regulating the Keap1 / Nrf2 / NQO1 antioxidant pathway. In vivo studies confirmed that compound 11b effectively inhibited tumor growth, achieving a tumor growth inhibition rate of 39.13%.

[0023] The chemical structural formulas of four rutin derivatives are as follows:

[0024] The four evodiamine derivatives mentioned above (2-NH-EVO, 3-NH-EVO, 2-NH-10-OCH3-EVO, and 3-NH-10-OCH3-EVO) are respectively combined with indolequinones via amide bonds. Specifically, the evodiamine-indolequinone composites are prepared by reacting amino-substituted evodiamines with indolequinones having different linkages; 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] The experimental section of this invention synthesized 16 novel compounds targeting NQO1, among which 11b and 12d showed good anti-non-small cell lung cancer activity in cytotoxicity experiments, with an IC50 concentration of 11b-12d ... 50 The effective concentrations were 2.72 µM and 3.66 µM, respectively. Flow cytometry results showed that 11b and 12d induced apoptosis in A549 cells, arrested the cell cycle at the G2 / M phase, and triggered a significant increase in reactive oxygen species. Western blotting analysis further showed that 11b and 12d, after 24 hours of treatment in A549 cells, inhibited Keap1 expression and promoted the expression of Nrf2, NQO1, and HO-1, indicating that these two compounds enhanced the cellular antioxidant defense capacity by regulating the Keap1 / Nrf2 / NQO1 antioxidant pathway. In vivo studies confirmed that compound 11b effectively inhibited tumor growth, with a tumor growth inhibition rate of 39.13%. Therefore, this study demonstrates that 11b and 12d exhibit remarkable anti-cancer potential by activating the NQO1 signaling pathway, providing new drug candidates for the treatment of non-small cell lung cancer.

[0029] This invention chemically bonds different raw materials to obtain NQO1 agonists with reduced toxicity and side effects, and additive or synergistic pharmacological effects. Unless otherwise specified, all raw materials and reagents used in the reaction process are of analytical grade and were purchased from Sigma Aldrich Trading Co., Ltd. The nuclear magnetic resonance spectrometers used were Bruker AVANCE400 and Bruker AVANCE500 models from Germany, with TMS as the internal standard and DMSO as the solvent. d 6 or CDCl3, chemical shift (d) and coupling constant ( J The units are ppm and Hz, respectively. ESI mass spectrometry was performed using an Aglient 7250 & JEOL-JMS-T100LP AccuTOF mass spectrometer, and TLC analysis was performed using silica gel GF. 254 (Qingdao Marine Chemistry, China). Cell lines A549, H460, PC9, PC9 / GR, and LO2 were purchased from Wuhan Pusel Biotechnology Co., Ltd., China. The specific experimental procedures and tests (including statistical data analysis) of this invention are standard techniques, and animal experiments comply with the regulations of Shaanxi University of Traditional Chinese Medicine.

[0030] This invention discloses a coupling reaction between aminoevodiamine and indolequinone to obtain an NQO1 agonist containing an evodiamine structural unit. The application of the above-mentioned NQO1 agonist containing the evodiamine structural unit in the preparation of antitumor drugs is also disclosed. The preferred tumor is lung cancer. The synthetic 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) Vilsmeier's reagent was prepared by adding POCl3 (0.85 mL, 9.28 mmol) to 3 mL of anhydrous DMF and stirring at 0 °C for 10 min. Then, 5-methoxy-2-methylindole (1.04 g, 6.45 mmol) was dissolved in 3 mL of anhydrous DMF, cooled to 0 °C, and the prepared Vilsmeier's reagent was added dropwise. After addition, the reaction mixture was stirred at 0 °C for 30 min. Subsequently, the reaction mixture was added dropwise to an ice-cold 2 M NaOH (50 mL) solution, followed by the addition of DCM (100 mL). The mixture was extracted and separated, and the aqueous layer was extracted again with DCM (50 mL). The organic layer remained bound, and the mixture was washed with brine, dried over anhydrous Na2SO4 to remove the solvent, and the residue was washed with glacial ethyl acetate to obtain 1.05 g (90%) of a pale 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] See Table 1 for the structural formulas of other compounds.

[0034] Table 1 Summary of Compound Structural Formulas ; ; ;

[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, followed by the addition of iodomethane (0.91 mL, 14.6 mmol) at 0 °C. The mixture was then stirred at room temperature for another 2 h. The mixture was dissolved in DCM and extracted sequentially with water and NaCl solutions. After drying with anhydrous Na2SO4 to remove the solvent, the mixture was purified by silica gel column chromatography and eluted with ethyl acetate / petroleum ether (1:2, Rf = 0.3) to give 3.68 g (97.0%) of 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-(methyl ethoxycarbonylcarbamate)-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, followed by the addition of iodomethane (0.91 mL, 14.6 mmol) at 0 °C. The mixture was then stirred at room temperature for another 2 h. The mixture was dissolved in DCM and extracted sequentially with water and NaCl solutions. After drying with anhydrous Na2SO4 to remove the solvent, the mixture was purified by silica gel column chromatography and eluted with ethyl acetate / petroleum ether (1:2, Rf = 0.3) to give 3.68 g (97.0%) of 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. Freund's salt (0.59 g, 2.2 mmol) was added with stirring. The reaction was allowed to proceed for 3 h at room temperature, monitored by TCL, and extracted with DCM (100 mL × 3). The organic layers were assembled and extracted sequentially with water and NaCl solutions, then dried over Na₂SO₄. After solvent removal, compound 6 (0.134 g, 84%) was obtained as a red solid without further 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 a mixture of anhydrous methanol and anhydrous tetrahydrofuran in equal proportions. NaBH4 (0.91 g, 2.40 mmol) was added under ice bath conditions, and the mixture was stirred for 8 min. The reaction was monitored by TCL, and then quenched with saturated NH4Cl solution. The mixture was extracted with DCM, washed successively with water and brine, dried over anhydrous Na2SO4, concentrated, purified by silica gel column chromatography, and eluted with EA / PE (1:1, Rf = 0.21) to give 0.41 g (67.7%) of compound 7 as an orange-red solid. 1 H NMR (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 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 oxaloyl 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 oxaloyl chloride, refluxed at 67 °C for 1.5 h, concentrated under reduced pressure to remove the solvent, and the residue was precipitated in cold diethyl ether (80 mL) for at least 4 h. The mixture was filtered to remove the diethyl 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-oxobutyric acid (8a) Succinic anhydride (85 mg, 0.85 mmol) and DMAP (51.9 mg, 0.43 mmol) were dissolved in anhydrous DCM. 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 and extracted successively with water and NaCl solution. The extract was dried over anhydrous Na2SO4, concentrated, purified by silica gel column chromatography, 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-oxopentane (8b) Glutaric anhydride (119.7 mg, 1.05 mmol) and DMAP (42.7 mg, 0.35 mmol) were dissolved in anhydrous DCM. 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 and extracted successively with water and NaCl solution. The extract was dried over anhydrous Na2SO4, concentrated, purified by silica gel column chromatography, and eluted with PE:EA (1:1, Rf=0.3) to give 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. 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 and extracted successively with water and NaCl solution. The extract was dried over anhydrous Na2SO4, concentrated, purified by silica gel column chromatography, 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)dithiopropionic 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 stirred at room temperature for 30 min. Then, compound 7 (84 mg, 0.35 mmol) was added, and the reaction was continued for 12 h. The solvent was removed, and the residue was dissolved in DCM and extracted successively with water and NaCl solution. The extract was dried over anhydrous Na2SO4, concentrated, purified by silica gel column chromatography, 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-hexahydroindolol[2,3':3,4]pyrido[2,1-b]quinazolin-2-yl)amino)-4-oxobutyrate (9a) 2-Aminoevorutin (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). The mixture was cooled to 0 °C, and DMAP (38.36 mg, 0.314 mmol) was added. The mixture was stirred at room temperature for 6–8 h under TCL monitoring. After the reaction was complete, the solvent was removed, and the mixture was dissolved in DCM. The solution was washed with water and saturated brine, dried over anhydrous Na₂SO₄, concentrated under reduced pressure, purified by silica gel column chromatography, and eluted with DCM / CH₃OH (6:1–1:1). A yellow solid, compound 9a (51% yield), 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)methyl5-(14-methyl-5-oxo-5,7,8,13,13b,14-hexahydroindolol[2,3:3,4]pyrido[2,1-b]quinazolin-2-yl)amino)-5-oxovalerate (9b) 2-Aminoevorutin (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). The mixture was cooled to 0 °C, and DMAP (38.36 mg, 0.314 mmol) was added. The mixture was stirred at room temperature for 6–8 h under TCL monitoring. After the reaction was complete, the solvent was removed, and the mixture was dissolved in DCM. The solution was washed with water and saturated brine, dried over anhydrous Na₂SO₄, concentrated under reduced pressure, purified by silica gel column chromatography, and eluted with DCM / CH₃OH (6:1–1:1). A yellow solid, compound 9b, was obtained (65% yield). 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-hexahydroindolol[2',3':3,4]pyrido[2,1-b]quinazolin-2-yl)amino)-6-oxohexanoate (9c) 2-Aminoevorutin (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). The mixture was cooled to 0 °C, and DMAP (38.36 mg, 0.314 mmol) was added. The mixture was stirred at room temperature for 6–8 h, monitored by TCL. After the reaction was complete, the solvent was removed, and the mixture was dissolved in DCM. The mixture was washed with water and saturated brine, dried over anhydrous Na₂SO₄, concentrated under reduced pressure, purified by silica gel column chromatography, and eluted with DCM / CH₃OH (6:1–1:1). A yellow solid, compound 9c (60% yield), was obtained. 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-hexahydroindolol[2',3':3,4]pyrido[2,1-b]quinazolin-2-yl)amino)-3-oxopropyl)dithionyl)propionate (9d) 2-Aminoevorutin (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). The mixture was cooled to 0 °C, and DMAP (38.36 mg, 0.314 mmol) was added. The mixture was stirred at room temperature for 6–8 h, monitored by TCL. After the reaction was complete, the solvent was removed, and the mixture was dissolved in DCM. The mixture was washed with water and saturated brine, dried over anhydrous Na₂SO₄, concentrated under reduced pressure, purified by silica gel column chromatography, and eluted with DCM / CH₃OH (6:1–1:1). A yellow solid, compound 9d (52% yield), was obtained. 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-hexahydroindolol[2',3':3,4]pyrido[2,1-b]quinazolin-2-yl)amino)-4-oxobutyrate (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). The mixture was cooled to 0 °C, and DMAP (35.06 mg, 0.287 mmol) was added. The mixture was stirred at room temperature for 6–8 h, monitored by TCL. After the reaction was complete, the solvent was removed, and the mixture was dissolved in DCM. The solution was washed with water and saturated brine, dried over anhydrous Na₂SO₄, concentrated under reduced pressure, purified by silica gel column chromatography, and eluted with DCM / CH₃OH (6:1–1:1). The resulting brownish-yellow solid compound 10a (64% yield) 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)methyl5-((10-methoxy-14-methyl-5-oxo-5,7,8,13,13b,14-hexahydroindolol[2',3':3,4]pyrido[2,1-b]quinazolin-2-yl)amino)-5-oxovalerate (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). The mixture was cooled to 0 °C, and DMAP (35.06 mg, 0.287 mmol) was added. The mixture was stirred at room temperature for 6–8 h, monitored by TCL. After the reaction was complete, the solvent was removed, and the mixture was dissolved in DCM. The solution was washed with water and saturated brine, dried over anhydrous Na₂SO₄, concentrated under reduced pressure, purified by silica gel column chromatography, and eluted with DCM / CH₃OH (6:1–1:1). The resulting brownish-yellow solid compound 10b (54% yield) 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-hexahydroindolol[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). The mixture was cooled to 0 °C, and DMAP (35.06 mg, 0.287 mmol) was added. The mixture was stirred at room temperature for 6–8 h, monitored by TCL. After the reaction was complete, the solvent was removed, and the mixture was dissolved in DCM. The solution was washed with water and saturated brine, dried over anhydrous Na₂SO₄, concentrated under reduced pressure, purified by silica gel column chromatography, and eluted with DCM / CH₃OH (6:1–1:1). The resulting brownish-yellow solid compound 10c (54% yield) 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-hexahydroindolol[2',3':3,4]pyrido[2,1-b]quinazolin-2-yl)amino)-3-oxopropyl)dithionyl)propionate (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). The mixture was cooled to 0 °C, and DMAP (35.06 mg, 0.287 mmol) was added. The mixture was stirred at room temperature for 6–8 h, monitored by TCL. After the reaction was complete, the solvent was removed, and the mixture was dissolved in DCM. The solution was washed with water and saturated brine, dried over anhydrous Na₂SO₄, concentrated under reduced pressure, purified by silica gel column chromatography, and eluted with DCM / CH₃OH (6:1–1:1). The resulting brownish-yellow solid compound 10d (57% yield) 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-hexahydroindolol[2',3':3,4]pyrido[2,1-b]quinazolin-3-yl)amino)-4-oxobutyrate (11a) 3-Aminoevorutin (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). The mixture was cooled to 0 °C, and DMAP (38.36 mg, 0.314 mmol) was added. The mixture was stirred at room temperature for 6–8 h, monitored by TCL. After the reaction was complete, the solvent was removed, and the mixture was dissolved in DCM. The mixture was washed with water and saturated brine, dried over anhydrous Na₂SO₄, concentrated under reduced pressure, purified by silica gel column chromatography, and eluted with DCM / CH₃OH (6:1–1:1). A yellow solid, compound 11a, was obtained (57% yield). 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-hexahydroindolol[2,3:3,4]pyrido[2,1-b]quinazolin-3-amino)-5-oxovalerate (11b) 3-Aminoevorutin (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). The mixture was cooled to 0 °C, and DMAP (38.36 mg, 0.314 mmol) was added. The mixture was stirred at room temperature for 6–8 h under TCL monitoring. After the reaction was complete, the solvent was removed, and the mixture was dissolved in DCM. The solution was washed with water and saturated brine, dried over anhydrous Na₂SO₄, concentrated under reduced pressure, purified by silica gel column chromatography, and eluted with DCM / CH₃OH (6:1–1:1). A yellow solid, compound 11b, was obtained (65% yield). 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-hexahydroindolol[2,3:3,4]pyrido[2,1-b]quinazolin-3-yl)amino)-6-oxohexanoate (11c) 3-Aminoevorutin (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). The mixture was cooled to 0 °C, and DMAP (38.36 mg, 0.314 mmol) was added. The mixture was stirred at room temperature for 6–8 h, monitored by TCL. After the reaction was complete, the solvent was removed, and the mixture was dissolved in DCM. The mixture was washed with water and saturated brine, dried over anhydrous Na₂SO₄, concentrated under reduced pressure, purified by silica gel column chromatography, and eluted with DCM / CH₃OH (6:1–1:1). A yellow solid, compound 11c, was obtained (70% yield). 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-hexahydroindolol[2',3':3,4]pyrido[2,1-b]quinazolin-3-yl)amino)-3-oxopropyl)dithionyl)propionate (11d) 3-Aminoevorutin (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). The mixture was cooled to 0 °C, and DMAP (38.36 mg, 0.314 mmol) was added. The mixture was stirred at room temperature for 6–8 h, monitored by TCL. After the reaction was complete, the solvent was removed, and the mixture was dissolved in DCM. The mixture was washed with water and saturated brine, dried over anhydrous Na₂SO₄, concentrated under reduced pressure, purified by silica gel column chromatography, and eluted with DCM / CH₃OH (6:1–1:1). A yellow solid, compound 11d (59% yield), was obtained. 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-hexahydroindolol[2',3':3,4]pyrido[2,1-b]quinazolin-3-yl)amino)-4-oxobutyrate (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). The mixture was cooled to 0 °C, and DMAP (35.06 mg, 0.287 mmol) was added. The mixture was stirred at room temperature for 6–8 h under TCL monitoring. After the reaction was complete, the solvent was removed, and the mixture was dissolved in DCM. The mixture was washed with water and saturated brine, dried over anhydrous Na₂SO₄, concentrated under reduced pressure, purified by silica gel column chromatography, and eluted with DCM / CH₃OH (6:1–1:1). The resulting brownish-yellow solid compound 12a (66% yield) 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)methyl5-(10-methoxy-14-methyl-5-oxo-5,7,8,13,13b,14-hexahydroindolol[2',3':3,4]pyrido[2,1-b]quinazolin-3-yl)amino)-5-oxovalerate (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). The mixture was cooled to 0 °C, and DMAP (35.06 mg, 0.287 mmol) was added. The mixture was stirred at room temperature for 6–8 h under TCL monitoring. After the reaction was complete, the solvent was removed, and the mixture was dissolved in DCM. The solution was washed with water and saturated brine, dried over anhydrous Na₂SO₄, concentrated under reduced pressure, purified by silica gel column chromatography, and eluted with DCM / CH₃OH (6:1–1:1). The resulting brownish-yellow solid compound 12b (59% yield) 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-hexahydroindolol[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). The mixture was cooled to 0 °C, and DMAP (35.06 mg, 0.287 mmol) was added. The mixture was stirred at room temperature for 6–8 h under TCL monitoring. After the reaction was complete, the solvent was removed, and the mixture was dissolved in DCM. The solution was washed with water and saturated brine, dried over anhydrous Na₂SO₄, concentrated under reduced pressure, purified by silica gel column chromatography, and eluted with DCM / CH₃OH (6:1–1:1). The resulting brownish-yellow solid compound 12c (64% yield) 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-hexahydroindolol[2',3':3,4]pyrido[2,1-b]quinazolin-3-yl)amino)-3-oxopropyl)dithionyl)propionate (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). The mixture was cooled to 0 °C, and DMAP (35.06 mg, 0.287 mmol) was added. The mixture was stirred at room temperature for 6–8 h under TCL monitoring. After the reaction was complete, the solvent was removed, and the mixture was dissolved in DCM. The solution was washed with water and saturated brine, dried over anhydrous Na₂SO₄, concentrated under reduced pressure, and purified by silica gel column chromatography using DCM / CH₃OH (6:1–1:1) elution. The resulting brownish-yellow solid compound 12d (67% yield) 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 Pusel Biotechnology Co., Ltd., China. A549, H460, PC9, and LO2 cells were stored in Roswell Park Memorial Institute (RPMI)-1640 medium. PC9 / GR cells were stored in DMEM medium containing 10% (v / v) fetal bovine serum (FBS), 100 U / mL penicillin, and 100 mg / mL streptomycin. 4000 cells were seeded per well of a 96-well plate for viability assay. After 12 hours, cells adhered, the supernatant was removed, and drugs were prepared according to a pre-defined concentration gradient. Adhering cells were treated with the corresponding drugs for 48 hours, followed by incubation with 0.5 mg / mL LMT for 4 hours, and then analyzed using OD... 492 Cell viability was measured. All experiments were performed in triplicate.

[0061] To analyze the cytotoxicity of these targeted compounds, compounds containing indolequinone nuclei (9a-9d, 10a-10d, 11a-11d, and 12a-12d) were initially screened using the MTT assay to evaluate their antiproliferative activity against human lung cancer cells A549, H460, PC9, and gefitinib-resistant PC9 / GR. Furthermore, the effects of these compounds on the normal human hepatocyte cell line LO2 were also evaluated within 48 hours. Evodiaein (EVO) was used as a positive control. As shown in Table 2, most compounds exhibited antitumor activity against these cancer cells, with an IC50 score of 9.9%. 50 Between 2.54 and 34.48 µM. Compared to EVO, IC... 50 Significantly reduced. Compounds 11b and 12d showed better effects on the A549 and PC9 cell lines. In A549 cells, IC50... 50 The concentrations were 2.72 µM and 3.66 µM, respectively, even higher than EVO's 19.65 µM. In PC9 cells, the IC50... 50 The concentrations were 3.80 µM and 2.54 µM, respectively, even higher than EVO's 15.21 µM. The selectivity index (SI) of the compound between normal and tumor cells was then calculated. SI was defined as the IC50 of LO2 in normal hepatocytes. 50 Values ​​and IC50 values ​​of A549 cells 50 The SI values ​​for EVO were 0.16, while those for compounds 11b and 12d increased to 3.07 (19.2-fold) and 1.80 (11.3-fold), respectively. Compounds 11b and 12d exhibited weak cytotoxicity against LO2 cells, indicating that these compounds selectively kill cancer cells rather than normal cells.

[0062] Table 2. Antiproliferative effects of the target compound in different cell lines at 48 h (IC50) 50 (Value) Summary

[0063] In studies on antiproliferative activity against tumor cells, compound 11b exhibited the lowest IC50 value against A549 cells. 50 The value (2.72±0.12 µM) indicates that it is considered a potential antitumor drug.

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

[0065] First, the toxicity of DIC to A549 cells was verified by exposing A549 cells to DIC for 48 hours. The results are as follows... Figure 1 As shown in Figure A. After 48 hours of culture, the effect of DIC on the IC50 of tumor cells... 50 The value was 140.5 ± 0.15 µM, indicating that pre-incubation of A549 cells with 10 µM DIC for 1 h had little effect on tumor cell proliferation. Therefore, A549 cells were co-incubated with 10 µM DIC, followed by the addition of compounds 11b and 12d. A549 cells without DIC incubation served as a control. Inhibitory effects were assessed at 24 h, 48 h, and 72 h. Figure 1 (B) IC 50 The values ​​are shown in Table 3. Without the addition of DIC, the IC50 values ​​of compound 11b at 24h, 48h, and 72h are... 50 The concentrations were 4.17 µM, 1.86 µM, and 1.25 µM, respectively. The IC50 values ​​of compound 12d at 24 h, 48 h, and 72 h were... 50 The concentrations were 5.52 µM, 3.00 µM, and 1.72 µM, respectively. Simultaneously, after 1 h of pretreatment with the NQO1 enzyme competitive inhibitor DIC, compound 11b was added. The IC50 values ​​of compound 11b at 24 h, 48 h, and 72 h were [not specified in the original text]. 50 The concentrations were 7.65 µM, 3.95 µM, and 1.84 µM, respectively. The anti-proliferative activity of compound 11b against A549 decreased by 1.83, 2.12, and 1.47 times, respectively (Table 3). The IC50 values ​​of compound 12d at 24 h, 48 h, and 72 h were... 50 At concentrations of 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-fold, respectively. This indicates that pretreatment of A549 cells with enzyme inhibitors reduced the antitumor activity of compounds 11b and 12d, suggesting that the antitumor proliferative activity of these compounds is closely related to NQO1.

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

[0067] Example 4: Effects of compounds 11b and 12d on apoptosis To better observe whether the inhibitory effects of compounds 11b and 12d on tumor cells in the MTT assay were related to their apoptosis-inducing ability, the Annexin V-FITC / PI kit was used to detect the effects of the compounds on cell apoptosis. The experimental results are as follows: Figure 2 As shown, compounds 11b and 12d induced apoptosis in A549 cells in a concentration-dependent manner. Treatment of A549 cells with compound 11b at 0, 2, 4, and 8 µM resulted in total apoptosis rates of 2.18%, 5.00%, 14.30%, and 27.86%, respectively. Compound 12d was measured at the same concentration gradient; treatment of A549 cells with compound 12d at 2, 4, and 8 µM resulted in total apoptosis rates of 5.98%, 11.49%, and 34.54%, respectively, indicating that compounds 11b and 12d effectively induced apoptosis in A549 cells.

[0068] Example 5: Effects of compounds 11b and 12d on the cell cycle Most antitumor drugs inhibit cell proliferation by inducing cell cycle arrest. To investigate the effect of compounds on the cell cycle of A549 cells, propidium iodide (PI) staining was used to detect the effects of 11b and 12d on the cell cycle. A549 cells were incubated for 48 h at 0, 2, 4, and 8 µM concentrations of 11b and 12d. Cell cycle distribution was detected by flow cytometry after PI staining alone. Results are as follows: Figure 3 As shown, compound 11b increased the percentage of G2 / M phase cells 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 G2 / M phase cells to 21.00%, consistent with previous literature reports. These results suggest that these compounds may inhibit cancer cell proliferation in the G2 / M phase by arresting the cell cycle. Simultaneously, the proportion of G1 phase cells decreased, while the proportion of S phase cells remained essentially unchanged.

[0069] Example 6: In vivo antitumor activity of compound 11b To evaluate the in vivo antitumor activity of compound 11b, this study used a mouse Lewis lung cancer (LLC) xenograft model. Figure 4 In the experiment, 6-week-old male C57BL / 6 mice were selected and injected subcutaneously with 1×10⁻⁶ mol / L mol / L. 6One LLC cell suspension (100 μL) was injected into the right axillary region of mice to induce tumor formation. Tumor growth was observed and measured daily after inoculation. Typically, after 7 days, the tumor volume reached approximately 100 mm³, at which point the formal treatment experiment began. Mice were randomly assigned to four groups: a control group, an EVO group, a compound 11b group, and a 5-fluorouracil (5-Fu) positive control group. During the treatment phase, each mouse received an intraperitoneal injection once daily. The EVO and compound 11b groups received 20 mg / kg of medication for 13 consecutive days; the 5-Fu group served as a positive control, receiving the same administration method, dosage, and duration. To assess the tumor growth inhibition effect, the length and width of the tumor were measured periodically using calipers, and the tumor volume was calculated using the formula (tumor volume = 0.5 × length × width²). Simultaneously, the weight changes of each mouse were recorded to assess the systemic toxicity of the drug and the overall health status of the mice.

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

[0071] Meanwhile, by monitoring changes in the body weight of the mice, the results showed that ( Figure 4 In the treatment of mice in group C, body weight increased slightly in all groups, but the differences were not significant. The liver and kidney index (liver and kidney weight to body weight ratio) showed that the EVO group had a slightly higher index than the control group, suggesting possible mild liver and kidney damage; while the compound 11b group had a lower index, indicating less damage to liver and kidney function and no obvious toxicity.

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

[0073] To further investigate the antitumor mechanism of compound 11b, immunohistochemical analysis of tumor tissue was performed, mainly detecting the expression of pro-apoptotic proteins Bax and Caspase3 and anti-apoptotic protein Bcl2. Figure 4 (H). The results showed that Bax and Caspase3 staining in the compound 11b group was significantly stronger than that in the control group and the EVO group, and also stronger than that in the 5-Fu group, indicating that compound 11b promotes tumor cell apoptosis by activating the apoptosis pathway. Conversely, Bcl2 staining in the compound 11b group was weaker, further verifying that it inhibits tumor cell survival by suppressing Bcl2 expression. In conclusion, compound 11b exhibits good anti-tumor effects in vivo with low toxicity and has good development potential.

[0074] in conclusion This invention involves reacting amino-substituted evodiamine with indolequinones containing different linkages to prepare the evodiamine-indolequinone composites (9a-12d) exhibiting NQO1 activating activity. The structures of these composites were analyzed using 1H NMR, 1C NMR, and mass spectrometry.

[0075] This invention relates to a novel therapeutic approach targeting tumor microenvironments with high NQO1 protein expression, specifically the design and synthesis of a series of EVO derivatives with NQO1 targeting specificity. In vitro antiproliferative activity assays showed that compounds 11b and 12d exhibited significant antitumor effects against A549 cells, with IC50 values ​​exceeding [value missing]. 50 The effective values ​​were 2.72 and 3.80 µM, respectively, significantly superior to the parent compound EVO. Flow cytometry analysis further confirmed that compounds 11b and 12d not only promoted apoptosis in A549 cells but also arrested the cell cycle at the G2 / M phase. These results indicate that compounds 11b and 12d show great potential in the treatment of non-small cell lung cancer. Therefore, this invention provides a novel compound with significant research value and clinical application prospects, worthy of further research and development.

Claims

1. An 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: ； 。 2. The method for preparing the NQO1 agonist based on evodiamine according to claim 1, characterized in that, The process includes the following steps: reacting amino-substituted evodiamine with indolequinone to prepare the above-mentioned evodiamine-based NQO1 agonist; the chemical structural formula of the amino-substituted evodiamine is as follows: ； ； The chemical structural formula of indolequinone is as follows: ； R1 is a substituent for the following: ； n is 3.

3. The method for preparing the NQO1 agonist based on evodiamine according to claim 2, characterized in that, The above-mentioned NQO1 agonist based on evodiamine was prepared by reacting amino-substituted evodiamine with indolequinone in the presence of an EDCI / DMAP system.

4. A pharmaceutical composition having the evodiamine-based NQO1 agonist of claim 1 as the active ingredient.

5. The use of the NQO1 agonist based on evodiamine of claim 1 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 4, in the preparation of the NQO1 agonist.

6. The use of the NQO1 agonist based on evodiamine of claim 1 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 4, in the preparation of an antitumor drug.

7. The use of the NQO1 agonist based on evodiamine of claim 1 or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of claim 4, in the preparation of an antiproliferative drug for drug-resistant cells, characterized in that... The resistant cells were gefitinib-resistant cells PC9 / GR.

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