17-hydroxy-euphorbia jolkinolide B coupled triphenylphosphine derivative as well as preparation method and application thereof

Abstract

The invention discloses a 17-hydroxy-jolkinolide B (HJB) coupled triphenylphosphine derivative as well as a preparation method and application thereof, and belongs to the technical field of antitumor drugs, the 17-hydroxy-jolkinolide B coupled triphenylphosphine derivative has the following structural formula: # imgabs0 #, and n is equal to 1-3. According to the invention, an effective component HJB extracted from euphorbia fischeriana is taken as a raw material, triphenylphosphine cations are introduced to C-17 site of HJB through esterification reaction and substitution reaction, and in-vivo and in-vitro antitumor activity evaluation of a novel derivative is carried out through an MTT method, laser confocal microscopy observation, flow cytometry and an animal transplantation tumor method. It is found that the novel derivative has relatively low toxicity to human normal mammary epithelial cells MCF-10A while retaining relatively good anti-tumor activity to human liver cancer HepG-2, human breast cancer MDA-MB-231, human lung cancer H1975 and human gastric cancer MNK45 cells, and can obviously inhibit growth of transplanted tumors in Balb / c mice. The 17-hydroxy-jolkinolide B coupled triphenylphosphine derivative disclosed by the invention has the potential of being developed into a new anti-tumor drug.

Description

Technical Field

[0001] The present invention belongs to the technical field of anti-tumor drugs, and particularly relates to a 17-hydroxy-lytophanolide B conjugated triphenylphosphine derivative, a preparation method thereof, and an application thereof. Background Art

[0002] In today's world, cancer is one of the diseases with the highest morbidity and mortality rates in the world, seriously affecting human health and life, and has become the second biggest killer threatening human current health. Chemotherapy is still one of the most effective cancer treatment methods at present, but adverse side effects limit the clinical use of many drugs. Therefore, developing new anti-tumor drugs with high efficiency and low toxicity is an important task.

[0003] Mitochondria play a key role in tumorigenesis. In addition to playing a core bioenergetic function, mitochondria provide a basis for tumor anabolism, control of redox and calcium homeostasis, participation in transcriptional regulation, and control of cell death. Therefore, tumor mitochondria have become a promising target organ for developing new anti-cancer drugs.

[0004] The mitochondrial membrane potential of tumor cells is significantly higher than that of normal cells. Utilizing this characteristic to specifically deliver anti-tumor drugs into tumor cell mitochondria and further induce apoptosis of tumor cells becomes possible. One of the strategies for selectively delivering anti-tumor drugs to tumor cell mitochondria is to covalently link the drug with a mitochondrial targeting vector to form a conjugate, thereby achieving targeting of mitochondria. The lipophilic triphenylphosphonium cation (TPP + ) has become the most widely used mitochondrial targeting vector at present because it is stable in biological systems and the synthesis and purification methods are relatively simple.

[0005] Euphorbia fischeriana was first recorded in "Shennong Ben Cao Jing". Both modern medicine and traditional Chinese medicine research have found its anti-tumor effect. 17-Hydroxy-lytophanolide B (HJB) is a diterpenoid compound extracted from Euphorbia fischeriana, which can significantly inhibit the proliferation of tumor cells. However, as a diterpenoid compound, 17-hydroxy-lytophanolide B still has many defects at present, such as poor water solubility and lack of selectivity, resulting in its difficulty in aggregating in tumor cells and limiting its further research and clinical application.

[0006] At present, the structural modification of existing 17-hydroxy-lytophanolide B is mainly located at the 17th carbon atom. This is because this position, as an allylic position, has high reactivity and is suitable for structural modification to prepare a series of structural derivatives. However, the existing structural modifications have problems such as high synthesis difficulty, numerous total synthesis steps, and low yields, and cannot meet the current scientific research and new drug development needs. Summary of the Invention

[0007] To solve the above technical problems, the present invention provides a 17-hydroxy-jatropholide B conjugated triphenylphosphine derivative, its preparation method and application. The present invention selects triphenylphosphine to modify the structure of 17-hydroxy-jatropholide B. On the basis of maintaining the original activity, the targeting ability and bioavailability of 17-hydroxy-jatropholide B to tumor cell mitochondria are significantly improved. The process of the present invention has the advantages of simple operation, short preparation cycle, low cost, easy scale-up and easy industrialization. And experiments also prove that the prepared derivative can be used for preventing or treating breast cancer, gastric cancer, liver cancer or other cancers.

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

[0009] One of the technical solutions of the present invention:

[0010] A 17-hydroxy-jatropholide B conjugated triphenylphosphine derivative (HJB-TPP) having the structure shown in Formula I:

[0011]

[0012] wherein, n = 1-3.

[0013] Further, the 17-hydroxy-jatropholide B conjugated triphenylphosphine derivative is selected from HJB-TPP-1, HJB-TPP-2 or HJB-TPP-3, and the structural formulas are as follows:

[0014]

[0015] As shown in the following structure: In most cases, during the structural modification of 17-hydroxy-jatropholide B, the two epoxy structures in part A are proved to be the functional groups necessary to ensure the activity of 17-hydroxy-jatropholide B, and the hydroxyl group at C-17 has a large space for structural modification. Therefore, the structural modification mainly focuses on the hydroxyl group at C-17. The present invention connects the anti-tumor active part A and the mitochondria-targeting part C through linkers with different carbon atom numbers. After parts A and C are connected in the manner of the present invention, a good tumor inhibitory effect can be maintained. The present invention selects triphenylphosphine to modify the structure of 17-hydroxy-jatropholide B. On the basis of maintaining the original activity, the targeting ability and bioavailability of 17-hydroxy-jatropholide B to tumor cell mitochondria are significantly improved.

[0016] The 17-hydroxy-jatropholide B conjugated triphenylphosphine derivative prepared by the present invention has better water solubility than 17-hydroxy-jatropholide B, and it can accumulate in the mitochondrial part of cells under the drive of mitochondrial transmembrane potential. Since the mitochondrial membrane potential of tumor cells is higher than that of normal cells, it can provide driving force for selective accumulation in tumor cell mitochondria, thus having mitochondrial targeting, and this compound (i.e., 17-hydroxy-jatropholide B conjugated triphenylphosphine derivative) exhibits obvious anti-tumor activity.

[0017] The second technical solution of the present invention:

[0018] The present invention also provides a preparation method of the above-mentioned 17-hydroxy-jatropholide B conjugated triphenylphosphine derivative. Using 17-hydroxy-jatropholide B as a raw material, through esterification reaction and substitution reaction, a triphenylphosphine cation is introduced at the C-17 position of the 17-hydroxy-jatropholide B to obtain the 17-hydroxy-jatropholide B conjugated triphenylphosphine derivative.

[0019] Further, the preparation method of the 17-hydroxy-jatropholide B conjugated triphenylphosphine derivative includes the following steps:

[0020] Dissolve 17-hydroxy-jatropholide B and bromoalkyl carboxylic acid in a first organic solvent, add a catalyst and a condensing agent, and carry out an esterification reaction to obtain intermediate A. The structural formula of the bromoalkyl carboxylic acid is where n = 1, 2 or 3, that is, the bromoalkyl carboxylic acid is 3-bromopropionic acid, 4-bromobutyric acid or 5-bromovaleric acid, and the specific structures are as follows:

[0021]

[0022] Dissolve the intermediate A and triphenylphosphine in a second organic solvent, heat under reflux for a substitution reaction. After the reaction is completed, evaporate the solvent and wash the precipitate to obtain the 17-hydroxy-jatropholide B conjugated triphenylphosphine derivative.

[0023] The catalyst is selected from one of 4-dimethylaminopyridine (DMAP), triethylamine (TEA) and N,N-diisopropylethylamine (DIPEA). The above catalysts can all reduce the occurrence of side reactions. In particular, the activity of the catalyst DMAP is 10 4 -10 6 times that of pyridine, and it can accurately catalyze the reaction at a specific position, and can further reduce the occurrence of side reactions, improving the selectivity and purity of the target product. Preferably DMAP; and / or

[0024] The condensing agent is selected from one of 1,3-dicyclohexylcarbodiimide (DCC), N,N'-diisopropylcarbodiimide (DIC), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI). The above condensing agents have good condensation effects on various types of carboxylic acids and amines. In particular, the condensing agent EDCI can rapidly promote the condensation reaction at room temperature, and the by-products generated are easy to remove, which can reduce the purification steps and costs, and improve the purity and yield of the product. Preferably, it is EDCI; and / or

[0025] The first organic solvent is selected from one of chloroform, N,N-dimethylformamide, and dichloromethane; and / or

[0026] The temperature of the esterification reaction is room temperature, and the time of the esterification reaction is 4 to 6 h; and / or

[0027] The second organic solvent is selected from one of dichloromethane, toluene, N,N-dimethylformamide, n-butanol, and acetonitrile; and / or

[0028] The temperature of the substitution reaction is 80 to 85 °C, and the time of the substitution reaction is 24 to 48 h.

[0029] When the catalyst is DMAP and the condensing agent is EDCI, the synthetic route of 17-hydroxy-jolkinolide B coupled with triphenylphosphine derivative is as follows:

[0030]

[0031] The molar ratio of 17-hydroxy-jolkinolide B, bromoalkyl carboxylic acid, catalyst, and condensing agent is 1∶(2 - 5)∶(0.6 - 1)∶(4 - 6), more preferably 1∶3∶0.8∶5.

[0032] The molar ratio of intermediate A and triphenylphosphine is 1∶(2 - 4), more preferably 1∶3.

[0033] 17-Hydroxy-jolkinolide B (HJB) is a diterpenoid compound with antitumor activity extracted from Euphorbia fischeriana. The two epoxy bridges between C-11 and C-12, and C-8 and C-14 in its structure have been proven to be functional groups necessary to ensure the antitumor activity of HJB. They can break in vivo and induce an increase in the level of reactive oxygen species, thereby causing apoptosis of tumor cells. The C-17 position in the HJB structure contains a hydroxyl (-OH) functional group, which has high reactivity and small steric hindrance, making it easier to be approached and attacked by reactants, and can greatly improve the selectivity of the reaction. Therefore, the present invention retains the two epoxy bridges between C-11 and C-12, and C-8 and C-14 in the HJB structure, that is, the functional groups necessary for exerting antitumor activity as the parent part, and uses the hydroxyl group at its C-17 position as the linking site, and selects three bromoalkyl carboxylic acids with different lengths (3-bromopropionic acid, 4-bromobutyric acid or 5-bromovaleric acid) as the linking arms. On the basis of maintaining the original antitumor activity, the bioavailability of 17-hydroxy-jolkinolide B has been significantly improved.

[0034] Meanwhile, the HJB parent part is connected to the mitochondrial targeting vector triphenylphosphine (TPP) through bromocarboxylic acid, further enhancing the tumor targeting effect of the compound. Triphenylphosphine carries a positive charge, and this charged property enables it to generate electrostatic interaction with the relatively high negative potential on the inner side of the tumor mitochondrial inner membrane. Driven by the electrostatic attraction, triphenylphosphine can move directionally along the electrochemical gradient, enrich from the outside of the membrane to the inside of the membrane, thereby achieving the targeted positioning of tumor mitochondria, and having less impact on normal cells, because the electrochemical gradient of the membrane potential of tumor mitochondria is higher than that of normal cell mitochondria, making triphenylphosphine selectively enriched in tumor cell mitochondria. Therefore, the tumor targeting of the three 17-hydroxy-jolkinolide B-coupled triphenylphosphine derivatives designed and synthesized in the present invention has also been significantly improved.

[0035] Technical solution three of the present invention:

[0036] The present invention also provides a drug carrier targeting mitochondria, comprising the above-mentioned 17-hydroxy-jolkinolide B-coupled triphenylphosphine derivative.

[0037] Technical solution four of the present invention:

[0038] The present invention also provides an antitumor drug targeting mitochondria. The components of the antitumor drug include the above-mentioned 17-hydroxy-jolkinolide B-coupled triphenylphosphine derivative, or its stereoisomer, prodrug and pharmaceutically acceptable salt.

[0039] Technical solution five of the present invention:

[0040] The present invention also provides the use of the 17-hydroxy-jatropholide B conjugated triphenylphosphine derivative in the preparation of an anti-tumor drug, and the anti-tumor drug includes an anti-hepatocarcinoma drug, an anti-breast cancer drug, an anti-lung cancer drug or an anti-gastric cancer drug.

[0041] Compared with the prior art, the present invention has the following advantages and technical effects:

[0042] (1) The present invention uses 17-hydroxy-jatropholide B as a lead compound. Under the guidance of the principle of combination, the triphenylphosphine group with mitochondrial targeting function is reacted with 17-hydroxy-jatropholide B to design and synthesize a series of mitochondrial targeting anti-tumor drugs, 17-hydroxy-jatropholide B conjugated triphenylphosphine derivatives, which make up for the disadvantages of 17-hydroxy-jatropholide B such as poor water solubility and low selectivity.

[0043] (2) Through MTT method, flow cytometry, laser confocal microscopy, etc., the present invention finds that the 17-hydroxy-jatropholide B conjugated triphenylphosphine derivative can significantly inhibit the proliferation of tumor cells, induce apoptosis of tumor cells significantly by inducing depolarization of mitochondrial membrane potential of breast cancer MDA-MB-231 cells and increasing the production of reactive oxygen species; through the mouse xenograft tumor experiment, it is found that the 17-hydroxy-jatropholide B conjugated triphenylphosphine derivative can significantly inhibit the growth of transplanted tumors in mice. BRIEF DESCRIPTION OF THE DRAWINGS

[0044] The drawings constituting a part of the present invention are used to provide a further understanding of the present invention. The schematic embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute an improper limitation of the present invention. In the drawings:

[0045] Figure 1 It is the result of the effect of HJB-TPP-3 (1 μM, 2 μM, 4 μM) on the morphological changes of breast cancer MDA-MB-231 cells detected by AO / EB method of the present invention (using HJB as the parent compound control (Ctrl));

[0046] Figure 2 It is the apoptosis situation of Annexin V-FITC / PI stained cells detected by flow cytometry after treating breast cancer MDA-MB-231 cells with the compounds HJB-TPP-3 (1 μM, 2 μM, 4 μM) and HJB (4 μM) for 24 h respectively;

[0047] Figure 3 It is the quantitative analysis result of the apoptosis of breast cancer MDA-MB-231 cells induced by the compounds HJB-TPP-3 and HJB; the data are expressed as mean ± SD (n = 3); *P < 0.05, **P < 0.01, ***P < 0.001 are the comparison results with the control group;

[0048] Figure 4 After treating breast cancer MDA-MB-231 cells with compounds HJB-TPP-3 (1 μM, 2 μM, 4 μM) and HJB (4 μM) for 24 h respectively, the changes in mitochondrial membrane potential were detected by flow cytometry (using HJB as the parent compound control);

[0049] Figure 5 Quantitative analysis results of the depolarization of the cell membrane potential of breast cancer MDA-MB-231 cells induced by compounds HJB-TPP-3 and HJB; Data are expressed as mean ± SD (n = 3); *P < 0.05, **P < 0.01, ***P < 0.001 compared with the control group (using HJB as the parent compound control);

[0050] Figure 6 To detect the effect of compound HJB-TPP-3 on the mitochondrial membrane potential of breast cancer MDA-MB-231 cells by JC-1 staining method, with compound HJB as the parent compound control;

[0051] Figure 7 After treating breast cancer MDA-MB-231 cells with compounds HJB-TPP-3 (1 μM, 2 μM, 4 μM) and HJB (4 μM) for 24 h respectively, the changes in the intracellular reactive oxygen species (ROS) level were detected by flow cytometry (using HJB as the parent compound control);

[0052] Figure 8 To detect the effect of HJB-TPP-3 on the reactive oxygen species (ROS) level of breast cancer MDA-MB-231 cells using a reactive oxygen species (ROS) detection kit, with compound HJB as the parent compound control;

[0053] Figure 9 Results of the effects of compounds HJB-TPP-3 and HJB on the body weight changes of Balb / c mice in the 4T1 tumor transplantation model;

[0054] Figure 10 Results of the effects of compounds HJB-TPP-3 and HJB on the tumor tissue growth rate;

[0055] Figure 11 Photographs of the tumor tissues of each mouse at the end of the anti-tumor experiment of compound HJB-TPP-3 on the 4T1 tumor transplantation model of Balb / c mice;

[0056] Figure 12 Statistical results of the weights of the tumor tissues of each mouse at the end of the anti-tumor experiment of compounds HJB-TPP-3 and HJB on the 4T1 tumor transplantation model of Balb / c mice;

[0057] Figure 13 The HE staining results of tumor tissue sections after detecting the anti-tumor effect of compound HJB-TPP-3 on the 4T1 transplanted tumor model in Balb / c mice (HJB was used as the parent compound control). Detailed implementation manners

[0058] The various exemplary implementation manners of the present invention will now be described in detail. This detailed description should not be considered as a limitation of the present invention, but rather as a more detailed description of certain aspects, characteristics, and implementation schemes of the present invention.

[0059] It should be understood that the terms used in the present invention are only for describing specific implementation manners and are not used to limit the present invention. Additionally, for the numerical ranges in the present invention, it should be understood that each intermediate value between the upper and lower limits of the range is also specifically disclosed. Each intermediate value within any stated value or stated range, as well as each smaller range between any other stated value or intermediate value within the stated range, is also included in the present invention. The upper and lower limits of these smaller ranges may be independently included or excluded from the range.

[0060] Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the art to which the present invention pertains. Although the present invention only describes preferred methods and materials, any methods and materials similar or equivalent to those described herein may also be used in the implementation or testing of the present invention. All documents mentioned in this specification are incorporated by reference to disclose and describe the methods and / or materials related to the documents. In case of conflict with any incorporated document, the content of this specification shall prevail.

[0061] Without departing from the scope or spirit of the present invention, various improvements and changes can be made to the specific implementation manners of the present invention specification, which are obvious to those skilled in the art. Other implementation manners obtained from the specification of the present invention are also obvious to those skilled in the art. The specification and examples of the present invention are merely exemplary.

[0062] Regarding the terms "comprising", "including", "having", "containing", etc. used herein, they are all open-ended terms, meaning including but not limited to.

[0063] Unless otherwise specified, the room temperature in the present invention is calculated as 25 ± 2 °C.

[0064] Each raw material and reagent used in the examples of the present invention was obtained by purchasing commercially. As an example, 4-dimethylaminopyridine (DMAP) was purchased from Shanghai Macklin Biochemical Co., Ltd.; 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI) was purchased from Shanghai Macklin Biochemical Co., Ltd.

[0065] It should be noted that, under the disclosure of the present invention, those skilled in the art can further optimize the reaction conditions on the basis of the present invention. For example, alternative solvents can be selected from more common reagents in the art. For example, the ratio can be further enlarged or reduced in the conventional adjustment according to the amount of substances disclosed in the present invention to obtain a higher yield or reduce the generation of impurities. Of course, it should also be understood that after each step of the reaction, those skilled in the art can choose to purify the product to make the next step of the reaction more accurate, and conventional purification methods can be selected or selected after conventional experiments.

[0066] In the examples of the present invention, 17-hydroxy-jatropholide B is used as the raw material, and a straight-chain aliphatic group containing a bromine atom is introduced at the C-17 position to obtain intermediate compound A. Then, intermediate compound A is subjected to a salt-forming reaction with triphenylphosphine to obtain the target product 17-hydroxy-jatropholide B-coupled triphenylphosphine derivative.

[0067] The examples of the present invention also provide a drug carrier targeting mitochondria, which contains the above-mentioned 17-hydroxy-jatropholide B-coupled triphenylphosphine derivative.

[0068] The examples of the present invention also provide an anti-tumor drug targeting mitochondria. The components of the anti-tumor drug include the above-mentioned 17-hydroxy-jatropholide B-coupled triphenylphosphine derivative, or stereoisomers, prodrugs and pharmaceutically acceptable salts of the above-mentioned 17-hydroxy-jatropholide B-coupled triphenylphosphine derivative.

[0069] The examples of the present invention also provide the application of the above-mentioned 17-hydroxy-jatropholide B-coupled triphenylphosphine derivative in the preparation of anti-tumor drugs. The tumor cell lines include human liver cancer cell line, human breast cancer cell line or human gastric cancer cell line.

[0070] It should be pointed out that the parts not detailedly described in the present invention are all conventional operation means in the art and are not the focus of the present invention.

[0071] The technical solution of the present invention will be further described below through examples.

[0072] Example 1

[0073] Preparation of 17-hydroxy-jatropholide B-coupled triphenylphosphine derivative HJB-TPP-1

[0074] (1) Preparation of intermediate compound 17-hydroxy-jatropholide B-17-substituted bromoester derivative A 1 Preparation:

[0075] Dissolve 0.3 mol of 17-hydroxy-jatropholide B and 0.6 mol of 3-bromopropionic acid in 10 mL of dichloromethane, add 0.18 mol of the catalyst DMAP and 1.2 mol of the condensing agent EDCI, and stir the mixture at room temperature for 5 h until the raw materials react completely. After the reaction is completed, the system is extracted with an equal volume of dichloromethane and distilled water, the organic phase is separated, dried with saturated brine and anhydrous sodium sulfate, and then concentrated by rotary evaporation. The crude product is purified by silica gel column chromatography, and the eluent ratio is V 二氯甲烷 ∶V 甲醇 =10∶1. Finally, a white solid intermediate A 1 is obtained, and its structural formula is as follows:

[0076]

[0077] (2) Preparation of the target product 17-hydroxy-jatropholide B-coupled triphenylphosphine derivative HJB-TPP-1:

[0078] Dissolve 0.3 mol of intermediate A 1 and 0.6 mol of triphenylphosphine in 10 mL of acetonitrile, heat the above system to 80 °C and react for 48 h until intermediate A 1 reacts completely and then terminate the reaction. After the reaction, evaporate the solvent, wash the precipitate with ether to obtain the final product HJB-TPP-1, and its structural formula is as follows:

[0079]

[0080] The 1 H NMR(600 MHz, CDCl 3 ) of HJB-TPP-1: δ 7.80 - 7.75 (m, 9H, Ar-H), 7.67 (td, J = 7.8, 3.5

[0081] Hz, 6H, Ar-H), 4.82 (d, J = 13.6 Hz, 1H, C 17 -H), 4.74 (d, J = 13.6 Hz, 1H, C 17 -H), 4.20 (s, 1H, C 14 -

[0082] H), 4.07 (s, 1H, C 11 -H), 4.02 (d, J = 14.7 Hz, 2H, C 3’ -CH 2 ), 2.98 (dd, J = 23.5, 16.9 Hz, 2H, C 2’ -

[0083] CH 2), 2.22 (s, 1H), 1.90 (d, J = 5.1 Hz, 1H), 1.85 (d, J = 11.3 Hz, 1H), 1.75 (d, J = 15.9 Hz, 1H),

[0084] 1.53 (s, 1H), 1.50 (s, 1H), 1.49 (s, 1H), 1.41 (d, J = 13.4 Hz, 1H), 1.26 (s, 1H), 1.20 (s, 2H), 1.04 (d, J = 9.7 Hz, 1H), 0.88 (s, 3H-CH 3 ), 0.79 (s, 3H-CH 3 ), 0.74 (s, 3H-CH 3 ). 13 C NMR (150 MHz, CDCl 3 ) δ 170.0, 167.4, 155.2, 135.4, 133.7, 130.6, 127.5, 118.0, 85.33, 67.7, 62.1, 55.1, 53.5, 50.4, 47.9, 41.3, 39.2, 39.1, 35.4, 33.5, 29.7, 27.2, 21.92, 20.8, 18.4, 15.5.

[0085] Example 2

[0086] Preparation of 17-hydroxy-jolkinolide B conjugated triphenylphosphine derivative HJB-TPP-2

[0087] (1) Preparation of intermediate compound 17-hydroxy-jolkinolide B-17-substituted bromoacid ester derivative A 2 :

[0088] Dissolve 0.3 mol of 17-hydroxy-jolkinolide B and 0.6 mol of 4-bromobutyric acid in 10 mL of dichloromethane. Add 0.18 mol of catalyst DMAP and 1.2 mol of condensing agent EDCI. The mixture is stirred at room temperature for 5 h until the raw materials react completely. After the reaction is completed, the system is extracted with an equal volume of dichloromethane and distilled water. The organic phase is separated, dried with saturated brine and anhydrous sodium sulfate, and then concentrated by rotary evaporation. The crude product is purified by silica gel column chromatography, and the eluent ratio is V 二氯甲烷 ∶V 甲醇 = 10∶1. Finally, white solid intermediate A 2 is obtained, and its structural formula is as follows:

[0089]

[0090] (2) Preparation of target product 17-hydroxy-jolkinolide B conjugated triphenylphosphine derivative HJB-TPP-2:

[0091] Dissolve 0.3 mol of intermediate A 2 and 0.6 mol of triphenylphosphine in 10 mL of acetonitrile. Heat the above system to 80 °C and react for 48 hours until intermediate A 2 completely reacts and then end the reaction. After the reaction, evaporate the solvent and wash the precipitate with ether to obtain the final product HJB-TPP-2, whose structural formula is as follows:

[0092]

[0093] For HJB-TPP-2 1 H NMR (600 MHz, CDCl 3 ₃): δ 7.79 (dd, J = 12.7, 7.6 Hz, 6H, Ar-H), 7.75 -

[0094] 7.72 (m, 3H, Ar-H), 7.67 (td, J = 7.6, 3.3 Hz, 6H, Ar-H), 4.91 (d, J = 14.0 Hz, 1H, C 17 -H), 4.84 (d, J = 14.0 Hz, 1H, C 17 -H), 3.98 (d, J = 10.8 Hz, 2H, C 14 -H, C 11 -H), 3.74 (s, 2H, C 4’ -CH 2 ₂), 2.48 (s, 2H, C 2’ -CH 2 ₂),, 2.22 (s, 1H), 1.98 (s, 2H, C 3’ -CH 2 ₂), 1.93 - 1.84 (m, 2H), 1.76 (s, 1H), 1.71 (s, 1H), 1.49 (s, 4H), 1.42 (d, J = 13.1 Hz, 1H), 1.22 (s, 2H), 1.05 (d, J = 9.8 Hz, 1H), 0.89 (s, 3H-CH 3 ₃), 0.80 (s, 3H-CH 3 ₃), 0.75 (s, 3H-CH 3 ₃). 13 C NMR (150 MHz, CDCl 3)δ172.6,167.4,154.1,135.0,133.8,130.5,128.5,118.6,85.4,67.5,61.9,55.5,55.2,53.6,47.9,41.3,39.3,39.2,35.6,33.5,33.0,29.7,25.2,22.0,20.9,18.5,15.4.

[0095] Example 3

[0096] Preparation of 17 - hydroxy - jolkinolide B conjugated triphenylphosphine derivative HJB - TPP - 3

[0097] (1) Preparation of intermediate compound 17 - hydroxy - jolkinolide B - 17 - substituted bromo - acid ester derivative A 3 :

[0098] Dissolve 0.3 mol of 17 - hydroxy - jolkinolide B and 0.6 mol of 5 - bromovaleric acid in 10 mL of dichloromethane. Add 0.18 mol of catalyst DMAP and 1.2 mol of condensing agent EDCI. Stir the mixture at room temperature for 5 h until the raw materials react completely. After the reaction, the system is extracted with equal volumes of dichloromethane and distilled water. Separate the organic phase, dry it with saturated brine and anhydrous sodium sulfate, and then rotary - evaporate to dryness. Purify the crude product by silica gel column chromatography, and the eluent ratio is V 二氯甲烷 ∶V 甲醇 = 10∶1. Finally, obtain intermediate white solid A 3 , and the structural formula is as follows:

[0099]

[0100] (2) Preparation of target product 17 - hydroxy - jolkinolide B conjugated triphenylphosphine derivative HJB - TPP - 3:

[0101] Dissolve 0.3 mol of intermediate A 3 and 0.6 mol of triphenylphosphine in 10 mL of acetonitrile. Heat the above - mentioned system to 80 °C and react for 48 h until intermediate A 3 reacts completely and then end the reaction. After the reaction, evaporate the solvent and wash the precipitate with ether to obtain the final product HJB - TPP - 3, and the structural formula is as follows:

[0102]

[0103] The 1 H NMR(600 MHz,CDCl 3 ): δ7.81 - 7.75(m,9H,Ar - H),7.68(td,J = 7.8,3.4

[0104] Hz, 6H, Ar-H), 4.94 (d, J = 14.0 Hz, 1H, C 17 -H), 4.85 (d, J = 14.0 Hz, 1H, C 17 -H), 3.98 (d, J = 36.1 Hz, 2H, C 14 -H, C 11 -H), 3.75 (d, J = 7.3 Hz, 2H, C 5’ -CH 2 ), 2.49 (s, 2H, C 2’ -CH 2 ), 2.24 (s, 1H), 1.96 (d, J = 9.3 Hz, 2H, C 3’ -CH 2 ), 1.93 (d, J = 5.0 Hz, 1H), 1.86 (d, J = 10.0 Hz, 1H), 1.78 (d, J = 11.5 Hz, 1H), 1.70 (d, J = 7.9 Hz, 2H, C 4’ -CH 2 ), 1.51 (d, J = 3.6 Hz, 2H), 1.48 (s, 1H), 1.43 (d, J = 11.7 Hz, 1H), 1.28 (s, 1H), 1.22 (s, 2H), 1.06 (d, J = 12.1 Hz, 1H), 0.90 (s, 3H-CH 3 ), 0.81 (s, 3H-CH 3 ), 0.76 (s, 3H-CH 3 ).

[0105] 13 C NMR (150 MHz, CDCl 3 ) δ 172.6, 167.4, 154.1, 135.1, 133.8, 130.6, 128.4, 118.6, 85.4, 67.5, 62.0, 55.5, 53.6, 50.6, 47.9, 41.3, 39.3, 39.2, 35.6, 33.5, 32.9, 29.8, 25.2, 22.0, 21.8, 20.9, 18.5, 15.4.

[0106] Antitumor Activity Test of the Novel 17-Hydroxy-jolkinolide B Coupled Triphenylphosphine Derivative HJB-TPP-(1 - 3) Prepared in Examples 1 - 3 of Application Example 1

[0107] Triphenylphosphine (TPP) and 17-hydroxy-jolkinolide B (HJB) were selected as the parent compound controls.

[0108] (1) Tested Cells

[0109] MDA-MB-231 (human breast cancer cell line), HepG-2 (human liver cancer cell line), H1975 (human lung cancer cell line), MNK45 (human gastric cancer cell line), and MCF-10A (human mammary epithelial cell line) were selected as the test cells.

[0110] (2) Specific experimental methods

[0111] Methyl thiazolyl tetrazolium (MTT) assay: Tumor cells in the logarithmic growth phase were collected and seeded in 96-well culture plates, with 1.0×10 5 / 100 μL of cells per well, and placed in an incubator at 37°C and 5% CO 2 2. The next day, the culture medium was removed, and 100 μL of compounds at different concentrations were added (the compound concentrations were serially diluted, with 5 - 6 concentrations set for each compound, 3 parallel wells set for each test, and repeated 3 times). The negative control group was not treated with drugs. After 48 h, 20 μL of MTT was added to each well, and the cells were further cultured for 4 h. Then, 150 μL of DMSO was added to each well to terminate the reaction, and the plates were shaken on a shaker for 10 min. The absorbance OD value at 490 nm of each well was measured using an enzyme-linked immunosorbent assay (ELISA) reader, and the cell growth inhibition rate was calculated.

[0112] The cell growth inhibition rate (%) = (average OD value of the solvent control group - average OD value of the drug-treated group) / average OD value of the control group. Then, the half-inhibitory concentration IC 50 .

[0113] (3) Experimental results of the antitumor activity test of compound HJB-TPP-(1 - 3)

[0114] Among the four tested tumor cell lines, the MDA-MB-231 and MNK45 cell lines were more sensitive to these derivatives. In the MDA-MB-231 cell line, all three derivatives (HJB-TPP-1 - HJB-TPP-3) showed significant cytotoxic effects, with IC 50 values ranging from 1.53 - 18.68 μM. In the MNK45 cell line, all three derivatives showed significant cytotoxicity, with IC 50 values ranging from 3.0 - 75.66 μM. In the HepG-2 cell line, all three derivatives also showed significant cytotoxicity, with IC 50 values ranging from 4.67 - 7.08 μM. In the H1975 cell line, all three derivatives showed relatively significant cytotoxicity, with IC 50 values ranging from 4.30 - 17.10 μM.

[0115] Among all three derivatives, the cytotoxic effects of compounds HJB-TPP-1, HJB-TPP-2, and HJB-TPP-3 on four cancer cell lines were higher than those of the HJB parent compound. In addition, compound HJB-TPP-3 exhibited higher cytotoxicity against MDA-MB-231 and MNK45 cell lines. The cytotoxic effect of compound HJB-TPP-3 against MDA-MB-231 cells was 12.83 times higher than that of HJB, indicating a significant enhancement in the cytotoxic effect of the modified compound HJB-TPP-3. The cytotoxic effect of compound HJB-TPP-3 against breast cancer MDA-MB-231 cells was 13.41 times higher than that against normal human breast epithelial cells MCF-10A. This indicates that compound HJB-TPP-3 has a relatively high selectivity for breast cancer cells.

[0116] Structure-activity relationship (SAR) analysis showed that the length of the carbon chain could affect the cytotoxic effects of all these derivatives. Compound HJB-TPP-3 contains a linker with 3 carbons and has better biological activity than HJB-TPP-1 and HJB-TPP-2. Using HJB-TPP-3 as a lead compound, studies on pharmacology and mechanism of action were carried out. The in vitro anti-tumor activities of compounds HJB-TPP-1 to HJB-TPP-3 are shown in Table 1.

[0117] Table 1 In vitro anti-tumor activities of compounds HJB-TPP-1 to HJB-TPP-3

[0118]

[0119] Note: IC 50 a : Concentration (μM) of the test compound that inhibits 50% cell growth. All data in Table 1 are the mean ± SD of three independent experiments.

[0120] Application Example 2 Detection of apoptosis induction of compound HJB-TPP-3 in human breast cancer MDA-MB-231 cells by AO / EB double fluorescence staining

[0121] (1) Experimental method:

[0122] Take MDA-MB-231 cells in the logarithmic growth phase and inoculate them at 1×10 6Inoculate the cells into the corresponding 6-well plates at a density of 1×10⁵ cells / well, and add 2 mL of L-15 culture medium containing 10% FBS (10% FBS means L-15 culture medium supplemented with 10% (v / v) fetal bovine serum (FBS)) to each well. When the cell confluence reaches 85%, discard the supernatant, and add the corresponding volume of HJB-TPP-3 (1 μM, 2 μM, 4 μM) respectively. Set 3 replicate wells for each group, and the group without adding HJB-TPP-3 (i.e., 0) is the control group. Continue to culture for 24 h. Add 1 mL of PBS buffer to wash the cells twice, and then add 500 μL of staining buffer. Add 5 μL of AO staining solution (i.e., acridine orange staining solution) and 5 μL of EB staining solution (i.e., ethidium bromide staining solution) respectively, mix well crosswise, and incubate at 4 °C in the dark for 20 min. Wash off the excess staining solution with the staining buffer, observe and take pictures under a fluorescence microscope.

[0123] (2) Experimental results:

[0124] The results are as Figure 1 shown. The nuclei of the cells in the blank control group were uniformly stained green and had normal structures. As the drug concentration increased, the number of apoptotic cells stained orange-red gradually increased. From the cell morphology, as the concentration of compound HJB-TPP-3 increased, the amount of tumor cell apoptosis gradually increased, showing a concentration-dependent relationship; at the same drug concentration, the number of apoptotic cells in the HJB-TPP-3 group was significantly higher than that in the HJB group.

[0125] Application Example 3 Detection of apoptosis induced by compound HJB-TPP-3 in MDA-MB-231 cells by Annexin V-FITC / PI double staining method

[0126] (1) Experimental method:

[0127] Take MDA-MB-231 cells in the logarithmic growth phase, at a density of 1×10 6 / well inoculated in the corresponding 6-well plate, add 2mL of L-15 culture medium containing 10% FBS to each well, when the cell confluence reaches 85%, discard the supernatant, add the corresponding volume of drug-containing culture medium (1μM, 2μM, 4μM), set up 3 replicates for each group, 0 is the control group (Control), and continue to culture for 24h. Add 2mL PBS to wash the cells, digest with 0.25% trypsin, collect the cells, centrifuge at 800r / min for 5min, get the precipitate, then rinse the cells with cell binding buffer, centrifuge at 800r / min for 5min, discard the supernatant and keep the precipitate. Add 100μL binding buffer to resuspend the cells and transfer them to the flow detection tube, add 5μL AnnexinV-FITC, protect from light, incubate at room temperature for 15min, centrifuge at 800r / min for 5min, and discard the dye solution. Add 100μL binding buffer to resuspend, add 5μL PI dye solution (propidium iodide), mix well in the dark, and incubate at room temperature for 20min. Centrifuge at 800r / min for 5min, discard the supernatant and keep the precipitate, add 400mL of annexin binding buffer, mix well, and perform quantitative analysis using FACS Calibur flow cytometer.

[0128] (2) Experimental results:

[0129] like Figures 2 to 3 As shown, the experimental results show that after the compound HJB-TPP-3 (1μM, 2μM, 4μM) treated the test cells for 24h, the apoptosis percentage of the test cells increased from 12.83% to 97.13%. At the same concentration, the apoptosis percentage of cells treated with HJB-TPP-3 (4μM) was 9.03 times that of cells treated with its parent compound HJB.

[0130] Application Example 4 Detection of mitochondrial membrane potential depolarization induced by compound HJB-TPP-3 in human breast cancer MDA-MB-231 cells

[0131] (1) Experimental methods:

[0132] MDA-MB-231 cells in the logarithmic growth phase were taken and 1×10 6Cells were inoculated into the corresponding 6-well plates at a density of [1×10⁵ cells / mL], and 2 mL of L-15 medium containing 10% FBS was added to each well. When the cell confluence reached 85%, the supernatant was discarded, and the corresponding volumes of HJB-TPP-3 (1 μM, 2 μM, 4 μM) were added. Each group was set with 3 replicate wells, and 0 was the control group (Control). The cells were cultured for another 24 h. The cells were washed with 2 mL of PBS, digested with 0.25% trypsin, collected, centrifuged at 800 r / min for 5 min to obtain a precipitate. Then the cells were washed with cell binding buffer and centrifuged at 800 r / min for 5 min, and the supernatant was discarded leaving the precipitate. 1 mL of pre-prepared JC-1 staining working solution was added to each centrifuge tube with a pipette, and after blowing evenly, it was placed in a carbon dioxide-free incubator at 37 °C for staining for 30 min, and mixed once every 5 min. After the staining was completed, the cells were centrifuged at 1000 r / min for 5 min in an ultra-low temperature centrifuge, and the supernatant was discarded leaving the precipitate. Then it was washed repeatedly twice with the pre-prepared JC-1 washing solution (1×), and the washing solution was carefully removed. 500 μL of basal medium was added to each tube to resuspend the cells and transferred to a flow tube, and stored in the dark for standby. Detection was performed on a flow cytometer.

[0133] (2) Experimental results:

[0134] As Figures 4 to 5 shown, the experimental results showed that the depolarized cell population of breast cancer MDA-MB-231 cells induced by the compound HJB-TPP-3 (1 μM, 2 μM, 4 μM) increased, showing a certain dose-dependence, increasing from 2.52% in the control group to 24.39%, 54.29% and 73.01% respectively. The maternal compound control HJB (4 μM) was 7.36%. After treatment with HJB-TPP-3 (4 μM), the proportion of JC-1 monomers in the cells was 9.92 times that of the maternal compound control HJB (4 μM). It was statistically significant compared with the control group (P < 0.05), proving that the mitochondrial membrane potential of breast cancer MDA-MB-231 cells after the action of the compound HJB-TPP-3 showed an obvious downward trend, and the effect was significantly better than its maternal compound HJB.

[0135] Application Example 5 Detection of Depolarization of Mitochondrial Membrane Potential of Human Breast Cancer Cells Induced by Compound HJB-TPP-3

[0136] (1) Experimental method

[0137] Take MDA-MB-231 cells in the logarithmic growth phase, at a density of 1×10 6 It should be noted that the value in "" is incomplete in the original text, so the translation here is also presented in an incomplete state. You can provide the complete original text for a more accurate translation.Inoculate cells at a density of 1×10 6 / well into the corresponding 6-well plates, and add 2 mL of L-15 culture medium containing 10% FBS to each well. When the cell confluence reaches 85%, discard the supernatant, and add the corresponding volumes of HJB-TPP-3 (1 μM, 2 μM, 4 μM) respectively. Set 3 replicate wells for each group, and the group without adding HJB-TPP-3 (i.e., 0 μM) is the blank control group (Control). Continue culturing for 24 h. Discard the culture medium and wash once with PBS. Add 1 mL of JC-1 working solution to each well, incubate at 37 °C for 20 min, and then discard the supernatant. Wash twice with JC-1 staining buffer, add 2 mL of serum-free L-15 culture medium, and observe the fluorescence color and intensity of cells in each group using a laser confocal microscope and take pictures.

[0138] (2) Experimental results

[0139] The results are as Figure 6 shown. As the concentration of HJB-TPP-3 increases, the amount of JC-1 monomers increases and the amount of JC-1 aggregates decreases, indicating that the mitochondrial membrane potential of MDA-MB-231 cells treated with HJB-TPP-3 decreases significantly.

[0140] Detection of the level of reactive oxygen species (ROS) generation in human breast cancer MDA-MB-231 cells induced by compound HJB-TPP-3 in Application Example 6

[0141] (1) Experimental method

[0142] Take MDA-MB-231 cells in the logarithmic growth phase, inoculate at a density of 1×10 6 / well into the corresponding 6-well plates, and add 2 mL of L-15 culture medium containing 10% FBS to each well. When the cell confluence reaches 85%, discard the supernatant, and add the corresponding volumes of HJB-TPP-3 (1 μM, 2 μM, 4 μM) respectively. Set 3 replicate wells for each group, and the group without adding HJB-TPP-3 (i.e., 0 μM) is the blank control group (Control). Continue culturing for 24 h. Add 2 mL of PBS to wash the cells, digest with 0.25% trypsin, collect the cells, centrifuge at 800 r / min for 5 min to obtain the precipitate. Add the DCFH-DA staining solution with a concentration of 10 μM to each centrifuge tube and mix well. Incubate in a 37 °C cell culture incubator for 20 minutes, invert and mix every 3 - 5 minutes to ensure full contact between the probe and the cells. After staining, centrifuge at 1000 r / min for 5 min, discard the supernatant and leave the precipitate, wash the cells 3 times with serum-free L-15 culture medium to remove the excess DCFH-DA. Transfer the suspension to a flow tube with a pipette and store in the dark for later use. Analyze the change in the ROS content of cells by flow cytometry.

[0143] (2) Experimental results

[0144] As Figure 7 ​​As shown, the experimental results indicate that compound HJB-TPP-3 can significantly induce ROS generation. Under the treatment of HJB-TPP-3 at the same drug concentration, the level of reactive oxygen species (ROS) is increased by 2.53 times compared with the parental control group.

[0145] Detection of the level of reactive oxygen species (ROS) generation induced by compound HJB-TPP-3 in human breast cancer cells - Application Example 7

[0146] (1) Experimental method

[0147] Take MDA-MB-231 cells in the logarithmic growth phase and inoculate them at 1×10 6 / well into the corresponding 6-well plates. Add 2 mL of L-15 culture medium containing 10% FBS to each well. When the cell confluence reaches 85%, discard the supernatant, and add the corresponding volume of HJB-TPP-3 (1 μM, 2 μM, 4 μM) respectively. Set 3 replicates for each group, and the group without adding HJB-TPP-3 (i.e., 0 μM) is the blank control group (Control). Continue to culture for 24 h. Add 1 mL of DCFH-DA staining solution with a concentration of 10 μM to each well and incubate in a 37 °C cell culture incubator for 20 min. Then wash the cells 3 times with serum-free L-15 medium to remove the excess DCFH-DA staining solution. Observe the fluorescence color and intensity of the cells in each group using a laser confocal microscope and take pictures.

[0148] (2) Experimental results

[0149] As Figure 8 shown, as the concentration of HJB-TPP-3 increases, the green fluorescence increases, indicating that HJB-TPP-3 can induce the generation of reactive oxygen species in human breast cancer MDA-MB-231 cells.

[0150] Detection of the anti-tumor effect of compound HJB-TPP-3 on the transplanted tumor model of Balb / c mice - Application Example 8

[0151] (1) Experimental method

[0152] Thirty healthy female Balb / c mice aged 4 - 6 weeks were randomly divided into a blank control group, a low-dose drug administration group of HJB-TPP-3 (10 mg / kg), a medium-dose drug administration group of HJB-TPP-3 (20 mg / kg), a high-dose drug administration group of HJB-TPP-3 (40 mg / kg), and a parental compound control group of HJB (40 mg / kg), with six mice in each group. The experimental period was 15 days, and the drugs were administered once every 2 days by intraperitoneal injection, for a total of 8 times. Record the body weight of the mice every day. After the experiment, collect the tumor tissues of the mice, weigh them, and take pictures for record. Make paraffin sections of the tumor tissues, perform HE staining, and evaluate the morphological effects of compound HJB-TPP-3 on the tumor tissues.

[0153] (2) Experimental results

[0154] As Figure 9 shown, within the experimental dosage range of the present invention, compound HJB-TPP-3 has no significant effect on the body weight of experimental mice compared with its parent compound HJB. After the experiment, tumor tissues of each mouse were collected and their volumes were measured. As Figures 10 to 12 shown, compared with the blank control group, the tumor volumes of all drug-administered groups decreased and the weights decreased; after HE staining, the morphological changes of the tumor tissues were observed. The results are as Figure 13 shown, the tumor cells in the drug-administered groups were plump, large, and closely arranged. As the drug concentration increased, the cell nuclei coagulated, the color deepened to purple-black, and a large area of cells died. In summary, compound HJB-TPP-3 can also significantly induce apoptosis of tumor cells in vivo; and the inhibitory effect of compound HJB-TPP-3 on tumor tissues is significantly better than that of its parent compound HJB.

[0155] In summary, the present invention uses 17-hydroxy-jatropholide B as a lead compound. Under the guidance of the principle of combination, triphenylphosphine with mitochondrial targeting function is combined with 17-hydroxy-jatropholide B, and a series of novel 17-hydroxy-jatropholide B-coupled triphenylphosphine derivatives with potential for clinical anti-tumor applications are designed and synthesized. The anti-tumor effect research shows that the synthesized target products HJB-TPP-(1-3) exhibit significant anti-tumor activities in cell experiments and have the potential for clinical applications.

[0156] The above is only a preferred specific embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any changes or substitutions that can be easily conceived by those skilled in the art within the technical scope disclosed by the present invention should be covered by the protection scope of the present invention. Therefore, the protection scope of the present invention should be subject to the protection scope of the claims.

Claims

1. A 17-hydroxy-euphorbia lactone B coupled with a triphenylphosphine derivative, characterized in that: It has the structure shown in formula I: Among them, n=1~3.

2. The 17-hydroxy-euphorbia lactone B coupled triphenylphosphine derivative according to claim 1, characterized in that: Selected from HJB-TPP-1, HJB-TPP-2 or HJB-TPP-3, the structural formula is as follows:

3. A method for preparing the 17-hydroxy-euphorbia lactone B coupled with a triphenylphosphine derivative according to any one of claims 1 to 2, characterized in that: With 17-hydroxy-euphorbia lactone B as a raw material, a triphenylphosphine cation is introduced into the C-17 position of the 17-hydroxy-euphorbia lactone B through an esterification reaction and a substitution reaction, so as to obtain the 17-hydroxy-euphorbia lactone B coupled with a triphenylphosphine derivative.

4. The method for preparing the 17-hydroxy-euphorbia lactone B coupled triphenylphosphine derivative according to claim 3, characterized in that: The following steps are involved: 17-Hydroxy-euphorbia lactone B and bromoalkyl carboxylic acid are dissolved in a first organic solvent, a catalyst and a condensing agent are added, and an esterification reaction is performed to obtain an intermediate A. The structural formula of the bromoalkyl carboxylic acid is Where n = 1 to 3; The intermediate A and triphenylphosphine are dissolved in a second organic solvent, heated under reflux for substitution reaction, and after the reaction is completed, the solvent is evaporated and the precipitate is washed to obtain the 17-hydroxy-euphorbia lactone B coupled with a triphenylphosphine derivative.

5. The method for preparing the 17-hydroxy-euphorbia lactone B coupled triphenylphosphine derivative according to claim 4, characterized in that: The catalyst is selected from one of 4-dimethylaminopyridine, triethylamine and N,N-diisopropylethylamine; and / or The condensing agent is selected from one of 1,3-dicyclohexylcarbodiimide, N,N'-diisopropylcarbodiimide and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; and / or The first organic solvent is selected from one of chloroform, N,N-dimethylformamide and dichloromethane; and / or The temperature of the esterification reaction is room temperature, and the time of the esterification reaction is 4 to 6 hours; and / or The second organic solvent is selected from one of dichloromethane, toluene, N,N-dimethylformamide, n-butanol and acetonitrile; and / or The temperature of the substitution reaction is 80-85° C., and the time of the substitution reaction is 24-48 hours.

6. The method for preparing 17-hydroxy-euphorbia lactone B coupled with triphenylphosphine derivative according to claim 4, characterized in that: The molar ratio of the 17-hydroxy-euphorbia lactone B, bromoalkyl carboxylic acid, catalyst and condensing agent is 1:(2-5):(0.6-1):(4-6).

7. The method for preparing 17-hydroxy-euphorbia lactone B coupled with triphenylphosphine derivative according to claim 4, characterized in that: The molar ratio of the intermediate A to triphenylphosphine is 1:(2-4).

8. A drug carrier targeting mitochondria, characterized in that: The invention comprises the 17-hydroxy-euphorbia lactone B coupled with a triphenylphosphine derivative as claimed in claim 1 or 2.

9. A mitochondrial-targeted anti-tumor drug, characterized in that: The components of the anti-tumor drug include the 17-hydroxy-euphorbia lactone B coupled triphenylphosphine derivative or its stereoisomer, prodrug and pharmaceutically acceptable salt as described in claim 1 or 2.

10. Use of the 17-hydroxy-euphorbia lactone B coupled with a triphenylphosphine derivative according to claim 1 or 2 in the preparation of anti-tumor drugs, characterized in that: The anti-tumor drugs include anti-liver cancer drugs, anti-breast cancer drugs, anti-lung cancer drugs or anti-gastric cancer drugs.

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