Guaiane type sesquiterpene dimer compound as well as preparation method and application thereof

Abstract

The invention provides a guaiane type sesquiterpenoid dimer compound, a preparation method thereof and application of the guaiane type sesquiterpenoid dimer compound in preparation of drugs for preventing or treating tumor-related diseases. The guaiane type sesquiterpenoid dimer compound 1-4 provided by the invention has an inhibiting effect on various cancer cells, can effectively inhibit the growth of tumor cells under the condition that the concentration is 40 mu M, and has a good practical application value.

Description

Technical Field

[0001] The present invention belongs to the field of anti-tumor drugs, and particularly relates to a guaiane-type sesquiterpene dimer compound, a preparation method thereof, and an application thereof. Background Art

[0002] The statements in this part only provide background technical information related to the present invention, and do not necessarily constitute prior art.

[0003] Hepatocellular carcinoma is a malignant tumor originating from hepatocytes and is one of the most serious complications of liver cancer. It has a high degree of malignancy, a poor prognosis, and is difficult to detect in the early stage; lung cancer is a malignant tumor originating from the bronchial mucosa or glands of the lungs, and its incidence and mortality rates are increasing rapidly; melanoma is a highly malignant tumor derived from melanocytes and is relatively common in skin cancer; cervical cancer, also known as uterine cervical cancer, is a malignant tumor occurring in the vaginal part of the uterus and the cervical canal and is one of the common tumors threatening women's health. Currently, common effective clinical chemotherapy drugs such as paclitaxel, afatinib, cisplatin, etc. still have disadvantages such as low response rates, severe side effects, and drug resistance. Therefore, searching for new effective anti-tumor drugs is one of the research focuses in the current medical field. Related research has found that sesquiterpenes and their dimers have different biological activities, such as anti-tumor, anti-inflammatory, anti-malaria, antibacterial, etc., and are a group of effective and low-toxic natural small molecules.

[0004] Artemisia stolonifera is a plant of the true Artemisia series in the genus Artemisia of the Compositae family. After systematic textual research on materia medica, it is considered that the medicinal material "Jiuniucao" recorded in the "Materia Medica Illustrated" in the Song Dynasty and the "Mengquan of Materia Medica" in the Ming Dynasty has the original plant of Artemisia stolonifera, which is one of the important original plants of the traditional Chinese medicine Artemisia argyi in ancient times. Compared with Artemisia argyi, its chemical composition and microscopic characteristics have certain differences, and its moxa floss has advantages such as "quick qi arrival, strong qi generation, high qi arrival rate, comfortable qi arrival feeling, and long post-qi arrival effect", and can be used as excellent moxa floss. However, there are few reports on the chemical composition research of Artemisia stolonifera at present, and it is unknown whether it can treat cancer. Summary of the Invention

[0005] The purpose of the present invention is to provide a guaiane-type sesquiterpene dimer compound, a preparation method thereof, and an application in the preparation of drugs for preventing or treating tumor-related diseases. The guaiane-type sesquiterpene dimer compounds 1-4 provided in the present invention have inhibitory effects on a variety of cancer cells, and can effectively inhibit the growth of tumor cells at a concentration of 40 μM, and have good practical application value.

[0006] In the first aspect of the present invention, there is provided a guaiane-type sesquiterpene dimer compound, and the structure of the compound is any one of the following 1-4:

[0007]

[0008] In a second aspect of the present invention, there is provided a method for preparing the guaiane-type sesquiterpene dimer compounds described in the first aspect, comprising:

[0009] After washing and drying Artemisia brachyloba, it is subjected to heating extraction with 60-80% ethanol, and the extract is concentrated to obtain a crude extract.

[0010] After dissolving the crude extract in water, it is successively extracted with petroleum ether and ethyl acetate, and the ethyl acetate extract is collected and concentrated to obtain a crude extract paste.

[0011] The crude extract paste is separated and purified by column chromatography and high performance liquid chromatography to obtain the compounds shown in any one of Formulas 1-4.

[0012] In a specific embodiment of the present invention, the temperature of the heating extraction is 55-65 °C, the time of the heating extraction is 8-12 h, and the number of times of the heating extraction is 2-4 times.

[0013] In a specific embodiment of the present invention, the column chromatography adopts one or two of normal phase silica gel column chromatography, reverse phase D101 macroporous resin column chromatography and gel column chromatography.

[0014] In a specific embodiment of the present invention, the elution system of the column chromatography includes methanol, dichloromethane-methanol, petroleum ether-ethyl acetate or methanol-water.

[0015] In a specific embodiment of the present invention, the specific steps of the column chromatography method are as follows: First, the ethyl acetate extraction part is adsorbed and mixed with 1-1.5 times its mass of silica gel with a mesh size of 80-100, and then dry loaded onto a normal phase silica gel column. Then, a dichloromethane-methanol system is used for gradient elution according to the gradients of 100:0, 100:1, 70:1, 50:1, 30:1, 10:1, 0:100. The gradient elution solutions of each gradient are collected and combined to obtain 6 components, Fr.1-6.

[0016] Fr.2 is adsorbed and mixed with 1-1.5 times its mass of D101 macroporous resin, and then dry loaded onto a reverse macroporous resin column. Then, gradient elution is carried out with 20%, 40%, 60%, 80%, 100% ethanol solutions. The gradient elution solutions of each gradient are collected, concentrated, and TLC spotted and combined to obtain 5 components, Fr.2-1 to Fr.2-5.

[0017] Fr.2-3 was loaded onto a gel column and eluted with a methanol or dichloromethane-methanol = 1:1 elution system. The eluates were collected and combined to obtain 4 fractions, Fr.2-3-1 to Fr.2-3-4. Fr.2-3-2 was dry-loaded onto a normal-phase silica gel column and then isocratically eluted with a dichloromethane-methanol system of 50:1. The eluates were collected and combined to obtain 3 sub-fractions. The second sub-fraction was purified by HPLC preparative chromatography to obtain Compounds 1 and 2;

[0018] Fr.2-4 was separated on a normal-phase silica gel column with gradient elution using petroleum ether-ethyl acetate systems of 6:1, 4:1, 3:1, 2:1, and 1:1 to obtain 4 fractions, Fr.2-4-1 to Fr.2-4-4. Fr.2-4-4 was further separated on a reverse column with gradient elution using methanol-water at 20%, 40%, 50%, 60%, 70%, and 80% to obtain 7 fractions. The second fraction was loaded onto a gel column and eluted with a methanol or dichloromethane-methanol = 1:1 elution system. The eluates were collected and combined to obtain 6 sub-fractions. The second sub-fraction was loaded onto a normal-phase silica gel column and then gradient eluted with petroleum ether-ethyl acetate systems of 6:1, 4:1, 3:1, 2:1, and 1:1. The gradient eluates of each gradient were collected and combined, and then purified by HPLC preparative chromatography to obtain Compounds 3 and 4;

[0019] In a specific embodiment of the present invention, high-performance liquid chromatography was performed using a reverse-phase C18 column, with an elution system of methanol-water or acetonitrile-water, a flow rate of 2 - 3 mL / min, a column temperature of 20 - 30 °C, a sample injection volume of 20 - 100 μL each time, and a UV detector with detection wavelengths of 210 nm and 254 nm;

[0020] In a third aspect of the present invention, there is provided the use of the guaiane-type sesquiterpene dimer compounds or pharmaceutically acceptable salts thereof described in the first aspect in the preparation of drugs for preventing and / or treating tumor-related diseases;

[0021] Preferably, the pharmaceutically acceptable salts are hydrochloride, phosphate, sulfate, acetate, maleate, citrate, benzenesulfonate, methylbenzenesulfonate, fumarate, or tartrate;

[0022] Preferably, the tumors include: liver cancer, lung cancer, melanoma, and cervical cancer.

[0023] In a fourth aspect of the present invention, there is provided an anti-tumor product, which includes the guaiane-type sesquiterpene dimer compounds or pharmaceutically acceptable salts thereof described in the first aspect;

[0024] The product further includes pharmaceutically acceptable excipients and / or carriers;

[0025] The product includes pharmaceutical preparations and experimental preparations.

[0026] In a fifth aspect of the present invention, there is provided the use of the guaiane-type sesquiterpene dimer compound described in the first aspect or a pharmaceutically acceptable salt thereof in the preparation of a product for inhibiting tumor cells;

[0027] The tumor cells include human melanoma A2058 cells, mouse melanoma B16 cells, human non-small cell lung cancer A549, human non-small cell lung cancer NCI-H1975 cells, human cervical cancer C-33A cells, human cervical squamous cell carcinoma SiHa cells, mouse liver cancer H22 cells, human liver cancer HuH-7 cells or human liver cancer Hep-G2 cells.

[0028] The above one or more technical solutions have the following beneficial effects:

[0029] (1) The present invention provides 4 new guaiane-type sesquiterpene dimer compounds, Artanomaloide, Arteminolide, Artanomaloide C, and Arteminolide C, whose structures are shown in Figures 1-4 respectively, filling the gaps in the prior art and further expanding the new applications of Artemisia stolonifera.

[0030] (2) The preparation method of the compound provided by the present invention is simple in operation and good in stability; the raw materials are easily available, easy to operate, with high yield, and suitable for industrial production.

[0031] (3) The guaiane-type sesquiterpene dimer compound provided by the present invention can effectively inhibit the growth of liver cancer, lung cancer, melanoma, and cervical cancer cells at a concentration of 40 μM, and can be used as a drug for preparing anti-tumor drugs.

[0032] The advantages of the additional aspects of the present invention will be partly given in the following description, partly will become obvious from the following description, or be understood through the practice of the present invention. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] The specification drawings forming 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 to the present invention.

[0034] Figure 1 For the 1 1H-NMR spectrum of Compound 1 in Example 2.

[0035] Figure 2 For the 13 13C-NMR spectrum of Compound 1 in Example 2.

[0036] Figure 3 For the 1 1H-NMR spectrum of Compound 2 in Example 2.

[0037] Figure 4 For the 13 C-NMR spectrum of Compound 2 in Example 2.

[0038] Figure 5 For the 1 H-NMR spectrum of Compound 3 in Example 2.

[0039] Figure 6 For the 13 C-NMR spectrum of Compound 3 in Example 2.

[0040] Figure 7 For the 13 C-NMR spectrum of Compound 4 in Example 2.

[0041] Figure 8 For the 13 C-NMR spectrum of Compound 4 in Example 2. Detailed implementation manners

[0042] It should be noted that the following detailed descriptions are all exemplary and are intended to provide further explanations of the present invention. Unless otherwise specified, all technical and scientific terms used herein have the same meaning as commonly understood by those of ordinary skill in the technical field to which the present invention belongs.

[0043] It should be noted that the terms used herein are only for describing specific implementation manners and are not intended to limit the exemplary embodiments according to the present invention.

[0044] Example 1

[0045] Preparation of the compound

[0046] (1) After washing and drying Artemisia stolonifera, it was extracted by heating with 70% ethanol, and the extract was concentrated to obtain a crude extract;

[0047] (2) After the crude extract was fully dissolved in water, it was successively extracted 6 times with petroleum ether and ethyl acetate according to a volume ratio of 1:1 of the crude extract aqueous solution to the extractant, and the ethyl acetate extract was combined and concentrated to obtain a crude extract (that is: the crude extract aqueous solution was first extracted multiple times with petroleum ether to remove small polar components; then the crude extract aqueous solution was extracted multiple times with ethyl acetate, and the ethyl acetate extract was collected);

[0048] (3) At room temperature, first, the ethyl acetate crude extract was adsorbed and mixed with 1.5 times its mass of silica gel of 80-100 mesh, and then dry-loaded onto a normal-phase silica gel column. Then, a dichloromethane-methanol system was used for gradient elution according to the gradients of 100:0, 100:1, 70:1, 50:1, 30:1, 10:1, 0:100. The gradient eluates of each gradient were collected and combined to obtain 6 fractions Fr.1-6;

[0049] Fr.2 was adsorbed with 1.5 times its mass of D101 macroporous resin and then loaded onto a reverse D101 macroporous resin column by dry loading. Then, gradient elution was carried out with 20%, 40%, 60%, 80%, and 100% ethanol solutions, and the gradient eluates of each gradient were collected, concentrated, and combined by TLC spotting to obtain 5 components, Fr.4-1 to Fr.4-5;

[0050] Fr.2-3 was loaded onto a gel column and eluted with a methanol or dichloromethane-methanol = 1:1 elution system. The eluates were collected and combined to obtain 4 components, Fr.2-3-1 to Fr.2-3-4. Fr.2-3-2 was loaded onto a normal-phase silica gel column by dry loading, and then isocratic elution was carried out with a dichloromethane-methanol system of 50:1. The eluates were collected and combined to obtain 3 sub-components. The second sub-component was subjected to HPLC preparation (55% acetonitrile, flow rate 3 mL / min) to obtain Compounds 1 and 2.

[0051] Fr.2-4 was subjected to gradient elution with a normal-phase silica gel column using a petroleum ether-ethyl acetate system of 6:1, 4:1, 3:1, 2:1, and 1:1 to obtain 4 components, Fr.2-4-1 to Fr.2-4-4. Fr.2-4-4 was subjected to gradient elution with a reverse column using 20%, 40%, 50%, 60%, 70%, and 80% methanol-water to obtain 7 components. The second component was loaded onto a gel column and eluted with a methanol or dichloromethane-methanol = 1:1 elution system. The eluates were collected and combined to obtain 6 sub-components. The second sub-component was loaded onto a normal-phase silica gel column, and then gradient elution was carried out with a petroleum ether-ethyl acetate system of 6:1, 4:1, 3:1, 2:1, and 1:1. The gradient eluates of each gradient were collected and combined, and then HPLC preparation (60% acetonitrile, flow rate 3 mL / min) was carried out to obtain Compounds 3 and 4.

[0052] Example 2

[0053] Compound Structure Identification

[0054] After all compounds were identified by NMR, the properties and spectral data of the obtained compounds were as follows:

[0055] Compound 1: Artanomaloide, white powder, molecular formula C 32 H 36 O8. 1 H NMR(500MHz,CDCl3):δ H6.19 (s, H-3), 3.48 (d, J = 10.1 Hz, H-5), 3.71 (t, J = 10.2 Hz, H-6), 2.87 (t, J = 10.2 Hz, H-7), 5.03 (td, J = 10.4, 2.5 Hz, H-8), 2.77 (dd, J = 13.2, 10.8 Hz, H-9a), 2.35 (m, H-9b), 2.32 (overlapped, H-13a), 1.53 (overlapped, H-13b), 2.41 (s, H-14), 2.31 (s, H-15), 5.80 (d, J = 5.5 Hz, H-2′), 6.39 (d, J = 5.5 Hz, H-3′), 1.93 (d, J = 9.9 Hz, H-5′), 4.18 (t, J = 9.7 Hz, H-6′), 3.08 (m, H-7′), 2.21 (m, H-8′a), 1.47 (m, H-8′b), 1.82 (m, H-9′), 6.11 (d, J = 3.5 Hz, H-13′a), 5.38 (d, J = 3.5 Hz, H-13′b), 1.29 (s, H-14′), 1.56 (s, H-15′), 2.03 (s, H-2″); 13 13C NMR (125 MHz, CDCl3): δ C 134.4 (C-1), 195.0 (C-2), 136.3 (C-3), 171.0 (C-4), 50.4 (C-5), 79.7 (C-6), 56.8 (C-7), 66.5 (C-8), 43.7 (C-9), 143.6 (C-10), 60.1 (C-11), 176.4 (C-12), 36.8 (C-13), 20.2 (C-14), 20.4 (C-15), 63.3 (C-1′), 132.0 (C-2′), 143.0 (C-3′), 57.4 (C-4′), 66.3 (C-5′), 79.3 (C-6′), 43.5 (C-7′), 23.8 (C-8′), 35.0 (C-9′), 72.8 (C-10′), 140.8 (C-11′), 170.6 (C-12′), 119.6 (C-13′), 23.0 (C-14′), 14.6 (C-15′), 170.3 (C-1″), 21.9 (C-2″). The above data were compared with the literature, so compound 1 was identified as Artanomaloide.

[0056] Compound 2: Arteminolide, a white powder, with the molecular formula C 32 H 36 O8. 1 1H NMR (500 MHz, CDCl3): δH 6.21 (s, H-3), 3.27 (d, J = 9.7 Hz, H-5), 3.99 (overlapped, H-6), 2.72 (t, J = 11.1 Hz, H-7), 4.79 (td, J = 10.7, 2.5 Hz, H-8), 2.50 (dd, J = 13.3, 10.5 Hz, H-9a), 2.26 (overlapped, H-9b), 2.62 (d, J = 11.6 Hz, H-13a), 1.91 (d, J = 11.6 Hz, H-13b), 2.34 (s, H-14), 2.41 (s, H-15), 5.87 (s, H-2′, 3′), 3.10 (d, J = 9.7 Hz, H-5′), 4.02 (overlapped, H-6′), 3.32 (m, H-7′), 2.26 (overlapped, H-8′a), 1.45 (m, H-8′b), 1.82 (m, H-9a′), 1.78 (m, H-9′b), 6.08 (d, J = 3.6 Hz, H-13′a), 5.35 (d, J = 3.4 Hz, H-13′b), 1.50 (s, H-14′), 1.32 (s, H-15′), 2.01 (s, H-2″); 13 13C NMR (125 MHz, CDCl3): δ C 134.3 (C-1), 195.0 (C-2), 136.4 (C-3), 170.3 (C-4), 52.1 (C-5), 80.2 (C-6), 59.5 (C-7), 68.5 (C-8), 44.7 (C-9), 143.9 (C-10), 178.6 (C-11), 61.5 (C-12), 40.4 (C-13), 20.5 (C-14), 20.6 (C-15), 63.1 (C-1′), 137.6 (C-2′), 136.7 (C-3′), 58.1 (C-4′), 67.1 (C-5′), 79.2 (C-6′), 43.3 (C-7′), 23.7 (C-8′), 34.8 (C-9′), 72.7 (C-10′), 140.7 (C-11′), 170.5 (C-12′), 119.0 (C-13′), 17.2 (C-14′), 30.0 (C-15′), 169.1 (C-1″), 21.8 (C-2″). Based on comparison with the literature data, compound 2 was identified as Arteminolide.

[0057] Compound 3: Artanomaloide C, a white powder, with the molecular formula C 35 H 40 O8. 11H NMR (500 MHz, CDCl3): δ H 6.20 (s, H-3), 3.51 (d, J = 9.9 Hz, H-5), 3.73 (t, J = 9.8 Hz, H-6), 2.97 (t, J = 10.2 Hz, H-7), 5.16 (td, J = 10.5, 2.1 Hz, H-8), 2.83 (dd, J = 10.5, 13.2 Hz, H-9a), 2.33 (d, J = 11.5 Hz, H-9b), 2.37 (overlapped, H-13a), 1.52 (d, J = 11.9 Hz, H-13b), 2.32 (s, H-14), 2.43 (s, H-15), 5.80 (d, J = 5.5 Hz, H-2′), 6.40 (d, J = 5.5 Hz, H-3′), 2.03 (d, J = 9.9 Hz, H-5′), 4.11 (t, J = 9.9 Hz, H-6′), 2.89 (m, H-7′), 2.18 (m, H-8′a), 1.43 (m, H-8′b), 1.80 (m, H-9′), 6.05 (d, J = 3.5 Hz, H-13′a), 5.32 (d, J = 3.5 Hz, H-13′b), 1.56 (s, H-14′), 1.28 (s, H-15′), 6.05 (m, H-3″), 1.91 (overlapped, H-4″), 1.89 (s, H-5″); 13 13C NMR (125 MHz, CDCl3): δ C 134.2 (C-1), 195.0 (C-2), 136.4 (C-3), 171.0 (C-4), 50.6 (C-5), 79.5 (C-6), 56.5 (C-7), 65.9 (C-8), 44.5 (C-9), 144.1 (C-10), 60.3 (C-11), 176.6 (C-12), 36.3 (C-13), 20.5 (C-14), 20.1 (C-15), 63.7 (C-1′), 132.1 (C-2′), 142.7 (C-3′), 57.5 (C-4′), 66.4 (C-5′), 79.4 (C-6′), 43.2 (C-7′), 23.9 (C-8′), 34.9 (C-9′), 72.5 (C-10′), 141.4 (C-11′), 170.1 (C-12′), 118.6 (C-13′), 14.9 (C-14′), 30.0 (C-15′), 166.5 (C-1″), 127.0 (C-2″), 142.7 (C-3″), 16.1 (C-4″), 20.3 (C-5″). The above data were compared with the literature, so compound 3 was identified as Artanomaloide C.

[0058] Compound 4: Arteminolide C, a white powder, with the molecular formula C 35 H 40 O8. 1 1H NMR (500 MHz, CDCl3): δ H 6.22 (s, H-3), 3.31 (d, J = 9.9 Hz, H-5), 4.05 (dd, J = 10.3, 2.7 Hz, H-6), 2.80 (t, J = 10.9 Hz, H-7), 4.89 (td, J = 10.5, 2.2 Hz, H-8), 2.56 (dd, J = 10.5, 5.2 Hz, H-9a), 2.37 (dd, J = 10.0, 2.4 Hz, H-9b), 2.60 (d, J = 11.5 Hz, H-13a), 1.87 (d, J = 11.5 Hz, H-13b), 2.34 (s, H-14), 2.43 (s, H-15), 5.67 (d, J = 5.6 Hz, H-2′), 5.85 (d, J = 5.6 Hz, H-3′), 3.08 (d, J = 9.9 Hz, H-5′), 3.99 (t, J = 9.9, H-6′), 3.30 (m, H-7′), 2.23 (m, H-8′a), 1.42 (m, H-8′b), 1.82 (m, H-9′a), 1.74 (m, H-9′b), 6.07 (d, J = 3.5 Hz, H-13′a), 5.34 (d, J = 3.5 Hz, H-13′b), 1.50 (s, H-14′), 1.24 (s, H-15′), 6.17 (dd, J = 7.3, 1.5 Hz, H-2″), 2.05 (d, J = 7.3, 1.5 Hz, H-4″a), 1.85 (s, H-4″b); 13 13C NMR (125 MHz, CDCl3): δ C134.3 (C-1), 195.1 (C-2), 136.2 (C-3), 170.3 (C-4), 52.1 (C-5), 80.4 (C-6), 59.7 (C-7), 66.8 (C-8), 44.9 (C-9), 144.0 (C-10), 61.4 (C-11), 178.7 (C-12), 40.7 (C-13), 20.5 (C-14), 20.7 (C-15), 63.1 (C-1′), 137.8 (C-2′), 136.2 (C-3′), 58.2 (C-4′), 66.8 (C-5′), 79.3 (C-6′), 43.3 (C-7′), 23.7 (C-8′), 34.8 (C-9′), 72.76 (C-10′), 140.8 (C-11′), 170.3 (C-12′), 118.9 (C-13′), 17.1 (C-14′), 29.9 (C-15′), 166.0 (C-1″), 127.6 (C-2″), 140.2 (C-3″), 16.1 (C-4″), 20.4 (C-5″). The above data was compared with the literature, so Compound 4 was identified as Arteminolide C.

[0059] Example 3

[0060] Antitumor Activity Experiment

[0061] Experimental method: Compounds 1-4 obtained in Example 2 and the positive drug (paclitaxel or dacarbazine) were prepared into 50 mM stock solutions with DMSO and stored at -20°C. The blank group contained no cells and the test sample, the control wells contained cells but no test sample, and the test wells contained cells and the test sample. The CCK8 method was used to detect the inhibition rate of the samples on tumor cells, and 3 replicate wells were set for each sample concentration.

[0062] Specific operation steps: Human melanoma A2058 cells, mouse melanoma B16 cells (purchased from Wuhan Ponuosai Co., Ltd.), mouse liver cancer H22 cells (gifted by the Traditional Chinese Medicine Resource Center of Hubei University of Chinese Medicine), human non-small cell lung cancer A549 and NCI-H1975 cells (purchased from the Cell Resource Center of Shanghai Institutes for Biological Sciences, Chinese Academy of Sciences) were cultured in RPMI 1640 complete medium (10% FBS, 1% penicillin, 1% streptomycin); human cervical cancer C-33A cells (purchased from Haixing Biotechnology Co., Ltd.), human liver cancer HuH-7 cells and human liver cancer Hep-G2 cells (gifted by the Traditional Chinese Medicine Resource Center of Hubei University of Chinese Medicine) were cultured in DMEM complete medium (10% FBS, 1% penicillin, 1% streptomycin); human cervical squamous cell carcinoma SiHa (purchased from Haixing Biotechnology Co., Ltd.) was cultured in MEM complete medium (10% FBS, 1% penicillin, 1% streptomycin).

[0063] The cells were cultured in an incubator at 37 °C with 5% CO2. The A2058 cells cultured to the logarithmic growth phase were evenly plated into a 96-well plate with 100 μL of cell suspension at a density of 3×10 3 . After culturing for 24 h until the cells adhered and grew, the test samples were respectively prepared into 200 μL of drug-containing medium at 40 μM, and the positive drug was prepared into 200 μL of drug-containing medium at 20 μM, and the medium was changed and the drugs were added. After 24 h of drug administration, the supernatant was discarded, and 100 μL of medium containing 10% CCK8 was added to each well. After culturing in the incubator for 1.5 - 2.5 h, the absorbance at 450 nm was measured with an enzyme-linked immunosorbent assay (ELISA) reader.

[0064] The B16 cells, H22 cells, A549, NCI-H1975 cells, C-33A cells, HuH-7 cells, Hep-G2 cells, and SiHa cells were treated according to the above method. The positive drug used for A2058 and B16 cells was dacarbazine, and the positive drug used for A549, NCI-H1975, H22, HuH-7, Hep-G2, C-33A, and SiHa cells was paclitaxel. The results obtained are shown in Table 1.

[0065] Table 1 shows the inhibitory results of compounds 1 - 4 on 9 tumor cell lines at 40 μM.

[0066] Table 1

[0067]

[0068] As can be seen from the data in the table, the inhibitions of the said Compound 1 on melanoma cells (B16, A2058) are 92.3% and 95.2% respectively, the inhibitions on cervical cancer cells (C-33A, SiHa) are 70.8 and 94.2% respectively, the inhibitions on liver cancer cells (Hep-G2, HuH-7, H22) are 100.6%, 94.9% and 82.7% respectively, and the inhibitions on lung cancer cells (A549, NCI-H1975) are 83.2% and 79.6% respectively; the inhibitions of Compound 2 on melanoma cells (B16, A2058) are 88.1% and 89.5% respectively, the inhibitions on cervical cancer cells (C-33A, SiHa) are 71.5 and 86.6% respectively, the inhibitions on liver cancer cells (Hep-G2, HuH-7, H22) are 81.6%, 74.5% and 65.7% respectively, and the inhibitions on lung cancer cells (A549, NCI-H1975) are 39.6% and 50.5% respectively; the inhibitions of Compound 3 on melanoma cells (B16, A2058) are 95.4% and 96.6% respectively, the inhibitions on cervical cancer cells (C-33A, SiHa) are 65.7 and 47.7% respectively, the inhibitions on liver cancer cells (Hep-G2, HuH-7, H22) are 76.5%, 62.3% and 98.4% respectively, and the inhibitions on lung cancer cells (A549, NCI-H1975) are 62.4% and 40.9% respectively; the inhibitions of Compound 4 on melanoma cells (B16, A2058) are 93.8% and 96.7% respectively, the inhibitions on cervical cancer cells (C-33A, SiHa) are 66.8 and 50.3% respectively, the inhibitions on liver cancer cells (Hep-G2, HuH-7, H22) are 102.3%, 97.8% and 96.9% respectively, and the inhibitions on lung cancer cells (A549, NCI-H1975) are 93.7% and 67.8% respectively.

[0069] It can be seen therefrom that Compounds 1-4 can effectively inhibit the growth of tumor cells at a concentration of 40 μM.

[0070] Although the specific embodiments of the present invention have been described above in conjunction with the accompanying drawings, it is not a limitation to the protection scope of the present invention. Those skilled in the art should understand that, based on the technical solutions of the present invention, various modifications or deformations that can be made by those skilled in the art without creative efforts are still within the protection scope of the present invention.

Claims

1. A guaiane-type sesquiterpene dimer compound, characterized in that, The structure of the said compound is any one of the following 1-4:

2. A method for preparing the guaiane-type sesquiterpene dimer compound according to claim 1, characterized in that, Including: After washing and drying Artemisia brachyloba, heat and extract it with 60-80% ethanol, concentrate the extractive solution to obtain a crude extract. After dissolving the said crude extract in water, extract it successively with petroleum ether and ethyl acetate, collect the ethyl acetate extractive solution and concentrate it to obtain a crude extract paste. Separate and purify the said crude extract paste by column chromatography and high performance liquid chromatography to obtain the compound shown in any of Formula 1-4.

3. The preparation method of the guaiane-type sesquiterpene dimer compound according to claim 2, wherein, The temperature of the said heat extraction is 55-65 °C, the time of heat extraction is 8-12 h, and the number of times of the said heat extraction is 2-4 times.

4. The preparation method of the guaiane-type sesquiterpene dimer compound according to claim 2, characterized in that, The said column chromatography adopts one or two of normal phase silica gel column chromatography, reverse phase D101 macroporous resin column chromatography and gel column chromatography.

5. The preparation method of the guaiane-type sesquiterpene dimer compound according to claim 2, characterized in that, The elution system of the said column chromatography includes methanol, dichloromethane-methanol, petroleum ether-ethyl acetate or methanol-water.

6. The preparation method of the guaiane-type sesquiterpene dimer compound according to claim 2, wherein The specific steps of the said column chromatography method are as follows: First, adsorb and mix the ethyl acetate extraction part with silica gel with a mass 1-1.5 times that of it and sample it dry onto a normal phase silica gel column, then elute it with a dichloromethane-methanol system according to the gradient of 100:0, 100:1, 70:1, 50:1, 30:1, 10:1, 0:100, collect the gradient eluate of each gradient and combine them to obtain 6 components Fr.1-6; Fr.2 is adsorbed and mixed with D101 macroporous resin with a mass 1-1.5 times that of it and sampled dry onto a reverse macroporous resin column, then elute it with 20%, 40%, 60%, 80%, 100% ethanol solutions for gradient elution, collect the gradient eluate of each gradient, concentrate it and combine it by TLC plate to obtain 5 components Fr.2-1 to Fr.2-5; Fr.2-3 is loaded onto a gel column and eluted with a methanol or dichloromethane-methanol = 1:1 elution system, collect the eluate and combine them to obtain 4 components Fr.2-3-1 to Fr.2-3-4. Fr.2-3-2 is sampled dry onto a normal phase silica gel column, then elute it isocratically with a dichloromethane-methanol system of 50:1, collect the eluate and combine them to obtain 3 sub-components. The second sub-component is purified by HPLC preparative chromatography to obtain compounds 1 and 2. Fr.2-4 adopts a normal phase silica gel column and is eluted with a petroleum ether-ethyl acetate system of 6:1, 4:1, 3:1, 2:1, 1:1 for gradient elution to obtain 4 components Fr.2-4-1 to Fr.2-4-4. Fr.2-4-4 adopts a reverse column and is eluted with 20%, 40%, 50%, 60%, 70%, 80% methanol-water for gradient elution to obtain 7 components. The second component is loaded onto a gel column and eluted with a methanol or dichloromethane-methanol = 1:1 elution system, collect the eluate and combine them to obtain 6 sub-components. The second sub-component is loaded onto a normal phase silica gel column, then eluted with a petroleum ether-ethyl acetate system of 6:1, 4:1, 3:1, 2:1, 1:1 for gradient elution, collect the gradient eluate of each gradient and combine them, and then carry out HPLC preparative chromatography purification to obtain compounds 3 and 4.

7. The preparation method of the guaiane-type sesquiterpene dimer compound according to claim 2, characterized in that, High performance liquid chromatography was performed using a reverse phase C18 column. The elution system was methanol-water or acetonitrile-water, with a flow rate of 2 - 3 mL / min, column temperature: 20 - 30 °C, injection volume each time: 20 - 100 μL. An ultraviolet detector was used, and the detection wavelengths were 210 nm and 254 nm.

8. Use of the guaiane-type sesquiterpene dimer compound or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a drug for preventing and / or treating tumor-related diseases; Preferably, the pharmaceutically acceptable salt is hydrochloride, phosphate, sulfate, acetate, maleate, citrate, benzenesulfonate, methylbenzenesulfonate, fumarate or tartrate; Preferably, the tumor includes: Liver cancer, lung cancer, melanoma, cervical cancer.

9. An anti-tumor product, characterized in that, The product comprises the guaiane-type sesquiterpene dimer compound or a pharmaceutically acceptable salt thereof according to claim 1; The product further comprises a pharmaceutically acceptable excipient and / or carrier; The product includes pharmaceutical preparations and experimental preparations.

10. Use of the guaiane-type sesquiterpene dimer compound or a pharmaceutically acceptable salt thereof according to claim 1 in the preparation of a product for inhibiting tumor cells; The tumor cells include human melanoma A2058 cells, mouse melanoma B16 cells, human non-small cell lung cancer A549, human non-small cell lung cancer NCI-H1975 cells, human cervical cancer C-33A cells, human cervical squamous cell carcinoma SiHa cells, mouse liver cancer H22 cells, human liver cancer HuH-7 cells or human liver cancer Hep-G2 cells.

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