Artemisinin derivatives and their applications in the preparation of anti-tumor drugs

Abstract

The present invention relates to an artemisinin derivative having at least one of the following structural formulas: [The text then abruptly shifts topics.] This invention uses indole iodide as a target to construct tumor-targeted mitochondria, achieving tumor-targeted delivery of artemisinin-based drugs. This invention uses Cy7 to link artemisinin, improving its targeting and bioavailability, and enhancing its anti-tumor effects.

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 its application in the preparation of anti-tumor drugs. Background Art

[0002] Cancer is currently one of the most common causes of death worldwide, with a high mortality rate. Cancer treatments primarily include surgery, chemotherapy, and radiotherapy. Existing technologies have discovered numerous anti-tumor drugs, and many chemotherapeutic agents have been used in clinical practice. However, many chemotherapeutic drugs have significant side effects and drug resistance, necessitating the development of effective drug delivery systems to enhance drug targeting and absorption within tumor cells.

[0003] Artemisinin and its derivatives are effective antimalarial drugs. Dihydroartemisinin and artesunate, as derivatives of artemisinin, exhibit good water solubility and antimalarial activity. These drugs contain peroxide bridges, which are their primary mechanism of anti-tumor activity. Previous studies have demonstrated that dihydroartemisinin and artesunate have significant toxic side effects against various malignancies through multiple mechanisms, including inhibition of tumor angiogenesis, promotion of apoptosis, cell cycle blockade, reversal of drug resistance, and cytotoxicity. Dihydroartemisinin and artesunate exert their anti-tumor effects by releasing reactive oxygen species and promoting oxidative stress. However, these drugs suffer from poor solubility, lack of targeting, low bioavailability, and the development of drug resistance, necessitating their combination with other drugs. Numerous studies have combined artemisinin-based drugs with other drugs to enhance their efficacy.

[0004] Recent studies have shown that near-infrared fluorescent probes such as indole iodide heptamethine cyanine (Cy7) exhibit excellent targeting properties in tumor cells. These compounds can be used for fluorescence imaging, photothermal therapy, and other cancer treatments. They also exhibit excellent biocompatibility and can form complexes with drugs, thereby improving drug delivery, reducing systemic toxicity, and increasing bioavailability.

[0005] This invention uses indole iodide as a target to construct tumor-targeted mitochondria, achieving tumor-targeted delivery of artemisinin-based drugs. This invention improves the targeting and bioavailability of artemisinin through the Cy7 linker, thereby enhancing its anti-tumor effects. 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 through Cy7, thereby improving the targeting and bioavailability of artemisinin and enhancing the anti-tumor effect of artemisinin.

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

[0008] To achieve the above object, the present invention adopts the following technical solutions:

[0009] An artemisinin derivative having at least one of the following structural formulas:

[0010] .

[0011] ART-Cy7, its relevant properties are as follows:

[0012] Molecular formula: C 61 H 82 N3O 10 S2; molecular weight: 1207.4492; detection method: 1 HNMR, 13 CNMR, HRMS; Properties: 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 primarily used to inhibit the growth of colorectal cancer cells, including DLD-1, SW480, and MC38.

[0013] DHA-Cy7, its relevant properties are as follows:

[0014] Molecular formula: C 53 H 70 N3O6I; molecular weight: 971.43; detection method: 1 HNMR, 13 CNMR, HRMS; Properties: 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 containing DHA-Cy7 as its active ingredient. The colorectal cancer cells described are MC38 cells, SW480 cells, or DLD-1 cells.

[0015] A method for preparing the above-mentioned artemisinin derivative, when the artemisinin derivative is ART-Cy7, comprises the following steps:

[0016] 1) Under an inert gas atmosphere, compound 1, compound 2, and sodium acetate were dissolved in acetic acid-acetic anhydride and reacted in the dark at 60-90°C for 10-20 hours. After the reaction, the mixture was precipitated with tert-butyl methyl ether, filtered, rotary evaporated, and purified on a silica gel column to obtain compound 3;

[0017]

[0018] 2) Under an inert gas atmosphere, compound 3 was dissolved in dimethylformamide, and compound 4 (hexaminohexanoic acid) and triethylamine were added. 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 rotary evaporated and purified on a silica gel column to obtain compound 5.

[0019]

[0020] 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. Compound 7 and 4-dimethylaminopyridine (DMAP) were then added and stirred at room temperature for 2-5 hours. After the reaction was completed, the mixture was filtered, rotary evaporated, and purified on a silica gel column to obtain intermediate compound 8.

[0021]

[0022] 4) Under an inert 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, the mixture was rotary evaporated and purified on a silica gel column to obtain compound 9.

[0023] .

[0024] 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.

[0025] A method for preparing the above-mentioned artemisinin derivative, when the artemisinin derivative is DHA-Cy7, comprises the following steps:

[0026] 1) Under an inert gas atmosphere, compound 1, compound 2, and sodium acetate were dissolved in acetic acid-acetic anhydride and reacted in the dark at 60-90°C for 10-20 hours. After the reaction, the mixture was precipitated with tert-butyl methyl ether, filtered, rotary evaporated, and purified on a silica gel column to obtain compound 3;

[0027]

[0028] 2) Under an inert gas atmosphere, compound 3 was dissolved in dimethylformamide, and compound 4 (hexaminohexanoic acid) and triethylamine were added. 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 rotary evaporated and purified on a silica gel column to obtain compound 5.

[0029]

[0030] 3) Under an inert 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 reacted at room temperature for 10-14 hours. The mixture was cooled to room temperature, rotary evaporated, and purified by column chromatography to obtain compound DHA-Cy7.

[0031] .

[0032] 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.

[0033] The present invention also provides a pharmaceutical composition comprising the artemisinin derivatives. Specifically, the artemisinin derivatives are used as active ingredients and are compounded with conventional excipients in the art to form a compound preparation, such as tablets, granules, pills, capsules, or injections.

[0034] The present invention provides the use of the above-mentioned artemisinin derivatives in the preparation of anti-tumor drugs.

[0035] 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.

[0036] 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.

[0037] Furthermore, the effective concentration of ART-Cy7 is 0.0975-50 μM. The ART-Cy7 inhibits the growth of subcutaneous tumors in BALB / C nude mice bearing the colorectal cancer cell line MC38 at a dose of 1 mg / kg for 3 days. The ART-Cy7 localizes 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.

[0038] The present invention provides a method for inhibiting tumor cell proliferation in vitro, comprising adding ART-Cy7 to a culture medium of tumor cells, wherein the final concentration of 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.

[0039] Furthermore, 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 bearing the colorectal cancer cell line MC38 is 3 mg / kg.2 days. The DHA-Cy7 is localized in the mitochondria of the colorectal cancer cell lines MC38, SW480, or DLD-1 and causes mitochondrial damage. The DHA-Cy7 induces the production of reactive oxygen species in the colorectal cancer cell lines MC38, SW480, or DLD-1. The DHA-Cy7 induces a decrease in the activity of the xCT-GPX4 antioxidant system in 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.

[0040] The present invention provides a method for inhibiting tumor cell proliferation in vitro, comprising adding DHA-Cy7 to a culture medium of tumor cells, wherein the final concentration of the added 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 a culture medium of tumor cells, wherein the final concentration of the added DHA-Cy7 is 0.02438-12.5 μM.

[0041] Compared with the prior art, the present invention has the following beneficial effects:

[0042] The present invention provides a novel artemisinin derivative ART-Cy7 and / or DHA-Cy7, a method for synthesizing the same, and its use 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-based drugs. The present invention connects artemisinin with Cy7, thereby improving the targeting and bioavailability of artemisinin and enhancing 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+Content detection results and Western Blot analysis of TFR1 showed that DHA-Cy7 can induce ferroptosis. The small molecule compounds ART-Cy7 and / or DHA-Cy7 of the present invention can be developed as new anti-tumor drugs, especially anti-colorectal tumor drugs, or auxiliary components thereof. They have significant tumor inhibition effects and will provide new approaches and means for treating and curing colorectal tumors. BRIEF DESCRIPTION OF THE DRAWINGS

[0043] Figure 1 :ART-Cy7 structure identification 1 H NMR (400 MHz, DMSO-d6) spectral data;

[0044] Figure 2 :ART-Cy7 structure identification 13 C NMR (100 MHz, DMSO-d6 ) spectral data;

[0045] Figure 3 : HRMS (MALDI-DHB) spectral data for ART-Cy7 structure identification;

[0046] 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;

[0047] Figure 5 :A: DLD-1, SW480, and MC38 cell morphology after ART-Cy7 administration; B: Plate cloning results;

[0048] Figure 6 : A: Tumor-bearing model test - tumor volume; B: Tumor-bearing model test - tumor weight; C: Toxicity test - images of nude mice in the Ctl, 5-Fu, and ART-Cy7 groups; D: Toxicity test - organ HE staining results; E: Tumor-bearing model test - tumor HE staining results; FG: Toxicity test - liver injury - AST and ALT determination; HI: Toxicity test - kidney injury - CR and BUN determination; J: Toxicity test - nude mouse body weight; KO are toxicity test - organ coefficients of heart, liver, spleen, lung, and kidney, respectively;

[0049] Figure 7 :A: mitochondrial co-localization results; BD: ATP detection results of DLD-1, SW480, and MC38 respectively;

[0050] Figure 8 : JC-1 staining results;

[0051] Figure 9 :A: ROS fluorescence detection results; BD: ROS flow cytometry detection results of DLD-1, SW480, and MC38 respectively;

[0052] Figure 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;

[0053] Figure 11 :DHA-Cy7 structure identification 1 H NMR (500 MHz, DMSO-d6) spectral data,

[0054] Figure 12 :DHA-Cy7 structure identification 13 C NMR (100 MHz, DMSO-d6) spectral data,

[0055] Figure 13 : DHA-Cy7 structure identification HRMS (MALDI-DHB) spectrum data,

[0056] Figure 14 :A, D, G: MTT results of MC38, SW480, and DLD-1 after DHA administration; B, E, H: MTT results of MC38, SW480, and DLD-1 after Cy7 administration; C, F, I: MTT results of MC38, SW480, and DLD-1 after DHA-Cy7 administration; JN: MTT results of DHA-Cy7 on other cancer cells A549, HEPG2, A375, EC9706, and 22RV1;

[0057] Figure 15 : Plate cloning results;

[0058] Figure 16 : A: Tumor-bearing model test - tumor weight; B: Tumor-bearing model test - tumor volume; C: Toxicity test - tumor images of NS group, 5-Fu group, and DHA-Cy7 group; D: Tumor-bearing model test - HE staining results of tumors; E: Toxicity test - body weight of nude mice; F: Toxicity test - HE staining results of organs; GK are the coefficients of heart, liver, spleen, lung, and kidney in toxicity test; LM are the AST and ALT measurements of liver damage in toxicity test; NO are the BUN and CR measurements of kidney damage in toxicity test; PT are the blood routine tests of white blood cells, red blood cells, lymphocytes, monocytes, and platelets;

[0059] Figure 17 : A to D are the effects of the combination of DHA-Cy7 with NAC, Z-VAD-FMK, Necrostatin-1, and DFO on the proliferation of DLD-1, MC38, and SW480 cells, respectively;

[0060] Figure 18 :A: Co-localization results of DHA-Cy7 in mitochondria of DLD-1, MC38, and SW480; BD: ATP detection results of DLD-1, MC38, and SW480 respectively;

[0061] Figure 19 : JC-1 staining results;

[0062] Figure 20 :A: ROS fluorescence detection results; BD are the results of ROS flow cytometric detection of DLD-1, MC38, and SW480; EG are the results of ROS flow cytometric detection of DLD-1, SW480, and MC38 after the combined action of DHA-Cy7 and NAC;

[0063] Figure 21 : AC are the GSH detection results of DLD-1, MC38, and SW480, respectively; DF are the GSSG detection results of DLD-1, MC38, and SW480, respectively; GI are the Cys detection results of DLD-1, MC38, and SW480, respectively; JL are the MDA detection results of DLD-1, MC38, and SW480, respectively; M: Western Blot analysis of the effect of DHA-Cy7 on the expression of XCT and GPX4;

[0064] Figure 22 : AC are the results of total iron ion detection in DLD-1, MC38 and SW480, respectively; DF are the results of MTT detection in DLD-1, MC38 and SW480 after the combined action of DHA-Cy7 and FAC, respectively; GI are the results of ROS flow cytometric detection in DLD-1, MC38 and SW480 after the combined action of DHA-Cy7 and FAC, respectively; JL are the results of ROS flow cytometric detection in DLD-1, MC38 and SW480 after the combined action of DHA-Cy7 and DFO, respectively; M: Western Blot detection result of the effect of DHA-Cy7 on the expression of ferritin, TRF1 and FTH1 in cells; In the accompanying figures, *, P<0.05; **, P<0.015; ***, P<0.001. DETAILED DESCRIPTION

[0065] In order to make the technical purpose, technical solution and beneficial effects of the present invention clearer, the technical solution of the present invention is further described below in conjunction with specific embodiments. However, the embodiments are intended to explain the present invention and are not to be construed 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 instructions shall be followed.

[0066] In the following examples, all raw materials used are commercially available products that can be purchased directly or prepared using conventional techniques in the art. Room temperature refers to 25±5°C.

[0067] Example 1 Synthesis of ART-Cy7

[0068] The present invention provides a synthetic route for the artemisinin derivative ART-Cy7, which is as follows:

[0069]

[0070] Under an Ar atmosphere, 1,2,3,3-tetramethyl-3H-indole iodide (2003 mg, 6.65 mmol, 2.0 eq), 2-chloro-3-(hydroxymethylene)-cyclohex-1-enecarbaldehyde (572 mg, 3.3 mmol, 1 eq), and sodium acetate (816.6 mg, 10 mmol, 3 eq) were dissolved in 60 mL of acetic acid and acetic anhydride (1:1, v:v) in a round-bottom flask. The mixture was reacted at 80°C in the dark for 14 h. After completion of the reaction, the product was precipitated with 300 mL of tert-butyl methyl ether, filtered, and rotary evaporated to obtain the crude product. Purification was performed on a silica gel column using a mobile phase of dichloromethane:methanol (20:1) to yield 201.5 mg of compound 3.

[0071]

[0072] Under an Ar atmosphere, compound 3 (600.3 mg, 1.242 mmol, 1 eq) was dissolved in 21 mL of dimethylformamide (DMF). Compound 4 (hexaminohexanoic acid) (651.4 mg, 4.996 mmol, 4 eq) and triethylamine (376.9 mg, 3.725 mmol, 3 eq) were then added to the solution. The reaction was incubated at 85°C for 3 h in the dark. Upon completion, 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. Purification was performed on a silica gel column using a mobile phase of dichloromethane:methanol (15:1) to yield 245.6 mg of compound 5.

[0073]

[0074] Under an Ar atmosphere, compound 6 (499.8 mg, 1.3 mmol, 1 eq) and DCC (348.9 mg, 1.7 mmol, 1.3 eq) were dissolved in dichloromethane and stirred at room temperature for 1 hour 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 mixture and stirred at room temperature overnight (12 hours). After the reaction, the white material was filtered and the dichloromethane was removed using a rotary evaporator to obtain the crude intermediate ART-SS. The crude intermediate was purified using a silica gel column with a mobile phase of dichloromethane:methanol (20:1). The crude intermediate ART-SS was purified using a silica gel column with a mobile phase of petroleum ether:ethyl acetate (2:1) to obtain 355 mg of the intermediate compound 8.

[0075]

[0076] Under an Ar atmosphere, compound 5 (100.3 mg, 1 mmol, 1 eq) and DCC (45.4 mg, 0.218 mmol, 1.3 eq) were dissolved in dichloromethane and stirred at room temperature for 1 hour to form a clear solution. Compound 8 CY7-COOH (270.1 mg, 0.52 mmol, 3 eq) and DMAP (21.3 mg, 0.17 mmol, 1 eq) were then added to the mixed solution, resulting in a blue color. The mixture was stirred at room temperature overnight (12 hours). After the reaction, the dichloromethane was removed using a rotary evaporator to obtain the crude product. The product was purified using a silica gel column with a mobile phase of dichloromethane:methanol (15:1) to obtain 69.8 mg of the final compound 9 ART-Cy7.

[0077] Figure 1 ART-Cy7 structure identification is given 1H 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).

[0078] Figure 2 ART-Cy7 structure identification is given 13 C 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.

[0079] Figure 3The HRMS (MALDI-DHB) spectrum data for the structural identification of ART-Cy7 are given, calcd for C61H82N3O10S2 [MI]+ 1080.5442, found [M-Br]+ 1080.5469.

[0080] Example 2 Synthesis of DHA-Cy7

[0081] The present invention provides a synthetic route for the artemisinin derivative DHA-Cy7, which is as follows:

[0082]

[0083] The synthesis of compound 3 was carried out according to Example 1.

[0084]

[0085] The synthesis of compound 5 was carried out according to Example 1.

[0086]

[0087] Under an Ar atmosphere, compound 6 (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 flask, 6 mL of dichloromethane was added, and the mixture was allowed to react 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 then added and allowed to react at room temperature overnight (12 h). The reaction was stopped, cooled to room temperature, and the solvent was removed by rotary evaporation to obtain the crude product. Column chromatography (dichloromethane:methanol = 20:1) afforded 80.28 mg of compound DHA-Cy7.

[0088] Figure 11 The structural identification of DHA-Cy7 is given 1H 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).

[0089] Figure 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.

[0090] Figure 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.

[0091] Pharmacological activity experiment 1

[0092] 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

[0093] DLD colorectal cancer cells (purchased from the Cell Bank of Type Culture Collection Committee of the Chinese Academy of Sciences) were cultured at a rate of 3 × 10 3 Cells were inoculated into 96-well plates at 4 °C for 12 h in 1640 complete medium containing 100 U / mL penicillin and 100 μg / mL streptomycin under 5% CO2. ART-Cy7 at different concentrations (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) was then 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.

[0094] The assay method was as follows: wash the cells once with serum-free medium, add 15 μL / well of pre-prepared MTT reaction solution, continue culturing for 4 h, aspirate and discard 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.

[0095] IC 50 Refers to the concentration of the inhibitor at which cell growth is inhibited by half. Here, it is the concentration of ART-Cy7 that produces half the number of DLD cells compared to the control group.

[0096] Results: The IC of ART-Cy7 on DLD cells was 50 The value is 2.102 μM (see Figure 4 AC).

[0097] 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).

[0098] The same method was used to determine the inhibitory effect of ART-Cy7 on other cancer cells: 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 Figure 10 A, B, C, D, E).

[0099] DLD cells were seeded at 500 cells / well in a 6-well cell culture plate. After the cells adhered, they were treated with a gradient of drugs and cultured in a 37°C, CO2 incubator for 48 hours. After that, fresh 1640 medium was replaced and continued to culture. After the cells in the blank control group grew to a certain number of visible colonies, they were stained with crystal violet and photographed.

[0100] Results: ART-Cy7 had a significant inhibitory effect on the proliferation of DLD cells (see Figure 5 B).

[0101] 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).

[0102] Application Example 2: Effects of ART-Cy7 on Subcutaneous Colorectal Cancer Tumors in Vivo and Toxicity Detection in Nude Mice

[0103] 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 at the injection site, the cell suspension was subcutaneously injected into the subcutaneous tissue of the right axilla of the nude mice. 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. Treatment was administered every three days for 30 days.

[0104] After 30 days, the three groups of nude mice were anesthetized, and blood was collected from the eyeballs. The heart, liver, spleen, lungs, kidneys, and tumors were removed, and the tumors and organs were weighed and recorded. After processing, the eyeball blood was analyzed using an animal analyzer for the following parameters: lymphocytes (LYM), white blood cells (WBC), hemoglobin (HGB), and red blood cells (RBC). Liver and kidney parameters such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), blood urea nitrogen (BUN), and creatinine (CR) were measured according to the kit instructions. Histomorphological examination of the organs was performed.

[0105] Results: ART-Cy7 did not cause substantial damage to the heart, liver, spleen, lung, and kidney of mice, and significantly inhibited tumor growth. The mice did not lose significant weight compared with the control group (see Figure 6 AO, A. The tumor volume decreased; B. The tumor weight decreased; C. The tumor size was photographed; D. No significant changes were observed in HE staining of the tumor; E. No significant changes were observed in HE staining of the heart, liver, spleen, lung, and kidney; F. Aspartate aminotransferase (AST); G. Alanine aminotransferase (ALT); H. Creatinine (CR); I. Blood urea nitrogen (BUN); J. Changes in mouse body weight; KO, respectively, are toxicity test-organ coefficients: heart; liver; spleen; lung; kidney).

[0106] Application Example 3: ART-Cy7 localizes to mitochondria in colorectal cancer cells and induces mitochondrial damage

[0107] Take 1×10 DLD cells 3 Seed cells onto glass-bottomed culture dishes and, after cells adhered, administer the drug (at a concentration of 3.12 μmol / L). After 48 h of culture, discard the culture medium and add 1 mL of pre-warmed Mito-Green staining solution (Mito-Green:1640 complete medium = 1:3000) to each dish. Incubate at 37°C for 40 min. Discard the staining solution, rinse twice with NaCl, and add 1 mL of fresh 1640 complete medium. Observe fluorescence using a laser confocal microscope and take photos. Protect from light throughout the process.

[0108] Results: The green fluorescence of Mito-Green probe and the red fluorescence stimulated by ART-Cy7 partially overlapped, indicating that ART-Cy7 can be localized in the mitochondria of DLD cells (see Figure 7 A).

[0109] 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 excited by ART-Cy7 partially overlapped, indicating that ART-Cy7 can be localized in the mitochondria of SW480 and MC38 cells (see Figure 7 A).

[0110] Take 1×10 DLD cells 4Cells were seeded into 6-well cell culture plates. After gradient drug treatment, cells were cultured in 1640 medium at 37°C in a CO2 incubator for 48 h. Digested with EDTA-free trypsin, the cells were collected in a centrifuge tube, and lysed on ice by adding 200 μL of ATP lysis buffer. After lysis, the cells were centrifuged at 12,000 g for 5 min at 4°C, and the supernatant was collected for analysis. An equivalent amount of ATP assay working solution was prepared using a ratio of ATP assay reagent: ATP assay reagent diluent of 1:9. 100 μL per well was added to the assay wells of a 96-well plate and allowed to stand at room temperature for 5 min to deplete background ATP. After 5 min, 20 μL of sample was added to each well and mixed quickly with a pipette. After a 2-s interval, the RLU value was measured using a chemiluminescence analyzer. Protect from light throughout the process.

[0111] Results: ART-Cy7 induced a decrease in mitochondrial ATP content in DLD cells (see Figure 7 B).

[0112] The same method was used to determine the effect of ART-Cy7 on the mitochondrial ATP content in colorectal cancer SW480 cells and colorectal cancer MC38 cells. The results showed that ART-Cy7 could induce a decrease in the mitochondrial ATP content in SW480 and MC38 cells (see 7C-D).

[0113] Take 1×10 DLD cells 3 Cells were seeded in 24-well cell culture dishes and treated with a gradient of agents after cells adhered. After 48 h of culture, the medium was discarded, the cells were washed once with PBS, and 500 μL of fresh 1640 complete medium was added to each well. Then, 500 μL of pre-prepared JC-1 staining working solution (JC-1 was diluted at a ratio of 8 mL of ultrapure water per 50 μL of JC-1 (200X)). Then, 2 mL of JC-1 staining buffer (5X) was added and mixed thoroughly to obtain the JC-1 staining working solution). The cells were incubated at 37°C in a CO2 incubator for 20 min. During the incubation period, an appropriate amount of JC-1 staining buffer (1X) was prepared by adding 4 mL of ultrapure water per 1 mL of JC-1 staining buffer (5X) and placed on ice. After the incubation period, the supernatant was removed, the cells were washed twice with JC-1 staining buffer (1X), and 500 μL of fresh 1640 complete medium was added to each well. The cells were observed under a fluorescence microscope.

[0114] Fluorescence microscopy results 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 ).

[0115] 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 ).

[0116] Take 1×10 DLD cells 4 Cells were seeded into 6-well cell culture plates. After gradient drug treatment, cells were cultured in 1640 complete medium at 37°C in a CO2 incubator for 48 hours. Flow cytometry analysis: Cells were digested with EDTA-free trypsin and collected in a centrifuge tube. DCFH-DA (purchased from Priligen) was diluted in serum-free 1640 medium to a final concentration of 10 μmol / L. The cells were resuspended and incubated at 37°C in a CO2 incubator for 20 minutes, inverting every 3-5 minutes to mix thoroughly. The cells were then analyzed by flow cytometry. 2) Fluorescence microscopy: Cell culture medium was removed, and 1 mL of diluted DCFH-DA was added. After incubation at 37°C in a CO2 incubator for 20 minutes, fluorescence intensity was measured using an excitation wavelength of 488 nm and an emission wavelength of 525 nm.

[0117] The results of flow cytometry and fluorescence detection showed that ART-Cy7 could induce the increase of ROS content in DLD cells (see Figure 9 AB ).

[0118] 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 Figure 9 A, C, D).

[0119] Pharmacological activity experiment 2

[0120] Application Example 1: MTT and plate cloning assays to determine the effect of DHA-Cy7 on colorectal cancer cell proliferation

[0121] MTT assay was performed according to the experimental procedures of ART-Cy7. Results: IC values ​​of DHA-Cy7 on DLD-1, SW480 and MC38 cells were 0. 50 The value was 0.8407 μM ( Figure 14 AC), 1.142 μM ( Figure 14 GI), 0.4616 μM ( Figure 14 DF). IC for 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 ( Figure 14 JN).

[0122] The effect of DHA-Cy7 on the proliferation of colorectal cancer cells MC38, SW480, and DLD-1 was determined by plate cloning assay using the experimental procedures of ART-Cy7. The results showed that DHA-Cy7 had a significant inhibitory effect on the proliferation of MC38, SW480, and DLD-1 cells (see Figure 15 ).

[0123] Application Example 2: Effect of DHA-Cy7 on subcutaneous colorectal cancer tumors in vivo and toxicity testing in nude mice with tumors

[0124] The experimental operation steps of ART-Cy7 were referred to above. The results showed that DHA-Cy7 did not cause substantial damage to the heart, liver, spleen, lungs, and kidneys of mice. Routine blood tests showed no significant abnormalities. It also significantly inhibited tumor growth, and the mice did not lose significant weight compared to the control group (see Figure 16 ).

[0125] Application Example 3: Effects of combined treatment with small molecule inhibitors and DHA-Cy7 on the proliferation of colorectal cancer cells

[0126] MC38 cells were cultured at 3×10 3 Cells were seeded into 96-well plates and cultured in 1640 complete medium containing 5% CO₂, 100 U / mL penicillin, and 100 μg / mL streptomycin at 37°C for 12 h. A medium containing NAC (1 mM N-acetylcysteine, purchased from Solebro) was prepared and used to dilute DHA-Cy7 to various 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, and 0.02438 μM). The cells were then dosed with DHA-Cy7, with four replicates per well. The culture was continued for 48 h, after which the medium was discarded and cell viability was determined using MTT reagent.

[0127] The same method was used to determine the inhibitory effect of DHA-Cy7 in combination 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 DHA-Cy7 had a significant inhibitory effect on the proliferation of MC38 cells, SW480 cells, and DLD-1 cells (see Figure 17 ).

[0128] Application Example 4: DHA-Cy7 localizes to the mitochondria of colorectal cancer cells and induces mitochondrial damage

[0129] Related experiments were performed according to the experimental procedures of ART-Cy7 described above.

[0130] 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 Figure 18 ).

[0131] The same method was used to determine the effect of DHA-Cy7 on the mitochondrial ATP content of colorectal cancer cells DLD-1, MC38, and SW480. The results showed that DHA-Cy7 could induce a decrease in the mitochondrial ATP content of DLD-1, MC38, and SW480 cells (see Figure 18 ).

[0132] 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. Fluorescence microscopy results showed that as the concentration of the compound increased, the red light of MC38, SW480, and DLD-1 cells gradually decreased and the green light gradually increased, indicating that DHA-Cy7 could induce a decrease in the mitochondrial membrane potential of MC38, SW480, and DLD-1 cells (see Figure 19 ).

[0133] 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 Figure 20 ).

[0134] Application Example 5: DHA-Cy7 induces ferroptosis in colorectal cancer cells

[0135] 1. GPX4 route

[0136] Take 1×10 MC38 cells 4 Cells were seeded into 6-well cell culture plates. After gradient drug treatment, cells were cultured in complete medium at 37°C in a CO2 incubator at 1640°C for 48 h. Cells were digested with EDTA-free trypsin and collected in centrifuge tubes. Cells were washed twice with PBS (subsequent procedures were performed according to the Solebol GSH Content Assay Kit instructions). Absorbance was measured at 412 nm (blank well A1, sample well A2), where ΔA = A2 - A1.

[0137] The same method was used to determine the effect of DHA-Cy7 on the GSH 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 GSH content in MC38, SW480, and DLD-1 cells (see Figure 21 AC).

[0138] Take 1×10 MC38 cells 4 Cells were seeded into 6-well cell culture plates. After gradient drug treatment, cells were cultured in complete medium at 37°C in a CO2 incubator at 1640°C for 48 h. Cells were digested with EDTA-free trypsin and collected in centrifuge tubes. The cells were washed twice with PBS. Subsequent procedures were performed according to the instructions for the Solebol GSSG Content Detection Kit.

[0139] The same method was used to determine the effect of DHA-Cy7 on the GSSG 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 GSSG content in MC38, SW480, and DLD-1 cells (see Figure 21 DF).

[0140] Take 1×10 MC38 cells 4 Cells were seeded into 6-well cell culture plates. After gradient drug treatment, cells were cultured in complete medium at 37°C in a CO2 incubator at 1640°C for 48 h. Cells were digested with EDTA-free trypsin and collected in centrifuge tubes. The cells were washed twice with PBS. Subsequent procedures were performed according to the instructions for the Nanjing Jiancheng Cysteine ​​(Cys) Detection Kit.

[0141] 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 Figure 21 GI).

[0142] Take 1×10 MC38 cells 4 Cells were seeded into 6-well cell culture plates. After gradient drug treatment, cells were cultured in complete medium at 37°C in a CO2 incubator at 1640°C for 48 h. Cells were digested with EDTA-free trypsin and collected in centrifuge tubes. The cells were washed twice with PBS. Subsequent procedures were performed according to the instructions of the Beyotime lipid oxidation (MDA) detection kit.

[0143] The same method was used to determine the effect of DHA-Cy7 on the MDA production in 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 Figure 21JL).

[0144] Take 1×10 DLD-1 cells 6 Cells were seeded in 60 mm culture dishes. After gradient drug treatment, cells were cultured in complete medium at 37°C in a CO2 incubator at 1640°C for 48 h, and protein was extracted. GPX4 expression was detected by Western blot.

[0145] The same method was used to determine the effect of DHA-Cy7 on GPX4 expression in colorectal cancer SW480 cells and colorectal cancer DLD-1 cells. Western Blot analysis showed that DHA-Cy7 induced a decrease in GPX4 expression in DLD-1 and SW480 cells (see Figure 21 M).

[0146] Summary: DHA-Cy7 mediated the decrease of GSH, GSSG, and Cys levels and the increase of MDA content in MC38, DLD-1, and SW480 cells through GPX4, further demonstrating that it can induce cell ferroptosis.

[0147] , TFR1 pathway

[0148] Take 1×10 MC38 cells 4 Cells were seeded into 6-well cell culture plates. After gradient drug treatment, cells were cultured in complete medium at 37°C in a CO2 incubator at 1640°C for 48 h. Cells were digested with EDTA-free trypsin and collected in centrifuge tubes. The cells were washed twice with PBS. Subsequent procedures were performed according to the instructions for the Nanjing Jiancheng Total Iron Detection Kit.

[0149] 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 Figure 22 AC).

[0150] Take 1×10 DLD-1 cells 6 Cells were seeded in 60 mm culture dishes. After gradient drug treatment, cells were cultured in complete medium at 37°C in a CO2 incubator at 1640°C for 48 h, and proteins were extracted. The expression of transferrin receptors (TFR1) and ferritin was detected by Western blot.

[0151] 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 Figure 22 M).

[0152] After adding the ferroptosis agonist FAC, the ROS production 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 treatment of DHA-Cy7 and FAC increased the ROS content of MC38, SW480, and DLD-1 cells compared with the treatment of DHA-Cy7 alone, and decreased the cell viability (see Figure 22 DI).

[0153] 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 Figure 22 JL).

[0154] 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: Having 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 in the dark at 60-90°C for 10-20 hours. After the reaction, the mixture was precipitated with tert-butyl methyl ether, filtered, rotary evaporated, and purified on a silica gel column to obtain compound 3; 2) Under an inert gas atmosphere, compound 3 was dissolved in dimethylformamide, and compound 4 (hexaminohexanoic acid) and triethylamine were added. 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 rotary evaporated and purified on a 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. Compound 7 and 4-dimethylaminopyridine were then added and stirred at room temperature for 2-5 hours. After the reaction was completed, the mixture was filtered, rotary evaporated, and purified on a 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, the mixture was rotary evaporated and purified on a silica gel column to obtain 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 in the dark at 60-90°C for 10-20 hours. After the reaction, the mixture was precipitated with tert-butyl methyl ether, filtered, rotary evaporated, and purified on a silica gel column to obtain compound 3; 2) Under an inert gas atmosphere, compound 3 was dissolved in dimethylformamide, and compound 4 (hexaminohexanoic acid) and triethylamine were added. 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 rotary evaporated and purified on a 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 reacted at room temperature for 10-14 hours. 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, wherein: 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 an artemisinin derivative according to claim 3, wherein: 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, wherein: The effective concentration of the artemisinin derivative is 0.02438-50 μM.

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