Artesunate-ebbeselenium derivative as well as preparation method and application thereof

Abstract

The invention discloses a series of artesunate-ebbeselenium derivatives which are novel in structure and good in biological activity, and discloses a preparation method and application of the artesunate-ebbeselenium derivatives. The preparation method comprises the following steps: taking compounds as shown in a formula (III) and a formula (IV), respectively mixing the compounds with artesunate, HATU, triethylamine or EDCI and DMAP, respectively putting the mixture into an organic solvent, reacting at room temperature, recovering the solvent from the obtained reaction materials, and respectively obtaining target compound crude products; # imgabs0 #; wherein R represents a hydrogen atom, a halogen atom or a methoxy group, or a C9-C12 alkyl group, a methoxy group or a halogen atom. Test results of the applicant show that part of the target compounds have good anti-tumor activity on various tumor cell strains, and are expected to be used for preparing anti-tumor drugs.

Description

Technical Field

[0001] The invention relates to artesunate-ebselen derivatives and preparation methods and applications thereof, belonging to the technical field of medicine. Background Art

[0002] Artesunate (ART) is a peroxide-bridged sesquiterpene lactone compound. It is a synthetic antimalarial drug derived by chemically modifying the traditional Chinese medicine artemisinin. It has a proven safety record in clinical use. As a naturally derived anti-tumor lead compound, artesunate lacks the cross-resistance associated with traditional drugs and exhibits broad-spectrum anti-tumor activity.

[0003] Recently, an increasing number of studies have shown that ferroptosis plays a crucial role in mediating the anti-cancer effects of ART. Inhibition of the intracellular antioxidant system is one of the reasons why ART induces ferroptosis in cancer cells. Numerous studies have found that glutathione peroxidase 4 (GPX4) can be used as an indicator of cellular ferroptosis. When ART exerts its tumor suppressor effect, the function of the GPX4-dependent defense system is significantly inhibited. However, the application of artesunate in the anti-tumor field has shown certain potential, but its anti-tumor activity is relatively limited and needs further optimization to improve its efficacy.

[0004] Glutathione peroxidase (GSH-Px) is an important peroxide-degrading enzyme widely present in the body. Ebselen (EBS) is considered the most successful small-molecule organoselenium compound that mimics GSH-Px and possesses multiple biological activities, including antioxidant, anti-inflammatory, and anti-tumor activities. EBS is an FDA-approved drug that has entered multiple clinical trials, covering areas such as neurological diseases, otological diseases, and viral infections. Although the drug has not yet been widely approved for marketing worldwide, it has been approved for specific therapeutic indications in Japan and has entered Phase III clinical trials for the treatment of ischemic stroke. Therefore, we introduced the ebselen structural unit into artesunate in the hope of obtaining a highly effective and low-toxic artesunate-ebselen compound. Summary of the Invention

[0005] The present invention provides a series of artesunate-ebselen derivatives with novel structures and good biological activities, and discloses preparation methods and applications thereof.

[0006] The artesunate-ebselen derivatives of the present invention are compounds having the structures represented by the following formulas (I) and (II):

[0007]

[0008] Wherein, R represents a hydrogen atom, a halogen atom, a methyl group or a methoxy group, and n represents 2, 4, 6, or C 37 ~C 40 Alkyl, methoxy, and halogen atoms;

[0009]

[0010] Wherein, R represents a hydrogen atom, a halogen atom, a methyl group or a methoxy group, and n represents 2, 4, 6, 8, or C 37 ~C 40 alkyl, methoxy, and halogen atoms.

[0011] The preparation method of the artesunate-ebselen derivative of the present invention comprises the following steps:

[0012] The compounds represented by the following formula (III) and formula (IV) are mixed with artesunate, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU), triethylamine or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide (EDCI), and 4-dimethylaminopyridine (DMAP), respectively, and then placed in an organic solvent, respectively, and reacted at room temperature. The solvent is recovered from the obtained reaction mass to obtain a crude target compound;

[0013]

[0014] Wherein, R represents a hydrogen atom, a halogen atom, a methoxy group, or a C9-C 12 alkyl, methoxy, and halogen atoms.

[0015] In the preparation method of the present invention, the dosage ratio of each raw material is a stoichiometric ratio. In actual operation, the molar ratio of the compound represented by formula (III) and the compound represented by formula (IV) to artesunate, HATU, triethylamine or EDCI, and DMAP is 1:0.4-4.

[0016] In the above preparation method, the organic solvent is dichloromethane or tetrahydrofuran, preferably dichloromethane.

[0017] The organic solvent is dehydrated with molecular sieves before use.

[0018] The amount of organic solvent used is preferably sufficient to dissolve the raw materials involved in the reaction. Usually, based on 1 mmol of the compound represented by formula (III) or (IV), all the raw materials involved in the reaction are usually dissolved in 3 to 5 mL of organic solvent.

[0019] In the above preparation method, the reaction is monitored by TLC until the reaction is complete. When dichloromethane is used as the organic solvent and the reaction is carried out at room temperature, the reaction time is preferably controlled within 3 to 6 hours.

[0020] The above method produces a crude product of the target compound. Therefore, the method of the present invention further comprises the step of purifying the crude product of the target compound. Specifically, the crude product can be purified by conventional purification methods to increase the purity of the target compound, such as by thin layer chromatography (TLC).

[0021] The developing solvent used to separate the compounds during purification was a mixed solvent consisting of ethyl acetate and petroleum ether.

[0022] In the mixed solvent, the volume ratio of petroleum ether (PE) to ethyl acetate (EA) is 1:2.

[0023] In the preparation method of the present invention, the raw material compound represented by formula (III) is an ebselenamino derivative, and the compound represented by formula (III) is prepared according to the following synthetic route (BOC in compound S2 and compound S3 in the synthetic route represents tert-butyloxycarbonyl):

[0024]

[0025] The specific method for preparing the compound represented by formula (III) comprises the following steps:

[0026] 1) Compound S1 and Compound S2 are placed in an organic solvent and reacted at room temperature to obtain Compound S3;

[0027] 2) Removing the protecting group (ie, tert-butyloxycarbonyl) in compound S3 to expose the NH group, thereby obtaining the compound represented by formula (III).

[0028] The organic solvent in step 1) of the above method for preparing the compound represented by formula (III) is dichloromethane or tetrahydrofuran.

[0029] The amount of the organic solvent can be determined as needed. Usually, based on 1 mmol of S1, all the reaction raw materials are dissolved in 3 to 5 mL of organic solvent.

[0030] In step 2) of the above-mentioned method for preparing the compound represented by formula (III), the protecting group in compound S3 is removed by conventional methods, such as placing compound S3 in a mixed solvent consisting of tetrahydrofuran and trifluoroacetic acid in a volume ratio of 2:1 and stirring for a certain period of time to remove the tert-butyloxycarbonyl group on compound S3.

[0031] In order to further expose the NH structure, an excess of dichloromethane as a reaction solvent and an excess of alkaline substances, such as inorganic bases (such as sodium hydroxide, potassium hydroxide, etc.) or organic bases (such as triethylamine, diethylamine, etc.) can be added to the reaction system to neutralize the excess acid.

[0032] The crude products obtained in steps 1) to 2) of the above-mentioned method for preparing the compound represented by formula (III) are all crude products of the compound, which can be purified according to conventional methods, usually using a silica gel column.

[0033] In the preparation method of the present invention, the raw material compound represented by formula (IV) is an ebselen hydroxy derivative, and the compound represented by formula (IV) is prepared according to the following synthetic route:

[0034]

[0035] A specific method for preparing the compound represented by formula (IV) is as follows: Compound S1 and Compound S4 are placed in dichloromethane and reacted at room temperature to obtain the compound.

[0036] In the above method for preparing the compound represented by formula (IV), the reaction solution is extracted with ethyl acetate, washed with water and brine, the organic phase is collected, dried over anhydrous sodium sulfate, and the residue obtained after spin drying is purified on a silica gel column (eluent is ethyl acetate and petroleum ether) to obtain a high-purity compound represented by formula (IV).

[0037] The applicant has also found through experiments that some of the derivatives of the present invention have good anti-tumor activity against various tumor cell lines.

[0038] The applicant has found through experiments that some of the derivatives of the present invention have good inhibitory activity against colorectal cancer cells.

[0039] Based on the above findings, the artesunate-ebselen derivatives of the present invention have potential application value in the preparation of drugs for treating tumors, and are expected to play an important role in the preparation of drugs for treating colon cancer.

[0040] Compared to the prior art, the present invention provides a series of novel artesunate-ebselen derivatives and their preparation methods. The applicant's experimental results indicate that some of the target compounds of the present invention exhibit good anti-tumor activity against various tumor cell lines and are expected to be used in the preparation of anti-tumor drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 This is a graph showing the in vivo anti-tumor effect of compound IIk in the HCT116 mouse model;

[0042] (A) Changes in tumor volume in different groups over 12 consecutive days; (B) Photos of tumor tissues after 12 days of drug administration;

[0043] (C) Effects of compounds on body weight in HCT116 mouse models; (D) Tumor weight in mice.

[0044] Figure 2The HE staining images of the main organs of mice inhibited by different concentrations of compound IIk in the examples (magnification, ×20).

[0045] Figure 3 The effect of compound IIk on CD4 + T / CD8 + Effects on T lymphocytes;

[0046] Among them, (A) Flow cytometry detection of compound IIk on CD4 + T / CD8 + The influence of T lymphocytes,

[0047] (B) Effect of Compound IIk on CD8 + T lymphocyte effect histogram; (C) Compound IIk on CD4 + Histogram of T lymphocyte impact.

[0048] Figure 4 This is an example of observing the effects of artesunate and compound IIk on ROS levels in HCT116 cells after 24 hours of action; wherein, (A) is a fluorescence image of the reactive oxygen species level in cells; and (B) is a flow cytometry image of the reactive oxygen species level.

[0049] Figure 5 This is an example of observing the effects of artesunate and compound IIk on JC-1 staining of HCT116 cells;

[0050] Among them, (A) Fluorescence image of mitochondrial membrane potential; (B) Flow cytometry detection of mitochondrial membrane potential changes.

[0051] Figure 6 This example shows the effects of artesunate and compound IIk on BODIPY 581 / 591C11 staining of HCT116 cells; (A) Fluorescence image of intracellular lipid peroxidation levels; (B) Flow cytometry detection of changes in intracellular lipid peroxidation levels.

[0052] Figure 7 In this example, electron microscopy experiments were performed to detect the effect of compound IIk on the mitochondrial structure of HCT116 cells.

[0053] Figure 8 This is a graph showing the effects of artesunate and compound IIk on the ferrous ion content in HCT116 cells.

[0054] Figure 9 This is a graph showing the effects of different concentrations of compound IIk on ferroptosis-related proteins in Example;

[0055] Among them, (A) expression of ferroptosis characteristic proteins GPX4, FTH1, ACSL4, and SLC7A11;

[0056] (B) Histogram of the relative expression levels of ferroptosis-characteristic proteins.

[0057] Figure 10 This is a graph showing the effects of different concentrations of compound IIk on GPX4-related qPCR. DETAILED DESCRIPTION

[0058] In order to better explain the technical solution of the present invention, the present invention is further described in detail below with reference to examples, but the embodiments of the present invention are not limited thereto.

[0059] The compounds represented by formula (III) involved in the following examples were prepared according to the following synthetic route:

[0060]

[0061] The specific preparation method comprises the following steps:

[0062] (1) Weigh 1 mmol of compound S1 and dissolve it in 3 mL of ultra-dry dichloromethane. Add triethylamine to adjust the pH of the solution to alkaline. Add 1 mL of compound S2 to the reaction and react for 3 h.

[0063] After the reaction was complete as monitored by TLC (PE:EA=1:2), the mixture was diluted with water (20 mL), the aqueous layer was extracted with ethyl acetate (15 mL×2), and then washed with brine (20 mL×2). The filtrate was purified by silica gel column chromatography using PE:EA=1:2 solution as eluent to obtain the target compound S3;

[0064] (2) 1 mmol of compound S3 was dissolved in 3 mL of ultra-dry tetrahydrofuran. Trifluoroacetic acid (1 mL) was added to the reaction at 0°C and the mixture was moved to room temperature for 5 h.

[0065] After the reaction was completed by TLC monitoring, the filtrate was washed with ethyl acetate (5 mL×2) to obtain a brown liquid III, which was used in the next reaction without purification.

[0066] The compounds represented by formula (IV) involved in the following examples were prepared according to the following synthetic route:

[0067]

[0068] Specific preparation method:

[0069] 1 mmol of compound S1 was weighed and dissolved in 3 mL of ultra-dry dichloromethane. Triethylamine was added to adjust the pH of the solution to alkaline. 1 mmol of compound S4 was added to the reaction and the reaction was allowed to proceed for 3 h. After the reaction was complete as monitored by TLC (PE:EA=1:2), the mixture was diluted with water (20 mL), the aqueous layer was extracted with ethyl acetate, washed with brine (20 mL×2), dried over sodium persulfate, filtered, and the filtrate was concentrated under reduced pressure. The residue was purified by silica gel column chromatography eluted with a PE:EA=1:2 solution to obtain the target compound IV.

[0070] Example 1

[0071] The artesunate-ebselen derivatives I and II of the present invention were synthesized according to the following synthetic route:

[0072]

[0073] 1.2 mmol of artesunate, 1 mmol of HATU, and 2 mmol of triethylamine were dissolved in 3 mL of ultra-dry dichloromethane. After reacting for 20 min, 1 mmol of compound III (adjust the pH to alkaline) was added and the reaction was continued at room temperature for 5 h.

[0074] After the reaction was completed as monitored by TLC (PE:EA=1:2), the mixture was diluted with water (20 mL), the aqueous layer was extracted with ethyl acetate (15 mL×2), and then washed with brine (20 mL×2). The filtrate was purified by silica gel column chromatography using PE:EA=1:2 solution as eluent to obtain the target compound I.

[0075]

[0076] 2 mmol of artesunate and 4 mmol of EDCI were dissolved in 3 mL of ultra-dry dichloromethane. After reacting for 1 h, 1 mmol of compound IV and 0.4 mmol of DMAP were added and the reaction was continued at room temperature for 5 h.

[0077] After the reaction was completed as monitored by TLC (PE:EA=1:2), the mixture was diluted with water (20 mL), the aqueous layer was extracted with ethyl acetate (15 mL×2), and then washed with brine (20 mL×2). The filtrate was concentrated under reduced pressure and purified by silica gel column chromatography eluting with PE:EA=1:2 solution to obtain the target compound II.

[0078] The different target products and their characterizations are as follows:

[0079]

[0080] (3R,5aS,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxolo[4,3-i]isochroman-10-yl 4-oxo-4-((4-(3-oxobenzo[d][1,2]selenazepin-2(3H)-yl)butyl)amino)butanoate (Ia); yield: 69%; white solid, melting point: 87-88°C; purity: 96.83%; 1 HNMR (400MHz, CDCl3) δ8.02 (dd, J=7.6, 1.2Hz, 1H, -CH), 7.67 (d, J=7.9Hz, 1H, -CH),7.62–7.58(m,1H,-CH),7.45–7.40(m,1H,-CH),6.61(t,J=5.6Hz,1H,-CH ),5.75(d,J=9.9Hz,1H,-CH),5.41(s,1H,-CH),3.96(td,J=5.7,2.5Hz,2H,-CH 2),3.57(q,J=5.7Hz,2H,-CH2),2.72(dt,J=9.7,6.8Hz,2H,-CH2),2.55–2.50( m,2H,-CH2),2.34(dd,J=13.9,4.0Hz,1H,-CH),2.04–1.99(m,1H,-CH),1.89(d q,J=10.4,3.3Hz,1H,-CH),1.77–1.67(m,2H,-CH2),1.63–1.57(m,1H,-CH),1. 50–1.43(m,1H,-CH),1.42(d,J=3.8Hz,3H,-CH3),1.35–1.24(m,4H,2-CH2),1. 04–0.97(m,1H,-CH),0.95(d,J=5.7Hz,3H,-CH3),0.83(d,J=7.1Hz,3H,-CH3). 13 C NMR (100MHz, CDCl3) δ171.9,171.8,167.9,138.4,132.2,128.7,126.68,126.3,124.1,104.5,92.1,91.5,80.1,5 1.5,45.2,44.2,40.2,37.2,36.17,34.0,31.7,30.6,29.5,25.9,24.5,21.9,20.2,12.0.HRMS(m / z)(ESI):calcd for C 28 H 36 N2NaO8Se 631.1530; [M+Na] + :found 631.1532.

[0081]

[0082] (3R,5aS,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxolo[4,3-i]isochroman-10-yl 4-oxo-4-((4-(3-oxobenzo[d][1,2]selenazepine) -2(3H)-1,3-dimethyl-2-(2-(3H)-1-amino)butyl)butanoate (Ib); yield: 70%; white solid, melting point: 78-79°C; purity: 98.33%; 1 HNMR(400MHz, CDCl3)δ8.03(dd,J=7.8,1.3Hz,1H),7.65(d,J=7.9Hz,1H),7.61–7.56(m,1H),7.46–7.39(m,1H),6.15(t,J=5.9Hz,1 H),5.78(d,J=9.8Hz,1H),5.42(s,1H),3.88(t,J=6.9Hz,2H),3.37–3.25(m,J=6.7Hz,2H),2.81–2.69(m,2H),2.53(ddd,J=14.5,7. 4,4.4Hz,2H),2.36(td,J=13.9,3.9Hz,1H),2.05–2.00(m,1H),1.88(dq,J=10.0,3.0Hz,1H),1.75(dq,J=11.8,5.9,4.5Hz,4H),1.6 3–1.55(m,3H),1.51–1.44(m,1H),1.42(s,3H),1.37–1.22(m,4H),1.05–0.97(m,1H),0.95(d,J=5.5Hz,3H),0.84(d,J=7.1Hz,3H). 13 C NMR (100MHz, CDCl3) δ171.8,171.4,167.3,137.8,131.9,128.8,127.35,126.2,124.0,104.5,92.1,91.5,80.1,51.5, 45.5,44.3,39.1,37.2,36.2,34.0,31.7,30.9,29.8,27.9,26.2,25.9,24.5,21.9,20.2,12.0.HRMS(m / z)(ESI):calcd for C 30 H 40 N2NaO8Se659.1843; [M+Na] + :found 659.1846.

[0083]

[0084] (3R,5aS,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxolo[4,3-i]isochroman-10-yl 4-oxo-4-((6-(3-oxobenzo[d][1,2]selenazepine) 2-(3H)-1,3-dimethyl-2-(3H)-1,3-hexyl)amino)butanoate (Ic); yield: 70%; white solid, melting point: 78-79°C; purity: 98.33%; 1 HNMR (400MHz, CDCl3) δ8.01(d,J=7.8Hz,1H),7.64(d,J=7.9Hz,1H),7.57(t,J=7.5Hz,1H),7.41(t,J=7.5Hz,1H),6.00(t,J=5.8Hz,1H),5.76( d,J=9.8Hz,1H),5.40(s,1H),3.85(t,J=6.9Hz,2H),3.20(q,J=7.0,6.1 Hz,2H),2.74(dt,J=16.0,7.0Hz,2H),2.56–2.49(m,2H),2.39–2.31(m, 1H),2.00(dt,J=14.4,3.7Hz,1H),1.86(dq,J=10.0,3.1Hz,1H),1.72(ddd,J=17.2,8.7,3.5Hz,4H),1.62–1.56(m,1H),1.48(t,J=6.9Hz,3H), 1.40(s,3H),1.37(dd,J=7.3,3.5Hz,4H),1.28(ddd,J=16.6,11.7,4.5Hz,4H),1.03–0.96(m,1H),0.93(d,J=5.7Hz,3H),0.82(d,J=7.1Hz,3H). 13 C NMR (100MHz, CDCl3) δ171.8,171.1,167.2,137.7,131.9,128.7,127.5,126.2,123.9,104.4,92.1,91.4,80.1,51.5,45.2,4 4.3,39.1,37.19,36.2,34.0,31.7,30.8,30.2,29.8,29.2,25.9,25.9,25.7,24.5,21.9,20.1,12.0.HRMS(m / z)(ESI):calcd for C 32 H 42N2NaO8Se 687.2156; [M+Na] + :found687.2155.

[0085]

[0086] (3R,5aS,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxolo[4,3-i]isochroman-10-yl 4-oxo-4-((4-((3-oxobenzo[d][1,2]selenazepine) -2(3H)-1,3-dimethylphenyl)butanoate (Id); yield: 65%; white solid, melting point: 104-105°C; purity: 97.55%; 1 H NMR (400MHz, CDCl3) δ8.64(s,1H),7.83(d,J=7.8Hz,1H),7.67(d,J=7.5Hz,1H),7.53(d,J=8.1Hz,2H),7.24–7.13( m,4H),6.48(t,J=5.9Hz,1H),5.72(d,J=9.8Hz,1H),4.30(d,J=5.6Hz,2H),2.74–2.61(m,2H),2.53–2.46(m,2H),2 .37–2.30(m,1H),1.99(dd,J=10.6,3.8Hz,1H),1.88–1.83(m,1H),1.73–1.66(m,2H),1.59–1.55(m,1H),1.45–1.4 0(m,1H),1.37(s,3H),1.26(d,J=4.5Hz,4H),1.00(d,J=11.3Hz,1H),0.93(d,J=5.3Hz,3H),0.80(d,J=7.1Hz,3H). 13 C NMR (100MHz, CDCl3) δ171.8,171.3,166.8,136.9,134.8,133.7,133.1,131.9,131.55,128.2,127.5,126.4,121.0,104.5,92.3,9 1.5,80.1,51.5,45.1,43.1,37.2,36.2,34.0,31.72,30.7,29.6,29.3,27.2,25.9,24.5,21.9,20.2,12.0.HRMS(m / z)(ESI):calcd for C 33 H 38 N2NaO8Se 693.1686; [M+Na]+ :found693.1633.

[0087]

[0088] (3R,5aS,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxolo[4,3-i]isochroman-10-yl 4-((4-(6-fluoro-3-oxobenzo[d][1,2]selenazepine) -2(3H)-1,3-dimethyl-2-(2-(3H)-1-butyl)amino)-4-oxobutanoate (Ie); yield: 67%; white solid, melting point: 80-81°C; purity: 95.79%; 1 H NMR (400MHz, CDCl3) δ7.97 (dd, J=8.6, 5.2Hz, 1H), 7.38 (dd, J=7.9, 2.3Hz, 1H), 7.12 (td, J=8.6, 2.3Hz, 1H), 6.23 (t, J=5.9Hz, 1H),5.76(d,J=9.9Hz,1H),5.41(s,1H),3.85(t,J=6.9Hz,2H),3.28(qd,J=6.8,4.2Hz,2H),2.76–2.68(m,2H),2.54–2.48(m, 2H),2.35(ddd,J=14.6,13.4,3.9Hz,1H),2.03–1.98(m,1H),1.87(ddd,J=13.7,6.3,3.3Hz,1H),1.77–1.66(m,4H),1.62–1.5 4(m,3H),1.49–1.41(m,1H),1.40(s,3H),1.35–1.21(m,4H),1.04–0.96(m,1H),0.94(d,J=5.4Hz,3H),0.82(d,J=7.1Hz,3H). 13 C NMR (100MHz, CDCl3) δ171.9, 171.5, 166.4, 165.0 (d, J = 253Hz), 139.5 (d, J = 10Hz), 130.6 (d, J = 10Hz), 123.7 (d, J = 2Hz), 114.9 (d, J = 24Hz), 111 .2(d,J=26Hz),104.5,92.2,91.5,80.1,51.5,45.1,44.3,38.9,37.2, 36.2,33.9,31.7,30.8,29.8,27.7,26.2,25.9,24.5,21.9,20.1,11.9. 19FNMR(376MHz,CDCl3)δ-106.5(s,–ArF).HRMS(m / z)(ESI):calcd for C 30 H 39 FN2NaO8Se677.1748; [M+Na] + :found 677.1745.

[0089]

[0090] (3R,5aS,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxolo[4,3-i]isochroman-10-yl 4-((4-(7-chloro-3-oxobenzo[d][1,2]selenazepine) -2(3H)-1,3-dimethyl-2-(2-(3H)-1-butyl)amino)-4-oxobutanoate (If); yield: 68%; white solid, melting point: 58-59°C; purity: 99.12%; 1 H NMR (400MHz, CDCl3) δ7.93(dd,J=7.7,1.1Hz,1H),7.56(dd,J=7.8,1.1Hz,1H),7.42(t,J=7.8Hz,1H),6.10(t,J=5.8Hz, 1H),5.77(d,J=9.8Hz,1H),5.40(s,1H),3.89(td,J=7.0,1.6Hz,2H),3.30(dq,J=13.1,6.8Hz,2H),2.79–2.71(m,2H),2 .55–2.48(m,2H),2.39–2.31(m,1H),2.03–1.98(m,1H),1.86(dq,J=9.9,3.1Hz,1H),1.79–1.68(m,4H),1.62–1.57(m,3 H),1.49–1.42(m,1H),1.40(s,3H),1.33–1.24(m,4H),1.04–0.97(m,1H),0.94(d,J=5.4Hz,3H),0.83(d,J=7.1Hz,3H). 13C NMR (100MHz, CDCl3) δ171.8,171.4,167.0,138.3,130.9,129.4,127.9,126.8,104.4,92.1,91.4,80.1,51.5,45.2,4 4.4,39.0,37.2,36.2,34.0,31.7,30.9,29.9,29.6,27.9,26.2,25.9,24.5,21.9,20.2,12.0.HRMS(m / z)(ESI):calcd for C 30 H 39 FN2NaO8Se 693.1453; [M+Na] + :found 693.1454.

[0091]

[0092] (3R,5aS,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxolo[4,3-i]isochroman-10-yl 4-((4-(6-chloro-3-oxobenzo[d][1,2]selenazepine) -2(3H)-1,3-dimethyl-2-(2-(3H)-1-butyl)amino)-4-oxobutanoate (1 g); yield: 58%; white solid, melting point: 98-99°C; purity: 97.36%; 1 H NMR (400MHz, CDCl3) δ7.91(d,J=8.4Hz,1H),7.67(d,J=1.8Hz,1H),7.38(dd,J=8.4,1.8Hz,1H),6.16(t,J=5.9Hz,1H),5 .76(d,J=9.8Hz,1H),5.40(s,1H),3.85(td,J=6.9,1.6Hz,2H),3.31–3.26(m,2H),2.78–2.71(m,2H),2.52–2.46(m,2H), 2.39–2.31(m,1H),2.03–1.98(m,1H),1.87(dt,J=10.8,2.9Hz,1H),1.74(dt,J=14.6,6.8Hz,4H),1.58(q,J=5.4,4.6Hz, 3H),1.49–1.42(m,1H),1.40(s,3H),1.35–1.23(m,4H),1.02–0.96(m,1H),0.94(d,J=5.5Hz,3H),0.82(d,J=7.1Hz,3H). 13C NMR (100MHz, CDCl3) δ171.8,171.4,166.4,138.9,138.5,129.6,127.0,125.9,123.9,104.5,92.1,91.5,80.1,51.5,4 5.1,44.3,38.9,37.2,36.2,34.0,31.7,30.9,29.8,27.8,26.3,25.9,24.5,21.9,20.2,12.0.HRMS(m / z)(ESI):calcd for C 30 H 39 ClN2NaO8Se 693.1453; [M+Na] + :found693.1458.

[0093]

[0094] (3R,5aS,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxolo[4,3-i]isochroman-10-yl 4-((4-(6-methoxy-3-oxobenzo[d][1,2]selenazepine) -2(3H)-1,3-dimethyl-2-(2-(3H)-1-butyl)amino)-4-oxobutanoate (Ih); yield: 68%; white solid, melting point: 70-71°C; purity: 95.68%; 1 H NMR (400MHz, CDCl3) δ7.88(d,J=8.7Hz,1H),7.12(d,J=2.2Hz,1H),6.94(dd,J=8.7,2.3Hz,1H),6.28(t,J=5.8Hz,1H),5.76(d, J=9.9Hz,1H),5.40(s,1H),3.87(s,3H),3.82(t,J=6.9Hz,2H),3.27(p,J=6.8Hz,2H),2.71(q,J=7.0Hz,2H),2.53–2.48(m,2H), 2.39–2.31(m,1H),2.01(ddd,J=14.6,4.7,2.7Hz,1H),1.87(dq,J=10.0,2.8Hz,1H),1.76–1.66(m,4H),1.57(ddd,J=14.6,9.3, 5.8Hz,3H),1.48–1.41(m,1H),1.40(s,3H),1.36–1.20(m,4H),1.03–0.96(m,1H),0.93(d,J=5.5Hz,3H),0.82(d,J=7.1Hz,3H). 13CNMR (100MHz, CDCl3) δ171.9,171.5,167.2,162.9,139.7,129.6,120.3,114.5,107.6,104.5,92.1,91.5,80.1,55.7,51. 5,45.1,44.1,38.9,37.2,36.2,34.0,31.7,30.8,29.8,27.8,26.0,25.9,24.5,21.9,20.2,11.9.HRMS(m / z)(ESI):calcd for C 31 H 42 N2NaO9Se689.1948; [M+Na] + :found 689.1946.

[0095]

[0096] (3R,5aS,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxolo[4,3-i]isochroman-10-yl 4-((4-(5,6-dimethoxy-3-oxobenzo[d][1,2]selenazepine) -2(3H)-1,2-dimethyl-2-butyl-1,2-diamino-2-oxobutanoate (Ii); yield: 68%; white solid, melting point: 89-90°C; purity: 95.32%; 1 H NMR (400MHz, CDCl3) δ7.60(s,1H),7.43(s,1H),6.43(t,J=5.7Hz,1H),5.92(d,J=9.8Hz,1H),5.56(s,1H),4.11(d,J=6.0 Hz,6H),4.01(td,J=6.9,2.0Hz,2H),3.45(dq,J=9.2,3.4,2.9Hz,2H),2.95–2.84(m,2H),2.74–2.66(m,2H),2.55–2.47(m ,1H),2.19–2.15(m,1H),2.04(ddd,J=10.6,5.0,2.9Hz,1H),1.89(dq,J=11.2,5.8Hz,4H),1.78–1.71(m,3H),1.65–1.58( m,1H),1.56(s,3H),1.43(ddd,J=15.5,10.0,2.6Hz,4H),1.20–1.12(m,1H),1.10(d,J=5.6Hz,3H),0.99(d,J=7.1Hz,3H). 13C NMR (100MHz, CDCl3) δ171.8,171.4,167.4,153.3,148.9,130.4,119.8,109.4,105.4,104.4,92.1,91.4,80.1,56.3,56.2,5 1.5,45.1,44.28,39.1,37.2,36.1,33.9,31.7,30.8,29.8,28.0,26.0,25.9,24.5,21.9,20.1,11.9.HRMS(m / z)(ESI):calcd for C 32 H 44 N2NaO 10 Se 719.2054; [M+Na] + :found719.2055.

[0097]

[0098] 2-(3-Oxobenzo[d][1,2]selenazepine -2(3H)-ethyl((3R,5aS,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxolo[4,3-i]isochroman-10-yl)succinate (IIa); yield: 60%; white solid, melting point: 69-70°C; purity: 99.53%; 1 H NMR (400MHz, CDCl3) δ8.05(d,J=7.8,1.4,0.7Hz,1H),7.67(d,J=8.0,0.9Hz,1H),7.60(t,J=8.1,7.1,1.3Hz,1H),7.42(t ,J=8.1,7.1,1.1Hz,1H),5.79(d,J=9.8Hz,1H),5.43(s,1H),4.41–4.34(m,2H),4.19–4.07(m,2H),2.78–2.69(m,4H),2.3 7(ddd,J=14.6,13.4,3.9Hz,1H),2.05–1.99(m,1H),1.91–1.86(m,1H),1.76–1.69(m,2H),1.63–1.58(m,1H),1.48(ddd,J =13.6,11.2,4.9Hz,1H),1.42(s,3H),1.37–1.24(m,4H),1.04–0.97(m,1H),0.95(d,J=5.9Hz,3H),0.83(d,J=7.1Hz,3H). 13C NMR (100MHz, CDCl3) δ171.6,171.1,167.4,138.4,132.2,128.8,126.6,126.2,123.9,104.5,92.2,91.5,80.1,6 3.8,51.5,45.2,43.4,37.24,36.2,34.0,31.8,29.1,28.9,25.9,24.5,21.9,20.2,12.0.HRMS(m / z)(ESI):calcd for C 28 H 35 NNaO9Se632.1370;[M+Na] + :found 632.1371.

[0099]

[0100] 4-(3-Oxobenzo([d][1,2])selenazepine -2(3H)-1-butyl((3R,5aS,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy([1,2])dioxolo([4,3-i])isochroman-10-yl)succinate (IIb); yield: 62%; white solid, melting point: 60-61°C; purity: 98.81%; 1 H NMR (400MHz, CDCl3) δ8.03(d,J=7.8Hz,1H),7.64(d,J=7.9Hz,1H),7.58(t,J=7.5Hz,1H),7.42(t,J=7.4Hz,1H),5.78(d,J=9. 8Hz,1H),5.42(s,1H),4.13(t,J=6.1Hz,2H),3.89(t,J=6.6Hz,2H),2.71(d,J=4.8Hz,2H),2.68–2.58(m,2H),2.36(td,J=14. 0,3.9Hz,1H),2.01(dd,J=14.8,4.1Hz,1H),1.90–1.85(m,1H),1.76(dq,J=21.7,8.7,7.3Hz,6H),1.63–1.57(m,1H),1.51–1. 44(m,1H),1.42(s,3H),1.31(ddd,J=29.0,12.3,5.0Hz,4H),1.05–0.97(m,1H),0.94(d,J=5.4Hz,3H),0.83(d,J=7.1Hz,3H). 13C NMR (100MHz, CDCl3) δ172.1,171.1,167.3,137.7,131.9,128.8,127.4,126.2,123.9,104.5,92.2,91.5,80.1,64.1,5 1.5,45.2,44.3,37.2,36.2,34.0,31.8,29.2,28.9,27.0,25.9,25.7,24.5,21.9,20.2,12.0.HRMS(m / z)(ESI):calcd for C 30 H 39 NNaO9Se 660.1683; [M+Na] + :found660.1686.

[0101]

[0102] 6-(3-Oxobenzo[d][1,2]selenazepine -2(3H)-yl)hexyl((3R,5aS,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxolo[4,3-i]isochroman-10-yl)succinate (IIc); yield: 64%; white solid, melting point: 50-51°C; purity: 99.76%; 1 H NMR (400MHz, CDCl3) δ8.03(d,J=7.8Hz,1H),7.64(d,J=8.0Hz,1H),7.58(t,J=7.6Hz,1H),7.42(t,J=7.5Hz,1H) ,5.78(d,J=9.8Hz,1H),5.42(s,1H),4.07(t,J=6.6Hz,2H),3.85(t,J=7.1Hz,2H),2.70(d,J=7.4Hz,2H),2.66–2 .56(m,2H),2.36(td,J=14.0,3.9Hz,1H),2.02(d,J=13.8Hz,1H),1.91–1.84(m,1H),1.79–1.56(m,8H),1.47(s ,1H),1.41(d,J=8.1Hz,7H),1.36–1.20(m,4H),1.05–0.97(m,1H),0.94(d,J=5.4Hz,3H),0.84(d,J=7.2Hz,3H). 13CNMR (100MHz, CDCl3) δ172.2,171.2,167.2,137.6,131.9,128.8,127.5,126.2,123.9,104.5,92.1,91.5,80.1,64.7,51.5,45.2,44.7 37.2,36.2,34.0,31.8,30.4,29.2,28.8,28.4,26.2,25.9,25.5,24.5,21.9,20.2,12.0.HRMS(m / z)(ESI):calcd for C 32 H 43 NNaO9Se688.1996; [M+Na] + :found 688.2000.

[0103]

[0104] 4-(3-Oxobenzo[d][1,2]selenazepine) -2(3H)-yl)benzyl((3R,5aS,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxolo[4,3-i]isochrom-10-yl)succinate (IId); yield: 59%; white solid, melting point: 79-80°C; purity: 95.09%; 1 H NMR (400MHz, CDCl3) δ8.11(d,J=7.8Hz,1H),7.73–7.57(m,4H),7.47(ddd,J=8.1,5.9,2.2Hz,1H),7.41(d,J=8.2H z,2H),5.78(d,J=9.9Hz,1H),5.43(s,1H),5.14(s,2H),2.76–2.73(m,2H),2.68(ddt,J=15.4,9.6,4.6Hz,2H),2.3 9–2.33(m,1H),2.02(dt,J=11.6,2.0Hz,1H),1.91–1.85(m,1H),1.78–1.68(m,2H),1.63–1.58(m,1H),1.52–1.45( m,1H),1.42(s,3H),1.34–1.24(m,4H),0.99(dd,J=11.7,3.2Hz,1H),0.95(d,J=5.8Hz,3H),0.83(d,J=7.1Hz,3H). 13C NMR (100MHz, CDCl3) δ171.9,171.1,165.7,139.0,137.5,134.2,132.6,129.4,129.2,127.4,126.6,125.3,123.7,121.9,104.5,9 2.2,91.5,80.1,65.9,51.5,45.2,37.2,36.2,34.0,31.8,29.7,29.1,28.9,25.9,24.5,21.9,20.2,12.0.HRMS(m / z)(ESI):calcd for C 33 H 37 NNaO9Se 694.1526; [M+Na] + :found 694.1528.

[0105]

[0106] 4-(6-fluoro-3-oxobenzo[d][1,2]selenazepine) -2(3H)-1,2-butyl((3R,5aS,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxolo[4,3-i]isochroman-10-yl)succinate (IIe); yield: 68%; white solid, melting point: 72-73°C; purity: 99.19%; 1H NMR (400MHz, CDCl3) δ8.00(dd,J=8.7,5.2Hz,1H),7.36(dd,J=7.8,2.2Hz,1H),7.14(td,J=8.7,2.2Hz,1H),5.78(d,J=9.8Hz,1H),5.42 (s,1H),4.13(td,J=6.0,1.9Hz,2H),3.87(t,J=6.7Hz,2H),2.75–2.70(m,2H),2.68–2.58(m,2H),2.37(td,J=14.0,3.9Hz,1H),2.03(d dd,J=14.6,4.9,3.0Hz,1H),1.89(ddd,J=13.9,6.3,3.4Hz,1H),1.76(tdd,J=14.3,9.4,6.9Hz,6H),1.63–1.58(m,1H),1.50–1.43(m,1 H),1.42(s,3H),1.29(ddd,J=19.8,12.5,3.9Hz,4H),1.01(td,J=12.1,11.3,3.2Hz,1H),0.95(d,J=5.6Hz,3H),0.84(d,J=7.2Hz,3H). 13 C NMR (100MHz, CDCl3) δ172.1, 171.2, 166.3, 165.0 (d, J = 252Hz), 139.2 (d, J = 10Hz), 130.7 (d, J = 10Hz), 123.7, 114.9 (d, J = 23Hz), 111.1 (d ,J=25Hz),104.5,92.2,91.5,80.1,64.0,51.5,45.2,44.3,37.2,36.2,34.0,31.8,29.2,28.8,26.9,25.9,25.7,24.5,21.9,20.2,12.0. 19 F NMR(376MHz, CDCl3)δ-106.5(s,–ArF).HRMS(m / z)(ESI):calcdfor C 30 H 38 FNNaO9Se 678.1589; [M+Na] + :found678.1586.

[0107]

[0108] 4-(7-chloro-3-oxobenzo[d][1,2]selenazepine) -2(3H)-1-butyl((3R,5aS,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxolo[4,3-i]isochroman-10-yl)succinate (IIf); yield: 62%; white solid, melting point: 73-74°C; purity: 99.29%; 1 H NMR (400MHz, CDCl3) δ7.92(d,J=7.7Hz,1H),7.54(d,J=7.8Hz,1H),7.41(t,J=7.7Hz,1H),5.76(d,J=9.8 Hz,1H),5.39(s,1H),4.11(td,J=6.2,2.2Hz,2H),3.88(t,J=6.8Hz,2H),2.74–2.59(m,4H),2.34(td,J= 14.0,3.9Hz,1H),2.02–1.97(m,1H),1.87–1.66(m,7H),1.61–1.55(m,1H),1.49–1.41(m,1H),1.40(s,3 H),1.28(ddd,J=32.0,12.5,4.7Hz,4H),1.02–0.95(m,1H),0.92(d,J=5.5Hz,3H),0.82(d,J=7.1Hz,3H). 13 C NMR (100MHz, CDCl3) δ172.0,171.1,166.87,138.2,130.9,129.4,129.3,127.9,126.8,104.4,92.1,91.4,80.0,63.9, 51.4,45.1,44.3,37.2,36.1,33.9,31.7,29.1,28.8,26.9,25.9,25.6,24.5,21.9,20.1,11.9.HRMS(m / z)(ESI):calcd for C 30 H 38 ClNNaO9Se 694.1293; [M+Na] + :found694.1290.

[0109]

[0110] 4-(6-chloro-3-oxobenzo[d][1,2]selenazepine) -2(3H)-1-butyl((3R,5aS,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxolo[4,3-i]isochroman-10-yl)succinate (II g); yield: 65%; white solid, melting point: 76-77°C; purity: 98.70%; 1 H NMR (400MHz, CDCl3) δ7.93(d,J=8.3Hz,1H),7.66(d,J=1.8Hz,1H),7.39(dd,J=8.4,1.8Hz,1H),5.78(d,J=9.8Hz,1H ),5.41(s,1H),4.13(td,J=6.1,1.4Hz,2H),3.87(t,J=6.7Hz,2H),2.74–2.70(m,2H),2.67–2.58(m,2H),2.41–2.32( m,1H),2.06–2.00(m,1H),1.89(ddd,J=13.7,6.5,3.4Hz,1H),1.81–1.69(m,6H),1.62–1.57(m,1H),1.47(ddd,J=13. 3,10.9,4.8Hz,1H),1.42(s,3H),1.36–1.24(m,4H),1.04–0.97(m,1H),0.95(d,J=5.5Hz,3H),0.83(d,J=7.1Hz,3H). 13 C NMR (100MHz, CDCl3) δ172.0,171.1,166.3,138.8,138.5,129.7,127.1,125.9,123.9,104.5,92.2,91.5,80.1,63.9,5 1.5,45.2,44.3,37.2,36.2,34.0,31.8,29.2,28.9,26.9,25.9,25.7,24.5,21.9,20.2,12.0.HRMS(m / z)(ESI):calcd forC 30 H 38 ClNNaO9Se 694.1293; [M+Na] + :found 694.1297.

[0111]

[0112] 4-(4-chloro-3-oxobenzo[d][1,2]selenazepine -2(3H)-1,2-butyl((3R,5aS,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxolo[4,3-iisochroman-10-yl)succinate (IIh); yield: 67%; white solid, melting point: 72-73°C; purity: 98.59%; 1 H NMR (400MHz, CDCl3) δ7.94(d,J=8.3Hz,1H),7.66(d,J=1.7Hz,1H),7.39(dt,J=8.3,1.6Hz,1H),5.78(d,J=9.8Hz,1H),5 .42(s,1H),4.13(t,J=6.0Hz,2H),3.87(t,J=6.7Hz,2H),2.71(d,J=6.0Hz,2H),2.64(dt,J=12.1,6.4Hz,2H),2.37(td,J =13.9,3.9Hz,1H),2.05–2.00(m,1H),1.91–1.86(m,1H),1.81–1.71(m,6H),1.62–1.58(m,1H),1.48(ddd,J=14.9,10.8 ,4.6Hz,1H),1.42(s,3H),1.37–1.25(m,4H),1.00(dt,J=11.2,6.1Hz,1H),0.95(d,J=5.4Hz,3H),0.83(d,J=7.1Hz,3H). 13 C NMR (100MHz, CDCl3) δ172.1,171.2,166.4,138.9,138.6,129.7,127.1,125.9,123.9,104.5,92.2,91.5,80.0,64.1,5 1.6,45.2,44.4,37.3,36.2,34.1,31.8,29.2,28.9,26.9,25.9,25.7,24.6,21.9,20.2,12.1.HRMS(m / z)(ESI):calcd for C 30 H 38 ClNNaO9Se 694.1293; [M+Na] + :found 694.1296.

[0113]

[0114] 4-(5-methoxy-3-oxobenzo[d][1,2]selenazepine -2(3H)-1,2-butyl((3R,5aS,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxolo[4,3-i]isochroman-10-yl)succinate (IIi); yield: 63%; white solid, melting point: 68-69°C; purity: 95.32%; 1 H NMR (400MHz, CDCl3) δ7.52–7.48(m,2H),7.21(dd,J=8.7,2.7Hz,1H),5.78(d,J=9.9Hz,1H),5.41(s,1H),4.13(t, J=6.0Hz,2H),3.88(d,J=2.8Hz,5H),2.74–2.70(m,2H),2.68–2.58(m,2H),2.36(ddd,J=14.6,13.4,4.0Hz,1H),2. 02(ddd,J=14.6,4.9,3.0Hz,1H),1.88(dq,J=10.0,3.0Hz,1H),1.81–1.71(m,6H),1.61(dd,J=13.7,4.5Hz,1H),1 .51–1.43(m,1H),1.42(s,3H),1.37–1.22(m,4H),1.03–0.96(m,1H),0.94(d,J=5.7Hz,3H),0.83(d,J=7.1Hz,3H). 13 C NMR (100MHz, CDCl3) δ172.1,171.1,167.1,158.9,128.4,128.37,124.7,121.9,110.5,104.5,92.1,91.5,80.1,64.1,55. 7,51.5,45.2,44.4,37.2,36.2,34.0,31.8,29.2,28.83,26.9,25.9,25.7,24.5,21.9,20.2,12.0.HRMS(m / z)(ESI):calcd forC 31 H 41 NNaO 10 Se 690.1788; [M+Na] + :found690.1788.

[0115]

[0116] 4-(5-methyl-3-oxobenzo[d][1,2]selenazepine -2(3H)-1-butyl((3R,5aS,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxolo[4,3-i]isochroman-10-yl)succinate (II j); yield: 59%; white solid, melting point: 70-71°C; purity: 99.43%; 1 H NMR (400MHz, CDCl3) δ7.86(s,1H,-CH),7.52(d,J=8.1Hz,1H,-CH),7.42(dd,J=8.2,1.9Hz,1H,-CH),5.79(d,J=9.8Hz,1H,-CH),5.43(s,1H,- CH),4.13(t,J=6.1Hz,2H,-CH2),3.88(t,J=6.7Hz,2H,-CH2),2.74–2.71(m,2H,-CH2),2.69–2.61(m,2H,-CH2),2.46(s,3H,-CH3),2.37(dd,J =13.8,9.7Hz,1H,-CH),2.06–2.01(m,1H,-CH),1.91–1.86(m,1H,-CH),1.78–1.69(m,6H,3-CH2),1.64–1.60(m,1H,-CH),1.52–1.46(m,1H,- CH),1.43(s,3H,-CH3),1.37–1.28(m,4H,2-CH2),1.03(dd,J=12.8,3.3Hz,1H,-CH),0.95(d,J=5.8Hz,3H,-CH3),0.84(d,J=7.1Hz,3H,-CH3). 13 C NMR (100MHz, CDCl3) δ172.1,171.1,167.4,136.4,134.2,133.3,128.9,127.4,123.7,104.5,92.2,91.5,80.1,64.1,51. 5,45.2,44.2,37.2,36.2,34.0,31.8,29.2,28.9,26.9,25.9,25.7,24.5,21.9,20.9,20.2,12.0.HRMS(m / z)(ESI):calcd for C 31 H 41 NNaO9Se 674.1839; [M+Na] + :found 674.1839.

[0117]

[0118] 4-(6-methoxy-3-oxobenzo[d][1,2]selenazepine -2(3H)-1,2-butyl((3R,5aS,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxolo[4,3-i]isochroman-10-yl)succinate (IIk); yield: 60%; white solid, melting point: 74-75°C; purity: 99.68%; 1 H NMR (400MHz, CDCl3) δ7.91(d,J=8.7Hz,1H),7.10(d,J=2.2Hz,1H),6.96(dd,J=8.7,2.2Hz,1H),5.78(d,J=9.9Hz,1H),5.42(s,1 H),4.12(t,J=6.0Hz,2H),3.88(s,3H),3.85(t,J=6.6Hz,2H),2.74–2.70(m,2H),2.67–2.57(m,2H),2.36(td,J=13.9,3.9Hz,1H ),2.04–1.99(m,1H),1.87(ddd,J=13.4,6.2,3.2Hz,1H),1.77–1.70(m,6H),1.60(dd,J=13.7,4.5Hz,1H),1.47(ddd,J=13.4,10 .9,4.8Hz,1H),1.42(s,3H),1.36–1.23(m,4H),1.00(td,J=12.1,11.3,3.3Hz,1H),0.94(d,J=5.7Hz,3H),0.83(d,J=7.1Hz,3H). 13 C NMR (100MHz, CDCl3) δ172.1,171.1,167.1,162.9,139.5,129.8,120.4,114.4,107.6,104.5,92.2,91.5,80.1,64.1,55. 7,51.5,45.2,44.1,37.2,36.2,34.0,31.8,29.2,28.9,26.9,25.9,25.7,24.5,21.9,20.2,12.0.HRMS(m / z)(ESI):calcd for C 31 H 41 NNaO 10 Se 690.1788; [M+Na] + :found 690.1783.

[0119]

[0120] 4-(5,6-dimethoxy-3-oxobenzo[d][1,2]selenazepine -2(3H)-1-butyl((3R,5aS,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxolo[4,3-i]isochroman-10-yl)succinate (III); yield: 62%; white solid, melting point: 93-94°C; purity: 99.11%; 1 HNMR (400MHz, CDCl3) δ7.64(s,1H),7.45(s,1H),5.97(d,J=9.8Hz,1H),5.60(s,1H),4.31(t,J=5.9Hz,2H),4.16–4.12( m,6H),4.05(t,J=6.7Hz,2H),2.92–2.88(m,2H),2.86–2.77(m,2H),2.55(td,J=14.0,3.9Hz,1H),2.20(dt,J=14.6,4.0H z,1H),2.07(ddd,J=14.0,6.4,3.4Hz,1H),1.93(qd,J=8.9,6.0,4.7Hz,6H),1.78(dt,J=13.9,4.5Hz,1H),1.70–1.61(m ,1H),1.60(s,3H),1.49(ddd,J=28.8,12.3,5.0Hz,4H),1.22–1.15(m,1H),1.13(d,J=5.6Hz,3H),1.02(d,J=7.1Hz,3H). 13 C NMR (100MHz, CDCl3) δ172.0,171.1,167.3,153.3,148.9,130.1,119.9,109.5,105.3,104.4,92.1,91.5,80.1,64.1,56.3, 56.2,51.5,45.2,44.3,37.2,36.234.0,31.8,29.2,28.9,27.1,25.9,25.7,24.5,21.9,20.2,12.0.HRMS(m / z)(ESI):calcd for C 32 H 43 NNaO 11 Se 720.1894; [M+Na] + :found 720.1898.

[0121]

[0122] 2-(6-methoxy-3-oxobenzo[d][1,2]selenazepine -2(3H)-yl)ethyl ((3R,5aS,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxolo[4,3-i]isochroman-10-yl)succinate (IIm); yield: 64%; white solid, melting point: 81-82°C; purity: 99.22%; 1 H NMR (400MHz, CDCl3) δ7.92 (dd, J=8.7, 1.6Hz, 1H), 7.13 (t, J=2.0Hz, 1H), 6.96 (dt, J=8.7, 2.0Hz, 1H), 5.78 (dd, J=9.9, 1.6Hz, 1H),5.43(d,J=1.7Hz,1H),4.35(td,J=5.1,4.6,2.0Hz,2H),4.19–3.99(m,2H),3.89(d,J=1.6Hz,3H),2.79–2.68(m,4H),2.4 0–2.32(m,1H),2.01(dd,J=15.6,3.4Hz,1H),1.88(dd,J=14.1,4.7Hz,1H),1.78–1.68(m,2H),1.63–1.57(m,1H),1.48(dq,J= 14.5,4.7Hz,1H),1.41(d,J=1.6Hz,3H),1.38–1.21(m,4H),1.05–0.98(m,1H),0.97–0.93(m,3H),0.83(dd,J=7.2,1.6Hz,3H). 13 C NMR (100MHz, CDCl3) δ171.6,171.1,167.3,163.1,140.4,129.8,119.6,114.5,107.4,104.5,92.2,91.51,80.1,63. 9,55.7,51.5,45.2,43.3,37.2,36.2,34.0,31.8,29.1,28.93,25.9,24.5,21.9,20.2,12.0.HRMS(m / z)(ESI):calcd for C 29 H 37 NNaO 10 Se 662.1475; [M+Na] + :found 662.1478.

[0123]

[0124] 6-(6-methoxy-3-oxobenzo[d][1,2]selenazepine -2(3H)-yl)hexyl((3R,5aS,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxolo[4,3-i]isochroman-10-yl)succinate (IIn); yield: 68%; white solid, melting point: 59-60°C; purity: 98.97%; 1 H NMR (400MHz, CDCl3) δ7.91(d,J=8.7Hz,1H),7.09(d,J=2.2Hz,1H),6.96(dd,J=8.7,2.2Hz,1H),5.78(d,J=9.9Hz,1H),5.42(s ,1H),4.06(t,J=6.6Hz,2H),3.88(s,3H),3.81(t,J=7.1Hz,2H),2.73–2.68(m,2H),2.61(ddd,J=15.6,7.4,5.5Hz,2H),2.40– 2.32(m,1H),2.04–1.98(m,1H),1.90–1.85(m,1H),1.78–1.67(m,6H),1.63–1.57(m,3H),1.51–1.45(m,1H),1.42(s,3H),1.3 9(d,J=3.8Hz,2H),1.28(dtd,J=17.2,12.0,11.1,6.4Hz,4H),1.04–0.97(m,1H),0.94(d,J=5.7Hz,3H),0.84(d,J=7.1Hz,3H). 13 C NMR (100MHz, CDCl3) δ172.2,171.2,166.9,162.9,139.5,129.7,120.6,114.3,107.5,104.4,92.1,91.5,80.1,64.7,55.7,51. 5,45.2,44.6,37.2,36.2,34.0,31.8,30.4,29.2,28.9,28.4,26.2,25.9,25.6,24.5,21.9,20.2,12.0.HRMS(m / z)(ESI):calcd for C 33 H 45 NNaO 10 Se 718.2101; [M+Na] + :found 718.2102.

[0125]

[0126] 8-(6-methoxy-3-oxobenzo[d][1,2]selenazepine -2(3H)-1,2-octyl((3R,5aS,6R,8aS,9R,10S,12R,12aR)-3,6,9-trimethyldecahydro-12H-3,12-epoxy[1,2]dioxolo[4,3-i]isochroman-10-yl)succinate (IIo); yield: 60%; white solid, melting point: 54-55°C; purity: 96.72%; 1 H NMR (400MHz, CDCl3) δ7.92(d,J=8.7Hz,1H),7.10(d,J=2.2Hz,1H),6.96(dd,J=8.7,2.2Hz,1H),5.79(d,J=9.8Hz,1H),5.43(s ,1H),4.06(t,J=6.7Hz,2H),3.89(s,3H),3.81(t,J=7.2Hz,2H),2.75–2.60(m,4H),2.37(td,J=14.0,4.0Hz,1H),2.03(dt,J=1 1.6,2.9Hz,1H),1.89(ddd,J=13.8,6.4,3.4Hz,1H),1.79–1.68(m,4H),1.61(dt,J=9.7,5.2Hz,3H),1.52–1.45(m,1H),1.43( s,3H),1.32(ddt,J=22.6,17.8,6.8Hz,12H),1.02(td,J=12.1,11.5,3.2Hz,1H),0.96(d,J=5.8Hz,3H),0.85(d,J=7.1Hz,3H). 13 C NMR (100MHz, CDCl3) δ172.2,171.1,166.9,162.8,139.4,129.7,120.7,114.3,107.5,104.4,92.1,91.5,80.1,64.8,55.7,51.5,45. 2,44.7,37.1,36.2,34.1,31.8,30.5,29.2,29.1,28.9,28.9,28.5,26.4,25.9,25.7,24.5,21.9,20.2,12.0.HRMS(m / z)(ESI):calcd for C 35 H 49 NNaO 10 Se746.2414; [M+Na] + :found 746.2417.

[0127] Experimental Example 1: In vitro antitumor activity test

[0128] Using ebselen and 5-fluorouracil as positive drugs, the MTT method was used to determine the anti-proliferative activity of the target compound of the present invention in HepG-2 glioma (original human liver cancer cells), MCF-7 human breast cancer cells, SW480 human colon cancer cells, SW620 human colon cancer cells, HCT116 human colon cancer cells, and HT29 human colon cancer cells.

[0129] Take cells in good growth state and use 6×10 4 Cells were seeded into 96-well plates at 100 μL per well and incubated in a 37°C incubator for 24 hours. After incubation, 20 μL of the test compound (starting at 80 μM, serially diluted by 2-fold dilution, with five replicates per well) was added and incubated in a cell culture incubator for 48 hours. 10 μL of 5 mg / mL MTT working solution was added to the 96-well plate and incubated in the incubator for 4 hours. The supernatant was carefully discarded, and 150 μL of DMSO was added to dissolve the reduced formazan purple crystals. The absorbance of each well was measured using a microplate reader. The cell proliferation inhibition rate was calculated for each dosing well, and the results are shown in Table 1 below.

[0130] Table 1 Inhibitory activity of the compounds of the present invention against different cell lines:

[0131]

[0132] a IC 50 The results are expressed as mean ± SD of three independent experiments.

[0133] The MTT results are shown in Table 1. Compounds I-II have significant in vitro anti-proliferative activity against the six cancer cell types, while the selected parent compound artesunate has no significant anti-tumor activity against all cells at the maximum dose of 50 μM, except for SW480 cells (IC 50 =8.55±0.16 μM) and SW620 cells (IC 50 =4.68±0.17μM). Most of the esterified derivatives of artesunate showed better in vitro antitumor activity than the acylated derivatives of artesunate. In SW480 cells, except for compounds Id, IIa, IIb, and IIc, which had no activity against them, the other compounds had good inhibitory effects on the cells. For SW620 cells, compounds I-II had strong inhibitory effects on the cells, among which compound IIb (IC 50 =4.3±0.18μM), IIc(IC 50 =3.74±0.16μM), IId(IC 50 =4.48±0.21μM), IIk(IC 50 =3.83±0.16μM) showed a higher 50=4.68±0.17μM) had better anti-proliferative activity. Among them, the combined administration had the best anti-tumor effect. In HCT116 cells, except for compounds Id and IIa, the other compounds had good inhibitory effects on the cells, among which compound IIk (IC 50 =0.81±0.29μM) showed better anti-proliferative activity than other compounds. In HT29 cells, compound I-II showed better anti-proliferative activity than other compounds. 50 =4.13±0.27μM), IId(IC 50 =5.41±0.31μM), IIk(IC 50 =5.96±0.31μM), IIo(IC 50 =3.57±0.31 μM) showed superior antiproliferative activity compared to other compounds. In HepG-2 cells, except for compounds IIb, IIc, and IId, which were inactive, all other compounds had good inhibitory effects on these cells. In MCF-7 cells, except for compounds Ia, Id, IIa, and IId, which were inactive, all other compounds showed moderate activity.

[0134] The above in vitro antitumor activity test shows that the artesunate-ebselen compound of the present invention can be used for the preparation of antitumor drugs.

[0135] Experimental Example 2: In vivo antitumor activity of compound IIk and HE staining pathological analysis

[0136] The experimental procedure for the inhibition of tumor transplantation in mice with compound IIk is as follows:

[0137] The mice were divided into four groups (one low-concentration group and one high-concentration group) with blank control and experimental groups, with 5 mice in each group. The colon cancer CT26 cells were then digested and centrifuged, collected into a centrifuge tube, and serum-free culture medium and matrix gel were added in the same proportion. The cells were injected subcutaneously into the left or right armpit of the mice and the tumors were grown to 100-150 mm. 3 Before administration, wipe and disinfect the injection site with 75% alcohol, turn the mouse head downward, insert the needle 1 cm to the left or right of the abdomen, pierce the epidermis first, insert the needle 0.5 cm at a 30-45° angle, slowly inject the drug into the abdominal cavity of the nude mouse, and then rotate the needle at a certain angle to slowly withdraw it. On the 12th day of administration, kill the mouse by dislocation. First, inject 0.2 mL of 1% sodium barbital solution and perfuse anesthetics into the mouse's abdominal cavity. Then, use the thumb and index finger of your left hand to press the mouse's head hard on the ground, grab the mouse's tail with your right hand, and slowly pull it outward with force to dislocate the animal's head and neck, causing the animal to die. Then dissect the nude mouse, cut the abdomen horizontally and then vertically, and remove tissues such as the heart, liver, spleen, lung, kidney, and brain, as well as tumors.

[0138] All mice were randomly divided into four groups (n=10 / group), blank group (intraperitoneal injection of drug, artesunate (50 mg / kg), compound IIk (low dose group, 25 mg / kg; high dose group, 50 mg / kg). Figure 1 As shown in Figure 2, compound IIk has an inhibition rate of 42.5% and 54.9% on tumor growth at a dose concentration of 25 mg / kg and 50 mg / kg, while the inhibition rate of parent nucleus artesunate on transplanted tumors is 28.9%. This indicates that compound IIk has good anti-tumor activity. From the changes in the body weight of mice ( Figure 1 C) It can be seen that IIk has lower toxicity.

[0139] like Figure 2 The results of HE staining pathological analysis of the heart, liver, spleen, lung, and kidney of mice treated with the compound are shown.

[0140] No significant pathological changes were observed in the five internal organs of mice in the control, artesunate, IIk (25 mg / kg), and IIk (50 mg / kg) groups. Based on the changes in mouse body weight, tumor weight, and staining pathology analysis, compound IIk exhibits strong in vivo antitumor activity and low toxicity, making it a promising antitumor agent.

[0141] Experimental Example 3: CD4 in mouse spleen + T / CD8 + T lymphocyte content detection

[0142] Based on relevant research reports, we conducted a study on + T / CD8 + T-related immune experiments verified that compound IIk has anti-tumor activity and also has the effect of enhancing immune response. Figure 3 By testing the spleen lymphocytes of mice in the blank group, artesunate group, and compound IIk (low concentration, high concentration) group, we found that compared with the blank group, after artesunate treatment, CD4 + The proportion of T lymphocytes increased by 3.5%, CD8 + The proportion of T lymphocytes increased by 0.9%; after the action of compound IIk (low concentration), CD4 + The proportion of T lymphocytes increased by 1.3%, CD8 + The proportion of T lymphocytes increased by 2.6%; after the action of compound IIk (high concentration), CD4 + The proportion of T lymphocytes increased by 6.2%, CD8 + The proportion of T lymphocytes increased by 7.2%. From the experimental results, it can be seen that compound IIk enhanced the CD4 + T / CD8 +The immune response of T lymphocytes, especially after the action of high concentrations of IIk, was significantly enhanced. It was preliminarily determined that compound IIk has the unique effect of enhancing the immune response of T lymphocytes while having anti-tumor effects.

[0143] Experimental Example 4: Intracellular Reactive Oxygen Species (ROS) Detection Experiment. The experimental process of compound IIk ROS is as follows:

[0144] HCT116 cells with good growth status were seeded in 6-well plates, with about 6×10 cells per well. 5 Cells were cultured in a 37°C incubator until ready for use. When the cell density reached 60-70%, compound IIk and artesunate were added. After incubation for 24 hours, the cell culture medium was removed and the cells were washed with PBS. 10 μM DCFH-DA was added to each well and the cells were cultured in a dark, 37°C incubator for 30 minutes. Afterwards, the cells were washed three times with serum-free medium and observed and photographed using an inverted fluorescence microscope.

[0145] We performed intracellular ROS level detection in HCT116 cells to study the changes in intracellular ROS under oxidative stress conditions. After compound IIk acted on HCT116 cells for 24 hours, the cells were labeled with DCFH-DA probe and their fluorescence intensity was detected using an inverted fluorescence microscope and flow cytometry. The results are shown in Figure 2. Figure 4 As shown in Figures A and 4B, compared with the blank control group and artesunate group, compound IIk induced a significant increase in the level of reactive oxygen species in HCT116 cells.

[0146] Experimental Example 5: Mitochondrial membrane potential (JC-1) detection experiment, compound IIk JC-1 staining experimental process is as follows:

[0147] HCT116 cells with good growth status were seeded into 6-well plates, with about 6×10 cells per well. 5 Cells were cultured in a constant temperature incubator at 37°C until use. When the cells were completely attached and the density reached 60-70%, fresh culture medium containing compound IIk or artesunate was replaced and cultured in the incubator for 24 hours. The culture medium was removed, washed twice with PBS buffer, digested with trypsin, and the cells were collected in a 15 mL centrifuge tube. After centrifugation, the supernatant was discarded and the cells were washed twice with serum-free medium. 500 μL of JC-1 staining working solution was added and incubated in the dark for 30 minutes. After the incubation, centrifugation was completed, the supernatant was discarded, and the cells were washed twice with JC-1 staining buffer. 500 μL of serum-free culture medium was added to suspend the cells and observed and photographed using a fluorescence inverted microscope.

[0148] JC-1 fluorescent probe was used to detect changes in mitochondrial membrane potential (MMP) in HCT116 cells. Figure 5As shown in Figure A, the probes in the experimental group mainly existed as green fluorescent monomers. In contrast, in the blank control group, they were converted into red fluorescent polymers, indicating that MMP in HCT116 cells was destroyed by compound IIk. Figure 5 B shows that the membrane potential of compound IIk (31.1%) was significantly reduced compared with the control group (9.68%) and artesunate (22.5%). These results indicate that IIk induces mitochondrial damage, resulting in the generation of a large amount of ROS in mitochondria and causing cell damage.

[0149] Experimental Example 6: Lipid peroxidation (BODIPY 581 / 591C11) level detection experiment, compound IIk BODIPY581 / 591C11 staining experiment process is as follows:

[0150] HCT116 cells with good growth status were seeded into 6-well plates, with about 6×10 cells per well. 5 Cells were cultured in a constant temperature incubator at 37°C until use. When the cells were completely attached and the density reached 60-70%, fresh culture medium containing compound IIk or artesunate was replaced and cultured in the incubator for 24 hours. The culture medium was removed, washed twice with PBS buffer, and trypsinized. The cells were collected in a 15 mL centrifuge tube, the supernatant was discarded after centrifugation, and the cells were washed twice with serum-free culture medium. 1 mL of diluted staining working solution was added and incubated in the dark for 30 minutes. After the incubation, the cells were centrifuged, the supernatant was discarded, and the cells were washed twice with PBS. 500 μL of serum-free culture medium was added to suspend the cells and observed and photographed using a fluorescence inverted microscope.

[0151] Image 6A obtained using the BODIPY-C11 581 / 591 fluorescent probe shows that the green fluorescence of HCT116 cells increased after treatment with compound IIk and artesunate, indicating that these cells produced more lipid ROS. Figure 6 B shows that the peak shifted to the right. Taken together, these results indicate that IIk damages cells by inducing ferroptosis.

[0152] Experimental Example 7: Transmission electron microscopy experiment. The electron microscopy experiment process of compound IIk is as follows:

[0153] Well-growing HCT116 cells were inoculated into 10 cm dishes and cultured in a 37°C incubator. Once cells were fully attached and reached a density of 60-70%, fresh medium containing compound IIk was replaced and cultured in the incubator for 24 h. The culture medium was removed, the cells were washed twice with PBS buffer, and trypsinized. The cells were collected in a 15 mL centrifuge tube and fixed with 3% glutaraldehyde. The cells were resuspended and allowed to stand at 4°C for 5 min. The cell suspension was transferred to a 1.5 mL EP tube and centrifuged at high speed (12,000 rpm / 10 min). The supernatant was gently discarded and 3% glutaraldehyde fixative was added. The cells were dehydrated using acetone in a stepwise manner, centrifuged to obtain cell spheres, and embedded in an embedding medium. The cells were cut using ultrafine microtome sections and mounted on copper foil. After drying, the cells were stained with heavy metals, first with uranyl acetate for 10-15 min, then with lead citrate for 1-2 min. Staining was performed at room temperature and finally examined by transmission electron microscopy.

[0154] To further confirm whether HCT116 cells treated with compound IIk undergo ferroptosis through the mitochondrial pathway, this study used transmission electron microscopy to observe the morphological changes of mitochondria. Figure 7 As shown, the mitochondria in the control cells were normal in morphology and clearly discernible in structure. In contrast, the mitochondria in HCT116 cells treated with compound IIk exhibited typical morphological features of ferroptosis: reduced mitochondrial size, rupture, and a significant decrease in cristae. These morphological changes provide important morphological evidence for further investigation into the mechanism of compound IIk-induced ferroptosis in HCT116 cells.

[0155] Experimental Example 8: Ferrous ions (Fe 2+ ) content determination experiment

[0156] Compound IIk Fe 2+ The experimental process is as follows:

[0157] Well-growing HCT116 cells were seeded into 10 cm dishes and cultured in a 37°C incubator. Once cells were fully attached and reached a density of 60-70%, fresh medium containing compound IIk or artesunate was replaced and cultured in the incubator for 24 hours. The culture medium was removed, the cells were washed twice with PBS buffer, and trypsinized. The cells were harvested into 15 mL centrifuge tubes and assayed according to the manufacturer's instructions.

[0158] We treated colon cancer cells HCT116 with compound IIk and artesunate for 24 h and used a cell ferrous colorimetric assay kit to detect the Fe 2+ Content. Figure 8As shown, compared with the blank group and artesunate group, the ferrous ion level in HCT116 cells treated with compound IIk was significantly increased. The above results indicate that compound IIk can significantly increase the ferrous ion level in HCT116 cells.

[0159] Experimental Example 9: Western blot experiment, compound IIk Wb experimental process is as follows:

[0160] Take HCT116 cells in good growth condition and inoculate them into 10cm dishes and culture them in a constant temperature incubator at 37℃ for later use. When the cells are completely attached and the density reaches 60-70%, replace with fresh culture medium containing different concentrations of compound IIk or carrier and culture in the incubator for 24h. Remove the culture medium, wash twice with PBS buffer, digest with trypsin, collect the cells in a 15mL centrifuge tube, add the pre-prepared lysis buffer, and lyse in an ice bath for 10min. Centrifuge at 1200rpm / 4min at 4℃, take the supernatant (record the supernatant volume), determine the relevant protein concentration with a BCA protein kit, and then add 5x protein loading buffer. Mix well, heat at 100℃ for 5min, cool on ice, and move to -20℃ for later use. The detection steps are carried out according to the instructions.

[0161] To verify whether ferroptosis is involved in the IIk anti-tumor pathway, we used Western Blot to detect the expression changes of ferroptosis-related proteins SLC7A11, GPX4, FTH1 and ACLS4. Figure 9 As shown in the results, the expression levels of SLC7A11 and GPX4 in HCT116 cells were significantly reduced after treatment with compound IIk, and the expression of SLC7A11 was enhanced, while there was no significant effect on ACSL4 expression. The results showed that compound IIk may induce ferroptosis through the Xc-GPX4 pathway.

[0162] Experimental Example 8: qPCR Experiment

[0163] Compound IIk Fe 2+ The experimental process is as follows:

[0164] Well-growing HCT116 cells were seeded into 10 cm dishes and cultured in a 37°C incubator. Once cells were fully attached and reached a density of 60-70%, fresh culture medium containing various concentrations of Compound IIk or vehicle was replaced and cultured in the incubator for 24 hours. The culture medium was removed, the cells were washed twice with PBS buffer, and trypsinized. The cells were harvested in 15 mL centrifuge tubes for RNA extraction, reverse transcription, and qPCR reaction setup for analysis.

[0165] After compound IIk acted on HCT116, the mRNA expression level of GPX4 was significantly reduced, indicating that the expression changes at the transcriptional level were consistent with the protein expression pattern. Figure 10 The results showed that compound IIk inhibited the GPX4 pathway and induced ferroptosis for cancer treatment.

Claims

1. Artesunate-ebselen derivatives having the structures represented by the following formulas (I) and (II): in, R represents a hydrogen atom, a halogen atom, a methyl group or a methoxy group, and n represents 2, 4, 6, or C 37 ~C 40 Alkyl, methoxy, and halogen atoms; Wherein, R represents a hydrogen atom, a halogen atom, a methyl group or a methoxy group, and n represents 2, 4, 6, 8, or C 37 ~C 40 alkyl, methoxy, and halogen atoms.

2. The method for preparing the artesunate-ebselen derivative according to claim 1, wherein: The following steps are involved: The compounds represented by the following formula (III) and formula (IV) are mixed with artesunate, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, triethylamine or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and 4-dimethylaminopyridine, respectively, and then placed in an organic solvent, respectively, and reacted at room temperature. The solvent is recovered from the obtained reaction mass to obtain a crude target compound; Wherein, R represents a hydrogen atom, a halogen atom, a methoxy group, or a C9-C 12 alkyl, methoxy, and halogen atoms.

3. The preparation method according to claim 2, wherein: The molar ratio of the compounds represented by formula (III) and formula (IV) to artesunate, 2-(7-azobenzotriazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate, triethylamine or 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide, and 4-dimethylaminopyridine is 1:0.4-4.

4. The preparation method according to claim 2, wherein: The organic solvent is dichloromethane or tetrahydrofuran.

5. The preparation method according to claim 2, wherein: The method further includes a step of purifying the crude target compound. During the purification, the developing solvent used to separate the compound is a mixed solvent consisting of ethyl acetate and petroleum ether, wherein the volume ratio of petroleum ether to ethyl acetate is 1:

2.

6. The preparation method according to claim 2, wherein: The synthetic route of the compound represented by formula (III) is: BOC in compound S2 and compound S3 in the synthetic route represents tert-butyloxycarbonyl; The synthesis method comprises the following steps: 1) Compound S1 and Compound S2 are placed in an organic solvent and reacted at room temperature to obtain Compound S3; 2) removing the tert-butyloxycarbonyl group in compound S3 to expose the NH, thereby obtaining a crude product of the compound represented by formula (III), which is then purified by conventional silica gel column purification; In step 1), the organic solvent is dichloromethane or tetrahydrofuran; In step 2), the tert-butyloxycarbonyl group in compound S3 is removed by placing compound S3 in a mixed solvent consisting of tetrahydrofuran and trifluoroacetic acid in a volume ratio of 2:1 and stirring for a certain period of time; In order to further expose the NH structure, an excess of dichloromethane is added to the reaction system as a reaction solvent, and an excess of alkaline substances or organic bases are added to neutralize the excess acid.

7. The preparation method according to claim 2, characterized in that: The synthetic route of the compound represented by formula (IV) is: The synthesis method is as follows: Compound S1 and Compound S4 are placed in dichloromethane and reacted at room temperature to obtain the product.

8. The preparation method according to claim 7, characterized in that: In the method for preparing the compound represented by formula (IV), the reaction liquid is extracted with ethyl acetate, washed with water and brine, the organic phase is collected, dried over anhydrous sodium sulfate, and the residue obtained after spin drying is purified on a silica gel column to obtain a high-purity compound represented by formula (IV). The purification eluent is ethyl acetate and petroleum ether.

9. Use of the artesunate-ebselen derivative according to claim 1 in the preparation of a drug for treating tumors.

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