Artemisia leucophylla lactone AK and its pharmaceutical composition as well as its preparation method and application

Abstract

The present invention provides 11 guaiacane-type sesquiterpene dimer compounds 1-11 (artemisinin A-K) represented by structural formula (I), as well as preparation methods, pharmaceutical compositions, and applications thereof, belonging to the field of pharmaceutical technology. These compounds exhibit inhibitory activity against human liver cancer cell lines HepG2, Huh7, and SK-Hep-1, can be combined with pharmaceutically acceptable carriers to form pharmaceutical compositions, and can be used to prepare anti-liver cancer drugs.

Description

Technical field:

[0001] The present invention belongs to the field of pharmaceutical technology, and specifically relates to a compound artemisinin AK (1-11), a preparation method and application thereof, and a pharmaceutical composition containing the same and application thereof. Background technology:

[0002] Hepatic carcinoma remains one of the most common and lethal malignancies. It is characterized by high malignancy, rapid progression, and a poor prognosis. Its mortality rate approaches its morbidity, and the five-year survival rate is only 12.1%. Currently, only nine drugs are clinically used to treat hepatic carcinoma, including the multikinase inhibitors sorafenib, lenvatinib, regorafenib, cabozantinib, and donafenib; the recombinant IgG1 monoclonal antibody ramucirumab; two immune checkpoint inhibitors, nivolumab and pembrolizumab; and the traditional Chinese medicine icaritin. However, these drugs, due to their limited targeting, are prone to drug resistance and have certain toxic side effects. Therefore, there is an urgent need to develop new, effective, and low-side-effect anti-hepatic drugs. Natural products are structurally diverse, possess diverse biological activities, and exhibit low toxicity. Some compounds, in particular, possess unique advantages in anti-tumor efficacy.

[0003] Artemisia annua is an annual or perennial herb of the genus Artemisia in the Asteraceae family. It is a variety of Artemisia annua and is mostly distributed in China (Guizhou, Qinghai, Sichuan, Tibet and Yunnan provinces), India, Myanmar and northern Thailand.

[0004] So far, there is no report on artemisinin AK(1-11) in the existing literature, no report on a pharmaceutical composition containing it as an active ingredient, and no report on the use of the pharmaceutical composition in the preparation or treatment of liver cancer drugs. Summary of the invention:

[0005] The present invention aims to provide a new class of artemisinin AK (1-11) represented by formula (I) with medicinal value, a preparation method and application thereof, a pharmaceutical composition containing artemisinin AK (1-11) and an application thereof. This class of compounds has significant inhibitory activity against liver cancer cell lines and can be used to prepare anti-liver cancer drugs.

[0006] In order to achieve the above-mentioned object of the present invention, the present invention provides the following technical solutions:

[0007] The present invention provides a series of sesquiterpene dimer compounds, artemisia lactone AK (1-11), having the structure shown in the following formula (I):

[0008]

[0009] The present invention provides a preparation method of the above-mentioned compound 1-11. The dried aerial part of Artemisia leucophylla (48.0 kg) was crushed and extracted three times with 90% ethanol cold soaking, each time for 3 days. The ethanol extracts were combined, and the ethanol extract was recovered under reduced pressure. Then, silica gel column chromatography was performed, and the elution was performed with acetone-petroleum ether gradient (0:100, 10:90, 20:80, 30:70, 40:60, 50:50). The fractions were combined by TLC to obtain seven fractions Fr.A-Fr.G; the fraction Fr.D (270.0 g) was subjected to MCI CHP 20P column chromatography, gradient elution with water-methanol (70:30, 50:50, 30:70, 10:90) to obtain four fractions Fr.D-1-Fr.D-4; fraction Fr.D-2 (45.0 g) was chromatographed on a silica gel column, gradient elution with ethyl acetate-petroleum ether (10:90, 20:80, 30:70, 40:60 and 50:50) to obtain five fractions Fr.D-2a-Fr.D-2b The fraction Fr.D-2b (29.7 g) was chromatographed on an RP-C18 column using a water-methanol gradient elution (70:30, 50:50, 30:70, 10:90) to obtain four fractions Fr.D-2b-1 to Fr.D-2b-4; the fraction Fr.D-2b-2 (7.0 g) was chromatographed on a silica gel column using an isocratic elution using acetone-petroleum ether (20:80) to obtain seven fractions F The fraction Fr.D-2b-2e (1.4 g) was subjected to silica gel column chromatography and isocratically eluted with ethyl acetate-petroleum ether (20:80), followed by semi-preparative HPLC to obtain compound 7 (12.0 mg); Fr.D-2b-2e (1.3 g) was subjected to silica gel column chromatography and then separated by HPLC to obtain compounds 1 (6.7 mg) and 4 (12 .0 mg); fraction Fr.D-2b-2f (36.0 mg) was purified by semi-preparative HPLC to give compound 8 (12.0 mg); fraction Fr.D-2b-3 (18.5 g) was purified by silica gel column chromatography and eluted isocratically with methanol-chloroform (5:95) to give four fractions Fr.D-2b-3a-Fr.D-2b-3d; fraction Fr.D-2b-3b (14.0 g) was purified by MCI After CHP 20P column chromatography, gradient elution with water-methanol (50:50, 40:60, 30:70, 20:80, 0:100), and subsequent purification by silica gel column chromatography and semi-preparative HPLC, compounds 9 (11.0 mg) and 10 (7.0 mg) were obtained. Fr.D-2b-3d (480.0 mg) was separated by silica gel column chromatography and semi-preparative HPLC to obtain compounds 3 (10.6 mg) and 6 (8.0 mg). Fraction Fr.D-2c (5.0 g) was subjected to two silica gel column chromatography cycles to obtain six fractions, Fr.D-2c-4c-1 to Fr.D-2c-4c-6; fraction Fr.F-2c-4c-2 (128.0 mg) was purified by semi-preparative HPLC to give compounds 2 (8.2 mg) and 5 (17.5 mg); fraction Fr.D-2c-4b (104 mg) was separated by semi-preparative HPLC to give compound 11 (12.0 mg).

[0010] The present invention provides the use of the above compounds 1-11 in the preparation of anti-liver cancer drugs. The present invention has no particular limitation on the method of the use, and any method well known in the art can be used.

[0011] The present invention also provides a pharmaceutical composition comprising at least one of the compounds 1-11 of formula (I) and a pharmaceutically acceptable carrier. The present invention also provides a method for preparing the pharmaceutical composition, wherein the compound 1-11 is obtained by the above-described method for preparing the compound, and at least one of the compounds 1-11 is added to a pharmaceutically acceptable carrier.

[0012] Furthermore, the invention also provides the use of the pharmaceutical composition in preparing anti-liver cancer drugs.

[0013] When at least one of the compounds 1-11 is used to prepare an anti-liver cancer drug, the present invention preferably uses the compound 1-11 directly or in the form of a pharmaceutical composition.

[0014] The pharmaceutical composition provided herein comprises at least one of the compounds 1-11 and a pharmaceutically acceptable carrier. In the present invention, the pharmaceutically acceptable carrier is preferably a solid, semisolid, or liquid diluent, a filler, or a pharmaceutical product adjuvant. The present invention does not particularly limit the pharmaceutically acceptable carrier; any pharmaceutically acceptable carrier well known in the art that is nontoxic and inert to humans and animals may be selected.

[0015] The present invention has no particular limitation on the preparation method of the pharmaceutical composition. At least one of compounds 1-11 can be directly mixed with a pharmaceutically acceptable carrier. The present invention has no particular limitation on the mixing process. A pharmaceutical composition can be obtained by selecting a process well known in the art.

[0016] The present invention provides the use of the pharmaceutical composition of the above technical solution in the preparation of an anti-liver cancer drug. The present invention has no particular limitation on the method of the application, and any method well known in the art can be used.

[0017] In the present invention, when the pharmaceutical composition is used to prepare an anti-liver cancer drug, the content of the composition in the drug is preferably 0.1-99%; in the pharmaceutical composition, the content of at least one of compounds 1-11 in the pharmaceutical composition is preferably 0.5-90%. The pharmaceutical composition of the present invention is preferably used in the form of a dosage per unit body weight. In the present invention, the prepared drug is preferably administered by injection (intravenous injection, intramuscular injection) and oral administration.

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

[0019] 1. The present invention provides a series of novel sesquiterpene dimer compounds, artemisia lactones AK (1-11). The present invention discovered that the ethanol extract of the dried aerial parts of Artemisia odorata has certain inhibitory activity against three liver cancer cell lines (HepG2, Huh7, and SK-Hep-1). Six sesquiterpene dimer compounds, artemisia lactones AK (1-11), with inhibitory activity against the three liver cancer cell lines were further isolated from the ethanol extract.

[0020] 2. The present invention provides a new method for preparing new compound 1-11, which has readily available raw materials, is easy to operate, and is suitable for industrial production.

[0021] 3. The present invention provides a pharmaceutical composition containing the new compound 1-11 as an active ingredient, which provides a new anti-liver cancer drug with better medicinal effects.

[0022] 4. Compounds 1-11 of the present invention have inhibitory activity against three liver cancer cell lines (HepG2, SK-HEP-1 and Huh7); for HepG2 cells, compounds 5 and 8 have strong inhibitory activity, and their IC 50 The values ​​were 14.2 and 18.8 μM, respectively. Compounds 6-7 and 9-10 also showed certain inhibitory activity, and their IC 50 The values ​​ranged from 20.5 to 26.7 μM. For Huh7 cells, compounds 1-2, 5-10 showed IC 50 The values ​​ranged from 7.9 to 20.5 μM. In particular, compound 7 exhibited stronger inhibitory activity than the positive drug sorafenib, with an IC 50 The inhibitory activity of compounds 5, 8-10 was comparable to that of sorafenib, and their IC 50 The value range is 9.0-11.5μM. For SK-Hep-1 cells, compound 5 exhibited stronger inhibitory activity than sorafenib, with an IC 50 The inhibitory activity of compounds 7-8 and 10 was comparable to that of sorafenib, with an IC 50The values ​​were 9.4, 11.3 and 9.1 μM, respectively. The above results indicate that compounds 1-11 isolated from Artemisia leucophylla can be used as drugs to treat liver cancer-related diseases. Description of the drawings:

[0023] Figure 1 Schematic diagram of the structural formula of Artemisia pubescens lactone compounds 1-11 of the present invention.

[0024] Figure 2 Schematic diagram of the X-ray single crystal diffraction structure of compound 2.

[0025] Figure 3 Schematic diagram of the X-ray single crystal diffraction structure of compound 8.

[0026] Figure 4 Schematic diagram of the X-ray single crystal diffraction structure of compound 9. Specific implementation method:

[0027] In order to better understand the essence of the present invention, the following is a description of the present invention in conjunction with the accompanying drawings, using the test examples and examples of the present invention to further illustrate the sesquiterpene dimer compounds of the present invention, Artemisia annua lactone compounds 1-11 and their preparation methods, structural identification, and pharmacological effects, but the present invention is not limited to these test examples and examples. The technical solutions of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the embodiments described are only some embodiments of the present invention, rather than all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of the present invention.

[0028] Example 1:

[0029] Preparation of guaiacyl sesquiterpene dimer compound 1-11 (artemisia lactone AK):

[0030] The dried aerial parts of Artemisia leucophylla (48.0 kg) were crushed and extracted with 90% ethanol for three times, each time for 3 days. The ethanol extracts were combined and the ethanol extract was recovered under reduced pressure. The extract was then chromatographed on a silica gel column using an acetone-petroleum ether gradient elution (0:100, 10:90, 20:80, 30:70, 40:60, 50:50). The fractions were combined by TLC to obtain seven fractions Fr.A-Fr.G. The fraction Fr.D (270.0 g) was purified by MCI CHP. 20P column chromatography, gradient elution with water-methanol (70:30, 50:50, 30:70, 10:90) to obtain four fractions Fr.D-1-Fr.D-4; fraction Fr.D-2 (45.0 g) was chromatographed on a silica gel column, gradient elution with ethyl acetate-petroleum ether (10:90, 20:80, 30:70, 40:60 and 50:50) to obtain five fractions Fr.D-2a-Fr.D-2b The fraction Fr.D-2b (29.7 g) was chromatographed on an RP-C18 column using a water-methanol gradient elution (70:30, 50:50, 30:70, 10:90) to obtain four fractions Fr.D-2b-1 to Fr.D-2b-4; the fraction Fr.D-2b-2 (7.0 g) was chromatographed on a silica gel column using an isocratic elution using acetone-petroleum ether (20:80) to obtain seven fractions F The fraction Fr.D-2b-2e (1.4 g) was subjected to silica gel column chromatography and isocratically eluted with ethyl acetate-petroleum ether (20:80), followed by semi-preparative HPLC to obtain compound 7 (12.0 mg); Fr.D-2b-2e (1.3 g) was subjected to silica gel column chromatography and then separated by HPLC to obtain compounds 1 (6.7 mg) and 4 (12 .0 mg); fraction Fr.D-2b-2f (36.0 mg) was purified by semi-preparative HPLC to give compound 8 (12.0 mg); fraction Fr.D-2b-3 (18.5 g) was purified by silica gel column chromatography and eluted isocratically with methanol-chloroform (5:95) to give four fractions Fr.D-2b-3a-Fr.D-2b-3d; fraction Fr.D-2b-3b (14.0 g) was purified by MCI After CHP 20P column chromatography, water-methanol gradient elution (50:50, 40:60, 30:70, 20:80, 0:100), and then purified by silica gel column chromatography and semi-preparative HPLC multiple times to give compounds 9 (11.0 mg) and 10 (7.0 mg); Fr.D-2b-3d (480.0 mg) was separated by silica gel column chromatography and semi-preparative HPLC to give compounds 3 (10.6 mg) and 6 (8.0 mg); fraction Fr.D-2c (5.0 g) was subjected to two silica gel column chromatography to give six fractions Fr.D-2c-4c-1-Fr.D-2c-4c-6; fraction Fr.F-2c-4c-2 (128.0 mg) was purified by semi-preparative HPLC to give compound 2 (8.0 mg).2 mg) and 5 (17.5 mg); fraction Fr.D-2c-4b (104 mg) was separated by semi-preparative HPLC to give compound 11 (12.0 mg).

[0031] 1. Instruments and Materials

[0032] The nuclear magnetic resonance spectrum was measured using an Avance III 600 (Bruker, Fallanden, Switzerland) with TMS (tetramethylsilane) as the internal standard. The optical rotation was measured using an Autopol VI polarimeter (Rudolph Research Analytical, Hackettstown, USA). The infrared spectrum (IR) was measured using a NICOLET iS10 infrared spectrometer (Thermo Fisher Scientific, Madison, USA) using the KBr pellet method. High-resolution mass spectrometry was measured using a Shimadzu LC-MS-IT-TOF (Shimadzu, Kyoto, Japan). CD and UV spectra were measured using a Chirascan instrument (Applied Photophysics, Surrey, UK). The melting point was measured using The melting point was determined using an X-4B micro-melting point apparatus purchased from Shanghai Precision Scientific Instrument Co., Ltd. The high-performance liquid chromatograph was manufactured by Shimadzu Corporation, with a CBM-20A controller, LC-20AR pump, SPD-M20A detector, and AT-350 column oven. The columns used were Agilent-Eclipse XDB-C20 (5 μm, 9.4 × 250 mm). The medium-pressure liquid chromatography (HPLC) system was manufactured by Beijing Chuangxin Hengtong Co., Ltd., and the MCI columns were manufactured by Mitsubishi Corporation of Japan, CHP-20P (75–150 μm). Thin-layer chromatography silica gel plates (HSGF254) and column chromatography silica gel (200–300 mesh) were purchased from Yantai Xinnuo Chemical Co., Ltd.; the Sephadex LH-20 column was purchased from GE Healthcare Bio-Sciences AB. Chromatographically pure acetonitrile and methanol were purchased from Merida (Beijing, China). Deionized water was purified using a MingChe™-D 24UV Merk Millipore system. Analytical-grade methanol and acetonitrile were purchased from Tianjin Damao Chemical Reagent Factory. The colorimetric reagent was a 10% H2SO4-EtOH solution.

[0033] Structural data of compound 1-11:

[0034]

[0035] Artemisia lactone A(1)

[0036] Molecular formula: C30 H 34 O6

[0037] Molecular weight: 490.24

[0038] Appearance: white powder

[0039] Optical rotation: (c 0.050, methanol)

[0040] HRESIMS(+)m / z: Found 491.2411[M+H] + , calculated value 491.2428[M+H] + ; HRESIMS (-) m / z: Observed value 535.2354 [M+HCOO] - , calculated value 535.2337 [M+HCOO] - .

[0041] IR(KBr)ν max :3438,1768,1688,1640,1619cm -1 .

[0042] ECD(Methanol)λ max (Δε):223(-1.92),249(+2.91),275(-0.88)nm.

[0043] 1 H NMR and 13 C NMR (DEPT) data are shown in Tables 1 and 3.

[0044]

[0045] Artemisia lactone B(2)

[0046] Molecular formula: C 30 H 37 O6Cl

[0047] Molecular weight: 528.23

[0048] Properties: Monoclinic crystal

[0049] Melting point: 201-202°C

[0050] Optical rotation: (c 0.062, methanol)

[0051] HRESIMS(+)m / z: Observed value 529.2343([M+H] + , calculated value 529.2351[M+H] +; HRESIMS (-) m / z: Observed value 573.2286 [M+HCOO] - , calculated value 573.2261[M+HCOO] - IR(KBr)ν max :3433,1761,1752,1633,1454cm -1 .

[0052] ECD(Methanol)λ max (Δε):224(-6.57)nm.

[0053] 1 H NMR and 13 C NMR (DEPT) data are shown in Tables 1 and 3.

[0054] Crystal data: C 30 H 37 ClO6·CHCl3,M=648.41, α=90°, β=107.233(2)°, γ=90°, T=150.(2)K, crystal space group P1211, Z=2, μ(Cu Kα)=3.911mm -1 The crystal data were measured using a D8 QUEST crystal diffractometer (copper target). The total number of diffractions was 30098, and 5459 were observed (R int =0.0741), I>2σ(I); wR(F 2 )=0.1519(I>2σ(I),R1=0.0536,wR(F 2 )=0.1543,F 2 =1.035. Flack parameter = 0.08 (6). The crystal parameters of compound 2 have been deposited in the Cambridge Crystallographic Data Center, accession number: CCDC 2266599. Website: https: / / www.ccdc.cam.ac.uk / . See the structure Figure 2 .

[0055]

[0056] Artemisia lactone C(3)

[0057] Molecular formula: C 30 H 38 O7

[0058] Appearance: white powder

[0059] Optical rotation: (c 0.073, methanol)

[0060] HRESIMS(+)m / z: Observed value 511.2673[M+H] + , calculated value 525.2690[M+H] + ; HRESIMS (-) m / z: Observed value 555.2602 [M+HCOO] - , calculated value 555.2600 [M+HCOO] - .

[0061] IR(KBr)ν max :3440,1750,1639,1457,1156cm -1 .

[0062] ECD(Methanol)λ max (Δε):196(+11.8),225(-6.51)nm.

[0063] 1 H NMR and 13 C NMR (DEPT) data are shown in Tables 1 and 3.

[0064]

[0065] Artemisia lactone D(4)

[0066] Molecular formula: C 30 H 38 O7

[0067] Appearance: white powder

[0068] Optical rotation: (c 0.070, methanol)

[0069] HRESIMS(+)m / z: Observed value 511.2696[M+H] + , calculated value 525.2690[M+H] + ; HRESIMS (-) m / z: Observed value 555.2587 [M+HCOO] - , calculated value 555.2600 [M+HCOO] - .

[0070] IR(KBr)ν max :3439,1753,1632,1458,1279,1158cm -1 .

[0071] ECD(Methanol)λ max(Δε):201(+4.32),215(-4.47)nm.

[0072] 1 H NMR and 13 C NMR (DEPT) data are shown in Tables 1 and 3.

[0073]

[0074] Artemisia leucophylla lactone E(5)

[0075] Molecular formula: C 30 H 33 O6Cl

[0076] Molecular weight: 524.21

[0077] Appearance: white powder

[0078] Optical rotation: (c 0.076, methanol)

[0079] HRESIMS(+)m / z: Observed value 525.2053[M+H] + , calculated value 525.2038[M+H] + ; HRESIMS (-) m / z: Observed value 569.1941 [M+HCOO] - , calculated value 569.1948 [M+HCOO] - .

[0080] IR(KBr)ν max :3448,1764,1693,1647,1621,1457cm -1 .

[0081] ECD(Methanol)λ max (Δε):218(-6.26),256(+1.20),282(+1.55)nm.

[0082] 1 H NMR and 13 C NMR (DEPT) data are shown in Tables 1 and 3.

[0083]

[0084] Artemisia scoparia lactone F(6)

[0085] Molecular formula: C 32 H 38 O8

[0086] Molecular weight: 550.27

[0087] Appearance: white powder

[0088] Optical rotation: (c 0.060, methanol)

[0089] HRESIMS(+)m / z: Observed value 551.2660[M+H] + , calculated value 551.2639[M+H] + ; HRESIMS (-) m / z: Observed value 595.2532 [M+HCOO] - , calculated value 595.2549 [M+HCOO] - IR(KBr)ν max :3445,1766,1639,1639,1263cm -1 .

[0090] ECD(Methanol)λ max (Δε): 231 (-1.52), 264 (+0.019) and 297 (+0.84) nm.

[0091] 1 H NMR and 13 C NMR (DEPT) data are shown in Tables 1 and 3.

[0092]

[0093] Artemisia scoparia lactone G(7)

[0094] Molecular formula: C 30 H 34 O6

[0095] Molecular weight: 490.24

[0096] Appearance: white powder

[0097] Optical rotation: (c 0.098, methanol)

[0098] HRESIMS(+)m / z: Found 491.2412[M+H] + , calculated value 491.2428[M+H] + ; HRESIMS (-) m / z: Observed value 535.2359 [M+HCOO] - , calculated value 535.2337 [M+HCOO] - IR(KBr)ν max :3441,1766,1688,1639,1619cm -1 .

[0099] ECD(Methanol)λ max (Δε):217(+17.6),274(-2.29)nm.

[0100] 1 H NMR and 13 C NMR (DEPT) data are shown in Tables 2 and 3.

[0101]

[0102] Artemisia scoparia lactone H(8)

[0103] Molecular formula: C 30 H 34 O6

[0104] Molecular weight: 490.24

[0105] Properties: oblique crystals

[0106] Melting point: 198-199°C

[0107] Optical rotation: (c 0.070, methanol)

[0108] HRESIMS(+)m / z: Found 491.2429([M+H] + , calculated value 491.2428[M+H] + ;

[0109] HRESIMS(-)m / z: Observed value 535.2333[M+HCOO] - , calculated value 535.2337 [M+HCOO] - IR(KBr)ν max :3436,1764,1680,1637,1620,1440,1384,1258,1153cm -1 ECD (methanol)λ max (Δε):209(+4.99),219(+7.01)nm.

[0110] 1 H NMR and 13 C NMR (DEPT) data are shown in Tables 2 and 3.

[0111] Crystal data: C 30 H 34 O6·CH3OH,M=490.57, α=90°, β=98.4150(10)°, γ=90°, T=100.(2)K, crystal space group P1211, Z=2, μ(Cu Kα)=0.730mm -1 The crystal data were measured using a D8 QUEST crystal diffractometer (copper target). The total number of diffractions was 23765, and 4891 were observed (R int =0.0392),I>2σ(I),wR(F 2 )=0.0668(I>2σ(I)),R1=0.0270,wR(F 2 )=0.0671,F 2 =1.045. Flack parameter = -0.05(4). Extraction number: CCDC 2266611. Website: https: / / www.ccdc.cam.ac.uk / The X-ray single crystal diffraction structure of compound 8 is shown in Figure 3 .

[0112]

[0113] Artemisia scoparia lactone I(9)

[0114] Molecular formula: C 30 H 34 O6

[0115] Molecular weight: 490.24

[0116] Properties: oblique crystals

[0117] Melting point: 198-199°C

[0118] Optical rotation: (c 0.050, methanol)

[0119] HRESIMS(+)m / z: Found 491.2428[M+H] + , calculated value 491.2428[M+H] + ; HRESIMS (-) m / z: Observed value 535.2334 [M+HCOO] - , calculated value 535.2337 [M+HCOO] - IR(KBr)ν max :3436,1769,1682,1633,1618,1442,1382,1257cm -1 ECD (methanol)λ max (Δε):216(+15.7),271(-1.03)nm.

[0120] 1 H NMR and 13 C NMR (DEPT) data are shown in Tables 2 and 3.

[0121] Crystal data: C 30 H 34 O6·CH3OH,M=490.57, α=90°, β=95.282(2)°, γ=90°, T=100.(2)K,space group P1211,Z=2,μ(Cu Kα)=0.738mm -1 The crystal data were measured using a D8 QUEST crystal diffractometer (copper target). The total number of diffractions was 24307, and 4593 were observed (R int =0.0490), I>2σ(I)), wR(F 2 )=0.0884(I>2σ(I)), R1=0.0351;wR(F 2 )=0.0889,F 2 =1.06. Flack parameter = 0.11(6). Extraction number: CCDC 2266594. Website: https: / / www.ccdc.cam.ac.uk / The X-ray single crystal diffraction structure of compound 9 is shown in Figure 4 .

[0122]

[0123] Artemisia scabracholide J(10)

[0124] Molecular formula: C 30 H 34 O6

[0125] Molecular weight: 490.24

[0126] Optical rotation: (c 0.070, methanol)

[0127] HRESIMS(+)m / z: Found 491.2419[M+H] + , calculated value 491.2428[M+H] + ; HRESIMS (-) m / z: Observed value 535.2331 [M+HCOO] - , calculated value 535.2337 [M+HCOO] - IR(KBr)ν max :3441,1760,1682,1634,1617cm -1 .

[0128] ECD(Methanol)λ max(Δε):205(-16.9),220(-1.17),232(-2.69)nm. 1 H NMR and 13 C NMR (DEPT) data are shown in Tables 2 and 3.

[0129]

[0130] Artemisia scoparia lactone K(11)

[0131] Molecular formula: C 30 H 40 O6

[0132] Molecular weight: 497.30

[0133] Optical rotation: (c 0.110, methanol)

[0134] HRESIMS(+)m / z: Found 497.2909[M+H] + , calculated value 497.2898[M+H] + ; HRESIMS (-) m / z: Observed value 541.2820 [M+HCOO] - , calculated value 541.2807 [M+HCOO] - IR(KBr)ν max :3437,1772,1633,1457,1382cm -1 .

[0135] ECD(Methanol)λ max (Δε):200(+14.6),226(-2.39)nm.

[0136] 1 H NMR and 13 C NMR (DEPT) data are shown in Tables 2 and 3.

[0137]

[0138]

[0139]

[0140] Example 2:

[0141] Inhibitory activity of compounds 1-11 against three liver cancer cell lines.

[0142] 1. Materials and Methods

[0143] 1.1 Materials

[0144] HepG2, Huh7, and SK-Hep-1 cell lines were purchased from Shanghai Jining Biotechnology Co., Ltd.; culture medium 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).

[0145] 1.2 Instruments

[0146] Flex Station 3 benchtop multifunctional microplate reader (Bio-RAD 680, USA); analytical balance (AG135, Metler Toledo, China); constant temperature box (DHP-9082, Shanghai).

[0147] 1.3 Experimental Procedure

[0148] 1) Take hepatocellular carcinoma cells in logarithmic phase, discard the old culture medium, wash twice with PBS, and discard the PBS;

[0149] 2) Digest the cells with 0.25% trypsin. When the cell outlines become darker and rounder under a microscope, quickly remove the trypsin.

[0150] 3) Terminate the digestion and resuspend the cells with DMEM complete medium containing 10% FBS, take 10 μL of the cell suspension, count the cells using a cell counter, and adjust the cell concentration to 1×10 4 / mL, seeded on a 96-well plate, added 100 μL of cell suspension to each well, and incubated in a 37°C, 5% CO2 incubator for 24 h to allow the cells to adhere;

[0151] 4) Aspirate the culture medium and add 100 μL of the diluted sample to each well of the plate. Set up 3 replicates for each concentration and continue incubating in the incubator for 48 hours.

[0152] 5) Aspirate the culture medium and add 100 μL of the prepared MTT solution (1 mg / mL) to each well. Incubate in the incubator for 4 h.

[0153] 6) Aspirate the MTT solution and add DMSO (100 μL per well) and incubate in the incubator for 10 min.

[0154] 7) Measure the absorbance at 490 nm using a microplate reader. Calculate the cell inhibition rate using the formula: inhibition rate = (negative - experimental group) / (negative - blank group) × 100%. Calculate the IC using the statistical software GraphPad Prism 5. 50 , the experiment was repeated 3 times.

[0155] 2. Results

[0156] The inhibitory activities of compounds 1-11 against three liver cancer cell lines (HepG2, Huh7 and SK-Hep-1) are shown in Table 4. For HepG2 cells, compounds 5 and 8 have strong inhibitory activities, with IC 50 The values ​​were 14.2 and 18.8 μM, respectively. Compounds 6-7 and 9-10 also showed certain inhibitory activity, and their IC 50 The values ​​ranged from 20.5 to 26.7 μM.

[0157] For Huh7 cells, compounds 1-2, 5-10 showed IC 50 The values ​​ranged from 7.9 to 20.5 μM. In particular, compound 7 exhibited stronger inhibitory activity than the positive drug sorafenib, with an IC 50 The inhibitory activity of compounds 5, 8-10 was comparable to that of sorafenib, and their IC 50 The values ​​ranged from 9.0 to 11.5 μM.

[0158] Compound 5 exhibited stronger inhibitory activity against SK-Hep-1 cells than sorafenib, with an IC 50 The inhibitory activity of compounds 7-8 and 10 was comparable to that of sorafenib, with an IC 50 The values ​​were 9.4, 11.3 and 9.1 μM, respectively.

[0159] Table 4 Inhibitory activity of compounds 1-11 against three liver cancer cell lines

[0160]

[0161] The value is expressed as IC 50 ±SD

[0162] 3. Conclusion

[0163] Based on the above experimental results, it can be concluded that compounds 1-11 showed certain inhibitory activity against three liver cancer cell lines (HepG2, Huh7 and SK-Hep-1). Compared with other compounds, compounds 5 and 8 showed stronger inhibitory activity against HepG2 cells, with their IC 50 The values ​​were 14.2 and 18.8 μM respectively; compound 7 showed stronger inhibitory activity against Huh7 cells than sorafenib; the inhibitory activity of four compounds (5 and 8-10) was comparable to that of the positive drug sorafenib; for SK-Hep-1 cells, compound 5 showed stronger inhibitory activity than sorafenib, and its IC 50The value was 8.8 μM; compounds 7-8 and 10 showed inhibitory activity comparable to that of sorafenib. The above results indicate that compounds 1-11 isolated from Artemisia leucophylla can be used as drugs for liver cancer-related diseases.

[0164] Preparation Example:

[0165] In the following formulation examples, conventional reagents are selected and the formulations are prepared according to conventional methods. These formulation examples merely demonstrate that at least one of the compounds 1-11 described herein can be prepared into different formulations, and the specific reagents and procedures are not specifically limited.

[0166] 1. Dissolve at least one of the compounds 1-11 of the present invention in DMSO, add water for injection according to conventional methods, filter thoroughly, and sterilize by filling to prepare an injection solution with a concentration of 0.5 to 5 mg / mL.

[0167] 2. Dissolve at least one of the compounds 1-11 of the present invention in DMSO, dissolve it in sterile water for injection, stir to dissolve it, filter it with a sterile suction funnel, and then filter it aseptically. The ampoule is divided into ampoules, freeze-dried at low temperature, and sealed aseptically to obtain a powder injection.

[0168] 3. Add at least one of the compounds 1-11 of the present invention to an excipient at a mass ratio of 9:1 to the excipient to prepare a powder.

[0169] 4. Add at least one of the compounds 1-11 of the present invention to the excipient at a mass ratio of 5:1, and granulate and tablet.

[0170] 5. Prepare at least one of the compounds 1-11 of the present invention into an oral solution according to a conventional oral solution preparation method.

[0171] 6. At least one of the compounds 1-11 of the present invention was added to an excipient at a mass ratio of 5:1 to the excipient to prepare a capsule.

[0172] 7. At least one of the compounds 1-11 of the present invention was added to an excipient at a mass ratio of 5:1 to the excipient to prepare granules.

[0173] As can be seen from the above examples, the present invention provides compounds from Artemisia leucophylla, their preparation methods and uses, and pharmaceutical compositions and uses thereof. The Artemisia leucophylla lactones provided by the present invention primarily include 11 compounds with novel structures. These compounds exhibit varying degrees of inhibitory activity against liver cancer cells and can be combined with pharmaceutically acceptable carriers to form pharmaceutical compositions for the preparation of anti-liver cancer drugs.

[0174] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principles of the present invention. These improvements and modifications should also be regarded as within the scope of protection of the present invention.

Claims

1. Compounds 1-7 and 10 represented by the following structural formula (I), 2. The method for preparing compounds 1-7 and 10 of formula (I) according to claim 1, characterized in that: The method comprises the following steps: crushing the dried aerial part of Artemisia leucophylla, cold-immersing and extracting it three times with 90% ethanol, each time for 3 days, combining the ethanol extracts, recovering the ethanol extract under reduced pressure, and then performing silica gel column chromatography, using acetone-petroleum ether gradient elution with a volume ratio of 0:100, 10:90, 20:80, 30:70, 40:60, and 50:50, developing the fractions by TLC and combining the fractions to obtain seven fractions Fr.A-Fr.G; the fraction Fr.D is subjected to MCI CHP 20P column chromatography, eluting with a water-methanol gradient of 70:30, 50:50, 30:70, and 10:90 by volume, afforded four fractions Fr.D-1 to Fr.D-4; fraction Fr.D-2 was chromatographed on a silica gel column, eluting with an ethyl acetate-petroleum ether gradient of 10:90, 20:80, 30:70, 40:60, and 50:50 by volume, afforded five fractions Fr.D-2a to Fr.D-2e; fraction Fr.D-2b was chromatographed on an RP-C18 column, eluting with a water-methanol gradient of 70:30, 50:50, 30:70, and 10:90 by volume, afforded four fractions Fr.D-2b-1 to Fr.D-2b-4; fraction Fr. D-2b-2 was subjected to silica gel column chromatography and isocratically eluted with acetone-petroleum ether in a volume ratio of 20:80 to obtain seven fractions Fr.D-2b-2a to Fr.D-2b-2g; fraction Fr.D-2b-2e was subjected to silica gel column chromatography and isocratically eluted with ethyl acetate-petroleum ether in a volume ratio of 20:80, and then subjected to semi-preparative HPLC to obtain compound 7; Fr.D-2b-2e was subjected to silica gel column chromatography and then separated by HPLC to obtain compounds 1 and 4; fraction Fr.D-2b-3 was subjected to silica gel column chromatography and isocratically eluted with methanol-chloroform in a volume ratio of 5:95 to obtain four fractions Fr.D-2b-3a to Fr.D-2b-3d; fraction Fr.D-2b-3b was subjected to MCI Compound 10 was obtained by CHP 20P column chromatography with a water-methanol gradient elution of 50:50, 40:60, 30:70, 20:80, and 0:100 by volume, followed by multiple purification by silica gel column chromatography and semi-preparative HPLC. Compounds 3 and 6 were obtained from Fr.D-2b-3d after separation by silica gel column chromatography and semi-preparative HPLC. The fraction Fr.D-2c was subjected to two silica gel column chromatography cycles to obtain six fractions, Fr.D-2c-4c-1 to Fr.D-2c-4c-6. The fraction Fr.F-2c-4c-2 was purified by semi-preparative HPLC to obtain compounds 2 and 5.

3. Use of compounds 1-7 and compound 10 of formula (I) according to claim 1 in the preparation of anti-liver cancer drugs.

4. A pharmaceutical composition comprising at least one of the compounds 1 to 7 and compound 10 of formula (I) according to claim 1 and a pharmaceutically acceptable carrier.

5. Use of the pharmaceutical composition according to claim 4 in the preparation of anti-liver cancer drugs.

6. The method for preparing the pharmaceutical composition according to claim 4, characterized in that: The method comprises the following steps: crushing the dried aerial part of Artemisia leucophylla, cold-immersing and extracting it three times with 90% ethanol, each time for 3 days, combining the ethanol extracts, recovering the ethanol extract under reduced pressure, and then performing silica gel column chromatography, using acetone-petroleum ether gradient elution with a volume ratio of 0:100, 10:90, 20:80, 30:70, 40:60, and 50:50, developing the fractions by TLC, combining the fractions, and obtaining seven fractions Fr.A-Fr.G; and subjecting the fraction Fr.D to MCI. After chromatography on a CHP20P column, the product was eluted with a water-methanol gradient of 70:30, 50:50, 30:70, and 10:90 by volume to obtain four fractions Fr.D-1 to Fr.D-4. Fraction Fr.D-2 was chromatographed on a silica gel column, and the product was eluted with an ethyl acetate-petroleum ether gradient of 10:90, 20:80, 30:70, 40:60, and 50:50 by volume to obtain five fractions Fr.D-2a to Fr.D-2e. Fraction Fr.D-2b was chromatographed on an RP-C18 column, and the product was eluted with a water-methanol gradient of 70:30, 50:50, 30:70, and 10:90 by volume to obtain four fractions Fr.D-2b-1 to Fr.D-2b-4. Fraction Fr .D-2b-2 was subjected to silica gel column chromatography and isocratically eluted with acetone-petroleum ether in a volume ratio of 20:80 to obtain seven fractions Fr.D-2b-2a to Fr.D-2b-2g; fraction Fr.D-2b-2e was subjected to silica gel column chromatography and isocratically eluted with ethyl acetate-petroleum ether in a volume ratio of 20:80, and then subjected to semi-preparative HPLC to obtain compound 7; Fr.D-2b-2e was subjected to silica gel column chromatography and then separated by HPLC to obtain compounds 1 and 4; fraction Fr.D-2b-3 was subjected to silica gel column chromatography and isocratically eluted with methanol-chloroform in a volume ratio of 5:95 to obtain four fractions Fr.D-2b-3a to Fr.D-2b-3d; fraction Fr.D-2b-3b was subjected to MCI After CHP20P column chromatography and gradient elution with water-methanol in a volume ratio of 50:50, 40:60, 30:70, 20:80, and 0:100, compound 10 was obtained by multiple silica gel column chromatography and semi-preparative HPLC purification; Fr.D-2b-3d was separated by silica gel column chromatography and semi-preparative HPLC to obtain compounds 3 and 6; the fraction Fr.D-2c was subjected to two silica gel column chromatography to obtain six fractions Fr.D-2c-4c-1 to Fr.D-2c-4c-6; the fraction Fr.F-2c-4c-2 was purified by semi-preparative HPLC to obtain compounds 2 and 5; then, at least one of the above compounds was added with a pharmaceutically acceptable carrier.

Images