Artemisia lactone dimer AG, its pharmaceutical composition, preparation method and application

Abstract

This invention provides seven guaiacol-type sesquiterpene dimers of structural formula (I), artemisinin dimers A-G (compounds 1-7), their preparation methods, pharmaceutical compositions thereof, and their applications, belonging to the field of pharmaceutical technology. These compounds exhibit inhibitory activity against human liver cancer cell lines HepG2, Huh7, and SK-Hep-1, and can be combined with pharmaceutically acceptable carriers to form pharmaceutical compositions, enabling the preparation of anti-liver cancer drugs.

Description

Technical fields:

[0001] This invention belongs to the field of pharmaceutical technology. Specifically, it relates to the compound artemisinin dimer AG(1-7), its preparation method and application, pharmaceutical compositions containing it and their applications. Background technology:

[0002] Hepatic carcinoma is the sixth most common cancer and the fourth leading cause of cancer-related death worldwide. It is characterized by high malignancy, rapid progression, and poor prognosis, with a mortality rate almost equal to its incidence rate. Currently, eight drugs are used clinically to treat hepatic carcinoma, including one VEGFR-2 inhibitor (ramucirumab), two PD-1 inhibitors (nivolumab and pembrolizumab), and five small molecule tyrosine kinase inhibitors (sorafenib, regorafenib, lenvatinib, cabozantinib, and donafenib). However, due to their similar structures and targets, drug resistance frequently occurs. Therefore, the search for new anti-hepatic carcinoma drugs with different structures and mechanisms is urgently needed.

[0003] Artemisia myriantha Wall. ex Bessser, also known as Artemisia argyi (Sichuan), Artemisia argyi, and Artemisia argyi (Yunnan), is mainly distributed in Shanxi (Zhongtiao Mountains), southern Gansu, Qinghai, Sichuan, Guizhou, Yunnan, and Guangxi in China. Except for Guangxi and southern Yunnan, where it is distributed at low altitudes up to 1000 meters, it is found at altitudes of 1000-2800 meters in other provinces. It commonly grows on hillsides, roadsides, and in thickets. It is also found in northern India, Bhutan, Nepal, Kashmir, northern Myanmar, and northern Thailand. In traditional Chinese medicine (Yunnan), Artemisia myriantha is used for its anti-inflammatory properties and as a catalyst for broad bean germination. It is bitter and cold in nature, possessing properties of clearing heat, relieving summer heat, cooling the blood, and stopping bleeding. It is mainly used for summer colds, heatstroke with fever, bone steaming, tidal fever, hematemesis, and epistaxis.

[0004] To date, there are no reports in existing literature regarding artemisia lactone dimer AG(1-7), no reports of pharmaceutical compositions containing it as an active ingredient, and no reports of its application in the preparation or treatment of liver cancer drugs. Summary of the Invention:

[0005] The purpose of this invention is to provide a new type of artemisinin dimer AG(1-7) of formula (I) with medicinal value, its preparation method and application, pharmaceutical compositions containing artemisinin dimer AG(1-7) and their applications. This type of compound has significant inhibitory activity against liver cancer cell lines and can be used to prepare anti-liver cancer drugs.

[0006] To achieve the above-mentioned objectives of the present invention, the present invention provides the following technical solution:

[0007] This invention provides a series of sesquiterpene dimer compounds, artemisia lactone dimer AG(1-7), having the structure shown in formula (I):

[0008]

[0009] This invention provides a method for preparing compounds 1-7. The leaves of *Artemisia annua* are dried, pulverized, and extracted three times with 90% ethanol at room temperature for three days each time. The ethanol extracts are combined, and the ethanol is recovered under reduced pressure to obtain 9.1 kg of extract. This extract is then subjected to silica gel column chromatography with a gradient elution of acetone-petroleum ether (v / v) (0:100, 10:90, 20:80, 30:70, 40:60, 50:50). After TLC development, the fractions are combined to obtain seven fractions Fr.A-Fr.G. Fraction Fr.F is subjected to MCI CHP. 20P column chromatography (945g, 7×50cm) was performed with water-methanol gradient elution (70:30, 50:50, 30:70, 10:90) to give five fractions Fr.F-1-Fr.F-5. Fraction Fr.F-4 was subjected to silica gel column chromatography with acetic acid-acetone-petroleum ether gradient elution (5:95, 10:90, 20:80, and 30:70) to give four fractions Fr.F-4a-Fr.F-4d. Fraction Fr.F-4b was subjected to silica gel column chromatography with ethyl acetate-petroleum ether (5:95) isocratic elution to give six fractions Fr.F-4b-1-Fr.F-4b-6. Fraction Fr.F-4b-3 was subjected to silica gel column chromatography and eluted isocratically with methanol-chloroform (5:95) to yield six fractions Fr.F-4b-3a-Fr.F-4b-3f. Fraction Fr.F-4b-3c was subjected to semi-preparative HPLC and eluted with water-acetonitrile (55:45) to yield compounds 1-3. Fraction Fr.F-4b-3d was subjected to semi-preparative HPLC and eluted with water-acetonitrile (52:48) to yield compounds 4 and 5. Fraction Fr.F-4b-3e was subjected to semi-preparative HPLC and eluted with water-acetonitrile (64:36) to yield compounds 6 and 7.

[0010] This invention provides the use of compounds 1-7 in the preparation of anti-liver cancer drugs. This invention does not specifically limit the method of application; any method well-known in the art may be used.

[0011] The present invention also provides a pharmaceutical composition comprising at least one of compounds 1-7 of formula (I) above and a pharmaceutically acceptable carrier. The present invention further provides a method for preparing the pharmaceutical composition, wherein compounds 1-7 are obtained by the above-described method for preparing compounds, and at least one of compounds 1-7 is added to a pharmaceutically acceptable carrier.

[0012] Furthermore, the use of the pharmaceutical composition described herein in the preparation of an anti-hepatocellular carcinoma drug is also provided.

[0013] When at least one of the compounds 1-7 is used to prepare an anti-liver cancer drug, the present invention preferably uses the compounds 1-7 directly or in the form of a pharmaceutical composition.

[0014] The pharmaceutical composition provided by this invention includes at least one pharmaceutically acceptable carrier of compounds 1-7 described above. In this invention, the pharmaceutically acceptable carrier is preferably a solid, semi-solid, or liquid diluent, filler, or pharmaceutical excipient. This invention does not impose any particular limitation on the pharmaceutically acceptable carrier; any pharmaceutically acceptable carrier well-known in the art that is non-toxic and inert to humans and animals may be selected.

[0015] The present invention does not impose any particular limitation on the preparation method of the pharmaceutical composition. At least one of compounds 1-7 can be directly mixed with a pharmaceutically acceptable carrier. The present invention does not impose any particular limitation on the mixing process. Any process well known in the art that can obtain the pharmaceutical composition can be selected.

[0016] This invention provides the application of the pharmaceutical composition described above in the preparation of anti-liver cancer drugs. This invention does not specifically limit the method of application; any method well-known in the art can be used.

[0017] In this invention, when the pharmaceutical composition is used to prepare an anti-liver cancer drug, the content of the composition in the drug is preferably 0.1% to 99%; in the pharmaceutical composition, the content of at least one of compounds 1-7 is preferably 0.5% to 90%. The pharmaceutical composition of this invention is preferably used in the form of a dose per unit body weight. In this invention, the prepared drug is preferably administered by both injection (intravenous injection, intramuscular injection) and oral administration.

[0018] Compared with the prior art, the present invention has the following advantages:

[0019] 1. This invention provides a series of novel sesquiterpene dimer compounds, artemisinin dimers AG(1-7). This invention discovered that the ethanol extract of the dried aerial parts of Artemisia annua exhibits certain inhibitory activity against three hepatocellular carcinoma cell lines (HepG2, Huh7, and SK-Hep-1), and further isolated six sesquiterpene dimer compounds, artemisinin dimers AG(1-7), which also exhibit inhibitory activity against these three hepatocellular carcinoma cell lines.

[0020] 2. This invention provides a new method for preparing new compounds 1-7, which uses readily available raw materials and is easy to operate.

[0021] 3. This invention provides a pharmaceutical composition with novel compounds 1-7 as active ingredients, providing a new drug with good medicinal effects for new anti-liver cancer drugs.

[0022] 4. Compounds 1-7 of the present invention exhibit inhibitory activity against three liver cancer cell lines (HepG2, SK-HEP-1, and Huh7); for HepG2 cells, compounds 1 and 4-7 show strong inhibitory activity, with an IC50 value of [missing information]. 50 The effective values ​​ranged from 7.4 to 12.6 μM; compounds 2 and 3 also exhibited good inhibitory activity, with IC50 values ​​of 7.4–12.6 μM. 50 The values ​​were 17.3 and 16.0 μM, respectively. For Huh7 cells, compound 4-7 exhibited inhibitory activity comparable to the positive control sorafenib, with an IC50 value of 17.3 and 16.0 μM. 50 The effective values ​​ranged from 6.9 to 9.7 μM; compounds 1 and 3 exhibited strong inhibitory activity against Huh7, with IC50 values ​​of 6.9–9.7 μM. 50 The values ​​were 18.2 and 13.8 μM, respectively; while compound 2 showed moderate inhibitory activity against Huh7, with an IC50 value of 18.2 and 13.8 μM. 50 The value was 26.5 μM. For SK-Hep-1 cells, compounds 1-2 and 4-7 exhibited inhibitory activity comparable to that of sorafenib, with an IC50 value of 26.5 μM. 50 The concentrations were 11.2, 10.3, 8.3, 9.8, 12.7, and 13.0 μM, respectively. These results indicate that compounds 1-7 isolated from Artemisia annua can be used as drugs to treat liver cancer-related diseases. Attached image description:

[0023] Figure 1 This is a schematic diagram of the structural formula of the compound Artemisia lactone dimer AG(1-7) of the present invention. Detailed implementation method:

[0024] To better understand the essence of the present invention, the following, in conjunction with the accompanying drawings, uses experimental examples and embodiments of the present invention to further illustrate the artemisinin dimers 1-7 of the present invention, their preparation methods, structural identification, and pharmacological effects, but these experimental examples and embodiments are not intended to limit the present invention.

[0025] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0026] Example 1:

[0027] Preparation of Artemisia lactone dimer AG(1-7):

[0028] The leaves of Artemisia annua (91 kg) were dried, pulverized, and extracted three times with 90% ethanol at room temperature for 3 days each time. The ethanol extracts were combined, and the ethanol was recovered under reduced pressure to obtain 9.1 kg of extract. This extract was then subjected to silica gel column chromatography (27.0 kg, 200-300 mesh, 30 × 90 cm) with a gradient elution of acetone-petroleum ether (v / v) (0:100, 10:90, 20:80, 30:70, 40:60, 50:50). After TLC development, the fractions were combined to obtain seven fractions Fr. A-Fr. G; fraction Fr. F (520 g) was analyzed by MCI CHP. Chromatography on a 20P column (945g, 7×50cm) yielded five fractions Fr.F-1-Fr.F-5 by gradient elution with water-methanol (v / v) (70:30, 50:50, 30:70, 10:90); fraction Fr.F-4 (71.0g) was chromatographically analyzed on a silica gel column (700g, 9×25cm) by gradient elution with acetic acid-acetone-petroleum ether (v / v) (5:95, 10:90, 20:80, 30:70) (Fr.F-4a-Fr.F-4d); fraction Fr.F-4b (7.2g) was chromatographically analyzed on a silica gel column (100g, 3.5×25cm) by isocratic elution with ethyl acetate-petroleum ether (5:95) (Fr.F-4b-1-Fr.F-4b-6). Fraction Fr.F-4b-3 (2.4 g) was subjected to silica gel column chromatography (30 g, 2 × 25 cm) and eluted isocratically with methanol-chloroform (5:95) to give six fractions Fr.F-4b-3a-Fr.F-4b-3f. Fraction Fr.F-4b-3c (240 mg) was subjected to semi-preparative HPLC and eluted with water-acetonitrile (55:45) to give compound 1 (8 mg, t). R =16.3min), 2(10.1mg,t) R =25.9min) and 3 (13mg,t) R =24.1 min). The fraction Fr.F-4b-3d (190 mg) was subjected to semi-preparative HPLC and eluted with water-acetonitrile (52:48) to give compound 4 (18 mg, t). R =27.8min) and 5 (11mg,t) R =24.6 min). The fraction Fr.F-4b-3e (160 mg) was subjected to semi-preparative HPLC and eluted with water-acetonitrile (64:36) to give compound 6 (12 mg, t). R =18.6min) and 7 (11mg,t) R =22.7min).

[0029] 1. Instruments and Materials

[0030] Nuclear magnetic resonance (NMR) spectra were determined using an Avance III 600 (Bruker, Fallanden, Switzerland), with TMS (tetramethylsilane) as an internal standard. Optical rotation was measured using an Autopol VI polarimeter (Rudolph Research Analytical, Hackettstown, USA). Infrared (IR) spectra were measured using a NICOLET iS10 infrared spectrometer (Thermo Fisher Scientific, Madison, USA) via the KBr pellet method. High-resolution mass spectrometry (HDMS) analysis was performed using a Shimadzu LC-MS-IT-TOF (Shimadzu, Kyoto, Japan). CD and UV spectra were measured using a Chirascan instrument (Applied Photophysics, Surrey, UK). Melting point was determined using... The X-4B micro melting point apparatus was used for determination, purchased from Shanghai Precision Scientific Instruments Co., Ltd. The high-performance liquid chromatograph (HPLC) was manufactured by Shimadzu Corporation, with a controller model of CBM-20A, a pump model of LC-20AR, a detector model of SPD-M20A, a column oven model of AT-350, and an Agilent-Eclipse XDB-C20 column (5μm, 9.4×250mm). The medium-pressure liquid chromatograph (LCP) was a product of Beijing Innovation Hengtong Co., Ltd., and the MCI column was a product of Mitsubishi Corporation of Japan, model CHP-20P (75~150μm). Thin-layer chromatography silica gel plates (HSGF254) and column chromatography silica gel (200~300 mesh) were purchased from Yantai Xinnuo Chemical Co., Ltd.; column chromatography dextran gel (LH-20) was purchased from GE Healthcare Bio-Sciences AB. Chromatographically pure acetonitrile and methanol were purchased from Merida Chemical Company (Beijing, China). Deionized water was purified using a MingChe™-D24UV Merk Millipore system. Analytically pure methanol and acetonitrile were purchased from Tianjin Damao Chemical Reagent Factory. The colorimetric reagent was a 10% H₂SO₄-EtOH solution.

[0031] 2. Structural data of compounds 1-7:

[0032]

[0033] Artemisia lactone dimer A(1)

[0034] Molecular formula: C 30 H 36 O7

[0035] Molecular weight: 508.25

[0036] Appearance: White powder

[0037] Optical rotation: (c 0.057, methanol)

[0038] HRESIMS(-)m / z: Experimental value 553.2437 [M+HCOO] - The calculated value is 553.2442[M+HCOO]. - .

[0039] IR(KBr)ν max :3450,1763,1634,1606,1511,1435cm -1 .

[0040] ECD (Methanol)λ max (Δε):216(+29.6),277(+0.77),245(-8.46)nm.

[0041] 1 H NMR and 13 C NMR (DEPT) data are shown in Tables 1 and 2.

[0042]

[0043] Artemisia lactone dimer B(2)

[0044] Molecular formula: C 30 H 36 O8

[0045] Molecular weight: 524.24

[0046] Appearance: White powder

[0047] Optical rotation: (c 0.052, methanol)

[0048] HRESIMS(+) m / z: Experimental value 525.2464 [M+H] + The calculated value is 525.2483 [M+H]. + IR(KBr)ν max :3444,1756,1633,1444,1383cm -1 .

[0049] ECD (Methanol)λ max (Δε):200(+8.55),229(-1.11)nm.

[0050] 1 H NMR and 13 C NMR (DEPT) data are shown in Tables 1 and 2.

[0051]

[0052] Artemisia lactone dimer C(3)

[0053] Molecular formula: C 30 H 38 O7

[0054] Molecular weight: 510.26

[0055] Appearance: White powder

[0056] Optical rotation: (c 0.110, methanol)

[0057] HRESIMS(+) m / z: Experimental value 533.2493 [M+Na] + The calculated value is 533.2510[M+Na]. + IR(KBr)ν max :3454,1746,1638,1453,1406cm -1 .

[0058] ECD (Methanol)λ max (Δε):232(+1.11),211(-9.61)nm.

[0059] 1 H NMR and 13 C NMR (DEPT) data are shown in Tables 1 and 2.

[0060]

[0061] Artemisia lactone dimer D(4)

[0062] Molecular formula: C 30 H 38 O7

[0063] Molecular weight: 510.26

[0064] Appearance: White powder

[0065] Optical rotation: (c 0.040, methanol)

[0066] HRESIMS(+)m / z: Experimental value 533.2521 ([M+Na]) + The calculated value is 533.2510[M+Na]. + ;IR(KBr)ν max :3448,1745,1640,1458,1381cm -1 .

[0067] ECD (Methanol)λ max (Δε):201(+11.2),226(-1.40)nm.

[0068] 1 H NMR and 13 C NMR (DEPT) data are shown in Tables 1 and 2.

[0069]

[0070] Artemisia lactone dimer E(5)

[0071] Molecular formula: C 30 H 34 O7

[0072] Molecular weight: 506.23

[0073] Appearance: White powder

[0074] Optical rotation: (c 0.050, methanol)

[0075] HRESIMS(+) m / z: Experimental value 507.2353 [M+H] + The calculated value is 507.2377 [M+H]. + IR(KBr)ν max :3449,1752,1685,1631,1459,1379cm -1 .

[0076] ECD (Methanol)λ max (Δε):212(+3.35),299(+0.36),231(-2.02),266(-4.49)nm. 1 HNMR and 13 C NMR (DEPT) data are shown in Tables 1 and 2.

[0077]

[0078] Artemisia lactone dimer F(6)

[0079] Molecular formula: C 30 H 34 O7

[0080] Molecular weight: 506.23

[0081] Optical rotation: (c 0.090, methanol)

[0082] HRESIMS(-)m / z: Experimental value 551.2301 [M+HCOO] -Calculated value: 551.2287[M+HCOO] - .

[0083] IR(KBr)ν max :3438,1769,1734,1637,1449cm -1 .

[0084] ECD (Methanol)λ max (Δε):201(+14.8),227(-1.24)nm.

[0085] 1 H NMR and 13 C NMR (DEPT) data are shown in Tables 1 and 2.

[0086]

[0087] Artemisia lactone dimer G(7)

[0088] Molecular formula: C 30 H 34 O7

[0089] Molecular weight: 506.23

[0090] Optical rotation: (c 0.055, methanol)

[0091] HRESIMS(-)m / z: Experimental value 551.2269 [M+HCOO] - Calculated value: 551.2287[M+HCOO] - .

[0092] IR(KBr)ν max :3445,1768,1711,1648,1449,1383cm -1 .

[0093] ECD (Methanol)λ max (Δε):200(+17.47),230(+2.77),216(-1.05)nm.

[0094] 1 H NMR and 13 C NMR (DEPT) data are shown in Tables 1 and 2.

[0095]

[0096] Table 2. Compounds 1-7 (δin ppm) 13 C NMR (150MHz) data

[0097]

[0098] a CDCl3 was used as the solvent. b Using C5D5N as solvent

[0099] Example 2:

[0100] Inhibitory activity of compounds 1-7 against three hepatocellular carcinoma cell lines.

[0101] 1. Materials and Methods

[0102] 1.1 Materials

[0103] HepG2, Huh7, and SK-Hep-1 cell lines were purchased from Shanghai Jining Biotechnology Co., Ltd.; culture media and serum (EME and FBS) were purchased from Shanghai Longtian Biotechnology Co., Ltd.; MTT was purchased from Guizhou Saiguo Biotechnology Co., Ltd.; sorafenib was purchased from Shanghai Aladdin Biochemical Technology Co., Ltd.; and DMSO was purchased from Soleberg Technology (Beijing, China).

[0104] 1.2 Instruments

[0105] Flex Station 3 benchtop multi-functional microplate reader (Bio-RAD 680, USA); analytical balance (AG135, Metler Toledo, China); incubator (DHP-9082, Shanghai).

[0106] 1.3 Experimental Procedure

[0107] 1) Take liver cancer cells in the logarithmic growth phase, discard the old culture medium, wash twice with PBS, and discard the PBS;

[0108] 2) Digest the cells with 0.25% trypsin. When the cell outlines are observed to darken and become rounded under a microscope, quickly remove the trypsin.

[0109] 3) Stop digestion with DMEM complete medium containing 10% FBS and resuspend the cells. Take 10 μL of the cell suspension, count the cells using a cell counter, and adjust the cell concentration to 1 × 10⁻⁶ cells with culture medium. 4 / mL, seeded into 96-well plates, add 100μL of cell suspension to each well, and incubate in a 37℃, 5% CO2 incubator for 24h to allow the cells to adhere;

[0110] 4) Remove the culture medium, add the diluted sample to the plate, 100 μL per well, set 3 replicates for each concentration, and continue incubation in the incubator for 48 h;

[0111] 5) Remove the culture medium, add the prepared MTT solution (1 mg / mL), add 100 μL to each well, and incubate in an incubator for 4 h;

[0112] 6) Remove the MTT solution, add 100 μL of DMSO to each well, and incubate in an incubator for 10 min;

[0113] 7) Measure the absorbance at 490nm using a microplate reader. Calculate the cell inhibition rate using the formula: Inhibition rate = (Negative - Experimental group) / (Negative - Blank group) × 100%. Calculate the IC50 using the statistical software GraphPad Prism 5. 50 The experiment was repeated 3 times.

[0114] 2. Results

[0115] The inhibitory activities of compounds 1-7 against three liver cancer cell lines (HepG2, Huh7, and SK-Hep-1) are shown in Table 3. For HepG2 cells, compounds 1 and 4-7 exhibited strong inhibitory activity, with IC50 values ​​of [missing information]. 50 The values ​​were 9.8, 9.2, 7.4, 11.4 and 12.6 μM, respectively.

[0116] For Huh7 cells, compound 4-7 exhibited inhibitory activity that was stronger or comparable to that of the positive control drug sorafenib, with an IC50 value of [missing information]. 50 The values ​​were 6.9, 9.7, 7.9, and 8.6 μM, respectively; the inhibitory activities of compounds 1 and 3 on Huh-7 cells, with IC50 values ​​of 6.9, 9.7, 7.9, and 8.6 μM. 50 The values ​​were 18.2 and 13.8 μM, respectively.

[0117] Compounds 1-2 and 4-7 exhibited strong inhibitory activity against SK-Hep-1 cells, with an IC50 value of [missing information]. 50 The values ​​were 11.2, 10.3, 8.3, 9.8, 12.7 and 13.0 μM, respectively.

[0118] Table 3 shows the inhibitory activity of compounds 1-7 against three liver cancer cell lines.

[0119]

[0120] a The numerical value is represented as IC. 50 ±SD

[0121] 3. Conclusion

[0122] Based on the above experimental results, compounds 1-7 exhibit certain inhibitory activity against three liver cancer cell lines (HepG2, Huh7, and SK-Hep-1). For HepG2 cells, compounds 1 and 4-7 showed strong inhibitory activity, with an IC50 value of [missing information]. 50The values ​​ranged from 7.4 to 12.6 μM, with compound 5 exhibiting inhibitory activity comparable to the positive control drug sorafenib, and its IC50 value was [missing value]. 50 The concentration was 7.7 μM; for Huh7 cells, compound 4-7 exhibited stronger or comparable inhibitory activity than the drug sorafenib, with an IC50 value of 7.7 μM. 50 The values ​​ranged from 6.9 to 9.7 μM; for SK-Hep-1 cells, compounds 1-2 and 4-5 exhibited inhibitory activity comparable to the positive control drug sorafenib, with IC50 values ​​of 6.9–9.7 μM. 50 The values ​​were 11.2, 10.3, 8.3, and 9.8 μM, respectively. These results indicate that compounds 1-7 isolated from *Artemisia annua* can be used as drugs for liver cancer-related diseases.

[0123] Formulation Examples:

[0124] In the following formulation examples, conventional reagents were selected and the formulations were prepared according to existing conventional methods. These formulation examples only demonstrate that at least one of the compounds 1-7 described in this invention can be prepared into different formulations, and no specific limitations are made on the specific reagents and operations:

[0125] 1. Dissolve at least one of the compounds 1-7 of the present invention in DMSO, add water for injection according to conventional methods, filter, fill and sterilize to prepare an injection solution, wherein the concentration of the injection solution is 0.5-5 mg / mL.

[0126] 2. Dissolve at least one of the compounds 1-7 of the present invention in DMSO, then dissolve it in sterile water for injection, stir to dissolve, filter with a sterile suction funnel, then filter aseptically, dispense into ampoules, freeze-dry at low temperature, and then seal aseptically to obtain a powder for injection.

[0127] 3. Add at least one of the compounds 1-7 of the present invention to the excipient at a mass ratio of 9:1 to prepare a powder.

[0128] 4. Add at least one of the compounds 1-7 of the present invention to the excipient at a mass ratio of 5:1, and granulate and compress the mixture into tablets.

[0129] 5. Prepare an oral liquid by taking at least one of the compounds 1-7 of the present invention according to conventional oral liquid preparation methods.

[0130] 6. At least one of the compounds 1-7 of the present invention is added to the excipient at a mass ratio of 5:1 to the excipient to form a capsule.

[0131] 7. Add at least one of the compounds 1-7 of the present invention to the excipient at a mass ratio of 5:1 to prepare granules.

[0132] As can be seen from the above embodiments, the present invention provides a compound from Artemisia annua, its preparation method and application, and a pharmaceutical composition and its application. The Artemisia annua lactone dimer provided by the present invention mainly includes seven compounds with novel structures. These compounds have different degrees of inhibitory activity against liver cancer cells and can be combined with pharmaceutically acceptable carriers to form pharmaceutical compositions, which can be used to prepare anti-liver cancer drugs.

[0133] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Compounds 1-4, 6, and 7, as shown in structural formula (I), ， ， (I)。 2. The method for preparing compounds 1-4, 6, and 7 as shown in structural formula (I) according to claim 1, characterized in that, The method includes the following steps: The leaves of *Artemisia annua* are dried, pulverized, and extracted three times with 90% ethanol at room temperature for three days each time. The ethanol extracts are combined, and the ethanol is recovered under reduced pressure to obtain a paste. This paste is then subjected to silica gel column chromatography, eluted with an acetone-petroleum ether gradient of v / v = 0:100, 10:90, 20:80, 30:70, 40:60, and 50:

50. After TLC development, the fractions are combined to obtain seven fractions Fr. A–Fr. G; fraction Fr. F is subjected to MCI CHP 20P column chromatography, eluted with a water-methanol gradient of v / v = 70:30, 50:50, 30:70, and 10:90 to obtain five fractions Fr. F-1–Fr. F-5; fraction Fr. F-4 is subjected to silica gel column chromatography, eluted with v / v = 5:95, 10:90, ... Elution with acetone-petroleum ether gradients of 20:80 and 30:70 yielded four fractions Fr.F-4a–Fr.F-4d; fraction Fr.F-4b was eluted by silica gel column chromatography with ethyl acetate-petroleum ether in a 5:95 ratio to yield six fractions Fr.F-4b-1–Fr.F-4b-6; fraction Fr.F-4b-3 was eluted by silica gel column chromatography with methanol-chloroform in a 5:95 ratio to yield six fractions Fr.F-4b-3a–Fr.F-4b-3f; fraction Fr.F-4b-3c was eluted by semi-preparative HPLC with water-acetonitrile in a 55:45 ratio to yield compounds 1–3; fraction Fr.F-4b-3d was eluted by semi-preparative HPLC with water-acetonitrile in a 52:48 ratio to yield compound 4; fraction Fr.F-4b-3e was eluted by semi-preparative HPLC with water-acetonitrile in a 64:36 ratio to yield compound 6. And 7.

3. The use of compounds 1-4, 6, and 7 of structural formula (I) as described in claim 1 in the preparation of anti-hepatocellular carcinoma drugs.

4. A pharmaceutical composition comprising at least one of compounds 1-4, 6, 7 of structural formula (I) as claimed in claim 1 and a pharmaceutically acceptable carrier.

5. The use of the pharmaceutical composition of claim 4 in the preparation of an anti-liver cancer drug.

6. The method for preparing the pharmaceutical composition according to claim 4, characterized in that, The method includes the following steps: The leaves of *Artemisia annua* are dried, pulverized, and extracted three times with 90% ethanol at room temperature for three days each time. The ethanol extracts are combined, and the ethanol is recovered under reduced pressure to obtain a paste. This paste is then subjected to silica gel column chromatography, eluted with an acetone-petroleum ether gradient of v / v = 0:100, 10:90, 20:80, 30:70, 40:60, and 50:

50. After TLC development, the fractions are combined to obtain seven fractions Fr. A–Fr. G; fraction Fr. F is subjected to MCI CHP 20P column chromatography, eluted with a water-methanol gradient of v / v = 70:30, 50:50, 30:70, and 10:90 to obtain five fractions Fr. F-1–Fr. F-5; fraction Fr. F-4 is subjected to silica gel column chromatography, eluted with v / v = 5:95, 10:90, ... Elution with acetone-petroleum ether gradients of 20:80 and 30:70 yielded four fractions Fr.F-4a–Fr.F-4d; fraction Fr.F-4b was eluted by silica gel column chromatography with ethyl acetate-petroleum ether in a 5:95 ratio to yield six fractions Fr.F-4b-1–Fr.F-4b-6; fraction Fr.F-4b-3 was eluted by silica gel column chromatography with methanol-chloroform in a 5:95 ratio to yield six fractions Fr.F-4b-3a–Fr.F-4b-3f; fraction Fr.F-4b-3c was eluted by semi-preparative HPLC with water-acetonitrile in a 55:45 ratio to yield compounds 1–3; fraction Fr.F-4b-3d was eluted by semi-preparative HPLC with water-acetonitrile in a 52:48 ratio to yield compound 4; fraction Fr.F-4b-3e was eluted by semi-preparative HPLC with water-acetonitrile in a 64:36 ratio to yield compound 6. 7; take any one of compounds 1-4, 6, 7 or any combination thereof, and add them to a pharmaceutically acceptable carrier.

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