Artemisinin derivative and application thereof in preparation of antitumor drugs

Abstract

The invention relates to an artemisinin derivative. The artemisinin derivative has at least one structural formula shown in the following formula: # imgabs0 #. According to the invention, indole iodized salt molecules are used as targets to construct tumor-targeted mitochondria, so that tumor-targeted delivery of artemisinin drugs is realized. According to the invention, the artemisinin is connected through Cy7, so that the targeting property and the bioavailability of the artemisinin are improved, and the anti-tumor effect of the artemisinin is improved.

Description

Technical Field

[0001] The present invention belongs to the technical field of chemical medicine, and specifically relates to an artemisinin derivative (ART-Cy7, DHA-Cy7) and an application thereof in the preparation of an anti-tumor drug. Background Art

[0002] Cancer is currently one of the most common causes of death worldwide, with a high mortality rate. The main ways to treat cancer are surgery, chemotherapy, radiotherapy, etc. Existing technologies have discovered many anti-tumor drugs, and many chemotherapeutic drugs have been applied to the clinical front line. However, many chemotherapeutic drugs have serious toxic side effects and drug resistance, so it is necessary to develop an effective drug delivery system to increase the targeting of drugs and enhance the absorption of drugs in tumor cells.

[0003] Artemisinin and its derivatives are effective antimalarial drugs. Dihydroartemisinin and artesunate, as derivatives of artemisinin, have good water solubility and antimalarial effects. This type of drug contains a peroxide bridge structure, which is the main anti-tumor mechanism of this type of drug. Existing literature has confirmed that dihydroartemisinin, artesunate, etc. have obvious toxic side effects on malignant tumors of various sources through multiple mechanisms such as inhibiting tumor angiogenesis, promoting cell apoptosis, blocking cell cycle, reversing drug resistance and cytotoxicity. Dihydroartemisinin, artesunate, etc. promote oxidative stress by releasing reactive oxygen to produce anti-tumor effects. However, this type of drug has poor solubility, no targeting, low bioavailability, and has already developed drug resistance, so it needs to be used in combination with other drugs. Many studies have also combined artemisinin drugs with other drugs to enhance the efficacy of the drugs.

[0004] Recent studies have found that near-infrared fluorescent probes such as indole iodide heptamethine cyanine (cy7) molecules show good targeting in tumor cells. Such compounds can be used for fluorescence imaging, photothermal therapy, etc. to assist in tumor treatment. Such compounds have good biocompatibility and can be coupled with drugs to form complexes, thereby improving drug delivery into the body, reducing drug systemic toxicity and increasing bioavailability.

[0005] The present invention uses indole iodide salt molecules as targets, constructs tumor-targeted mitochondria, and realizes tumor-targeted delivery of artemisinin drugs. The present invention connects artemisinin through cy7, thereby improving the targeting and bioavailability of artemisinin and improving the anti-tumor effect of artemisinin. Summary of the invention

[0006] The purpose of the present invention is to overcome the defects of the prior art and provide an artemisinin derivative (ART-Cy7, DHA-Cy7, etc.), which connects artemisinin via Cy7, thereby improving the targeting and bioavailability of artemisinin and enhancing the anti-tumor effect of artemisinin.

[0007] The present invention also provides a method for preparing the above-mentioned artemisinin derivatives (ART-Cy7, DHA-Cy7, etc.) and their use in preparing anti-tumor drugs.

[0008] To achieve the above object, the present invention adopts the following technical solution: An artemisinin derivative having at least one of the following structural formulas: .

[0009] ART-Cy7, its relevant properties are as follows: Molecular formula: C 61 H 82 N3O 10 S2; molecular weight: 1207.4492; detection method: 1 HNMR, 13 CNMR, HRMS; Properties: This product is a blue powder; Source: Designed and synthesized by our research group. Pharmacological properties: Insoluble in water, soluble in DMSO. Application of ART-Cy7 in the preparation of anti-colorectal cancer drugs. This compound is mainly used to fight colorectal cancer DLD-1 cells, colorectal cancer SW480 cells, colorectal cancer MC38 cells, etc.

[0010] DHA-Cy7, its relevant properties are as follows: Molecular formula: C 53 H 70 N3O6I; Molecular weight: 971.43; Detection method: 1 HNMR, 13 CNMR, HRMS; Properties: This product is a blue powder; Source: Designed and synthesized by this research group. Pharmacological properties: Insoluble in water, soluble in DMSO. The present invention also provides an anti-colorectal cancer drug, the active ingredient of which is DHA-Cy7. The colorectal cancer cells are MC38 cells, SW480 cells or DLD-1 cells.

[0011] A method for preparing the above-mentioned artemisinin derivative, when the artemisinin derivative is ART-Cy7, comprises the following steps: 1) Under an inert gas atmosphere, compound 1, compound 2, and sodium acetate were dissolved in acetic acid-acetic anhydride, and reacted at 60-90° C. in the dark for 10-20 hours. After the reaction, tert-butyl methyl ether was used for precipitation, filtration, rotary evaporation, and silica gel column purification to obtain compound 3; 2) Under an inert gas atmosphere, compound 3 was dissolved in dimethylformamide, and compound 4 hexaminocaproic acid and triethylamine were added, and the mixture was reacted at 70-90°C for 2-5 hours in the dark. After the reaction, dimethylformamide was removed by extraction, and the mixture was subjected to rotary evaporation and purified by silica gel column to obtain compound 5; 3) Under an inert gas atmosphere, compound 6 and N,N'-dicyclohexylcarbodiimide (DCC) were dissolved in dichloromethane and stirred at room temperature to form a milky white suspension, and then compound 7 and 4-dimethylaminopyridine (DMAP) were added and stirred at room temperature for 2-5 hours. After the reaction was completed, the mixture was filtered, rotary evaporated, and purified by silica gel column to obtain intermediate compound 8; 4) Under an inert gas atmosphere, compound 5 and N,N'-dicyclohexylcarbodiimide (DCC) were dissolved in dichloromethane and stirred at room temperature to form a transparent solution. Compound 8 and 4-dimethylaminopyridine (DMAP) were then added and stirred at room temperature for 10-14 h. After the reaction was completed, the solution was rotary evaporated and purified by silica gel column to obtain the final compound 9; .

[0012] Specifically, in the preparation process of the above-mentioned artemisinin derivative ART-Cy7, the molar ratio of compound 1 to compound 2 is 2-2.5:1; the molar ratio of compound 3 to compound 4 is 1:4-4.5; the molar ratio of compound 6 to compound 7 is 1:1.2-2.0; and the molar ratio of compound 5 to compound 8 is 1:2-4.

[0013] A method for preparing the above-mentioned artemisinin derivative, when the artemisinin derivative is DHA-Cy7, comprises the following steps: 1) Under an inert gas atmosphere, compound 1, compound 2, and sodium acetate were dissolved in acetic acid-acetic anhydride, and reacted at 60-90° C. in the dark for 10-20 hours. After the reaction, tert-butyl methyl ether was used for precipitation, filtration, rotary evaporation, and silica gel column purification to obtain compound 3; 2) Under an inert gas atmosphere, compound 3 was dissolved in dimethylformamide, and compound 4 hexaminocaproic acid and triethylamine were added, and the mixture was reacted at 70-90°C for 2-5 hours in the dark. After the reaction, dimethylformamide was removed by extraction, and the mixture was subjected to rotary evaporation and purified by silica gel column to obtain compound 5; 3) Under an inert gas atmosphere, compound 6, N,N'-dicyclohexylcarbodiimide (DCC) and dichloromethane were reacted at room temperature for 1-2 hours, compound 5 and 4-dimethylaminopyridine (DMAP) were added, and the reaction was carried out at room temperature for 10-14 hours, cooled to room temperature, rotary evaporated, and purified by column chromatography to obtain compound DHA-Cy7; .

[0014] Specifically, in the preparation process of the above-mentioned artemisinin derivative DHA-Cy7, the molar ratio of compound 1 to compound 2 is 2-2.5:1; the molar ratio of compound 3 to compound 4 is 1:4-4.5; and the molar ratio of compound 5 to compound 6 is 1:2-4.

[0015] The present invention also provides a pharmaceutical composition, which comprises the artemisinin derivative, that is, the artemisinin derivative is used as an active ingredient and is compounded with conventional adjuvants in the art to form a compound preparation, such as tablets, granules, pills, capsules or injections.

[0016] The present invention provides the use of the above-mentioned artemisinin derivatives in the preparation of anti-tumor drugs.

[0017] In the above application, further, the tumor includes but is not limited to at least one of colorectal cancer, lung cancer, liver cancer, prostate cancer, malignant melanoma, esophageal cancer, etc.

[0018] Specifically, the artemisinin derivative can inhibit the activity of colorectal cancer cells (rectal cancer cell lines DLD-1, SW480 or MC38, etc.), induce cell apoptosis and produce reactive oxygen species. The effective concentration of the artemisinin derivative is 0.02438-50 μM.

[0019] Further, the effective concentration of ART-Cy7 is 0.0975-50 μM. The dose of ART-Cy7 to inhibit the growth of subcutaneous tumors in BALB / C nude mice with a colorectal cancer cell line MC38 is 1 mg / kg.3d. The ART-Cy7 is localized in the mitochondria of the colorectal cancer cell lines DLD-1, SW480 or MC38 and causes mitochondrial damage. The ART-Cy7 induces the production of reactive oxygen species in the colorectal cancer cell lines HCT116, SW480 or DLD-1.

[0020] The present invention provides a method for inhibiting the proliferation of tumor cells in vitro, wherein ART-Cy7 is added to the culture medium of the tumor cells, and the final concentration of the added ART-Cy7 is 0.0975-50 μM. The tumor cells may be colorectal cancer DLD-1 cells, colorectal cancer SW480 cells, or colorectal cancer MC38 cells.

[0021] Further, the effective concentration of DHA-Cy7 is 0.02438-12.5 μM. The dose of DHA-Cy7 to inhibit the growth of subcutaneous tumors in BALB / C nude mice with a colorectal cancer cell line MC38 is 3 mg / kg.2d. The DHA-Cy7 is localized in the mitochondria of the colorectal cancer cell line MC38, SW480 or DLD-1 and causes mitochondrial damage. The DHA-Cy7 induces the production of reactive oxygen species in the colorectal cancer cell line MC38, SW480 or DLD-1. The DHA-Cy7 induces a decrease in the activity of the xCT-GPX4 antioxidant system of colorectal cancer cells. The DHA-Cy7 induces an increase in the total iron content of colorectal cancer cells. The DHA-Cy7 induces ferroptosis in colorectal cancer cells.

[0022] The present invention provides a method for inhibiting the proliferation of tumor cells in vitro, wherein DHA-Cy7 is added to the culture fluid of tumor cells, and the final concentration of DHA-Cy7 is 0.02438-12.5 μM. The present invention also provides a method for inducing ferroptosis of tumor cells in vitro, wherein DHA-Cy7 is added to the culture fluid of tumor cells, and the final concentration of DHA-Cy7 is 0.02438-12.5 μM.

[0023] Compared with the prior art, the present invention has the following beneficial effects: The present invention provides a novel artemisinin derivative ART-Cy7 and / or DHA-Cy7, a synthesis method thereof, and use thereof in the preparation of anti-tumor drugs. The present invention uses indole iodide molecules as targets to construct tumor-targeted mitochondria, thereby achieving tumor-targeted delivery of artemisinin drugs. The present invention connects artemisinin through Cy7, thereby improving the targeting and bioavailability of artemisinin and improving the anti-tumor effect of artemisinin. MTT results show that ART-Cy7 can not only significantly inhibit the proliferation of colorectal tumor cells, but also inhibit other tumor cells (liver cancer, lung cancer, breast cancer, etc.), and can co-localize with mitochondria. DHA-Cy7 can significantly inhibit the proliferation of colorectal tumor cells and can co-localize with mitochondria. ROS and Fe 2+ The results of content detection and Western Blot detection of TFR1 showed that DHA-Cy7 can induce cell ferroptosis. The small molecule compound ART-Cy7 and / or DHA-Cy7 of the present invention can be developed as a new anti-tumor drug, especially an anti-colorectal tumor drug or its auxiliary component, with significant tumor inhibition effect, which will provide a new way and means for treating and curing colorectal tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 :ART-Cy7 structure identification 1 H NMR (400 MHz, DMSO-d6) spectral data; Figure 2 :ART-Cy7 structure identification 13 C NMR (100 MHz, DMSO-d6 ) spectral data; Figure 3 : ART-Cy7 structure identification HRMS (MALDI-DHB) spectrum data; Figure 4 : MTT results of DLD-1 (AC: Cy7, ART, ART-Cy7), SW480 (DF: Cy7, ART, ART-Cy7), and MC38 (GI: Cy7, ART, ART-Cy7) cells after ART-Cy7 administration; Figure 5 :A: DLD-1, SW480, MC38 cell morphology results after ART-Cy7 administration; B: Plate cloning results; Figure 6 :A: Tumor-bearing model test-tumor volume; B: Tumor-bearing model test-tumor weight; C: Toxicity test-nude mouse pictures in Ctl group, 5-Fu group, and ART-Cy7 group; D: Toxicity test-results of organ HE staining; E: Tumor-bearing model test-results of tumor HE staining; FG: Toxicity test-AST and ALT determination in liver injury; HI: Toxicity test-CR and BUN determination in kidney injury; J: Toxicity test-nude mouse weight; KO are organ coefficients in toxicity test-heart, liver, spleen, lung, and kidney, respectively; Figure 7 :A: mitochondrial co-localization results; BD: ATP detection results of DLD-1, SW480, and MC38 respectively; Figure 8 :JC-1 staining results; Fig. 9 :A: ROS fluorescence detection results; BD: ROS flow cytometry detection results of DLD-1, SW480, and MC38 respectively; Fig.10 : Detection of the anti-tumor activity of ART-Cy7 in other cancer cells, A549 cells (A), Hepa1-6 cells (B), 22RV1 cells (C), A375 cells (D), EC9706 cells (E); in the figure, *, P<0.05; **, P<0.015; ***, P<0.001; Fig.11 :DHA-Cy7 structure identification 1 H NMR (500 MHz, DMSO-d6) spectral data, Fig.12 :DHA-Cy7 structure identification 13C NMR (100 MHz, DMSO-d6) spectral data, Fig.13 : DHA-Cy7 structure identification HRMS (MALDI-DHB) spectrum data, Fig.14 :A, D, G: MTT results of MC38, SW480, DLD-1 after DHA administration; B, E, H: MTT results of MC38, SW480, DLD-1 after cy7 administration; C, F, I: MTT results of MC38, SW480, DLD-1 after DHA-Cy7 administration; JN: MTT results of DHA-Cy7 on other cancer cells A549, HEPG2, A375, EC9706, 22RV1; Fig.15 :Plate cloning results; Fig.16 :A: Tumor-bearing model test-tumor weight; B: Tumor-bearing model test-tumor volume; C: Toxicity test-tumor pictures of NS group, 5-Fu group, and DHA-Cy7 group; D: Tumor-bearing model test-tumor HE staining results; E: Toxicity test-weight of nude mice; F: Toxicity test-organ HE staining results; GK are the coefficients of toxicity test-organ heart, liver, spleen, lung, and kidney; LM are the toxicity test-liver injury AST and ALT determination; NO are the toxicity test-kidney injury BUN and CR determination; PT are the blood routine tests of white blood cells, red blood cells, lymphocytes, monocytes, and platelets; Fig.17 : A to D are the effects of the combined action of DHA-Cy7 with NAC, Z-VAD-FMK, Necrostatin-1 and DFO on the proliferation of DLD-1, MC38 and SW480 cells; Fig.18 :A: The co-localization results of DHA-Cy7 in mitochondria of DLD-1, MC38, and SW480; BD: The ATP detection results of DLD-1, MC38, and SW480 respectively; Fig.19 :JC-1 staining results; Fig. 20 :A: ROS fluorescence detection results; BD are the results of ROS flow cytometry detection of DLD-1, MC38, and SW480; EG are the results of ROS flow cytometry detection of DLD-1, SW480, and MC38 after the combined action of DHA-Cy7 and NAC; Fig.21: AC are the GSH detection results of DLD-1, MC38, and SW480; DF are the GSSG detection results of DLD-1, MC38, and SW480; GI are the Cys detection results of DLD-1, MC38, and SW480; JL are the MDA detection results of DLD-1, MC38, and SW480; M: Western Blot detection of the effect of DHA-Cy7 on the expression of XCT and GPX4; Fig. 22 : AC are the results of total iron ion detection of DLD-1, MC38 and SW480, respectively; DF are the results of MTT detection of DLD-1, MC38 and SW480 after the combined action of DHA-Cy7 and FAC, respectively; GI are the results of ROS flow cytometric detection of DLD-1, MC38 and SW480 after the combined action of DHA-Cy7 and FAC, respectively; JL are the results of ROS flow cytometric detection of DLD-1, MC38 and SW480 after the combined action of DHA-Cy7 and DFO, respectively; M: Western Blot detection of the effect of DHA-Cy7 on the expression of ferritin, TRF1 and FTH1 in cells; In the accompanying drawings, *, P<0.05; **, P<0.015; ***, P<0.001. DETAILED DESCRIPTION

[0025] In order to make the technical purpose, technical scheme and beneficial effects of the present invention clearer, the technical scheme of the present invention is further described below in conjunction with specific embodiments. However, the embodiments are intended to explain the present invention and cannot be understood as limiting the present invention. If no specific technology or conditions are specified in the embodiments, the technology or conditions described in the literature in the field or the product manual shall be followed.

[0026] In the following examples, the raw materials used are all common commercial products that can be directly purchased or can be prepared using conventional techniques in the art. Room temperature refers to 25±5°C.

[0027] Example 1 Synthesis of ART-Cy7 The present invention provides a synthetic route of artemisinin derivative ART-Cy7, which is as follows: Under Ar gas atmosphere, 1,2,3,3-tetramethyl-3H-indole iodide compound 1 (2003 mg, 6.65 mmol, 2.0 eq), 2-chloro-3-(hydroxymethylene)-cyclohex-1-enecarboxaldehyde compound 2 (572 mg, 3.3 mmol, 1 eq), and sodium acetate (816.6 mg, 10 mmol, 3 eq) were dissolved in a round-bottom bottle containing 60 mL acetic acid-acetic anhydride (1:1, v:v), and reacted at 80°C in the dark for 14 h. After the reaction, 300 mL tert-butyl methyl ether was used for precipitation, filtration, and rotary evaporation to obtain a crude product. The mobile phase was dichloromethane: methanol = 20:1, and the compound 3 was purified by silica gel column to obtain 201.5 mg.

[0028] Under Ar gas atmosphere, compound 3 (600.3 mg, 1.242 mmol, 1 eq) was dissolved in 21 mL of dimethylformamide (DMF), and compound 4 hexaminocaproic acid (651.4 mg, 4.996 mmol, 4 eq) and triethylamine (376.9 mg, 3.725 mmol, 3 eq) were added to the above solution. The reaction was carried out at 85 ° C for 3 h under light-proof conditions. After the reaction, the color of the solution turned blue. DMF was removed by extraction with a mixture of ethyl acetate and water (1:3, v:v), and the crude product was obtained by rotary evaporation. The mobile phase was dichloromethane: methanol = 15:1, and the compound 5 was purified by silica gel column to obtain 245.6 mg.

[0029] Under Ar gas atmosphere, compound 6 (499.8 mg, 1.3 mmol, 1 eq) and DCC (348.9 mg, 1.7mmol, 1.3 eq) were dissolved in dichloromethane and stirred at room temperature for 1h to form a milky white suspension. Compound 7 bis(2-hydroxyethyl) disulfide (301.6 mg, 1.96 mmol, 1.5 eq) and DMAP (47.7 mg, 0.4 mmol, 0.3 eq) were then added to the mixed solution and stirred at room temperature overnight (12 h). After the reaction was completed, the white substance was filtered off and dichloromethane was removed by rotary evaporation to obtain the intermediate ART-SS crude product. The first purification was carried out using a silica gel column with dichloromethane: methanol = 20:1 as the mobile phase; the second purification was carried out using a silica gel column with petroleum ether: ethyl acetate = 2:1 as the mobile phase, and the intermediate compound 8 was finally obtained: 355 mg.

[0030] Under Ar gas atmosphere, compound 5 (100.3 mg, 1 mmol, 1 eq) and DCC (45.4 mg, 0.218mmol, 1.3 eq) were dissolved in dichloromethane and stirred at room temperature for 1h to form a transparent solution. Compound 8 CY7-COOH (270.1 mg, 0.52 mmol, 3 eq) and DMAP (21.3 mg, 0.17 mmol, 1eq) were added to the mixed solution, and the color was blue. Stir at room temperature overnight (12 h). After the reaction was completed, dichloromethane was removed by rotary evaporation to obtain a crude product. Using dichloromethane: methanol = 15:1 as the mobile phase, the final compound 9 ART-Cy7 was purified by silica gel column to obtain 69.8 mg.

[0031] Figure 1 ART-Cy7 structural identification is given 1 H NMR (400 MHz, DMSO-d6) spectral data, 1H NMR (400 MHz, DMSO-d6) δ 8.60 (s, 1H), 7.61-7.59 (d, J = 10.4 Hz, 2H), 7.44-7.43(d, J = 6.4 Hz, 2H), 7.31-7.28 (t, J = 6.0 Hz, 2H), 7.15-7.14 (d, J = 6.4 Hz,2H), 7.07-7.04 (t, J = 6.0 Hz, 2H), 5.73-5.71 (d, J = 10.4 Hz, 2H), 5.66-5.64(d, J = 8 Hz, 2H),5.54 (s, 1H), 4.25-4.22 (m, 4H), 3.68-3.67 (m, 2H), 3.42(s,6H), 2.96-2.92 (m, 4H), 2.65-2.58 (m, 4H), 1.59 (s, 12H),1.27 (s, 3H), 0.88-0.87 (d, J = 5.6 Hz, 3H), 0.75-0.74 (d, J = 5.6 Hz, 3H).

[0032] Figure 2 ART-Cy7 structural identification is given 13C NMR (100 MHz, DMSO-d6 ) spectral data, 13C NMR (100 MHz, DMSO-d6) δ 172.6, 171.7, 170.8, 169.2, 167.3, 143.5, 139.6, 137.8,128.1, 122.3, 121.9, 119.9, 108.9, 103.6, 94.2, 91.8, 90.6, 79.8, 64.9, 62.0,61.6, 51.1, 49.5, 47.0, 44.5, 36.4, 36.2, 36.0, 35.9, 33.7, 33.3, 31.6, 28.6,28.3, 28.2, 25.6, 25.5, 24.7, 24.2, 24.1, 21.3, 21.0, 20.0, 15.2, 11.7.

[0033] Figure 3 The HRMS (MALDI-DHB) spectrum data for the structure identification of ART-Cy7 were given, calcd for C61H82N3O10S2 [MI]+ 1080.5442, found [M-Br]+ 1080.5469.

[0034] Example 2 Synthesis of DHA-Cy7 The present invention provides a synthetic route of artemisinin derivative DHA-Cy7, which is as follows: The synthesis of compound 3 was carried out according to Example 1.

[0035] The synthesis of compound 5 was carried out according to Example 1.

[0036] Under Ar gas atmosphere, compound 6 artemisinin (294.7 mg, 1.036 mmol, 3.0 eq) and DCC (92.7 mg, 0.449 mmol, 1.3 eq) were placed in a 50 mL Schlenk bottle, 6 ml of dichloromethane was added, and the mixture was reacted at room temperature for 1 h. Compound 5 (200.7 mg, 0.347 mmol, 1.0 eq) and DMAP (35.9 mg, 0.294 mmol, 0.85 eq) were added, and the mixture was reacted at room temperature overnight (12 h). The reaction was stopped, cooled to room temperature, and the solvent was removed by rotary evaporation to obtain a crude product, which was then purified by column chromatography (dichloromethane: methanol = 20:1) to obtain compound DHA-Cy7: 80.28 mg.

[0037] Fig.11 The structural identification of DHA-Cy7 is given 1 H NMR (500 MHz, DMSO-d6) spectral data, 1H NMR (400 MHz, DMSO-d6) δ 8.63 (s, 1H), 7.61-7.59 (d, J = 10.4 Hz, 2H), 7.44-7.43(d, J = 6.4 Hz, 2H),7.31-7.28 (t, J = 6.0 Hz, 2H), 7.15-7.14 (d, J = 6.4 Hz,2H), 7.07-7.04 (t, J = 6.0 Hz, 2H), 5.73-5.70 (d, J = 10.4 Hz, 2H), 5.66-5.64(d, J = 8 Hz, 2H),5.53(s, 1H), 3.69-3.68 (m, 2H), 3.42 (s, 6H), 1.59 (s,12H), 1.24 (s, 3H), 0.88-0.87 (d, J = 5.6 Hz, 3H), 0.74-0.73 (d, J = 5.6 Hz, 3H).

[0038] Fig.12 The structural identification of DHA-Cy7 is given 13 C NMR (100 MHz, DMSO-d6) spectral data, 13C NMR (100 MHz, DMSO-d6) δ 171.6, 169.2, 167.2, 143.6, 139.6, 137.7, 129.6, 128.1,122.3, 121.9, 119.8,108.9, 103.5, 94.2, 91.5, 90.5, 79.8, 51.1, 49.4, 47.0,44.5, 35.9, 35.8, 33.7, 33.3, 31.5, 26.8, 25.6, 25.5, 24.8, 24.2, 23.9, 21.3,21.0, 20.0, 11.8.

[0039] Fig.13 The HRMS (MALDI-DHB) spectrum data for the structural identification of DHA-Cy7 are given, HRMS (ESI): calculated for C53H70N3O6 [MI]+ 844.5265, found [M-Br]+ 844.5289.

[0040] Pharmacological activity experiment 1 Application Example 1: MTT and plate cloning assays to determine the effect of ART-Cy7 on the proliferation of colorectal cancer cells and other cancer cells DLD colorectal cancer cells (purchased from the Cell Bank of Type Culture Collection Committee of the Chinese Academy of Sciences) were cultured at 3×10 3 / well were inoculated into 96-well plates and cultured at 37°C for 12 h in 1640 complete medium containing 100 U / mL penicillin and 100 μg / mL streptomycin in a 5% CO2 environment. Then, different concentrations of ART-Cy7 (50 μM, 25 μM, 12.5 μM, 6.25 μM, 3.12 μM, 1.56 μM, 0.78 μM, 0.39 μM, 0.195 μM, and 0.0975 μM) were added. Five replicate wells were set for each concentration. The cells were cultured for 48 h and the cell growth status was observed after taking pictures (see Figure 5 A) The culture medium was discarded and the cell viability was determined by MTT reagent.

[0041] The determination method is as follows: wash the cells once with serum-free medium, add 15 μL / well of the pre-prepared MTT reaction solution, continue to culture for 4 h, aspirate the supernatant, add 100 μL / well of DMSO to dissolve the reduction product, shake on a shaker for 10 min, read the absorbance value at a wavelength of 490 nm, calculate the cell survival rate, and determine the absorbance value of the ART-Cy7 intervention well / the absorbance value of the control well as the value of the cell survival rate, and use this to calculate the IC value of ART-Cy7 for DLD cells. 50 value.

[0042] IC 50 Refers to the concentration of the inhibitor when cell growth is inhibited by half. Here, it is the concentration of ART-Cy7 when the number of DLD cells is half of that of the control group.

[0043] Results: The IC of ART-Cy7 on DLD cells was 50 The value is 2.102 μM (see Figure 4 AC).

[0044] The same method was used to determine the inhibitory effect of ART-Cy7 on colorectal cancer SW 480 cells and colorectal cancer MC38 cells. The results showed that the IC 50 The values ​​were 2.752 μM (see Figure 4 DF), 2.167 μM (see Figure 4 GI).

[0045] The same method was used to determine the inhibitory effect of ART-Cy7 on other cancer cells, including A549 lung cancer cells, Hepa-1 liver cancer cells, 22RV1 prostate cancer cells, A375 malignant melanoma cells, and EC9706 esophageal cancer cells. 50 The values ​​were 2.783μM, 2.582μM, 2.012μM, 4.522μM, and 5.324μM (see Fig.10 A, B, C, D, E).

[0046] DLD cells were inoculated into 6-well cell culture plates at 500 cells / well. After the cells adhered to the wall, they were treated with gradient drugs and cultured in a 37°C, CO2 incubator for 48 h. Fresh 1640 medium was replaced and continued to be cultured. After the cells in the blank group grew into a certain number of clones visible to the naked eye, they were stained with crystal violet and photographed.

[0047] Results: ART-Cy7 had a significant inhibitory effect on the proliferation of DLD cells (see Figure 5 B).

[0048] The same method was used to determine the inhibitory effect of ART-Cy7 on the proliferation of colorectal cancer SW480 cells and colorectal cancer MC38 cells. The results showed that ART-Cy7 had a significant inhibitory effect on the proliferation of SW480 cells and MC38 cells (see Figure 5 B).

[0049] Application Example 2: Effect of ART-Cy7 on subcutaneous colorectal cancer tumors in vivo and toxicity detection in nude mice with tumors MC38 cells in the logarithmic growth phase were collected and resuspended in physiological saline to a density of 1×10 7 After disinfecting the skin of the nude mouse injection site, the cell suspension was injected subcutaneously into the subcutaneous tissue of the right armpit of the nude mouse. Seven days after inoculation, the blank control group, 5-fluorouracil group and experimental group were treated with normal saline, 3.48 mg / mL 5-fluorouracil and 1 mg / kg ART-Cy7 respectively. The treatment was once every 3 days for 30 days.

[0050] After 30 days, the three groups of nude mice were anesthetized, blood was collected from the eyeballs, and the heart, liver, spleen, lung, kidney and tumor were removed. The tumors and organs were weighed and recorded. After the eyeball blood was processed, the following indicators were detected using an animal-specific analyzer: lymphocytes (LYM), white blood cells (WBC), hemoglobin (HGB) and red blood cells (RBC). According to the instructions of the kit, liver and kidney indicators were detected: alanine aminotransferase (ALT), aspartate aminotransferase (AST), urea nitrogen (BUN), creatinine (CR). Organs were subjected to tissue morphology.

[0051] Results: ART-Cy7 did not cause substantial damage to the heart, liver, spleen, lung, and kidney of mice, and could significantly inhibit the growth of tumors. Moreover, the mice did not lose significant weight compared with the control group (see Figure 6 AO, A tumor volume decreased; B tumor weight decreased; C tumor size photographed; D tumor HE staining showed no significant changes; E heart, liver, spleen, lung and kidney HE staining showed no significant changes; F aspartate aminotransferase AST; G alanine aminotransferase ALT; H creatinine CR; I urea nitrogen BUN; J mouse body weight changes; KO respectively are toxicity test-organ coefficients: heart; liver; spleen; lung; kidney).

[0052] Application Example 3: ART-Cy7 localizes to mitochondria in colorectal cancer cells and induces mitochondrial damage Take 1×10 DLD cells 3 Inoculate in glass-bottomed cell culture dishes, and after the cells adhere to the wall, administer drugs (concentration of 3.12μmol / L) to the cells. After continuing to culture for 48 hours, discard the culture medium, add 1mL of the pre-incubated Mito-Green staining solution (Mito-Green: 1640 complete culture medium = 1:3000) to each culture dish and incubate at 37℃ for 40 minutes, then discard the staining solution, wash twice with NaCl, add 1mL of a certain amount of fresh 1640 complete culture medium, observe fluorescence with a laser confocal microscope and take pictures. Keep away from light throughout the process.

[0053] Results: The green fluorescence of Mito-Green probe and the red fluorescence stimulated by ART-Cy7 partially overlap, indicating that ART-Cy7 can be localized in the mitochondria of DLD cells (see Figure 7 A).

[0054] The same method was used to determine the effect of ART-Cy7 on mitochondrial co-localization in colorectal cancer SW480 cells and colorectal cancer MC38 cells. The results showed that the green fluorescence of the Mito-Green probe and the red fluorescence stimulated by ART-Cy7 partially overlapped, indicating that ART-Cy7 can be localized in the mitochondria of SW480 and MC38 cells (see Figure 7 A).

[0055] Take 1×10 DLD cells 4Inoculate in 6-well cell culture plates. After gradient drug treatment, culture in 1640 medium at 37°C and CO2 incubator for 48 h. Digest with EDTA-free trypsin and collect cells in centrifuge tubes, add 200 μL ATP lysis solution and lyse on ice. After lysis, centrifuge at 4°C and 12000g for 5 min, and take the supernatant for testing. Prepare an equivalent ATP detection working solution according to the ratio of ATP detection reagent: ATP detection reagent diluent = 1: 9, add 100 μL per well to the detection wells of the 96-well plate, and leave at room temperature for 5 min to consume background ATP. After 5 min, add 20 μL of sample to each well, quickly mix with a gun, and measure the RLU value with a chemiluminometer after an interval of 2 s. Pay attention to light protection throughout the process.

[0056] Results: ART-Cy7 induced a decrease in mitochondrial ATP content in DLD cells (see Figure 7 B).

[0057] The same method was used to determine the effect of ART-Cy7 on mitochondrial ATP content in colorectal cancer SW480 cells and colorectal cancer MC38 cells. The results showed that ART-Cy7 could induce a decrease in mitochondrial ATP content in SW480 and MC38 cells (see 7C-D).

[0058] Take 1×10 DLD cells 3 Inoculate in a 24-well cell culture dish, and after the cells adhere to the wall, administer the cell gradient drug. After continuing to culture for 48 hours, discard the medium, wash once with PBS, add 500 μL of fresh 1640 complete medium to each well, and then add 500 μL of the pre-prepared JC-1 staining working solution (JC-1 is diluted by adding 8 mL of ultrapure water to every 50 μL of JC-1 (200 X). Then add 2 mL of JC-1 staining buffer (5 X), and mix well to obtain the JC-1 staining working solution). Mix well. Incubate at 37°C, CO2 incubator for 20 min. During the incubation period, prepare an appropriate amount of JC-1 staining buffer (1 X) by adding 4 mL of ultrapure water to every 1 mL of JC-1 staining buffer (5 X) and place it in an ice bath. After the incubation, remove the supernatant, wash twice with JC-1 staining buffer (1 X), and then add 500 μL of fresh 1640 complete medium and observe under a fluorescence microscope.

[0059] The results of fluorescence microscopy showed that as the concentration of the compound increased, the red light of DLD-1 cells gradually weakened and the green light gradually increased, indicating that ART-Cy7 could induce a decrease in the mitochondrial membrane potential of DLD cells (see Figure 8 ).

[0060] The same method was used to determine the effect of ART-Cy7 on the mitochondrial membrane potential of colorectal cancer SW480 cells and colorectal cancer MC38 cells. The results showed that as the concentration of the compound increased, the red light of SW480 and MC38 cells gradually weakened and the green light gradually increased, indicating that ART-Cy7 can induce a decrease in the mitochondrial membrane potential of SW480 and MC38 cells (see Figure 8 ).

[0061] Take 1×10 DLD cells 4 Inoculate in 6-well cell culture plates. After gradient drug treatment, culture in 1640 complete medium at 37°C, CO2 incubator for 48 h. Flow cytometer detection: Digest with EDTA-free trypsin and collect cells in a centrifuge tube, dilute DCFH-DA (purchased from Prilep) with serum-free 1640 medium to a final concentration of 10 μmol / L, resuspend the cells, incubate in 37°C, CO2 incubator for 20 min, invert and mix every 3-5 min, and then measure with flow cytometer. 2) Fluorescence microscopy detection: Remove cell culture medium, add 1 mL of diluted DCFH-DA, incubate in 37°C, CO2 incubator for 20 min, and use 488 nm excitation wavelength and 525 nm emission wavelength to detect fluorescence intensity.

[0062] The results of flow cytometry and fluorescence detection showed that ART-Cy7 could induce an increase in ROS content in DLD cells (see Fig. 9 AB).

[0063] The same method was used to determine the effect of ART-Cy7 on the ROS production in colorectal cancer SW480 cells and colorectal cancer MC38 cells. The results showed that ART-Cy7 could induce an increase in the ROS content in SW480 and MC38 cells (see Fig. 9 A, C, D).

[0064] Pharmacological activity experiment 2 Application Example 1: MTT and plate cloning assays to determine the effect of DHA-Cy7 on the proliferation of colorectal cancer cells MTT assay was performed according to the experimental operation steps of ART-Cy7. Results: IC values ​​of DHA-Cy7 on DLD-1, SW480, and MC38 cells 50 The value is 0.8407 μM ( Fig.14 AC)、1.142 μM( Fig.14 GI)、0.4616 μM( Fig.14 DF). IC of A549 cells, HEPG2 cells, A375 cells, EC9706 cells, 22RV1 cells 50The values ​​were 0.2867 μM, 0.4389 μM, 0.5658 μM, 2.607 μM, and 0.3854 μM, indicating that DHA-Cy7 can also inhibit other cancer cells ( Fig.14 JN).

[0065] Referring to the experimental operation steps of ART-Cy7, the plate cloning method was used to determine the effect of DHA-Cy7 on the proliferation of colorectal cancer cells MC38, SW480, and DLD-1. The results showed that DHA-Cy7 had a significant inhibitory effect on the proliferation of MC38 cells, SW480 cells, and DLD-1 cells (see Fig.15 ).

[0066] Application Example 2: Effect of DHA-Cy7 on subcutaneous colorectal cancer tumors in vivo and toxicity detection in nude mice with tumors The experimental operation steps of ART-Cy7 were referred to above. The results showed that DHA-Cy7 had no substantial damage to the heart, liver, spleen, lung, and kidney of mice, and there was no obvious abnormality in the blood routine test results. It could significantly inhibit the growth of tumors, and the mice did not lose weight significantly compared with the control group (see Fig.16 ).

[0067] Application Example 3: Effect of combined treatment of small molecule inhibitors and DHA-Cy7 on the proliferation of colorectal cancer cells MC38 cells were cultured at 3×10 3 / well were inoculated into 96-well plates, and cultured at 37 ℃ for 12 h using 1640 complete medium containing 5% CO2, 100 U / mL penicillin and 100 μg / mL streptomycin. A culture medium containing NAC (N-acetylcysteine, purchased from Solebol, 1 mM) was prepared, and DHA-Cy7 was diluted to different concentrations (12.5 μM, 6.25 μM, 3.125 μM, 1.56 μM, 0.78 μM, 0.39 μM, 0.195 μM, 0.0975 μM, 0.04875 μM, 0.02438 μM) with this culture medium, and then administered to the cells. Four replicate wells were set for each concentration, and the culture was continued for 48 h. The culture medium was discarded, and the cell survival rate was determined by MTT reagent.

[0068] The same method was used to determine the inhibitory effect of DHA-Cy7 combined with NAC, Z-VAD-FMK, Necrostatin-1, and DFO on the proliferation of colorectal cancer SW480 cells and colorectal cancer DLD-1 cells. The results showed that it had a significant inhibitory effect on the proliferation of MC38 cells, SW480 cells, and DLD-1 cells (see Fig.17 ).

[0069] Application Example 4: DHA-Cy7 localizes to mitochondria in colorectal cancer cells and induces mitochondrial damage Refer to the above ART-Cy7 experimental operation steps for related experiments.

[0070] The results showed that the green fluorescence of Mito-Green probe and the red fluorescence stimulated by DHA-Cy7 partially overlapped, indicating that DHA-Cy7 can be localized in the mitochondria of MC38, SW480, and DLD-1 cells (see Fig.18 ).

[0071] The same method was used to determine the effect of DHA-Cy7 on mitochondrial ATP content in colorectal cancer cells DLD-1, MC38, and SW480 cells. The results showed that DHA-Cy7 could induce a decrease in mitochondrial ATP content in DLD-1, MC38, and SW480 cells (see Fig.18 ).

[0072] The same method was used to determine the effect of DHA-Cy7 on the mitochondrial membrane potential of colorectal cancer cells MC38, SW480, and DLD-1 cells. The results of fluorescence microscopy showed that as the concentration of the compound increased, the red light of MC38, SW480, and DLD-1 cells gradually weakened and the green light gradually increased, indicating that DHA-Cy7 can induce a decrease in the mitochondrial membrane potential of MC38, SW480, and DLD-1 cells (see Fig.19 ).

[0073] The same method was used to determine the effect of DHA-Cy7 on the ROS production in colorectal cancer cells MC38, SW480, and DLD-1. The results of flow cytometry and fluorescence detection showed that DHA-Cy7 could induce an increase in the ROS content in MC38, SW480, and DLD-1 cells (see Fig. 20 ).

[0074] Application Example 5: DHA-Cy7 induces ferroptosis in colorectal cancer cells 1. GPX4 pathway Take 1×10 MC38 cells 4 Inoculate in 6-well cell culture plates. After gradient drug treatment, culture in complete medium at 37°C, CO2 incubator 1640 for 48 h. Digest with EDTA-free trypsin and collect cells in centrifuge tubes, wash twice with PBS (refer to the instructions of the Solebao GSH content detection kit for subsequent operation steps), and detect the absorbance at 412 nm (blank well A1, sample well A2), △A=A2-A1.

[0075] The same method was used to determine the effect of DHA-Cy7 on the GSH content of colorectal cancer SW480 cells and colorectal cancer DLD-1 cells. The OD value determination results showed that DHA-Cy7 could induce a decrease in the GSH content in MC38, SW480, and DLD-1 cells (see Fig.21 AC).

[0076] Take 1×10 MC38 cells 4 Inoculate in 6-well cell culture plates. After gradient drug treatment, culture in complete medium at 37°C, CO2 incubator 1640 for 48 h. Digest with EDTA-free trypsin and collect cells in centrifuge tubes, wash twice with PBS, and follow the instructions of the Solebow GSSG content detection kit for subsequent operation steps.

[0077] The same method was used to determine the effect of DHA-Cy7 on the GSSG content in colorectal cancer SW480 cells and colorectal cancer DLD-1 cells. The OD value determination results showed that DHA-Cy7 could induce a decrease in the GSSG content in MC38, SW480, and DLD-1 cells (see Fig.21 DF).

[0078] Take 1×10 MC38 cells 4 Inoculate in 6-well cell culture plates. After gradient drug treatment, culture in complete medium at 37°C, CO2 incubator 1640 for 48 h. Digest with EDTA-free trypsin and collect cells in centrifuge tubes, wash twice with PBS, and follow the instructions of Nanjing Jiancheng Cysteine ​​(Cys) Detection Kit for subsequent operation.

[0079] The same method was used to determine the effect of DHA-Cy7 on the Cys production in colorectal cancer SW480 cells and colorectal cancer DLD-1 cells. The OD value determination results showed that DHA-Cy7 could induce a decrease in the Cys content in MC38, SW480, and DLD-1 cells (see Fig.21 GI).

[0080] Take 1×10 MC38 cells 4 Inoculate in 6-well cell culture plates. After gradient drug treatment, culture in complete medium at 37°C, CO2 incubator 1640 for 48 h. Digest with EDTA-free trypsin and collect cells in centrifuge tubes, wash twice with PBS, and follow the instructions of the Bio-Tech lipid oxidation (MDA) detection kit for subsequent operation steps.

[0081] The same method was used to determine the effect of DHA-Cy7 on the MDA content of colorectal cancer SW480 cells and colorectal cancer DLD-1 cells. The OD value determination results showed that DHA-Cy7 could induce an increase in the MDA content in MC38, SW480, and DLD-1 cells (see Fig.21JL).

[0082] Take 1×10 DLD-1 cells 6 The cells were inoculated in 60 mm cell culture dishes. After gradient drug treatment, the cells were cultured in complete medium at 37°C and CO2 incubator for 48 h, and then the proteins were extracted. The expression of GPX4 was detected by Western Blot.

[0083] The same method was used to determine the effect of DHA-Cy7 on the expression of GPX4 in colorectal cancer SW480 cells and colorectal cancer DLD-1 cells. Western Blot results showed that DHA-Cy7 induced a decrease in the expression of GPX4 in DLD-1 and SW480 cells (see Fig.21 M).

[0084] Summary: DHA-Cy7 mediated the decrease of GSH, GSSG, and Cys contents and the increase of MDA content in MC38, DLD-1, and SW480 cells through GPX4, further indicating that it can induce cell ferroptosis.

[0085] , TFR1 pathway Take 1×10 MC38 cells 4 Inoculate in 6-well cell culture plates. After gradient drug treatment, culture in complete medium at 37°C, CO2 incubator 1640 for 48 h. Digest with EDTA-free trypsin and collect cells in centrifuge tubes, wash twice with PBS, and follow the instructions of Nanjing Jiancheng Total Iron Detection Kit for subsequent operation.

[0086] The same method was used to determine the effect of DHA-Cy7 on the total iron content of colorectal cancer SW480 cells and colorectal cancer DLD-1 cells. The OD value determination results showed that DHA-Cy7 could induce an increase in the total iron content of MC38, SW480, and DLD-1 cells (see Fig. 22 AC).

[0087] Take 1×10 DLD-1 cells 6 The cells were inoculated in 60 mm cell culture dishes. After gradient drug treatment, the cells were cultured in complete medium at 37°C and CO2 incubator for 48 h, and then proteins were extracted. The expression of transferrin receptor TFR1 and Ferritin was detected by Western Blot.

[0088] The same method was used to determine the effect of DHA-Cy7 on the expression of TFR1 and Ferritin in colorectal cancer SW480 cells. The results showed that DHA-Cy7 could induce the increase of TFR1 and Ferritin expression in DLD-1 and SW480 cells (see Fig. 22 M).

[0089] After adding the ferroptosis agonist FAC, the ROS production content and cell viability of MC38, SW480, and DLD-1 cells were measured again according to the above method. The OD value measurement results showed that the combined action of DHA-Cy7 and FAC increased the ROS content of MC38, SW480, and DLD-1 cells compared with the action of DHA-Cy7 alone, and the cell viability decreased (see Fig. 22 DI).

[0090] After adding the iron chelator DFO, the ROS production content of MC38, SW480, and DLD-1 cells was measured again according to the above method. The OD value measurement results showed that the combined effect of DHA-Cy7 and DFO reduced the ROS content of MC38, SW480, and DLD-1 cells compared with the effect of DHA-Cy7 alone (see Fig. 22 JL).

[0091] Summary: DHA-Cy7 mediated the increase of total iron and ROS contents in MC38, SW480, and DLD-1 cells through TFR1, further inducing cell ferroptosis.

Claims

1. An artemisinin derivative, characterized in that: Has at least one of the following structural formulas: 。 2. The method for preparing the artemisinin derivative according to claim 1, characterized in that: When the artemisinin derivative is ART-Cy7, the method comprises the following steps: 1) Under an inert gas atmosphere, compound 1, compound 2, and sodium acetate were dissolved in acetic acid-acetic anhydride, and reacted at 60-90° C. in the dark for 10-20 hours. After the reaction, tert-butyl methyl ether was used for precipitation, filtration, rotary evaporation, and silica gel column purification to obtain compound 3; 2) Under an inert gas atmosphere, compound 3 was dissolved in dimethylformamide, and compound 4 hexaminocaproic acid and triethylamine were added, and the mixture was reacted at 70-90°C for 2-5 hours in the dark. After the reaction, dimethylformamide was removed by extraction, and the mixture was subjected to rotary evaporation and purified by silica gel column to obtain compound 5; 3) Under an inert gas atmosphere, compound 6 and N,N'-dicyclohexylcarbodiimide were dissolved in dichloromethane and stirred at room temperature to form a milky white suspension, and compound 7 and 4-dimethylaminopyridine were added and stirred at room temperature for 2-5 hours. After the reaction was completed, the mixture was filtered, rotary evaporated, and purified by silica gel column to obtain intermediate compound 8; 4) Under an inert gas atmosphere, compound 5 and N,N'-dicyclohexylcarbodiimide were dissolved in dichloromethane and stirred at room temperature to form a transparent solution. Compound 8 and 4-dimethylaminopyridine were then added and stirred at room temperature for 10-14 h. After the reaction was completed, the solution was rotary evaporated and purified by silica gel column to obtain the final compound 9; 。 3. The method for preparing the artemisinin derivative according to claim 1, characterized in that: When the artemisinin derivative is DHA-Cy7, the method comprises the following steps: 1) Under an inert gas atmosphere, compound 1, compound 2, and sodium acetate were dissolved in acetic acid-acetic anhydride, and reacted at 60-90° C. in the dark for 10-20 hours. After the reaction, tert-butyl methyl ether was used for precipitation, filtration, rotary evaporation, and silica gel column purification to obtain compound 3; 2) Under an inert gas atmosphere, compound 3 was dissolved in dimethylformamide, and compound 4 hexaminocaproic acid and triethylamine were added, and the mixture was reacted at 70-90°C for 2-5 hours in the dark. After the reaction, dimethylformamide was removed by extraction, and the mixture was subjected to rotary evaporation and purified by silica gel column to obtain compound 5; 3) Under an inert gas atmosphere, compound 6, N,N'-dicyclohexylcarbodiimide and dichloromethane were reacted at room temperature for 1-2 hours, compound 5 and 4-dimethylaminopyridine were added, and the reaction was carried out at room temperature for 10-14 hours, and the mixture was cooled to room temperature, rotary evaporated, and purified by column chromatography to obtain compound DHA-Cy7; 。 4. The method for preparing an artemisinin derivative according to claim 2, characterized in that: The molar ratio of compound 1 to compound 2 is 2-2.5:1; the molar ratio of compound 3 to compound 4 is 1:4-4.5; the molar ratio of compound 6 to compound 7 is 1:1.2-2.0; and the molar ratio of compound 5 to compound 8 is 1:2-4.

5. The method for preparing the artemisinin derivative according to claim 3, characterized in that: The molar ratio of compound 1 to compound 2 is 2-2.5:1; the molar ratio of compound 3 to compound 4 is 1:4-4.5; and the molar ratio of compound 5 to compound 6 is 1:2-4.

6. A pharmaceutical composition, characterized in that Comprising the artemisinin derivative according to claim 1.

7. Use of the artemisinin derivative according to claim 1 or the pharmaceutical composition according to claim 6 in the preparation of anti-tumor drugs.

8. The use of the artemisinin derivative according to claim 7 in the preparation of an anti-tumor drug, characterized in that: The tumor includes at least one of colorectal cancer, lung cancer, liver cancer, prostate cancer, malignant melanoma, and esophageal cancer.

9. The use of the artemisinin derivative according to claim 8 in the preparation of an anti-tumor drug, characterized in that: The artemisinin derivatives can inhibit the vitality of colorectal cancer cells, induce cell apoptosis and generate reactive oxygen species.

10. The use of the artemisinin derivative according to claim 8 in the preparation of an anti-tumor drug, characterized in that: The effective concentration of the artemisinin derivative is 0.02438-50 μM.

Images