CN116284036A - Artemisia reevesii lactone A, B, D-P and pharmaceutical composition thereof, preparation method and application thereof - Google Patents

Abstract

The invention provides 15 novel compounds shown in a structural formula (I), namely, artelactone A, B, D-P (artemiprinolides A, B, D-P, 1-15), a pharmaceutical composition thereof, a preparation method and application thereof, and belongs to the technical field of medicines. The compound has remarkable cytotoxic activity on human liver cancer cell strains HepG2, huh7 and SK-Hep-1, can form a pharmaceutical composition with a pharmaceutically acceptable carrier, and can be used for preparing anti-liver cancer drugs.

Description

Artemisia reevesii lactone A, B, D-P and pharmaceutical composition thereof, preparation method and application thereof

Technical field:

the invention belongs to the technical field of medicines. In particular to a medical composition taking the kui artelactone A, B, D-P (artemiprinolides A, B, D-P, 1-15) as an active ingredient, a preparation method thereof and application thereof in pharmacy.

The background technology is as follows:

liver cancer (liver cancer) is one of the main causes of malignant tumor death in China, and has hidden morbidity, complex etiology and difficult early diagnosis, and the liver cancer often enters middle and late stages when found, so that the prognosis is poor, which brings great challenges to a new treatment method. The latest cancer report issued in the scientific progress of cancer (JNCC) of 2022 month shows that 38.9 thousands of new cases of liver cancer in China are listed at the 4 th place of malignant tumor, 33.6 thousands of annual death cases are listed at the second place of malignant tumor death. The HCC disease is heavily burdened, and early epidemiological and mechanistic studies indicate that chronic HBV infection is a major risk factor for HCC in our country, and that HBV infection has heterogeneity in HCC occurrence. HCC places a very heavy disease burden worldwide, but the pattern of disease varies greatly from region to region. Currently, there are 7 clinical drugs for treating liver cancer patients: sorafenib (sorafenib), regorafenib (revafenib), lenvatinib (lenvatinib), caboztinib (cabozantinib), nivolumab (nivolumab), pamumab (pembrolizumab) and Lei Molu monoclonal antibody (ramucirumab), but with resistance and some toxic side effects. Although autonomous research and development of medicines in China also rapidly develop, such as apatinib mesylate, carilizumab, tirelizumab and the like, enrich liver cancer treatment schemes, some problems still exist. Therefore, development of novel effective anti-liver cancer drugs is urgently required. Natural products have unique structure and low toxicity, and are important groups for finding novel anti-liver cancer active ingredients.

Artemisia (Artemisia L.) is a perennial herb plant, with about 380 species in the world, 186 species in China, 44 varieties, 82 unique species in China, and all over the country. A great deal of research at home and abroad shows that the plant has wide modern pharmacological activities such as antimalarial, anti-inflammatory, anticancer, antifungal infection, antiviral and the like, and a plurality of varieties of the plant have important medical value in folks. The representative sesquiterpene molecular artemisinin from the Artemisia annua of the genus plant creates a new generation of antimalarial drugs, is an excellent example of successful research and development from the treasury of traditional Chinese drugs, and has good antimalarial activity, and also has certain anti-tumor, antifungal and immunoregulatory effects. The plant is more and more focused by students at home and abroad because of its wide medicinal value, novel and diverse secondary metabolites and diverse biological activities.

The Artemisia princeps (Artemisia princeps) is a perennial herb plant, and is also called Artemisia rupestris, artemisia annua, artemisia princeps, artemisia annua, and distributed in northern, eastern and western provinces of China, and is mainly used at low altitude or on roadside, hillside, shrub, forest margin and ditch side of Zhonghai region. Japanese and Korean are also known. The Chinese medicinal composition is used as substitute for folium Artemisiae Argyi, and has effects of dispelling cold and dampness, regulating qi and blood, regulating menstruation, preventing miscarriage, stopping bleeding, and relieving inflammation.

To date, the prior art has no report of brothers lactone A, B, D-P (artemiprinolides A, B, D-P, 1-15), no report of pharmacological activity, no report of pharmaceutical composition taking the brothers lactone A, B, D-P,1-15 as an active ingredient, and no report of application of the brothers lactone A, B, D-P,1-15 in preparing medicines for treating liver cancer.

The invention comprises the following steps:

the invention aims to provide novel kui-artelactone A, B, D-P (artemiprinolides A, B, D-P, 1-15) with medicinal value as well as a pharmaceutical composition, a preparation method and application thereof.

In order to achieve the above object of the present invention, the present invention provides the following technical solutions:

the invention provides a series of sesquiterpene dimer compounds, namely, brothers' artelactone A, B and D-P (artemiprinolides A, B, D-P and 1-15), which have the structure shown in the following formula (I):

the invention provides a preparation method of the compound 1-15, crushing the dried overground part of artemisia kui, carrying out cold leaching extraction twice by using 90% ethanol with the amount of 10 times, combining ethanol extract, concentrating under reduced pressure until no ethanol smell exists, obtaining ethanol extract, dispersing the extract in water, extracting for 3 times by using ethyl acetate to obtain an ethyl acetate extract part, carrying out silica gel column chromatography on the ethyl acetate extract part, and carrying out gradient elution by using acetone-petroleum ether (10:90, 20:80,30:70,40:60, v/v) and acetone to obtain 5 fractions Fr.1-Fr.5; fr.2 is subjected to medium pressure MCI CHP 20P column chromatography, and water-methanol (50:50, 30:70,10:90, 0:100) gradient elution is carried out to obtain 4 components Fr.2-1-Fr.2-4; fr.2-3 was subjected to silica gel column chromatography (ethyl acetate-petroleum ether, 20:80,30:70,40:60,100:0, v/v) to give 4 fractions Fr.2-3-1-Fr.2-3-4; fr.2-3-2 is subjected to medium pressure RP-C8 column chromatography and is subjected to gradient elution by water-methanol (40:60, 20:80, 10:90) to obtain 3 components Fr.2-3-2-1-Fr.2-3-2-3; subjecting Fr.2-3-2-1 to Sephadex LH-20 gel column chromatography (methanol-chloroform, 50:50) to obtain 3 components Fr.2-3-2-1a-Fr.2-3-2-1c; fr.2-3-2-1a was purified by semi-preparative HPLC (water-methanol, 32:68) to give compound 5; fr.2-3-2-1b was purified by semi-preparative HPLC (water-methanol, 33:67) to give compounds 6,7,8 and 11; fr.2-3-3 is subjected to medium pressure RP-C18 column chromatography and is subjected to gradient elution by water-methanol (40:60, 20:80, 0:100) to obtain 3 components Fr.2-3-3-1-Fr.2-3-3-3; subjecting Fr.2-3-3-1 to Sephadex LH-20 gel column chromatography (methanol-chloroform, 50:50) to obtain 3 components Fr.2-3-3-1a-Fr.2-3-3-1c; fr.2-3-3-1b was purified by semi-preparative HPLC (water-acetonitrile, 45:55) to give compounds 1,2 and 3; fr.3 is subjected to medium pressure MCI CHP 20P column chromatography, and water-methanol (50:50, 30:70,10:90, 0:100) gradient elution is carried out to obtain 4 components Fr.3-1-Fr.3-4; fr.3-2 is subjected to medium pressure RP-C18 column chromatography and is subjected to gradient elution by water-methanol (50:50, 40:60, 30:70) to obtain 3 components Fr.3-2-1-Fr.3-2-3; fr.3-2-1 is subjected to medium pressure RP-C18 column chromatography and is subjected to gradient elution by water-acetonitrile (75:25, 70:30,65:35, 60:40) to obtain 4 components Fr.3-2-1-1-Fr.3-2-1-4; fr.3-2-1-3 was subjected to silica gel column chromatography (ethyl acetate-petroleum ether, 40:60 to 100:0) to give 3 fractions Fr.3-2-1-3a-Fr.3-2-1-3c; fr.3-2-1-3c was purified by semi-preparative HPLC (water-methanol, 32:68) to give compound 15; fr.3-2-1-3a was purified by semi-preparative HPLC (water-acetonitrile, 60:40) to give compounds 9 and 12; fr.3-2-2 is subjected to medium pressure RP-C18 column chromatography and is eluted with a water-acetonitrile (70:30 to 50:50) gradient to obtain 5 components Fr.3-2-2-1-Fr.3-2-2-5; fr.3-2-2-2 was purified by preparative HPLC (water-acetonitrile, 60:40) to give 5 subfractions Fr.3-2-2-2a-3-2-2-2e; fr.3-2-2-2b was purified by semi-preparative HPLC (water-methanol, 42:58) to give compound 4; fr.3-2-2-2d was purified by semi-preparative HPLC (water-acetonitrile, 62:38) to give 5 subfractions Fr.3-2-2-2d-1-Fr.3-2-2-2d-5; fr.3-2-2-2d-1 was purified by semi-preparative HPLC (water-methanol, 50:50) to give compound 10; fr.3-2-2-2d-3 was purified by semi-preparative HPLC (water-methanol, 50:50) to give compounds 13 and 14.

The invention provides application of the compounds 1-15 in preparing anti-liver cancer drugs and in preparing anti-tumor inhibitors. The method of the present invention is not particularly limited, and methods well known in the art may be used.

The invention also provides a pharmaceutical composition comprising at least one of the compounds 1-15 of formula (I) and a pharmaceutically acceptable carrier.

And the application of the pharmaceutical composition in preparing anti-liver cancer drugs and in preparing anti-tumor inhibitors is also provided.

Meanwhile, the invention also provides a preparation method of the pharmaceutical composition, which comprises the steps of preparing the compounds 1-15, taking at least one of the compounds 1-15, and adding a pharmaceutically acceptable carrier.

When at least one of the compounds 1 to 15 is used for preparing an anti-liver cancer drug, the present invention preferably uses the compound 1 to 15 directly or in the form of a pharmaceutical composition.

The invention provides a pharmaceutical composition comprising at least one of the above compounds 1-15 and a pharmaceutically acceptable carrier or excipient. In the present invention, the pharmaceutically acceptable carrier or excipient is preferably a solid, semi-solid or liquid diluent, filler and pharmaceutical preparation adjuvant. The pharmaceutically acceptable carrier or excipient is not particularly limited, and pharmaceutically acceptable carriers and/or excipients which are well known in the art, nontoxic and inert to human and animals can be selected.

The preparation method of the pharmaceutical composition is not particularly limited, at least one of the compounds 1-15 is directly mixed with a pharmaceutically acceptable carrier or excipient, the mixing process is not particularly limited, and the pharmaceutical composition can be obtained by selecting processes well known in the art.

The invention provides application of the pharmaceutical composition in preparation of anti-liver cancer drugs. The method of the present invention is not particularly limited, and methods well known in the art may be used.

In the present invention, when the pharmaceutical composition is used for preparing 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 the compounds 1 to 15 in the pharmaceutical composition is preferably 0.5 to 90%. The pharmaceutical composition of the present invention is preferably used in the form of a unit weight dose. In the present invention, the prepared medicine may be preferably administered in both injection (intravenous injection, intramuscular injection) and oral administration.

Compared with the prior art, the invention has the following advantages:

1. the invention provides a series of novel sesquiterpene dimer compounds, namely, brotheron A, B, D-P (artemiprinolides A, B, D-P, 1-15).

2. The invention provides a novel method for preparing novel compounds 1-15, which has the advantages of easily available raw materials, simple process and easy operation.

3. The invention provides a pharmaceutical composition with novel compounds 1-15 as active ingredients, and provides a novel drug with better medicinal effect for a novel anti-liver cancer drug.

4. The compounds 1-15 of the invention have stronger activity on the cytotoxicity of three liver cancer cells (HepG 2, SK-HEP-1 and Huh 7), and the compound 1 has stronger cytotoxicity on the HepG2 cells and IC thereof ₅₀ The value is 5.4 mu M, which is better than that of the positive medicine sorafenib; compounds 3,11 and 15 also exhibit a certain cytotoxic activity, IC ₅₀ The values were 11.6,13.0 and 11.8. Mu.M, respectively, which are comparable to the positive drug sorafenib. For the followingHuh7 cells, compound 1 has stronger cytotoxic activity, IC thereof ₅₀ The values are 7.7 mu M respectively, which is better than the positive medicine sorafenib; compound 15 also has a cytotoxic activity, IC ₅₀ The value was 13.4. Mu.M, which is comparable to the positive drug sorafenib. For SK-Hep-1 cells, compounds 1 and 15 have potent cytotoxic activity, IC ₅₀ The values are 11.8,14.3 mu M respectively, which is superior to the positive drug sorafenib; compounds 2,3 and 7 also have a certain cytotoxic activity, IC ₅₀ The values were 18.7,20.1 and 19.0. Mu.M, respectively, which are comparable to the positive drug sorafenib. Compounds 3 and 11 showed significant cytotoxic activity only against HepG2 cells, IC ₅₀ Values were 11.6 and 13.0 μm, respectively; interestingly, compound 1 had strong cytotoxic activity against all three liver cancer cells, its IC ₅₀ The values were 5.4,7.7 and 11.8. Mu.M, respectively.

5. Sesquiterpene dimer brotherin A, B and D-P (1-15) separated from brotherin can be used as medicines for treating liver cancer related diseases.

Description of the drawings:

FIG. 1 is a schematic representation of the structural formula of compounds 1-15 of the present invention;

FIG. 2 is a schematic diagram of the X-single crystal diffraction structure of Compound 1 of the present invention.

The specific embodiment is as follows:

in order to better understand the essence of the present invention, the sesquiterpene dimers of the present invention, artelactone a, B, D-P (artemiprinolides A, B, D-P, 1-15), and the preparation method, structure identification, pharmacological effects thereof will be further described with reference to the accompanying drawings, but the present invention is not limited to this example.

The technical solutions of the present invention will be clearly and completely described in the following in connection with the embodiments of the present invention. It will be apparent that the described embodiments are only some, but not all, embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.

Example 1:

preparation of Compounds 1-15:

the aerial parts (49.5 kg) of the artemisia kui are dried, crushed, subjected to cold leaching with 90% ethanol with the amount of 10 times for two times, the ethanol extract is combined and concentrated under reduced pressure until no ethanol smell exists, the ethanol extract is obtained, the extract is dispersed in water, ethyl acetate is used for extraction for 3 times, an ethyl acetate extraction part (2.0 kg) is obtained, the ethyl acetate extraction part is subjected to silica gel column chromatography, and gradient elution is carried out by acetone-petroleum ether (10:90, 20:80,30:70,40:60, v/v) and acetone to obtain 5 fractions Fr.1-Fr.5 (867, 205,147,90,415 g); fr.2 is subjected to medium pressure MCI CHP 20P column chromatography, and water-methanol (50:50, 30:70,10:90, 0:100) gradient elution is carried out to obtain 4 components Fr.2-1-Fr.2-4; fr.2-3 was subjected to silica gel column chromatography (ethyl acetate-petroleum ether, 20:80,30:70,40:60,100:0, v/v) to give 4 fractions Fr.2-3-1-Fr.2-3-4; fr.2-3-2 is subjected to medium pressure RP-C8 column chromatography and is subjected to gradient elution by water-methanol (40:60, 20:80, 10:90) to obtain 3 components Fr.2-3-2-1-Fr.2-3-2-3; subjecting Fr.2-3-2-1 to Sephadex LH-20 gel column chromatography (methanol-chloroform, 50:50) to obtain 3 components Fr.2-3-2-1a-Fr.2-3-2-1c; fr.2-3-2-1a was purified by semi-preparative HPLC (water-methanol, 32:68) to give compound 5 (4 mg); fr.2-3-2-1b was purified by semi-preparative HPLC (water-methanol, 33:67) to give compounds 6 (13 mg), 7 (16 mg), 8 (20 mg) and 11 (6 mg); fr.2-3-3 is subjected to medium pressure RP-C18 column chromatography and is subjected to gradient elution by water-methanol (40:60, 20:80, 0:100) to obtain 3 components Fr.2-3-3-1-Fr.2-3-3-3; subjecting Fr.2-3-3-1 to Sephadex LH-20 gel column chromatography (methanol-chloroform, 50:50) to obtain 3 components Fr.2-3-3-1a-Fr.2-3-3-1c; fr.2-3-3-1b was purified by semi-preparative HPLC (water-acetonitrile, 45:55) to give compounds 1 (8 mg), 2 (10 mg) and 3 (11 mg); fr.3 is subjected to medium pressure MCI CHP 20P column chromatography, and water-methanol (50:50, 30:70,10:90, 0:100) gradient elution is carried out to obtain 4 components Fr.3-1-Fr.3-4; fr.3-2 is subjected to medium pressure RP-C18 column chromatography and is subjected to gradient elution by water-methanol (50:50, 40:60, 30:70) to obtain 3 components Fr.3-2-1-Fr.3-2-3; fr.3-2-1 is subjected to medium pressure RP-C18 column chromatography and is subjected to gradient elution by water-acetonitrile (75:25, 70:30,65:35, 60:40) to obtain 4 components Fr.3-2-1-1-Fr.3-2-1-4; fr.3-2-1-3 was subjected to silica gel column chromatography (ethyl acetate-petroleum ether, 40:60 to 100:0) to give 3 fractions Fr.3-2-1-3a-Fr.3-2-1-3c; fr.3-2-1-3c was purified by semi-preparative HPLC (water-methanol, 32:68) to give compound 15 (5 mg); fr.3-2-1-3a was purified by semi-preparative HPLC (water-acetonitrile, 60:40) to give compounds 9 (35 mg) and 12 (23 mg); fr.3-2-2 is subjected to medium pressure RP-C18 column chromatography and is eluted with a water-acetonitrile (70:30 to 50:50) gradient to obtain 5 components Fr.3-2-2-1-Fr.3-2-2-5; fr.3-2-2-2 was purified by preparative HPLC (water-acetonitrile, 60:40) to give 5 subfractions Fr.3-2-2-2a-3-2-2-2e; fr.3-2-2-2b was purified by semi-preparative HPLC (water-methanol, 42:58) to give compound 4 (5 mg); fr.3-2-2-2d was purified by semi-preparative HPLC (water-acetonitrile, 62:38) to give 5 subfractions Fr.3-2-2-2d-1-Fr.3-2-2-2d-5; fr.3-2-2-2d-1 was purified by semi-preparative HPLC (water-methanol, 50:50) to give compound 10 (22 mg); fr.3-2-2-2d-3 was purified by semi-preparative HPLC (water-methanol, 50:50) to give compounds 13 (5 mg) and 14 (7 mg).

Structural data for compounds 1-15:

nuclear magnetic resonance spectroscopy was performed using Avance III 600 (Bruker,

switzerland) or Avance III HD 400 (Bruker, bremerhaven, germany) as an internal standard. High resolution mass spectrometry was performed using Shimadzu LC-MS-IT-TOF (Shimadzu, kyoto, japan). Infrared spectroscopy (IR) was determined by KBr tabletting method by NICOLET iS10 infrared spectrometer (Thermo Fisher Scientific, madison, USA). ECD spectra were measured using a Chirascan-type instrument (Applied Photophysics, surrey, UK). The optical rotation was determined by an Autopol VI polarimeter (Rudolph Research Analytical, hackettstown, USA). Melting point->

The measurements were made by a microscopic melting point apparatus, available from Shanghai precision scientific instruments Inc. The thin layer chromatography silica gel plate HSGF254 is a product of Nicotiana tabacum Jiang you silica gel development Co., ltd; column chromatography silica gel (200-300 meshes) is produced by Yi Ling Shang Hai Xiang chemical industry Co., ltd; column chromatography Sephadex LH-20 is available from GE Healthcare Bio-Sciences AB. The high performance liquid chromatograph is manufactured by Shimadzu corporation, the controller model is CBM-20A, the pump model is LC-20AR, the detector model is SPD-M20A, the column temperature box model is AT-350, and the used chromatographic column model is Agilent-Eclipse XDB-C18 (5 μm, 9.4X1250 mm). Chromatographic purityAcetonitrile was purchased from merida and deionized water was purified by the mingchem-D24 UV Merk Millipore system. The medium pressure liquid phase (Dr Flash-II) is the product of Shanghai Lisui company, mitsubishi corporation of Japan, MCI column, model CHP-20P (75-150 μm). Analytically pure methanol and acetonitrile were purchased from Tianjin metallocene chemical reagent plant. The color-developing agent is 10% H ₂ SO ₄ -EtOH solution.

Artemisia princeps lactone A (1)

The molecular formula: c (C) ₃₅ H ₄₀ O ₈

Molecular weight: 588.27

Traits: monoclinic crystal

Melting point: 232-233 DEG C

Optical rotation:

HRESIMS (+) m/z Experimental values 589.2793[ M+H ]] ⁺ Calculated 589.2796[ M+H ]] ⁺ 。

IR(KBr)ν _max :3435,1769,1697,1650,1383,1287,1072,1042cm ^-1 。

ECD (methanol) lambda _max (Δε):208(+3.42),219(+6.81),233(+3.46),243(+4.51),275(–0.79)nm。

Crystal data: c (C) ₃₅ H ₄₀ O ₈ ·2(H ₂ O),M＝624.70,

α＝90°,β＝117.2390(10)°,γ＝90°,/>

T=100. (2) K, lattice dimensions P1211, z=2, μ (Cu kα) =0.782 mm ^-1 The crystal data were measured using a D8 QUEST type crystal diffractometer (copper target) with a total diffraction order of 29440, where 625 is observed4 times (R) _int ＝0.0295),I>2σ(I),R ₁ ＝0.0279,wR(F ² )＝0.0730,F ² = 1.049,Flack parameter = -0.02 (3). The crystal parameters of compound 1 have been stored in the cambridge crystal data center, extract No.: CCDC 2207150. Web site: https:// www.ccdc.cam.ac.uk.

The X-single crystal diffraction structure of compound 1 is shown in FIG. 2.

¹ H NMR ¹³ The C NMR (DEPT) data are shown in tables 1 and 4.

Artemisia princeps lactone B (2)

The molecular formula: c (C) ₃₅ H ₄₀ O ₈

Molecular weight: 588.27

Traits: white amorphous powder

Optical rotation:

HRESIMS (+) m/z Experimental values 589.2791[ M+H ]] ⁺ Calculated 589.2796[ M+H ]] ⁺ 。

IRν _max :3435,1768,1714,1648,1379,1257,1153,1028cm ^-1 。

ECD (methanol) lambda _max (Δε):207(+4.30),217(+6.25),235(–0.62),247(+0.34),270(–1.01)nm。

¹ H NMR ¹³ The C NMR (DEPT) data are shown in tables 1 and 4.

Artemisia princeps lactone D (3)

The molecular formula: c (C) ₃₄ H ₄₀ O ₈

Molecular weight: 576.27

Traits: white amorphous powder

Optical rotation:

HRESIMS (-) m/z Experimental value 621.2694[ M+HCOO ]]Calculated value 621.2705[ M+HCOO ]] ^– 。

IR(KBr)ν _max :3434,1768,1738,1696,1650,1383,1266,1150,1086cm ^-1 。

ECD (methanol) lambda _max (Δε):212(–17.18),240(+2.31),269(–1.94)nm。

¹ H NMR ¹³ The C NMR (DEPT) data are shown in tables 1 and 4.

Artemisia princeps lactone E (4)

The molecular formula: c (C) ₃₀ H ₃₄ O ₇

Molecular weight: 506.23

Traits: white amorphous powder

Optical rotation:

HRESIMS (-) m/z Experimental value 551.2279[ M+HCOO ]] ^– Calculated 551.2287[ M+HCOO] ^– 。

IRν _max :3435,1762,1687,1622,1441,1266,1218,1145,1092cm ^-1 。

ECD (methanol) lambda _max (Δε):212(–13.79),234(+15.43),268(–20.01),351(+4.54)nm。

¹ H NMR ¹³ The C NMR (DEPT) data are shown in tables 1 and 4.

Artemisia princeps lactone F (5)

The molecular formula: c (C) ₃₄ H ₄₀ O ₈

Molecular weight: 576.27

Traits: white amorphous powder

Optical rotation:

HRESIMS (-) m/z Experimental value 621.2700[ M+HCOO ]] ^– Calculated 621.2705[ M+HCOO] ^– 。

IRν _max :3436,1768,1739,1697,1652,1383,1219,1204,1187cm ^-1 。

ECD (methanol) lambda _max (Δε):220(–4.19),244(+0.35),270(–1.95)nm。

¹ H NMR ¹³ The C NMR (DEPT) data are shown in tables 1 and 4.

Artemisia princeps lactone G (6)

The molecular formula: c (C) ₃₅ H ₄₂ O ₈

Molecular weight: 590.28

Traits: white amorphous powder

Optical rotation:

HRESIMS (-) m/z Experimental value 635.2667[ M+HCOO ]] ^– Calculated 635.2663[ M+HCOO] ^– 。

IR(KBr)ν _max :3469,1767,1737,1697,1650,1406,1379,1205,1180cm ^-1 。

ECD (methanol) lambda _max (Δε)220(–4.44),244(+0.34),270(–2.01)nm。

¹ H NMR ¹³ The C NMR (DEPT) data are shown in tables 2 and 4.

Artemisia princeps lactone H (7)

The molecular formula: c (C) ₃₅ H ₄₀ O ₈

Molecular weight: 588.27

Traits: white amorphous powder

Optical rotation:

HRESIMS (+) m/z Experimental values 589.2779[ M+H ]] ⁺ Calculated 589.2796[ M+H ]] ⁺ 。

IRν _max :3468,1766,1715,1697,1646,1378,1343,1223,1137cm ^-1 。

ECD (methanol) lambda _max (Δε):216(–11.26),237(+1.53),273(–1.79)nm。

¹ H NMR ¹³ The C NMR (DEPT) data are shown in tables 2 and 4.

Artemisia princeps lactone I (8)

The molecular formula: c (C) ₃₅ H ₄₀ O ₈

Molecular weight: 588.27

Traits: white amorphous powder

Optical rotation:

HRESIMS (-) m/z Experimental value 633.2695[ M+HCOO ]] ^– Calculated 633.2705[ M+HCOO] ^– 。

IRν _max :3501,1766,1698,1648,1381,1261,1154,1068cm ^-1 。

ECD (methanol) lambda _max (Δε):222(–8.23),246(+2.53),274(–1.53)nm。

¹ H NMR ¹³ The C NMR (DEPT) data are shown in tables 2 and 4.

Artemisia princeps lactone J (9)

The molecular formula:C ₃₀ H ₃₄ O ₇

molecular weight: 506.23

Traits: white amorphous powder

Optical rotation:

HRESIMS (+) m/z, experimental value 507.2399[ M+H ]] ⁺ Calculated value 507.2386[ M+H ]] ⁺ 。

IRν _max :3437,1758,1681,1634,1384,1313,1296,1012cm ^-1 。

ECD (methanol) lambda _max (Δε):197(+1.81),208(+5.15),219(+3.77),226(+3.95),268(–8.63),350(+1.50)nm。

¹ H NMR ¹³ The C NMR (DEPT) data are shown in tables 2 and 5.

Artemisia princeps lactone K (10)

The molecular formula: c (C) ₃₅ H ₄₄ O ₉

Molecular weight: 608.29

Traits: white amorphous powder

Optical rotation:

HRESIMS (-) m/z Experimental values 653.2717[ M+HCOO ]]Calculated 653.2704[ M+HCOO ]] ^– 。

IR(KBr)ν _max :3438,1762,1714,1646,1455,1230,1201,1148cm ^-1 。

ECD (methanol) lambda _max (Δε):218(–2.12),246(+0.73),264(+0.22)nm。

¹ H NMR ¹³ The C NMR (DEPT) data are shown in tables 2 and 5.

Artemisia princeps lactone L (11)

The molecular formula: c (C) ₃₅ H ₄₂ O ₈

Molecular weight: 590.28

Traits: white amorphous powder

Optical rotation:

HRESIMS (-) m/z Experimental value 635.2649[ M+HCOO ]]Calculated 635.2654[ M+HCOO ]] ^– 。

IR(KBr)ν _max :3447,1766,1714,1663,1379,1229,1153,1039cm ^-1 。

ECD (methanol) lambda _max (Δε):219(–3.16),248(+0.60),270(+0.01)nm。

¹ H NMR ¹³ The C NMR (DEPT) data are shown in tables 3 and 5.

Artemisia princeps lactone M (12)

The molecular formula: c (C) ₃₀ H ₃₈ O ₉

Molecular weight: 542.25

Traits: white amorphous powder

Optical rotation:

HRESIMS (+) m/z Experimental No. 543.2116[ M+H ]] ⁺ Calculated 543.2123[ M+H ]] ⁺ 。

IR(KBr)ν _max :3455,1755,1663,1632,1377,1266,1245,1159cm ^-1 。

ECD (methanol) lambda _max (Δε):229(–2.28)nm。

¹ H NMR ¹³ The C NMR (DEPT) data are shown in tables 3 and 5.

Artemisia princeps lactone N (13)

The molecular formula: c (C) ₃₀ H ₃₆ O ₆

Molecular weight: 490.25

Traits: white amorphous powder

Optical rotation:

HRESIMS (-) m/z Experimental value 537.2461[ M+HCOO ]] ^– Calculated 537.2494[ M+HCOO] ^– 。

IR(KBr)ν _max :3455,1754,1634,1383,1221,1130,1022cm ^-1 。

ECD (methanol) lambda _max (Δε):200(+18.59),225(–1.28)nm。

¹ H NMR ¹³ The C NMR (DEPT) data are shown in tables 3 and 5.

Artemisia princeps lactone O (14)

The molecular formula: c (C) ₃₂ H ₃₈ O ₈

Molecular weight: 550.25

Traits: white amorphous powder

Optical rotation:

HRESIMS (-) m/z Experimental values 595.2348[ M+HCOO ]] ^– Calculated 595.2352[ M+HCOO] ^– 。

IR(KBr)ν _max :3477,1754,1642,1344,1265,1239,1135cm ^-1 。

ECD (methanol) lambda _max (Δε):203(+25.15),225(–6.53)nm。

¹ H NMR ¹³ The C NMR (DEPT) data are shown in tables 3 and 5.

Artemisia princeps Pampanini (15)

The molecular formula: c (C) ₃₂ H ₃₆ O ₈

Molecular weight: 548.24

Traits: white amorphous powder

Optical rotation:

HRESIMS (-) m/z Experimental value 593.2403[ M+HCOO ]] ^– Calculated 593.2392[ M+HCOO] ^– 。

IR(KBr)ν _max :3495,1764,1712,1653,1401,1290,1265,1126cm ^-1 。

ECD (methanol) lambda _max (Δε):207(+3.83),217(+7.10),231(+4.39),268(–1.72)nm。

¹ H NMR ¹³ The C NMR (DEPT) data are shown in tables 3 and 5.

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TABLE 5 Compounds 9-15 ¹³ C NMR (DEPT) data (150 MHz, CDCl ₃ ,δin ppm)

Example 2

Cytotoxic activity of Compounds 1-15 against three liver cancer cell lines.

1. Materials and methods

1.1 materials

HepG2 cell lines were given by the Kunming plant institute of China academy of sciences active screening center, and Huh7 and SK-Hep-1 cell lines were purchased from Shanghai Ji Ning Biotechnology Co., ltd; medium (Dulbecco's Modified Eagle Medium, DMEM) was purchased from Thermo Fisher Scientific (Suzhou, china); serum (fetal bovine serum, FBS) was purchased from Life Technologies (NY, USA); RPMI-1640 was purchased from ThermoFisher Biochemical Products (Beijing, china).

1.2 instruments

Flex Station 3 bench-top multifunctional microplate reader (Bio-RAD 680, USA); analytical balances (AG 135, metler Toledo, china); incubator (DHP-9082, shanghai).

1.3 Experimental procedure

1) Taking liver cancer cells growing in log phase, discarding old culture medium, washing twice with PBS, discarding PBS;

2) Digesting the cells with 0.25% trypsin, and rapidly absorbing trypsin when the outline of the cells is deepened and the rounding trend is observed under a microscope;

3) The cells were stopped and resuspended in DMEM complete medium containing 10% FBS, 10 μl of cell suspension was taken, counted with a cytometer, and the cell concentration was adjusted to 1×10 with medium ⁴ Per mL, plated on 96-well plates with 100. Mu.L of cell suspension added to each well at 37℃with 5% CO ₂ Incubating for 24 hours in an incubator of (2) to adhere cells;

4) Sucking the culture medium, adding diluted samples into the plate, adding 100 mu L of the diluted samples into each hole, setting 3 compound holes for each concentration, and continuously incubating in an incubator for 48 hours;

5) Sucking out the culture medium, adding the prepared MTT solution (1 mg/mL), adding 100 mu L of the solution into each hole, and incubating in an incubator for 4 hours;

6) Sucking MTT solution, adding DMSO, adding 100 μl per well, and incubating in incubator for 10min;

7) Absorbance values were measured at 490nm wavelength using a microplate reader by the formula: inhibition ratio =Cell inhibition was calculated as (negative-experimental)/(negative-blank) ×100% and IC was calculated with statistical software GraphPad prism 5 ₅₀ Experiments were repeated 3 times.

2. Results

The cytotoxic activity of compounds 1-15 against three liver cancer cells (HepG 2, huh7 and SK-Hep-1) is shown in Table 6, and for HepG2 cells, compound 1 has strong cytotoxic activity and IC ₅₀ The value is 5.4 mu M, which is better than that of the positive medicine sorafenib; compounds 3,11 and 15 also exhibit a certain cytotoxic activity, IC ₅₀ The values were 11.6,13.0 and 11.8. Mu.M, respectively, which are comparable to the positive drug sorafenib.

For Huh7 cells, compound 1 has stronger cytotoxic activity, IC ₅₀ The values are 7.7 mu M respectively, which is better than the positive medicine sorafenib; compound 15 also has a cytotoxic activity, IC ₅₀ The value was 13.4. Mu.M, which is comparable to the positive drug sorafenib.

For SK-Hep-1 cells, compounds 1 and 15 have potent cytotoxic activity, IC ₅₀ The values are 11.8,14.3 mu M respectively, which is superior to the positive drug sorafenib; compounds 2,3 and 7 also have a certain cytotoxic activity, IC ₅₀ The values were 18.7,20.1 and 19.0. Mu.M, respectively, which are comparable to the positive drug sorafenib. Compounds 3 and 11 showed significant cytotoxic activity only against HepG2 cells, IC ₅₀ Values were 11.6 and 13.0 μm, respectively; interestingly, compound 1 had strong cytotoxic activity against all three liver cancer cells, its IC ₅₀ The values were 5.4,7.7 and 11.8. Mu.M, respectively.

TABLE 6 results of cytotoxic Activity of Compounds 1-15 against three liver cancer cells

^a The numerical value is expressed as IC ₅₀ ±SD

3. Conclusion(s)

Experimental results show that the cytotoxic activity of the compounds 1-15 on three liver cancer cells (HepG 2, SK-HEP-1 and Huh 7) is shown in Table 6, and the compound 1 has stronger fineness on the HepG2 cellsCytotoxic activity of its IC ₅₀ The value is 5.4 mu M, which is better than that of the positive medicine sorafenib; compounds 3,11 and 15 also exhibit a certain cytotoxic activity, IC ₅₀ The values were 11.6,13.0 and 11.8. Mu.M, respectively, which are comparable to the positive drug sorafenib. For Huh7 cells, compound 1 has stronger cytotoxic activity, IC ₅₀ The values are 7.7 mu M respectively, which is better than the positive medicine sorafenib; compound 15 also has a cytotoxic activity, IC ₅₀ The value was 13.4. Mu.M, which is comparable to the positive drug sorafenib. For SK-Hep-1 cells, compounds 1 and 15 have potent cytotoxic activity, IC ₅₀ The values are 11.8 and 14.3 mu M respectively, which is superior to the positive drug sorafenib; compounds 2,3 and 7 also have a certain cytotoxic activity, IC ₅₀ The values were 18.7,20.1 and 19.0. Mu.M, respectively, which are comparable to the positive drug sorafenib. Compounds 3 and 11 showed significant cytotoxic activity only against HepG2 cells, IC ₅₀ Values were 11.6 and 13.0 μm, respectively; interestingly, compound 1 had strong cytotoxic activity against all three liver cancer cells, its IC ₅₀ The values were 5.4,7.7 and 11.8. Mu.M, respectively. The results show that the compounds 1-15 separated from the artemisia kui can be used as medicaments for treating liver cancer related diseases.

Formulation examples 1-7

In the following formulation examples, conventional reagents are selected and formulation preparation is performed according to the conventional methods, and this application example only embodies that at least one of the compounds 1 to 15 of the present invention can be prepared into different formulations, and specific reagents and operations are not particularly limited:

1. dissolving at least one of the compounds 1-15 with DMSO, adding water for injection according to a conventional method, fine filtering, packaging and sterilizing to prepare injection, wherein the concentration of the injection is 0.5-5 mg/mL.

2. Dissolving at least one of the compounds 1-15 with DMSO, dissolving in sterile water for injection, stirring to dissolve, filtering with sterile suction filter funnel, sterile fine filtering, packaging in ampoule, freeze drying at low temperature, and sealing under sterile condition to obtain powder for injection.

3. Adding excipient into at least one of the compounds 1-15 according to the mass ratio of the compound to the excipient of 9:1, and preparing into powder.

4. At least one of the compounds 1-15 is added with excipient according to the mass ratio of 5:1, and the mixture is granulated and tabletted.

5. At least one of the compounds 1-15 is prepared into oral liquid according to the conventional oral liquid preparation method.

6. Adding excipient into at least one of the compounds 1-15 according to the mass ratio of 5:1, and making into capsule.

7. Adding excipient into at least one of the compounds 1-15 according to the mass ratio of the compound to the excipient of 5:1, and making into granule.

From the above examples, the present invention provides a compound of artemisia kui, its preparation method and application, pharmaceutical composition and its application. The kui artelactone provided by the invention mainly comprises 15 compounds with novel structures, the compounds have different degrees of cytotoxic activity on liver cancer cells, can form a pharmaceutical composition with a pharmaceutically acceptable carrier or excipient, and can be used for preparing anti-liver cancer drugs.

The foregoing is merely a preferred embodiment of the present invention and it should be noted that modifications and adaptations to those skilled in the art may be made without departing from the principles of the present invention, which are intended to be comprehended within the scope of the present invention.

Claims (8)

1. Compounds 1 to 15, brotheron A, B, D-P, shown in the following structural formula (I),

2. a process for the preparation of compounds 1-15 of formula (I) according to claim 1, characterized in that it comprises the following steps: pulverizing the dried aerial parts of the artemisia kui, carrying out cold leaching extraction twice by using 90% ethanol with the amount of 10 times, combining ethanol extract, concentrating under reduced pressure until no ethanol smell exists, obtaining ethanol extract, dispersing the extract in water, extracting for 3 times by using ethyl acetate, obtaining an ethyl acetate extraction part, carrying out silica gel column chromatography on the ethyl acetate extraction part, and carrying out gradient elution by using acetone-petroleum ether 10:90,20:80,30:70,40:60, v/v and acetone to obtain 5 fractions Fr.1-Fr.5; fr.2 is subjected to medium pressure MCI CHP 20P column chromatography, and is subjected to gradient elution by water-methanol 50:50,30:70,10:90 and 0:100 to obtain 4 components Fr.2-1-Fr.2-4; subjecting Fr.2-3 to silica gel column chromatography, and subjecting ethyl acetate-petroleum ether, 20:80,30:70,40:60,100:0, v/v to obtain 4 components Fr.2-3-1-Fr.2-3-4; fr.2-3-2 is subjected to medium pressure RP-C8 column chromatography, and water-methanol 40:60,20:80 and 10:90 are used for gradient elution to obtain 3 components Fr.2-3-2-1-Fr.2-3-2-3; subjecting Fr.2-3-2-1 to Sephadex LH-20 gel column chromatography, and subjecting to methanol-chloroform 50:50 to obtain 3 components Fr.2-3-2-1a-Fr.2-3-2-1c; fr.2-3-2-1a was purified by semi-preparative HPLC, water-methanol 32:68 to give compound 5; fr.2-3-2-1b was purified by semi-preparative HPLC, water-methanol 33:67 to give compounds 6,7,8 and 11; fr.2-3-3 is subjected to medium pressure RP-C18 column chromatography, and water-methanol 40:60,20:80 and 0:100 are used for gradient elution to obtain 3 components Fr.2-3-3-1-Fr.2-3-3-3; subjecting Fr.2-3-3-1 to Sephadex LH-20 gel column chromatography, and subjecting to methanol-chloroform 50:50 to obtain 3 components Fr.2-3-3-1a-Fr.2-3-3-1c; fr.2-3-3-1b was purified by semi-preparative HPLC, water-acetonitrile 45:55 to give compounds 1,2 and 3; fr.3 is subjected to medium pressure MCI CHP 20P column chromatography, and is subjected to gradient elution by water-methanol 50:50,30:70,10:90 and 0:100 to obtain 4 components Fr.3-1-Fr.3-4; subjecting Fr.3-2 to medium pressure RP-C18 column chromatography, and gradient eluting with water-methanol 50:50,40:60,30:70 to obtain 3 components Fr.3-2-1-Fr.3-2-3; fr.3-2-1 is subjected to medium pressure RP-C18 column chromatography, and is subjected to gradient elution by water-acetonitrile 75:25,70:30,65:35 and 60:40 to obtain 4 components Fr.3-2-1-1-Fr.3-2-1-4; subjecting Fr.3-2-1-3 to silica gel column chromatography, and obtaining 3 fractions Fr.3-2-1-3a-Fr.3-2-1-3c from ethyl acetate-petroleum ether 40:60 to 100:0; fr.3-2-1-3c was purified by semi-preparative HPLC, water-methanol 32:68 to give compound 15; fr.3-2-1-3a was purified by semi-preparative HPLC, water-acetonitrile 60:40 to give compounds 9 and 12; subjecting Fr.3-2-2 to medium pressure RP-C18 column chromatography, and gradient eluting with water-acetonitrile 70:30 to 50:50 to obtain 5 components Fr.3-2-2-1-Fr.3-2-2-5; fr.3-2-2-2 was purified by preparative HPLC water-acetonitrile, 0:40 to give 5 subfractions Fr.3-2-2-2a-3-2-2-2e; fr.3-2-2-2b was purified by semi-preparative HPLC water-methanol 42:58 to give compound 4; fr.3-2-2-2d was purified by semi-preparative HPLC, water-acetonitrile 62:38 to give 5 subfractions Fr.3-2-2-2d-1-Fr.3-2-2-2d-5; fr.3-2-2-2d-1 was purified by semi-preparative HPLC, water-methanol 50:50 to give compound 10; fr.3-2-2-2d-3 was purified by semi-preparative HPLC, water-methanol 50:50 to give compounds 13 and 14.

3. The use of compounds 1-15 of formula (I) as defined in claim 1 for the preparation of an anti-hepatoma medicament.

4. The use of compounds 1-15 of formula (I) as claimed in claim 1 for the preparation of anti-liver cancer inhibitors.

5. A pharmaceutical composition comprising at least one of the compounds of formula (I) 1-15 according to claim 1 and a pharmaceutically acceptable carrier.

6. The use of the pharmaceutical composition of claim 5 in the preparation of anti-liver cancer drugs.

7. The use of the pharmaceutical composition of claim 5 for preparing an anti-liver cancer inhibitor.

8. A process for the preparation of a pharmaceutical composition according to claim 5, comprising the steps of: pulverizing the dried aerial parts of the artemisia kui, carrying out cold leaching extraction twice by using 90% ethanol with the amount of 10 times, combining ethanol extract, concentrating under reduced pressure until no ethanol smell exists, obtaining ethanol extract, dispersing the extract in water, extracting for 3 times by using ethyl acetate, obtaining an ethyl acetate extraction part, carrying out silica gel column chromatography on the ethyl acetate extraction part, and carrying out gradient elution by using acetone-petroleum ether 10:90,20:80,30:70,40:60, v/v and acetone to obtain 5 fractions Fr.1-Fr.5; fr.2 is subjected to medium pressure MCI CHP 20P column chromatography, and is subjected to gradient elution by water-methanol 50:50,30:70,10:90 and 0:100 to obtain 4 components Fr.2-1-Fr.2-4; subjecting Fr.2-3 to silica gel column chromatography, and subjecting ethyl acetate-petroleum ether, 20:80,30:70,40:60,100:0, v/v to obtain 4 components Fr.2-3-1-Fr.2-3-4; fr.2-3-2 is subjected to medium pressure RP-C8 column chromatography, and water-methanol 40:60,20:80 and 10:90 are used for gradient elution to obtain 3 components Fr.2-3-2-1-Fr.2-3-2-3; subjecting Fr.2-3-2-1 to Sephadex LH-20 gel column chromatography, and subjecting to methanol-chloroform 50:50 to obtain 3 components Fr.2-3-2-1a-Fr.2-3-2-1c; fr.2-3-2-1a was purified by semi-preparative HPLC, water-methanol 32:68 to give compound 5; fr.2-3-2-1b was purified by semi-preparative HPLC, water-methanol 33:67 to give compounds 6,7,8 and 11; fr.2-3-3 is subjected to medium pressure RP-C18 column chromatography, and water-methanol 40:60,20:80 and 0:100 are used for gradient elution to obtain 3 components Fr.2-3-3-1-Fr.2-3-3-3; subjecting Fr.2-3-3-1 to Sephadex LH-20 gel column chromatography, and subjecting to methanol-chloroform 50:50 to obtain 3 components Fr.2-3-3-1a-Fr.2-3-3-1c; fr.2-3-3-1b was purified by semi-preparative HPLC, water-acetonitrile 45:55 to give compounds 1,2 and 3; fr.3 is subjected to medium pressure MCI CHP 20P column chromatography, and is subjected to gradient elution by water-methanol 50:50,30:70,10:90 and 0:100 to obtain 4 components Fr.3-1-Fr.3-4; subjecting Fr.3-2 to medium pressure RP-C18 column chromatography, and gradient eluting with water-methanol 50:50,40:60,30:70 to obtain 3 components Fr.3-2-1-Fr.3-2-3; fr.3-2-1 is subjected to medium pressure RP-C18 column chromatography, and is subjected to gradient elution by water-acetonitrile 75:25,70:30,65:35 and 60:40 to obtain 4 components Fr.3-2-1-1-Fr.3-2-1-4; subjecting Fr.3-2-1-3 to silica gel column chromatography, and obtaining 3 fractions Fr.3-2-1-3a-Fr.3-2-1-3c from ethyl acetate-petroleum ether 40:60 to 100:0; fr.3-2-1-3c was purified by semi-preparative HPLC, water-methanol 32:68 to give compound 15; fr.3-2-1-3a was purified by semi-preparative HPLC, water-acetonitrile 60:40 to give compounds 9 and 12; subjecting Fr.3-2-2 to medium pressure RP-C18 column chromatography, and gradient eluting with water-acetonitrile 70:30 to 50:50 to obtain 5 components Fr.3-2-2-1-Fr.3-2-2-5; fr.3-2-2-2 was purified by preparative HPLC water-acetonitrile, 0:40 to give 5 subfractions Fr.3-2-2-2a-3-2-2-2e; fr.3-2-2-2b was purified by semi-preparative HPLC water-methanol 42:58 to give compound 4; fr.3-2-2-2d was purified by semi-preparative HPLC, water-acetonitrile 62:38 to give 5 subfractions Fr.3-2-2-2d-1-Fr.3-2-2-2d-5; fr.3-2-2-2d-1 was purified by semi-preparative HPLC, water-methanol 50:50 to give compound 10; fr.3-2-2-2d-3 was purified by semi-preparative HPLC, water-methanol 50:50 to give compounds 13 and 14; and then adding a pharmaceutically acceptable carrier to at least one of the compounds 1-15.

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