Artemordosins A-L as well as pharmaceutical composition, preparation method and application of Artemordosins A-L

Abstract

The invention provides artemordosins AL (compounds 1-12) separated from Artemisia ordosins, a pharmaceutical composition of the artemordosins AL, a preparation method of the artemordosins AL and an application of the artemordosins AL, and belongs to the technical field of medicines. The 12 sesquiterpenoids artemordosins AL as shown in the structural formula (I) have inhibitory activity on human hepatoma cell lines HepG2, Huh7 and SK-Hep-1 cells, can form a pharmaceutical composition with a pharmaceutically acceptable carrier or excipient, and can be used for preparing anti-hepatoma drugs.

Description

Technical field:

[0001] The present invention belongs to the field of pharmaceutical technology. Specifically, it relates to 12 sesquiterpenoid compounds with new structures, artemordosins AL (compounds 1-12), and preparation methods and applications thereof, as well as pharmaceutical compositions with compounds 1-12 as active ingredients, their preparation methods, and applications thereof in the preparation of anti-liver cancer drugs. Background technology:

[0002] Patients with early-stage liver cancer can choose radical treatments such as liver resection, liver transplantation, and radiofrequency ablation to prolong their survival. However, due to the insidious onset, rapid development, and difficulty in early diagnosis of hepatocellular carcinoma, most patients have reached a local advanced stage or have metastasized when diagnosed, and are not suitable for the above treatments, and can only be treated with drugs.

[0003] There are about 380 species of Artemisia in the Asteraceae family, distributed all over the world, and 186 species and 44 varieties are distributed in my country. Among them, some plants are well-known folk and traditional Chinese medicines, such as Artemisia capillaris, Artemisia annua, etc., which are used to treat various diseases such as malaria, hepatitis, cancer, eczema, diarrhea, bruises and rheumatism. Phytochemical studies have shown that sesquiterpenes are one of the most important chemical components in Artemisia plants, and the common ones are eucalyptane type, guaiacane type, gemmaran type, cadinane type, etc. Literature reports that some of these sesquiterpenes have significant anti-tumor, anti-inflammatory, antiviral, and immunomodulatory effects.

[0004] Black Artemisia (A.ordosica Krasch.) is a perennial herbaceous plant of the genus Artemisia in the Asteraceae family, mainly distributed in the northwest and northern regions of my country. Black Artemisia is bitter and slightly warm in nature. Its whole plant can be used to treat urinary retention; its roots are used to stop bleeding; its stems, leaves and flower buds can be used to treat rheumatoid arthritis and sores, carbuncles and swellings; its fruits can reduce inflammation, relieve swelling, relieve chest tightness and promote qi, and kill insects. It can be applied to the affected area to treat mumps and sores, carbuncles and swellings, and taken internally to treat hernia, etc. Mongolian medicine uses it as an anti-inflammatory, hemostatic, wind-dispelling and heat-clearing medicine. Black Artemisia has rich chemical components, the main chemical components are phenols, coumarins, enynes, in addition, there are terpenes, sterols and other components. However, there have been no reports on the sesquiterpenoid components and anti-liver cancer activity in black Artemisia.

[0005] As a continuation of previous research, the present invention has isolated 12 new sesquiterpenoid compounds, artemordosins AL (compounds 1-12), from Artemisia sphaerocephala. So far, there is no report on artemordosins AL (compounds 1-12) in the prior art, nor on pharmaceutical compositions containing them as active ingredients, nor on the use of the compounds and their pharmaceutical compositions in the preparation or treatment of liver cancer drugs. Summary of the invention:

[0006] The object of the present invention is to provide a new class of artemordosins AL (compounds 1-12) with medicinal value as shown in formula (I), and a preparation method, a pharmaceutical composition and an application thereof. The present invention separates and identifies 12 sesquiterpenoid compounds with new structures, artemordosins AL (compounds 1-12), from Artemisia sphaerocephala. The compounds have obvious inhibitory activity on human liver cancer cell lines HepG2, Huh7 and SK-Hep-1 cells, and can be used to prepare anti-liver cancer drugs.

[0007] In order to achieve the above-mentioned object of the present invention, the present invention provides the following technical solutions:

[0008] The present invention provides a series of sesquiterpenoid compounds, artemordosins AL (compounds 1-12), the structures of which are shown in the following formula (I):

[0009]

[0010] The present invention further provides a method for preparing compound 1-12 shown in formula I,

[0011] The dried aerial part of Artemisia sphaerocephala was taken and crushed, and extracted twice with 3 times the amount of 90% ethanol. The crude extract obtained after the combined extracts were concentrated under reduced pressure was dispersed in water, and extracted with ethyl acetate to obtain the ethyl acetate extraction part; then, the ethyl acetate extraction part was subjected to silica gel column chromatography, and gradient eluted with acetone-petroleum ether at a volume ratio of 0:100, 5:95, 10:90, 20:80, 40:60 and 100:0 to obtain six fractions Frs.AF; the fraction Fr.D was subjected to silica gel column chromatography, and eluted with ethyl acetate-petroleum ether at a volume ratio of 10:90, 20:80, 30:70, 50:50 and 0:100 to obtain four fractions Fr.D1-Fr.D4. Fr.D2 was purified by MCI gel CHP20P column chromatography and eluted with methanol-water at 40:60, 50:50, 70:30, 90:10 and 100:0 to obtain four sub-fractions Frs.D2.1-D2.4. Fr.D2.2 was further purified by Rp-C 18 Column chromatography was performed with methanol-water at 50:50, 70:30, 90:10 and 100:0 to obtain three fractions Fr.D2.2.1-D2.2.3; Fr.D2.2.1 was eluted with methanol-chloroform at 50:50 on a Sephadex LH-20 dextran gel column, and then eluted with acetone-petroleum ether at a gradient of 2:98-100:0 on a silica gel column to obtain three fractions Fr.D2.2.1a-D2.2.1c; Fr.D2.2.1b was finally purified by semi-preparative HPLC on an Agilent XDB-C 18Compounds 9 and 10 (artemordosins I and J) were purified by elution with 34:66 acetonitrile-water on the column; Fr.D2.2.1c was finally purified by semi-preparative HPLC on Agilent XDB-C 18 Compounds 11 and 12 (artemordosins K and L) were purified by eluting with acetonitrile-water in a ratio of 32:68. Fr.D2.2.2 was purified by Sephadex LH-20 dextran gel column with methanol-chloroform 50:50, and then chromatographed on a silica gel column with ethyl acetate-petroleum ether 10:90-100:0 gradient elution to obtain three fractions Fr.D2.2.2a-D2.2.2c; Fr.D2.2.2a was finally purified by semi-preparative HPLC on Agilent XDB-C 18 Compound 8 (artemordosin H) was purified by elution with 36:64 acetonitrile-water on the column; Fr.D2.2.2b was finally purified by semi-preparative HPLC on Agilent XDB-C 18 Compound 7 (artemordosin G) was purified by eluting with 40:60 acetonitrile-water on the column. 18 Column chromatography was performed with methanol-water at 50:50, 70:30, 90:10 and 100:0 to obtain four fractions Fr.D2.3.1-D2.3.4; Fr.D2.3.2 was subjected to silica gel column chromatography and eluted with methanol-chloroform at a gradient of 2:98-15:85 to obtain four fractions Fr.D2.3.2a-D2.3.2d; Fr.D2.3.2b was finally purified by semi-preparative HPLC on Agilent XDB-C 18 Compound 1 (artemordosin A) was purified by eluting with 70:30 methanol-water on the column; Fr.D2.3.2c was finally purified by semi-preparative HPLC on Agilent XDB-C 18 Compound 2 (artemordosin B) was purified by eluting with acetonitrile-water at a ratio of 60:40. Fr.D3 was chromatographed on an MCI gel CHP 20P column using methanol-water at a ratio of 40:60, 50:50, 70:30, 90:10 and 100:0 to obtain four subfractions, Frs.D3.1-D3.4. Fr.D3.1 was further purified by Rp-C 18Column chromatography was performed with methanol-water at 40:60, 50:50, 60:40, 70:30 and 100:0 to obtain three fractions Fr.D3.1.1-D3.1.3; Fr.D3.1.1 was chromatographed on a Sephadex LH-20 column, eluted with methanol-chloroform at 50:50, and then chromatographed on a silica gel column with ethyl acetate-petroleum ether at 10:90-50:50 to obtain three fractions Fr.D3.1.1a-D3.1.1c; Fr.D3.1.1b was finally purified by semi-preparative HPLC on an Agilent XDB-C 18 Compound 6 (artemordosin F) was purified by eluting with 38:62 methanol-water on the column; Fr.D3.1.1c was finally purified by semi-preparative HPLC on Agilent XDB-C 18 Compounds 4 and 5 (artemordosins D and E) were purified by eluting with acetonitrile-water in a ratio of 29:71. Fr.D3.1.3 was purified by Sephadex LH-20 column chromatography with methanol-chloroform 50:50, and then by silica gel column chromatography with methanol-chloroform 2:98-15:85 to obtain three fractions Fr.D3.1.3a-D3.1.3c; Fr.D3.1.3b was finally purified by semi-preparative HPLC on Agilent XDB-C 18 The column was purified by eluting with 43:57 acetonitrile-water to obtain compound 3 (artemordosin C). The present invention provides the use of compounds 1-12 shown in formula I in the preparation of anti-liver cancer drugs. The present invention has no particular limitation on the method of the use, and any method known in the art can be used.

[0012] The present invention also provides a pharmaceutical composition, which comprises at least one of the compounds 1-12 represented by formula (I) and a pharmaceutically acceptable carrier.

[0013] In addition, the invention also provides the use of the pharmaceutical composition in the preparation of anti-liver cancer drugs. Also provided is a method for preparing the pharmaceutical composition: the compounds 1-12 of the invention are prepared by the above-mentioned method for preparing compounds, and then a pharmaceutically acceptable carrier is added.

[0014] When at least one of the compounds 1-12 is used to prepare an anti-liver cancer drug, the present invention preferably uses the compounds 1-12 directly or in the form of a pharmaceutical composition.

[0015] The pharmaceutical composition provided by the present invention comprises at least one of the above compounds 1-12 and a pharmaceutically acceptable carrier. In the present invention, the pharmaceutically acceptable carrier is preferably a solid, semisolid or liquid diluent, a filler and a pharmaceutical product adjuvant. The present invention has no particular limitation on the pharmaceutically acceptable carrier, and any pharmaceutically acceptable carrier well known in the art, non-toxic and inert to humans and animals can be selected.

[0016] The present invention has no particular limitation on the method for preparing the pharmaceutical composition. At least one of compounds 1-12 may be directly mixed with a pharmaceutically acceptable carrier. The present invention has no particular limitation on the mixing process. A pharmaceutical composition may be obtained by selecting a process well known in the art.

[0017] The present invention provides the use of the pharmaceutical composition described in the above technical solution in the preparation of anti-liver cancer drugs. There is no special limitation on the method of the application, and any method well known in the art can be used.

[0018] 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 the compounds 1-12 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 can preferably be administered in two forms: injection (intravenous injection, intramuscular injection) and oral administration.

[0019] Compared with the prior art, the present invention has the following advantages:

[0020] 1. The present invention provides 12 sesquiterpenoid compounds with new structures, artemordosins A–L (compounds 1-12).

[0021] 2. The present invention provides a new method for preparing new compounds 1-12, which has readily available raw materials and is easy to operate.

[0022] 3. The present invention provides a pharmaceutical composition with new compounds 1-12 as active ingredients, providing a new anti-liver cancer drug with better medicinal effects.

[0023] 4. Compounds 1-12 of the present invention have cytotoxic activity against HepG2, Huh7, and SK-Hep-1 at 200 μM. Among them, compounds 1, 2, 9, 10, and 11 have inhibitory effects on three liver cancer cells, and their IC 50The values ​​were 120.8, 37.1, 37.0, 42.0 and 38.7 μM (HepG2); 14.5, 27.7, 35.8, 58.4 and 52.7 μM (Huh7); 125.2, 39.6, 40.2, 87.6 and 82.4 μM (SK-Hep-1). The above results indicate that compounds 1-12 isolated from Artemisia sphaerocephala can be used as drugs to treat liver cancer-related diseases. Description of the drawings:

[0024] Figure 1 Schematic diagram of the structural formula of compound 1-12 of the present invention. Specific implementation method:

[0025] In order to better understand the essence of the present invention, the test examples and embodiments of the present invention are used below in conjunction with the accompanying drawings to further illustrate the preparation method, structural identification, pharmacological effects, and preparation method and drug composition of the present invention for artemordosins A-L (compounds 1-12), but the present invention is not limited to these test examples and embodiments.

[0026] The technical solutions in the present invention will be described clearly and completely below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0027] Embodiment 1:

[0028] Preparation of sesquiterpenoid compounds of the present invention, artemordosins A-L (compounds 1-12):

[0029] 51.5 kg of the above-ground part of dried Artemisia sphaerocephala was taken and crushed, and extracted twice with 3 times the amount of 90% ethanol. The crude extract obtained after the combined extracts were concentrated under reduced pressure was dispersed in water, and extracted with ethyl acetate to obtain an ethyl acetate extraction part; then, the ethyl acetate extraction part was subjected to silica gel column chromatography, and gradient eluted with acetone-petroleum ether at a volume ratio of 0:100, 5:95, 10:90, 20:80, 40:60 and 100:0 to obtain six fractions Frs.AF; fraction Fr.D (295 g) was subjected to silica gel column chromatography, and eluted with ethyl acetate-petroleum ether at a volume ratio of 10:90, 20:80, 30:70, 50:50 and 0:100 to obtain four fractions Fr.D1-Fr.D4; Fr.D2 (85 g) was subjected to MCI gel CHP 20P column chromatography, eluted with methanol-water 40:60, 50:50, 70:30, 90:10 and 100:0, to obtain four sub-fractions Frs.D2.1-D2.4; Fr.D2.2 (22 g) was further purified by Rp-C 18 Column chromatography was performed with methanol-water 50:50, 70:30, 90:10 and 100:0 to obtain three fractions Fr.D2.2.1-D2.2.3; Fr.D2.2.1 (6 g) was eluted with methanol-chloroform 50:50 on a Sephadex LH-20 dextran gel column, and then chromatographed on a silica gel column with a gradient elution of acetone-petroleum ether 2:98-100:0 to obtain three fractions Fr.D2.2.1a-D2.2.1c; Fr.D2.2.1b (504 mg) was finally purified by semi-preparative HPLC on an Agilent XDB-C 18 Compounds 9 (artemordosin I, 12 mg) and 10 (artemordosin J 16 mg) were purified by elution with 34:66 acetonitrile-water on the column. Fr.D2.2.1c (110 mg) was finally purified by semi-preparative HPLC on Agilent XDB-C 18 Compounds 11 (artemordosin K, 6 mg) and 12 (artemordosin L, 4 mg) were purified by eluting with acetonitrile-water in a ratio of 32:68 on the column. Fr.D2.2.2 (7 g) was purified by Sephadex LH-20 dextran gel column with methanol-chloroform 50:50, and then purified by silica gel column chromatography with ethyl acetate-petroleum ether 10:90-100:0 gradient elution to obtain three fractions Fr.D2.2.2a-D2.2.2c; Fr.D2.2.2a (680 mg) was finally purified by semi-preparative HPLC on Agilent XDB-C 18 Compound 8 (artemordosin H, 31 mg) was purified by elution with 36:64 acetonitrile-water on the column; Fr.D2.2.2b (420 mg) was finally purified by semi-preparative HPLC on Agilent XDB-C18 Compound 7 (artemordosin G, 37 mg) was purified by eluting with 40:60 acetonitrile-water on the column. Fr.D2.3 (25 g) was then purified by Rp-C 18 Column chromatography was performed with methanol-water 50:50, 70:30, 90:10 and 100:0 to obtain four fractions Fr.D2.3.1-D2.3.4; Fr.D2.3.2 was subjected to silica gel column chromatography and gradient elution with methanol-chloroform 2:98-15:85 to obtain four fractions Fr.D2.3.2a-D2.3.2d; Fr.D2.3.2b (98 mg) was finally purified by semi-preparative HPLC on Agilent XDB-C 18 Compound 1 (artemordosin A, 16.1 mg) was purified by eluting with 70:30 methanol-water on the column; Fr.D2.3.2c (78 mg) was finally purified by semi-preparative HPLC on Agilent XDB-C 18 Compound 2 (artemordosin B, 3.3 mg) was purified by eluting with 60:40 acetonitrile-water on the column. Fr.D3 (70 g) was chromatographed on an MCI gel CHP 20P column with methanol-water 40:60, 50:50, 70:30, 90:10 and 100:0 to obtain four sub-fractions Frs.D3.1-D3.4; Fr.D3.1 (24 g) was then purified by Rp-C 18 Column chromatography was performed with methanol-water at 40:60, 50:50, 60:40, 70:30 and 100:0 to obtain three fractions Fr.D3.1.1-D3.1.3; Fr.D3.1.1 (5 g) was eluted with methanol-chloroform at 50:50 on a Sephadex LH-20 column and then eluted with ethyl acetate-petroleum ether at 10:90-50:50 on a silica gel column to obtain three fractions Fr.D3.1.1a-D3.1.1c; Fr.D3.1.1b (310 mg) was finally purified by semi-preparative HPLC on an Agilent XDB-C 18 Compound 6 (artemordosin F, 34 mg) was purified by eluting with 38:62 methanol-water on the column; Fr.D3.1.1c was finally purified by semi-preparative HPLC on Agilent XDB-C 18Compounds 4 (artemordosin D, 29 mg) and 5 (artemordosin E, 6 mg) were purified by eluting with acetonitrile-water in a ratio of 29:71. Fr.D3.1.3 (4 g) was purified by Sephadex LH-20 column chromatography with methanol-chloroform 50:50 and then by silica gel column chromatography with methanol-chloroform 2:98-15:85 to obtain three fractions Fr.D3.1.3a-D3.1.3c; Fr.D3.1.3b (380 mg) was finally purified by semi-preparative HPLC on Agilent XDB-C 18 The product was purified by column elution with 43:57 acetonitrile-water to obtain compound 3 (artemordosin C, 34 mg).

[0030] Structural data of compounds 1-12:

[0031] Optical rotation was measured by Autopol VI polarimeter (Rudolph, USA); infrared spectrum was measured by Bio-Rad FTS-135 infrared spectrometer (Bio-Rad, USA) using KBr tablet method; UV spectrum was measured by UV-2401PC UV spectrometer (Shimadzu, Japan); circular dichroism spectrum was measured by Applied Photophysics circular dichroism spectrometer (Agilent, USA); nuclear magnetic resonance spectrum (one-dimensional and two-dimensional) was measured by Avance III-600 superconducting nuclear magnetic resonance instrument (Bruker, Germany), using CDCl3 as solvent; high-resolution mass spectrum was measured by Shimadzu LCMS-IT-TOF mass spectrometer (Shimadzu, Japan); thin layer chromatography silica gel plate HSGF254 was a product of Yantai Jiangyou Silica Gel Development Co., Ltd.; column chromatography silica gel (200-300 mesh) was produced by Linyi Haixiang Chemical Co., Ltd.; Sephadex LH-20 was purchased from GE Healthcare Bio-Sciences AB Company; the high performance liquid chromatograph was produced by Shimadzu Corporation, the controller model was CBM-20A, the pump model was LC-20AR, the detector model was SPD-M20A, the column oven model was AT-350, and the chromatographic column model used was Agilent-EClipse XDB-C18 (5μm, 9.4×250mm); chromatographically pure acetonitrile was purchased from Merida Company; MCI gel CHP20P (75~150μm) was purchased from Mitsubishi Chemical Corporation of Japan; the color developer was 10% H2SO4-EtOH solution.

[0032]

[0033] Artemordosin A(1)

[0034] Molecular formula: C 25 H37 O3

[0035] Molecular weight: 384

[0036] Properties: Colorless oil;

[0037] Positive ion mode high resolution mass spectrometry m / z 385.2727 [M+H] + (Calculated as C 25 H 37 O3,385.2737).

[0038] Infrared (KBr)v max 3429,1731,1659,1631,1444,1376,1233,1195,1125,1073cm -1

[0039] Circular dichroism (MeOH)λ max (Δε)208(+2.1)nm,236(-1.3)nm,274(+0.2)nm;

[0040] (c 0.10, MeOH);

[0041] The H NMR and C NMR data are shown in Table 1.

[0042]

[0043] Artemordosin B(2)

[0044] Molecular formula: C 25 H 38 O4

[0045] Molecular weight: 424

[0046] Properties: Colorless oil;

[0047] Positive ion mode high resolution mass spectrometry m / z 425.2657[M+H] + (Calculated as C 25 H 39 O4,425.2662).

[0048] Infrared (KBr)v max 3450,1716,1635,1412,1382,1165,1112,1097,1062,1013cm -1

[0049] Circular dichroism (MeOH)λ max (Δε)218(-1.02),265(-0.34)nm;

[0050] (c 0.12, MeOH)

[0051] The H-NMR and C-NMR data are shown in Table 2.

[0052]

[0053] Artemordosin C(3)

[0054] Molecular formula: C 15 H 24 O2

[0055] Molecular weight: 236

[0056] Properties: White amorphous powder;

[0057] Positive ion mode high resolution mass spectrum 237.1840 [M+H] + , (calculated as C 15 H 25 O2,237.1849).

[0058] Infrared (KBr)v max 3434,1695,1644,1448,1379,1287,1194,1085,1042cm -1

[0059] Circular dichroism (MeOH)λ max (Δε)235(+0.21),292(+1.44)nm;

[0060] (c 0.10, MeOH)

[0061] The H NMR and C NMR data are shown in Tables 2 and 3.

[0062]

[0063] Artemordosin D(4)

[0064] Molecular formula: C 15 H 24 O3

[0065] Molecular weight: 252

[0066] Properties: Colorless oil;

[0067] Positive ion mode high resolution mass spectrometry m / z 275.1617 [M+Na] + (Calculated as C 15 H 24O3Na,275.1618).

[0068] Infrared (KBr)v max 3450-2871,1702,1626,1450,1381,1261,1174,1124,1066cm -1

[0069] Circular dichroism (MeOH)λ max (Δε)198(+1.29),218(-1.15),241(+1.60)nm;

[0070] (c 0.30, MeOH)

[0071] The H NMR and C NMR data are shown in Tables 2 and 3.

[0072]

[0073] Artemordosin(5)

[0074] Molecular formula: C 15 H 18 O3

[0075] Molecular weight: 246

[0076] Properties: Yellow oil;

[0077] Positive ion mode high resolution mass spectrometry m / z 247.1324 [M+H] + (Calculated as C 15 H 19 O3,247.1329).

[0078] Infrared (KBr)v max 3430-2875,1716,1668,1613,1451,1384,1254,1180,1094cm -1

[0079] Circular dichroism (MeOH)λ max (Δε)214(-1.84),303(+5.44)nm;

[0080] (c 0.12, MeOH)

[0081] The H NMR and C NMR data are shown in Tables 2 and 3.

[0082]

[0083] Artemordosin F(6)

[0084] Molecular formula: C 15 H 20 O3

[0085] Molecular weight: 248

[0086] Properties: Colorless oil;

[0087] Positive ion mode high resolution mass spectrometry m / z 249.1510 [M+H] + (Calculated as C 15 H 21 O3,249.1485)

[0088] Infrared (KBr)v max 3444-2871,1708,1668,1626,1456,1381,1250,1172,1028cm -1

[0089] UV(MeOH)λ max (logε):237(0.35)

[0090] Circular dichroism (MeOH)λ max (Δε)217(-2.23),285(+0.28),332(-0.18)nm;

[0091] [α] 2 D 2 -81.3 (c 0.11, MeOH)

[0092] The H NMR and C NMR data are shown in Tables 2 and 3.

[0093]

[0094] Artemordosin G(7)

[0095] Molecular formula: C 15 H 26 O2

[0096] Molecular weight: 238

[0097] Properties: White amorphous powder;

[0098] Positive ion mode high resolution mass spectrometry m / z 261.1824 [M+Na] + (Calculated as C 15 H 26 O2Na,261.1825).

[0099] Infrared (KBr)v max3323,1748,1651,1473,1446,1374,1335,1304,1284,1211,1165,1136,1075cm -1

[0100] Circular dichroism (MeOH)λ max (Δε) 197(+4.46)nm;

[0101] [α] 2 D 5 -75.2(c 0.12,MeOH)

[0102] The H NMR and C NMR data are shown in Tables 2 and 3.

[0103]

[0104] Artemordosin H(8)

[0105] Molecular formula: C 15 H 22 O3

[0106] Molecular weight: 250

[0107] Properties: White amorphous powder;

[0108] Positive ion mode high resolution mass spectrometry m / z 273.1466 [M+Na] + (Calculated as C 15 H 22 O3Na,273.1461)

[0109] Infrared (KBr)v max 3492,1712,1634,1454,1378,1297,1208,1192,1160,1120,1082,1068,1052cm -1

[0110] Circular dichroism (MeOH)λ max (Δε)201(+2.13),218(-3.18)nm

[0111] [α] 2 D 1 +14.5(c 0.13,MeOH)

[0112] The H NMR and C NMR data are shown in Tables 2 and 3.

[0113]

[0114] Artemordosin I(9)

[0115] Molecular formula: C 15 H 20 O3

[0116] Molecular weight: 248

[0117] Properties: White amorphous powder;

[0118] Positive ion mode high resolution mass spectrometry m / z 249.1474 [M+H] + (Calculated as C 15 H 21 O3,249.1485).

[0119] Infrared (KBr)v max 3442,1769,1628,1453,1380,1348,1250,1223,1198,1170,1049,1026,1005cm -1

[0120] Circular dichroism (MeOH)λ max (Δε) 215 (+0.42) nm;

[0121] [α] 2 D 5 -96.4(c 0.45MeOH)

[0122] The H NMR and C NMR data are shown in Tables 2 and 3.

[0123]

[0124] Artemordosin J(10)

[0125] Molecular formula: C 15 H 20 O3

[0126] Molecular weight: 248

[0127] Properties: White amorphous powder;

[0128] Positive ion mode high resolution mass spectrometry m / z 271.1293 [M+Na] + (Calculated as C 15 H 20 O3Na,271.1305).IR(KBr)v max 3446,1744,1672,1449,1279,1149,1112,1082,1053,1013cm -1Circular dichroism (MeOH)λ max (Δε)219(-1.42),264(-0.68)nm

[0129] [α] 2 D 2 +56.6(c 0.15,MeOH)

[0130] The H NMR and C NMR data are shown in Tables 2 and 3.

[0131]

[0132] Artemordosin K(11)

[0133] Molecular formula: C 15 H 18 O4

[0134] Molecular weight: 262

[0135] Properties: White amorphous powder;

[0136] Positive ion mode high resolution mass spectrometry m / z 263.1279 [M+H] + (Calculated as C 15 H 19 O4,263.1278).IR (KBr)v max 3442,1767,1680,1455,1381,1266,1190,1172,1080,1054cm -1 UV(MeOH)λ max (logε):199(0.47),226(0.50)nm

[0137] Circular dichroism (MeOH)λ max (Δε)215(+4.97),248(-1.07),350(+0.38)nm

[0138] [α] 2 D 2 +59.6(c 0.05,MeOH)

[0139] The H NMR and C NMR data are shown in Tables 2 and 3.

[0140]

[0141] Artemordosin L(12)

[0142] Molecular formula: C 15 H 20 O3

[0143] Molecular weight: 248

[0144] Properties: Colorless oil;

[0145] Positive ion mode high resolution mass spectrometry m / z 249.1481 ([M+H] + , calculated as C 15 H 21 O3,249.1485).IR (KBr)v max 3438,1746,1685,1635,1449,1380,1260,1169cm -1

[0146] UV(MeOH)λ max (logε):218(0.70)nm

[0147] Circular dichroism (MeOH)λ max (Δε):208(+0.40),230(+0.86),246(-0.73)nm[α] 2 D 5 -84.6(c0.25,MeOH)

[0148] The H NMR and C NMR data are shown in Tables 2 and 3.

[0149] Table 1 NMR data of compound 1-2 (CDCl3, hydrogen spectrum 600MHz, carbon spectrum 150MHz)

[0150]

[0151]

[0152]

[0153] Embodiment 2:

[0154] Inhibitory activity of compounds 1-12 against three liver cancer cell lines.

[0155] 1. Materials and Methods

[0156] 1.1 Materials

[0157] HepG2, SK-Hep-1 and Huh7 cell lines were purchased from Shanghai Jining Biotechnology Co., Ltd.; cell culture medium (Dulbecco's Modified Eagle Medium, DMEM) was purchased from Thermo Fisher Scientific (Suzhou, China); serum (fetal bovine serum, FBS) was purchased from Thermo Fisher Scientific; RPMI-1640 was purchased from Thermo Fisher Biochemical Products (Beijing) Co., Ltd.

[0158] 1.2 Instruments

[0159] Flex Station 3 multifunctional microplate reader (Bio-RAD 680, USA); analytical balance (AG135, Metler Toledo, China); constant temperature box (DHP-9082, Shanghai).

[0160] 1.3 Experimental process

[0161] 1). Take the hepatoma cells in logarithmic phase, discard the old culture medium, wash twice with PBS, and discard the PBS;

[0162] 2) Digest the cells with 0.25% trypsin. When the cell outline becomes darker and tends to become round under the microscope, quickly remove the trypsin.

[0163] 3) Use DMEM complete medium containing 10% serum to stop digestion and resuspend the cells. Take 10 μL of cell suspension, count with a cell counter, and adjust the cell concentration to 1×10 4 / mL, inoculated on a 96-well plate, 100 μL of cell suspension was added to each well, and incubated in a 37°C, 5% CO2 incubator for 24 h to allow the cells to adhere;

[0164] 4). Aspirate the culture medium and add the diluted sample to the plate, add 100 μL to each well, set up 3 replicate wells for each concentration, and continue incubating in the incubator for 48 hours;

[0165] 5). Aspirate the culture medium, add the prepared MTT solution (1 mg / mL), add 100 μL to each well, and incubate in the incubator for 4 hours;

[0166] 6). Aspirate the MTT solution, add DMSO, add 100 μL to each well, and incubate in the incubator for 10 minutes;

[0167] 7). Use an enzyme-labeled instrument to measure the absorbance at a wavelength of 490 nm, and use the formula inhibition rate = (negative - experimental group)

[0168] / (negative-blank group)×100% to calculate the cell inhibition rate, and the IC was calculated using the statistical software GraphPad prism 550 , the experiment was repeated 3 times.

[0169] Table 4 Inhibitory activity of compounds on liver cancer cells

[0170]

[0171] Table 5 Inhibitory activity of compounds 1-12 against liver cancer cells at 200 and 100 μM

[0172]

[0173] Note: Sorafenib is a positive control, IC 50 8.65 (HepG2), 7.24 (Huh7), 10.97 (SK-Hep-1) μg / mL.

[0174] 2. Results

[0175] All the isolated compounds were evaluated for their anti-liver cancer cell cytotoxicity, and the experimental results are shown in Tables 4 and 5: Compounds 1-12 have cytotoxic activity against three liver cancer cell lines HepG2, Huh7, and SK-Hep-1 at 200 μM. Among them, compounds 1, 2, 9, 10, and 11 have inhibitory effects on the three liver cancer cell lines, and their IC 50 The values ​​were 120.8, 37.1, 37.0, 42.0 and 38.7 μM (HepG2); 14.5, 27.7, 35.8, 58.4 and 52.7 μM (Huh7); 125.2, 39.6, 40.2, 87.6 and 82.4 μM (SK-Hep-1). In summary, compound 2 has the best cytotoxic activity against three liver cancer cell lines HepG2, Huh7 and SK-Hep-1, with an IC 50 37.0, 27.7 and 39.6 μM respectively.

[0176] 3. Conclusion

[0177] The experimental results showed that compounds 1-12 had cytotoxic activity against three liver cancer cell lines: HepG2, Huh7, and SK-Hep-1. Compound 2 had moderate cytotoxic activity against HepG2 cells, with an IC 50 The value is 37.0 μM; Compound 2 has moderate cytotoxic activity against Huh7 cells, and its IC 50 The value was 27.7 μM; Compound 2 had moderate cytotoxic activity against SK-Hep-1 cells, and its IC 50 The value was 39.6 μM. The remaining compounds had certain inhibitory activity against the three liver cancer cells at 200 μM. In summary, compound 2 had the best cytotoxic activity against the three liver cancer cells HepG2, Huh7 and SK-Hep-1, and its IC50 The above results indicate that compounds 1-12 isolated from Artemisia sphaerocephala can be used as drugs to treat liver cancer-related diseases.

[0178] Formulation Examples

[0179] In the following formulation examples, conventional reagents are selected and the formulations are prepared according to conventional methods. This formulation example only reflects that at least one of the compounds 1-12 described in the present invention can be prepared into different formulations, and the specific reagents and operations are not specifically limited:

[0180] 1. Dissolve at least one of the compounds 1-12 of the present invention in a small amount of DMSO, add water for injection, filter, and sterilize the mixture to prepare an injection solution, wherein the concentration of the injection solution is 0.5-5 mg / mL.

[0181] 2. Dissolve at least one of the compounds 1-12 of the present invention in a small amount of DMSO, dissolve it in sterile water for injection, stir to dissolve, filter with a sterile suction funnel, filter aseptically, dispense into ampoules, freeze-dry at low temperature, and aseptically seal to obtain a powder injection.

[0182] 3. Add at least one of the compounds 1-12 of the present invention to an excipient at a weight ratio of 9:1 to the excipient to prepare a powder.

[0183] 4. Add at least one of the compounds 1-12 of the present invention to an excipient at a weight ratio of 5:1, and granulate and tablet.

[0184] 5. Prepare at least one of the compounds 1-12 of the present invention into an oral liquid according to a conventional oral liquid preparation method.

[0185] 6. At least one of the compounds 1-12 of the present invention is added to an excipient at a weight ratio of 5:1 to the excipient to prepare a capsule.

[0186] 7. At least one of the compounds 1-12 of the present invention is added to an excipient at a weight ratio of 5:1 to the excipient to prepare granules.

[0187] As can be seen from the above embodiments, the present invention provides a compound from Artemisia sphaerocephala and its preparation method and application, a pharmaceutical composition and its application. The artemordosins provided by the present invention mainly include 12 sesquiterpenoid compounds with new structures, which have different degrees of cytotoxic activity against liver cancer cells, can be combined with pharmaceutically acceptable carriers or excipients to form a pharmaceutical composition, and can be used to prepare anti-liver cancer drugs.

[0188] 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 principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. The compound artemordosins AL represented by the following structural formula (I), namely compounds 1-12, 2. A method for preparing the compound artemordosins AL represented by the structural formula (I) as claimed in claim 1, i.e., compounds 1-12, characterized in that The method comprises the following steps: taking the dried aerial part of Artemisia sphaerocephala, crushing it, extracting it twice with 3 times the amount of 90% ethanol, combining the extracts and concentrating them under reduced pressure to obtain a crude extract dispersed in water, and extracting it with ethyl acetate to obtain an ethyl acetate extraction part; subsequently, subjecting the ethyl acetate extraction part to silica gel column chromatography, and gradient eluting with acetone-petroleum ether at a volume ratio of 0:100, 5:95, 10:90, 20:80, 40:60 and 100:0 to obtain six fractions Frs.AF; subjecting the fraction Fr.D to silica gel column chromatography, and eluting with ethyl acetate-petroleum ether at a volume ratio of 10:90, 20:80, 30:70, 50:50 and 0:100 to obtain four fractions Fr.D1-Fr.D4; subjecting Fr.D2 to MCI gel CHP 20P column chromatography, eluted with methanol-water 40:60, 50:50, 70:30, 90:10 and 100:0, to obtain four sub-fractions Frs.D2.1-D2.4; Fr.D2.2 was further purified by Rp-C 18 Column chromatography was performed with methanol-water at 50:50, 70:30, 90:10 and 100:0 to obtain three fractions Fr.D2.2.1-D2.2.3; Fr.D2.2.1 was eluted with methanol-chloroform at 50:50 on a Sephadex LH-20 dextran gel column, and then eluted with acetone-petroleum ether at a gradient of 2:98-100:0 on a silica gel column to obtain three fractions Fr.D2.2.1a-D2.2.1c; Fr.D2.2.1b was finally purified by semi-preparative HPLC on an Agilent XDB-C 18 Compounds 9 and 10 were purified by elution with 34:66 acetonitrile-water on the column; Fr.D2.2.1c was finally purified by semi-preparative HPLC on Agilent XDB-C 18 Compounds 11 and 12 were purified by eluting with acetonitrile-water in a ratio of 32:68 on the column; Fr.D2.2.2 was purified by Sephadex LH-20 dextran gel column with methanol-chloroform 50:50, and then by silica gel column chromatography with ethyl acetate-petroleum ether 10:90-100:0 gradient elution to obtain three fractions Fr.D2.2.2a-D2.2.2c; Fr.D2.2.2a was finally purified by semi-preparative HPLC on Agilent XDB-C 18 Compound 8 was purified by elution with 36:64 acetonitrile-water on the column; Fr.D2.2.2b was finally purified by semi-preparative HPLC on Agilent XDB-C 18 Compound 7 was purified by eluting with 40:60 acetonitrile-water on the column; Fr.D2.3 was then purified by Rp-C 18 Column chromatography was performed with methanol-water at 50:50, 70:30, 90:10 and 100:0 to obtain four fractions Fr.D2.3.1-D2.3.4; Fr.D2.3.2 was subjected to silica gel column chromatography and eluted with methanol-chloroform at a gradient of 2:98-15:85 to obtain four fractions Fr.D2.3.2a-D2.3.2d; Fr.D2.3.2b was finally purified by semi-preparative HPLC on Agilent XDB-C 18 Compound 1 was purified by eluting with 70:30 methanol-water on the column; Fr.D2.3.2c was finally purified by semi-preparative HPLC on Agilent XDB-C 18 Compound 2 was purified by eluting with 60:40 acetonitrile-water on the column; Fr.D3 was chromatographed on an MCI gel CHP 20P column and eluted with methanol-water 40:60, 50:50, 70:30, 90:10 and 100:0 to obtain four subfractions Frs.D3.1-D3.4; Fr.D3.1 and then Rp-C 18 Column chromatography, treated with methanol-water 40:60, 50:50, 60:40, 70:30 and 100:0, obtained three fractions Fr.D3.1.1-D3.1.3; Fr.D3.1.1 was purified by Sephadex LH-20 column chromatography, eluted with methanol-chloroform 50:50, and then purified by silica gel column chromatography, eluted with ethyl acetate-petroleum ether 10:90-50:50, to obtain three fractions Fr.D3.1.1a-D3.1.1c; Fr.D3.1.1b was finally purified by semi-preparative HPLC on Agilent XDB-C 18 Compound 6 was purified by eluting with 38:62 methanol-water on the column; Fr.D3.1.1c was finally purified by semi-preparative HPLC on Agilent XDB-C 18 Compounds 4 and 5 were purified by eluting with acetonitrile-water in a ratio of 29:

71. Fr.D3.1.3 was purified by Sephadex LH-20 column chromatography with methanol-chloroform 50:50 and then by silica gel column chromatography with methanol-chloroform 2:98-15:85 to obtain three fractions Fr.D3.1.3a-D3.1.3c. Fr.D3.1.3b was finally purified by semi-preparative HPLC on Agilent XDB-C 18 Compound 3 was obtained by eluting the column with 43:57 acetonitrile-water.

3. Use of the compound black artemordosins AL, i.e. compounds 1-12, represented by structural formula (I) as claimed in claim 1 in the preparation of anti-liver cancer drugs.

4. A pharmaceutical composition comprising the compound artemordosins AL represented by the structural formula (I) as claimed in claim 1, that is, at least one of compounds 1-12 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: taking the dried aerial part of Artemisia sphaerocephala, crushing it, extracting it twice with 3 times the amount of 90% ethanol, combining the extracts and concentrating them under reduced pressure to obtain a crude extract dispersed in water, and extracting it with ethyl acetate to obtain an ethyl acetate extraction part; subsequently, subjecting the ethyl acetate extraction part to silica gel column chromatography, and gradient eluting with acetone-petroleum ether at a volume ratio of 0:100, 5:95, 10:90, 20:80, 40:60 and 100:0 to obtain six fractions Frs.AF; subjecting the fraction Fr.D to silica gel column chromatography, and eluting with ethyl acetate-petroleum ether at a volume ratio of 10:90, 20:80, 30:70, 50:50 and 0:100 to obtain four fractions Fr.D1-Fr.D4; subjecting Fr.D2 to MCI gel CHP 20P column chromatography, eluted with methanol-water 40:60, 50:50, 70:30, 90:10 and 100:0, to obtain four sub-fractions Frs.D2.1-D2.4; Fr.D2.2 was further purified by Rp-C 18 Column chromatography was performed with methanol-water at 50:50, 70:30, 90:10 and 100:0 to obtain three fractions Fr.D2.2.1-D2.2.3; Fr.D2.2.1 was eluted with methanol-chloroform at 50:50 on a Sephadex LH-20 dextran gel column, and then eluted with acetone-petroleum ether at a gradient of 2:98-100:0 on a silica gel column to obtain three fractions Fr.D2.2.1a-D2.2.1c; Fr.D2.2.1b was finally purified by semi-preparative HPLC on an Agilent XDB-C 18 Compounds 9 and 10 were purified by elution with 34:66 acetonitrile-water on the column; Fr.D2.2.1c was finally purified by semi-preparative HPLC on Agilent XDB-C 18 Compounds 11 and 12 were purified by eluting with acetonitrile-water in a ratio of 32:68 on the column; Fr.D2.2.2 was purified by Sephadex LH-20 dextran gel column with methanol-chloroform 50:50, and then by silica gel column chromatography with ethyl acetate-petroleum ether 10:90-100:0 gradient elution to obtain three fractions Fr.D2.2.2a-D2.2.2c; Fr.D2.2.2a was finally purified by semi-preparative HPLC on Agilent XDB-C 18 Compound 8 was purified by elution with 36:64 acetonitrile-water on the column; Fr.D2.2.2b was finally purified by semi-preparative HPLC on Agilent XDB-C 18 Compound 7 was purified by eluting with 40:60 acetonitrile-water on the column; Fr.D2.3 was then purified by Rp-C 18 Column chromatography was performed with methanol-water at 50:50, 70:30, 90:10 and 100:0 to obtain four fractions Fr.D2.3.1-D2.3.4; Fr.D2.3.2 was subjected to silica gel column chromatography and eluted with methanol-chloroform at a gradient of 2:98-15:85 to obtain four fractions Fr.D2.3.2a-D2.3.2d; Fr.D2.3.2b was finally purified by semi-preparative HPLC on Agilent XDB-C 18 Compound 1 was purified by eluting with 70:30 methanol-water on the column; Fr.D2.3.2c was finally purified by semi-preparative HPLC on Agilent XDB-C 18 Compound 2 was purified by eluting with 60:40 acetonitrile-water on the column; Fr.D3 was chromatographed on an MCI gel CHP 20P column and eluted with methanol-water 40:60, 50:50, 70:30, 90:10 and 100:0 to obtain four subfractions Frs.D3.1-D3.4; Fr.D3.1 and then Rp-C 18 Column chromatography, treated with methanol-water 40:60, 50:50, 60:40, 70:30 and 100:0, obtained three fractions Fr.D3.1.1-D3.1.3; Fr.D3.1.1 was purified by Sephadex LH-20 column chromatography, eluted with methanol-chloroform 50:50, and then purified by silica gel column chromatography, eluted with ethyl acetate-petroleum ether 10:90-50:50, to obtain three fractions Fr.D3.1.1a-D3.1.1c; Fr.D3.1.1b was finally purified by semi-preparative HPLC on Agilent XDB-C 18 Compound 6 was purified by eluting with 38:62 methanol-water on the column; Fr.D3.1.1c was finally purified by semi-preparative HPLC on Agilent XDB-C 18 Compounds 4 and 5 were purified by eluting with acetonitrile-water in a ratio of 29:

71. Fr.D3.1.3 was purified by Sephadex LH-20 column chromatography with methanol-chloroform 50:50 and then by silica gel column chromatography with methanol-chloroform 2:98-15:85 to obtain three fractions Fr.D3.1.3a-D3.1.3c. Fr.D3.1.3b was finally purified by semi-preparative HPLC on Agilent XDB-C 18 Compound 3 was purified by eluting with 43:57 acetonitrile-water on the column; then any one of the above compounds 1-12 or any combination thereof was added with a pharmaceutically acceptable carrier.

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