CN112225746A

CN112225746A - Artemisia sphaerocephala lactone A-R and pharmaceutical composition and application thereof

Info

Publication number: CN112225746A
Application number: CN202011274544.9A
Authority: CN
Inventors: 陈纪军; 苏丽花; 耿长安; 张心湉; 马云保; 黄晓燕; 李天泽; 胡敬; 张雪梅
Original assignee: Kunming Institute of Botany of CAS
Current assignee: Kunming Institute of Botany of CAS
Priority date: 2020-11-15
Filing date: 2020-11-15
Publication date: 2021-01-15
Anticipated expiration: 2040-11-15
Also published as: CN112225746B

Abstract

The invention provides 18 new compounds shown in a structural formula (I), namely artematrolides A-R (1-18), a preparation method and application thereof, a pharmaceutical composition and application thereof, and relates to the technical field of medicines. The compound of the incarvillea delavayi lactone A-R has obvious cytotoxic activity on human liver cancer cell strains HepG2, Huh7 and SMMC-7721, can form a pharmaceutical composition with a pharmaceutically acceptable carrier or excipient, and can be used for preparing anti-liver cancer drugs.

Description

Artemisia sphaerocephala lactone A-R and pharmaceutical composition and application thereof

The technical field is as follows:

the invention belongs to the technical field of medicines. In particular to artesunolide A-R (artemimatrolides A-R,1-18), a preparation method and application thereof, a pharmaceutical composition and application thereof.

Background art:

hepatocellular carcinoma (HCC) is the most common primary liver cancer and the second most common cause of cancer-related death worldwide. The latest data of the international agency for research on cancer (IARC) show that the number of new liver cancer in 2018 worldwide exceeds 84 ten thousand, and the number of deaths caused by liver cancer is about 78 ten thousand. Among them, about 50% of new cases of liver cancer occur in China. In china and south-east asia, most hepatitis is caused by hepatitis b virus, while in the west, nonalcoholic steatosis appears to be one of the major causes. HCC is a complex tumor type with multiple genetic aberrations. Many signal paths are activated during the initiation and development of HCC. HCC contributes to an extremely heavy disease burden globally, but the incidence patterns vary greatly in different regions. At present, the most effective radical treatment method of HCC is surgical resection, but 80 percent of cases are discovered to belong to middle and advanced stages, and liver cirrhosis and liver function abnormality are mostly combined, so the best time for surgical resection treatment is lost. Chemotherapy is an important treatment for liver cancer, but it exhibits severe cardiac and renal toxicity. The natural product has unique structure and low toxicity, and various scholars aim to search the anti-liver cancer active component from the natural product.

Artemisia (Artemisial) family Compositae (Asteraceae) is perennial herbaceous plant, and has about 380 species globally, 186 species, 44 varieties and 82 special species in China all over the country. The plants of this genus have a wide range of pharmacological activities such as anti-inflammatory, anti-cancer, anti-fungal infection and anti-viral effects, and some species of the plants of this genus are common folk herbs such as artemisia annua (a. annua), artemisia argyi (a. argyi), artemisia capillaris (a. capillaris), artemisia anomala (a. anomala), artemisia nanasiana (a. nilagirica), artemisia alba (a. sacrotum), artemisia mongolica (a. mongolica fisch) and artemisia anomala (a. kekanaamiq), and have anti-inflammatory, anti-malarial, diarrhea, wind-cold, bruise and rheumatic diseases. The sesquiterpene compound artemisinin is the most representative natural molecule of the plant, and has good antimalarial activity and certain antitumor activity. The plant is more and more concerned by scholars at home and abroad due to wide medicinal use, novel and various secondary metabolites and various biological activities.

Dark green wormwood (Artemisia atrovirens) is perennial herb of Artemisia of Compositae, and is also named as Artemisia princeps (Yunnan), Artemisia sphaerocephala (Sichuan), Artemisia princeps (Hunan), and Artemisia alba (Henan). Mainly distributed in southern Shaanxi, southern Gansu, Anhui, Zhejiang, Jiangxi, northern Fujian, southern Henan, northern Hubei, Hunan, northern Guangdong, northern Guangxi, Sichuan, Guizhou and Yunnan. Furthermore, thailand has a distribution. Artemisia scoparia is recorded as a medicinal plant with medical value in the chemical composition and development and utilization of medicinal plants in the Compositae of China (1998), but the research on the chemical composition and the pharmacological activity of Artemisia scoparia is less, and only the literature reports the analysis of the chemical composition of volatile oil.

The invention discovers that the ethanol extract of the dried overground part of artemisia apiacea has stronger cytotoxic activity on three hepatoma carcinoma cells (HepG2, Huh7 and SMMC-7721), and 18 sesquiterpene dimer compounds with cytotoxic activity on three hepatoma carcinoma cell lines, namely artemisia apiacea lactone A-R (artemimatritides A-R,1-18), are further separated from the artemisia apiacea extract. So far, in the prior art, there are no reports of artesunolide A-R (artemimatrolides A-R,1-18), no reports of pharmaceutical compositions of artesunolide A-R as an active ingredient, and no reports of applications of the pharmaceutical compositions in preparation of or treatment of liver cancer drugs.

The invention content is as follows:

the invention aims to provide a new artemisia anomala lactone A-R (artemimatrolides A-R,1-18) shown as a formula (I) with medicinal value, a preparation method and application thereof, a medicinal composition and application thereof.

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

the invention provides a series of sesquiterpene dimer compounds, namely artemisia anomala lactone A-R (artemimatrolides A-R,1-18), which have a structure shown as the following formula (I):

the invention provides a preparation method of the compounds 1-18, which comprises the steps of crushing dry overground parts of artemisia apiacea, carrying out cold extraction twice by 10 times of 90% ethanol, combining ethanol extracting solutions, carrying out vacuum concentration until no alcohol smell exists, obtaining an ethanol extract, dispersing the extract in water, extracting for 3 times by using ethyl acetate, obtaining an ethyl acetate extracting part, carrying out silica gel column chromatography on the ethyl acetate extracting part, and carrying out gradient elution by using acetone-petroleum ether (10:90,30:70,50:50, v/v) and acetone to obtain 3 fractions Fr.1-Fr.3; fr.2 is subjected to silica gel column chromatography (ethyl acetate-petroleum ether, 10:90,20:80,40:60,100:0, v/v) to obtain 4 components Fr.2-1-Fr.2-4; subjecting Fr.2-1 to medium pressure MCI CHP 20P column chromatography, and gradient eluting with water-methanol (50:50,30:70,10:90,0:100) to obtain 4 components Fr.2-1-4; fr.2-1-3 is subjected to silica gel column chromatography (ethyl acetate-petroleum ether, 10:90 to 40:60) to obtain 5 fractions Fr.2-1-3-1 to Fr.2-1-3-5; fr.2-1-3-4 is chromatographed by silica gel column (acetone-petroleum ether, 15:85 to 30:70) to obtain 3 fractions Fr.2-1-3-4 a-Fr.2-1-3-4 c; fr.2-1-3-4b by recrystallization (MeOH-CH)₂Cl₂10:90) to give compound 1, the remainder was purified by semi-preparative HPLC (water-acetonitrile, 44:56) to give

compounds

2,10 and 17; fr.2-1-3-5 by silica gel column chromatography (ethyl acetate-chloroform, 2:98 to 10:90) to obtain 3 fractions Fr.2-1-3-5 a-Fr.2-1-3-5 c; fr.2-1-3-5a was purified by preparative HPLC (water-acetonitrile, 44:56) and semi-preparative HPLC (water-methanol, 20:80) to give

compounds

5,15,16 and 18; fr.2-1-3-5b purification by semi-preparative HPLC (water-acetonitrile, 48:52) gave compound 11; performing medium-pressure MCI CHP 20P column chromatography on Fr.2-2, and performing gradient elution with water-methanol (50:50,30:70,10:90,0:100) to obtain 4 fractions Fr.2-2-1-Fr.2-2-4; fr.2-2-2 silica gel column chromatography (ethyl acetate-petroleum ether, 20: 80-50: 50) to obtain 4 fractions Fr.2-2-2-1EFr.2-2-2-4; fr.2-2-2-2 is subjected to silica gel column chromatography (acetone-petroleum ether, 15:85,20:80,30:70) to obtain 3 fractions Fr.2-2-2 a-Fr.2-2-2 c; fr.2-2-2-2a is purified by preparative TLC (ethyl acetate-chloroform, 50:50) using Sephadex LH-20 gel column chromatography (methanol-chloroform, 50:50) to give compound 4; fr.2-2-2-2b was first purified by silica gel column chromatography (ethyl acetate-chloroform, 15:85) by preparative HPLC (water-acetonitrile, 52:48), semi-preparative HPLC (water-methanol, 37:63) to give

compounds

7,8 and 9; subjecting Fr.2-2-2-2c to Sephadex LH-20 gel column chromatography (methanol-chloroform, 50:50) to obtain two fractions Fr.2-2-2-2c-1-Fr.2-2-2 c-2; fr.2-2-2-2c-1 was purified by semi-preparative HPLC (water-methanol, 35:65) to give

compounds

12 and 13; fr.2-2-2-2c-2 was purified by semi-preparative HPLC (water-methanol, 40:60) to give

compounds

6 and 14; fr.2-2-2-3 by silica gel column chromatography (acetone-petroleum ether, 20:80), preparative HPLC (water-acetonitrile, 45:55), semi-preparative HPLC (water-acetonitrile, 53:47) to give Compound 3.

The invention provides application of the compounds 1-18 in preparing anti-liver cancer drugs. The method of the present invention is not particularly limited, and any method known in the art may be used.

The present invention also provides a pharmaceutical composition comprising at least one of the compounds 1 to 18 of formula (I) as described above and a pharmaceutically acceptable carrier or excipient.

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

When at least one of the compounds 1 to 18 is used for preparing an anti-liver cancer medicament, the compounds 1 to 18 are preferably used directly or in the form of a pharmaceutical composition.

The invention provides a pharmaceutical composition, which comprises at least one of the compounds 1-18 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 product adjuvant. The pharmaceutically acceptable carrier or excipient is not particularly limited in the present invention, and may be selected from pharmaceutically acceptable carriers and/or excipients which are well known in the art, are non-toxic and inert to humans and animals.

The preparation method of the pharmaceutical composition is not particularly limited, at least one of the compounds 1 to 18 can be 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 the process well known in the art.

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

In the invention, when the pharmaceutical composition is used for preparing an anti-liver cancer drug, the content of the composition in the drug is preferably 0.1-99%; in the pharmaceutical composition, the content of at least one of the compounds 1-18 in the pharmaceutical composition is preferably 0.5-90%. The pharmaceutical composition of the present invention is preferably used in the form of a dose per unit body weight. In the present invention, the prepared drug can be administered preferably by both injection (intravenous injection, intramuscular injection) and oral administration.

Description of the drawings:

FIG. 1 is a structural formula of compounds 1-18 of the present invention;

FIG. 2 is an X-single crystal diffraction structure of Compound 1 of the present invention;

FIG. 3 is an X-single crystal diffraction structure of Compound 7 of the present invention;

FIG. 4 is an X-single crystal diffraction structure of Compound 9 of the present invention;

FIG. 5 is an X-single crystal diffraction structure of Compound 15 of the present invention;

FIG. 6 shows the X-single crystal diffraction structure of Compound 16 of the present invention;

FIG. 7 shows the X-single crystal diffraction structure of Compound 17 of the present invention.

The specific implementation mode is as follows:

in order to better understand the essence of the present invention, the sesquiterpene dimer compounds, incarvillea atroviride A-R (1-18) and their preparation method, structural identification and pharmacological action are further illustrated by the following experimental examples and examples of the present invention, which are not intended to limit the present invention.

The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. It is to be understood that the described embodiments are merely exemplary of the invention, and not restrictive of the full scope of the invention. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

Example 1:

preparation of Compounds 1-18:

crushing dry aerial parts of artemisia scoparia, extracting twice with 10 times of 90% ethanol, combining ethanol extracting solutions, concentrating under reduced pressure until no ethanol smell exists to obtain an ethanol extract, dispersing the extract in water, extracting for 3 times with ethyl acetate to obtain an ethyl acetate extracting part, performing silica gel column chromatography on the ethyl acetate extracting part, and performing gradient elution with acetone-petroleum ether (10:90,30:70,50:50, v/v) and acetone to obtain 3 fractions Fr.1-Fr.3; fr.2 is subjected to silica gel column chromatography (ethyl acetate-petroleum ether, 10:90,20:80,40:60,100:0, v/v) to obtain 4 components Fr.2-1-Fr.2-4; subjecting Fr.2-1 to medium pressure MCI CHP 20P column chromatography, and gradient eluting with water-methanol (50:50,30:70,10:90,0:100) to obtain 4 components Fr.2-1-4; fr.2-1-3 is subjected to silica gel column chromatography (ethyl acetate-petroleum ether, 10:90 to 40:60) to obtain 5 fractions Fr.2-1-3-1 to Fr.2-1-3-5; fr.2-1-3-4 is chromatographed by silica gel column (acetone-petroleum ether, 15:85 to 30:70) to obtain 3 fractions Fr.2-1-3-4 a-Fr.2-1-3-4 c; fr.2-1-3-4b by recrystallization (MeOH-CH)₂Cl₂10:90) to give compound 1, the remainder was purified by semi-preparative HPLC (water-acetonitrile, 44:56) to give compounds 2,10 and 17; fr.2-1-3-5 by silica gel column chromatography (ethyl acetate-chloroform, 2:98 to 10:90) to obtain 3 fractions Fr.2-1-3-5 a-Fr.2-1-3-5 c; fr.2-1-3-5a was purified by preparative HPLC (water-acetonitrile, 44:56) and semi-preparative HPLC (water-methanol, 20:80) to give compounds 5,15,16 and 18; fr.2-1-3-5b purification by semi-preparative HPLC (water-acetonitrile, 48:52) gave compound 11; performing medium-pressure MCI CHP 20P column chromatography on Fr.2-2, and performing gradient elution with water-methanol (50:50,30:70,10:90,0:100) to obtain 4 fractions Fr.2-2-1-Fr.2-2-4; fr.2-2-2 is subjected to silica gel column chromatography (ethyl acetate-petroleum ether, 20:80 to 50:50) to obtain 4 fractions Fr.2-2-2-1 to Fr.2-2-4; fr.2-2-2-2 is subjected to silica gel column chromatography (acetone-petroleum ether, 15:85,20:80,30:70) to obtain 3 fractions Fr.2-2-2 a-Fr.2-2-2 c; fr.2-2-2-2a is purified by preparative TLC (ethyl acetate-chloroform, 50:50) using Sephadex LH-20 gel column chromatography (methanol-chloroform, 50:50) to give compound 4; fr.2-2-2-2b was first purified by silica gel column chromatography (ethyl acetate-chloroform, 15:85) by preparative HPLC (water-acetonitrile, 52:48), semi-preparative HPLC (water-methanol, 37:63) to give compounds 7,8 and 9; subjecting Fr.2-2-2-2c to Sephadex LH-20 gel column chromatography (methanol-chloroform, 50:50) to obtain two fractions Fr.2-2-2-2c-1-Fr.2-2-2 c-2; fr.2-2-2-2c-1 was purified by semi-preparative HPLC (water-methanol, 35:65) to give compounds 12 and 13; fr.2-2-2-2c-2 was purified by semi-preparative HPLC (water-methanol, 40:60) to give compounds 6 and 14; fr.2-2-2-3 by silica gel column chromatography (acetone-petroleum ether, 20:80), preparative HPLC (water-acetonitrile, 45:55), semi-preparative HPLC (water-acetonitrile, 53:47) to give Compound 3.

Structural data for compounds 1-18:

nmr spectra were obtained using Avance III 600(Bruker,

switzerland) or Avance III HD 400(Bruker, Bremerhaven, Germany) superconducting nmr apparatus, TMS (tetramethylsilane) as internal standard. High resolution mass spectrometry was performed using Shimadzu LC-MS-IT-TOF (Shimadzu, Kyoto, Japan). Infrared spectroscopy (IR) was measured by KBr pellet procedure using a NICOLET iS10 type infrared spectrometer (Thermo Fisher scientific, Madison, USA). ECD spectra were determined using a Chirascan-type instrument (Applied Photophysics, Surrey, UK). The optical rotation was determined by an Autopol VI polarimeter (Rudolph Research Analytical, Hackettstown, USA). For melting point

X-4B micro melting point apparatus, available from Shanghai precision scientific instruments, Inc. The thin-layer chromatography silica gel plate HSGF254 is a product of silica gel development Limited of Yangtze river friend of cigarette Tai; column chromatography silica gel (200-300 mesh) produced by Haixiang chemical industry Co., Ltd in Linyi City(ii) a Column chromatography Sephadex LH-20 was purchased from GE Healthcare Bio-Sciences AB. The HPLC 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 oven model is AT-350, and the used chromatographic column model is Agilent-Eclipse XDB-C18(5 μ M,9.4 × 250 mm). Chromatographically pure acetonitrile was purchased from Mirrida and the deionized channel water was purified by the MingCheTM-D24UV Merk Millipore system. The medium-pressure liquid phase (Dr Flash-II) is a product of Shanghai Lisui company, and the model of MCI column Mitsubishi company, namely CHP-20P (75-150 mu m). Analytically pure methanol and acetonitrile were purchased from Tianjin Daloco chemical reagent works. Developer of 10% H₂SO₄-EtOH solution.

Artemisia sphaerocephala lactone A (1)

The molecular formula is as follows: c₃₀H₃₆O₆

Molecular weight: 492.25

The characteristics are as follows: orthorhombic crystal

Melting point: 239-240 DEG C

And (3) optical rotation:

(c 0.03, methanol)

HRESIMS (+) M/z Experimental value 515.2411[ M + Na]⁺Calculated value 515.2404[ M + Na]⁺。

IR(KBr)ν_max:3476,1767,1744,1629,1404,1257,1121,1031cm^-1。

ECD (methanol) lambda_max(Δε):203(-0.25),224(+1.64),259(-0.17)nm。

Crystal data: c₃₀H₃₆O₆,M＝492.59,

α＝90°,β＝90°,γ＝90°,

100.(2) K, lattice size P2 ₁2₁2₁,Z＝4,μ(CuKα)＝0.722 mm^-1The data of the crystals were measured using a D8 QUEST type crystal diffractometer (copper target) for a total number of diffraction orders of 43964, of which 4948 were observed (R_int＝0.0243),I>2σ(I),R₁＝0.0316,wR(F²)＝0.1103,F²1.111, and-0.009 (17). The crystal parameters for compound 1 have been stored in the cambridge crystal data center, with the extraction numbers: CDCC 1999120. Website address: https:// www.ccdc.cam.ac.uk. The X-single crystal diffraction structure of Compound 1 is shown in FIG. 2.

¹HNMR and¹³the CNMR (DEPT) data are shown in tables 1 and 4.

Artemisia sphaerocephala lactone B (2)

The molecular formula is as follows: c₃₀H₃₈O₆

Molecular weight: 494.27

The characteristics are as follows: white amorphous powder

And (3) optical rotation:

(c 0.05, methanol)

HRESIMS (+) M/z Experimental value 517.2561[ M + Na]⁺Calculated value 517.2561[ M + Na]⁺。

IRν_max:3510,3437,1776,1755,1630,1357,1256,1165,1018 cm^-1。

ECD (methanol) lambda_max(Δε):228(+0.84),203(-0.39)nm。

¹H NMR and¹³the C NMR (DEPT) data are shown in tables 1 and 4.

Artemisia sphaerocephala lactone C (3)

The molecular formula is as follows: c₃₀H₃₆O₆

Molecular weight: 492.25

The characteristics are as follows: white amorphous powder

And (3) optical rotation:

(c 0.04, methanol)

HRESIMS (+) M/z Experimental value 493.2577[ M + H]⁺Calculated value 493.2585[ M + H]⁺。

IR(KBr)ν_max:3450,1766,1640,1440,1288,1092cm^-1。

ECD (methanol) lambda_max(Δε):213(+0.65),199(+0.29)nm。

¹H NMR and¹³the C NMR (DEPT) data are shown in tables 1 and 4.

Artemisia sphaerocephala lactone D (4)

The molecular formula is as follows: c₃₀H₃₈O₆

Molecular weight: 494.27

The characteristics are as follows: white amorphous powder

And (3) optical rotation:

(c 0.06, methanol)

HRESIMS (+) M/z Experimental value 495.2726[ M + H]⁺Calculated value 495.2741[ M + H]⁺。

IR(KBr)ν_max:3449,1772,1636,1439,1236,1082,1009cm^-1。

ECD (methanol) lambda_max(Δε):213(+1.06),200(+0.51)nm。

¹HNMR and¹³the CNMR (DEPT) data are shown in tables 1 and 4.

Artemisia sphaerocephala lactone E (5)

The molecular formula is as follows: c₃₀H₃₄O₅

Molecular weight: 474.24

The characteristics are as follows: white amorphous powder

And (3) optical rotation:

(c 0.08, methanol)

HRESIMS (+) M/z Experimental value 475.2473[ M + H]⁺Calculated value 475.2479[ M + H]⁺。

IRν_max:3446,1759,1637,1384,1226,1184cm^-1。

UV/Vis (methanol) lambda_max(logε):240(2.65),260(2.59)nm。

ECD (methanol) lambda_max(Δε):205(-0.19),215(-0.12),233(-0.10),259(-0.35)nm。

¹HNMR and¹³the CNMR (DEPT) data are shown in tables 1 and 4.

Artemisia sphaerocephala lactone F (6)

The molecular formula is as follows: c₃₀H₃₄O₆

Molecular weight: 490.24

The characteristics are as follows: white amorphous powder

And (3) optical rotation:

(c 0.16, methanol)

HRESIMS (+) M/z Experimental value 491.2413[ M + H]⁺Calculated value 491.2428[ M + H]⁺。

IR ν_max:3444,1766,1687,1640,1618,1321,1150cm^-1。

UV/Vis (methanol) lambda_max(logε):206(3.30),231(2.90),256(3.14)nm。

ECD (methanol))λ_max(Δε):224(+0.02),246(+0.47)nm。

¹HNMR and¹³the CNMR (DEPT) data are shown in tables 1 and 4.

Artemisia sphaerocephala lactone G (7)

The molecular formula is as follows: c₃₀H₃₆O₆

Molecular weight: 492.25

The characteristics are as follows: monoclinic crystal

Melting point: 200-201 DEG C

And (3) optical rotation:

(c 0.05, methanol)

HRESIMS (+) M/z Experimental value 493.2538[ M + H]⁺Calculated value 493.2544[ M + H]⁺。

IR(KBr)ν_max:3453,1760,1653,1633,1258,1055 cm^-1。

ECD (methanol) lambda_max(Δε)214(+2.18),255(-0.13)nm。

Crystal data: c₃₀H₃₆O₆,M＝492.59,

α＝90°,β＝97.9230(10)°,γ＝90°,

T100. (2) K, lattice size P1211, Z2, μ (Cu K α) 0.691 mm^-1The data of the crystals were measured using a D8 QUEST type crystal diffractometer (copper target) for a total number of 24243 diffraction orders, of which 5160 orders were observed (R_int＝0.0243),I>2σ(I),R₁＝0.0256,wR(F²)＝0.0662,F²1.061 and 0.07 (3). Process for preparation of Compound 7The crystal parameters are stored in a Cambridge crystal data center, and the number is extracted: CDCC 1999123. Website address: https:// www.ccdc.cam.ac.uk. The X-single crystal diffraction structure of Compound 7 is shown in FIG. 3.

¹HNMR and¹³the CNMR (DEPT) data are shown in tables 2 and 4.

Artemisia sphaerocephala lactone H (8)

The molecular formula is as follows: c₃₀H₃₆O₆

Molecular weight: 492.25

The characteristics are as follows: white amorphous powder

And (3) optical rotation:

(c 0.02, methanol)

HRESIMS (+) M/z Experimental value 493.2516[ M + H]⁺Calculated value 493.2526[ M + H]⁺。

IRν_max:3439,1760,1632,1446,1221,1061cm^-1。

ECD (methanol) lambda_max(Δε):198(-1.09),211(-0.22),222(-0.75),265(+0.13)nm。

¹H NMR and¹³the C NMR (DEPT) data are shown in tables 2 and 4.

Artemisia sphaerocephala lactone I (9)

The molecular formula is as follows: c₃₀H₃₆O₆

Molecular weight: 492.25

The characteristics are as follows: monoclinic crystal

Melting point: 205-206 DEG C

And (3) optical rotation:

(c 0.10, methanol))

HRESIMS (+) M/z Experimental value 493.2069[ M + H]⁺Calculated value 493.2068[ M + H]⁺。

IRν_max:3440,1760,1631,1443,1312,1090cm^-1。

ECD (methanol) lambda_max(Δε):218(-0.50),241(-0.05)nm。

Crystal data: c₃₀H₃₆O₆,M＝492.59,

α＝90°,β＝108.0790(10)°,γ＝90°,

T100. (2) K, lattice size P1211, Z4, μ (Cu K α) 0.699mm^-1The data of the crystals were measured using a D8 QUEST type crystal diffractometer (copper target) with a total number of diffraction orders of 57080, of which 10194 orders were observed (R_int＝0.0603).(I>2σ(I).R₁＝0.0407,wR(F²)＝0.1037,F²1.035.Flack parameter 0.03 (6). The crystal parameters for compound 9 have been stored in the cambridge crystal data center, with the extraction numbers: CDCC 2042988. Website address: https:// www.ccdc.cam.ac.uk. The X-single crystal diffraction structure of Compound 9 is shown in FIG. 4.

¹HNMR and¹³the CNMR (DEPT) data are shown in tables 2 and 4.

Dark green artemisia lactone J (10)

The molecular formula is as follows: c₃₀H₃₆O₆

Molecular weight: 492.25

The characteristics are as follows: white amorphous powder

And (3) optical rotation:

(c 0.04, methanol)

HRESIMS (+) M/z Experimental value 493.2594[ M + H]⁺Calculated value 493.2585[ M + H]⁺。

IRν_max:3446,1760,1739,1631,1400,1272,1133 cm^-1。

ECD (methanol) lambda_max(Δε):208(+3.75),229(-0.27)nm。

¹HNMR and¹³CNMR (DEPT) data are shown in tables 2 and 5.

Artemisia sphaerocephala lactone K (11)

The molecular formula is as follows: c₃₀H₃₆O₆

Molecular weight: 492.25

The characteristics are as follows: white amorphous powder

And (3) optical rotation:

(c 0.08, methanol)

HRESIMS (+) M/z Experimental value 493.2550[ M + H]⁺Calculated value 493.2561[ M + H]⁺。

IR(KBr)ν_max:3442,1761,1631,1451,1339,1177,1016 cm^-1。

ECD (methanol) lambda_max(Δε):207(-1.88),235(+0.07)nm。

¹HNMR and¹³CNMR (DEPT) data are shown in tables 2 and 5.

Artemisia sphaerocephala L (12)

The molecular formula is as follows: c₃₀H₃₆O₆

Molecular weight: 492.25

The characteristics are as follows: white amorphous powder

And (3) optical rotation:

(c 0.06, methanol)

HRESIMS (+) M/z Experimental value 493.2583[ M + H]⁺Calculated value 493.2585[ M + H]⁺。

IR(KBr)ν_max:3442,1763,1632,1402,1221,1064 cm^-1。

ECD (methanol) lambda_max(Δε):196(+1.48),230(-0.30)nm。

¹HNMR and¹³CNMR (DEPT) data are shown in tables 2 and 5.

Artemisia sphaerocephala lactone M (13)

The molecular formula is as follows: c₃₀H₃₆O₆

Molecular weight: 492.25

The characteristics are as follows: white amorphous powder

And (3) optical rotation:

(c 0.12, methanol)

HRESIMS (+) M/z Experimental value 493.2551[ M + H]⁺Calculated value 493.2561[ M + H]⁺。

IR(KBr)ν_max:3445,1766,1632,1406,1344,1219,1015 cm^-1。

ECD (methanol) lambda_max(Δε):196(+1.28),229(-0.19)nm。

¹HNMR and¹³CNMR (DEPT) data are shown in tables 3 and 5.

Artemisia sphaerocephala lactone N (14)

The molecular formula is as follows: c₃₀H₃₆O₆

Molecular weight: 490.24

The characteristics are as follows: white amorphous powder

And (3) optical rotation:

(c 0.08, methanol)

HRESIMS (+) M/z Experimental value 491.2435[ M + H]⁺Calculated value 491.2428[ M + H]⁺。

IR(KBr)ν_max:3441,1762,1686,1638,1618,1219,1146cm^-1。

UV/Vis (methanol) lambda_max(logε):232(2.93),256(3.12)nm。

ECD (methanol) lambda_max(Δε):212(-1.62),239(+0.39)nm。

¹HNMR and¹³CNMR (DEPT) data are shown in tables 3 and 5.

Artemisia sphaerocephala lactone O (15)

The molecular formula is as follows: c₃₀H₃₈O₆

Molecular weight: 494.27

The characteristics are as follows: orthorhombic crystal

Melting point: 195-196 DEG C

And (3) optical rotation:

(c 0.06, methanol)

HRESIMS (+) M/z Experimental value 517.2558[ M + Na]⁺Calculated value 517.2561[ M + Na]⁺。

IR(KBr)ν_max:3498,1775,1721,1677,1632,1329,1261,1153cm^-1。

ECD (methanol) lambda_max(Δε):199(-3.81),222(+1.63)nm。

Crystal data: c₃₀H₃₈O₆,M＝494.60,

α＝90°,β＝90°,γ＝90°,

100.(2) K, lattice size P2 ₁2₁2₁,Z＝4,μ(Cu Kα)＝0.704mm^-1The data of the crystals were measured using a D8 QUEST type crystal diffractometer (copper target) and the total number of diffraction was 48650, of which 5081 (R) was observed_int＝0.0251),I>2σ(I),R₁＝0.0265,wR(F²)＝0.0688,F²1.048 and-0.003 (17). The crystal parameters for compound 15 have been stored in the cambridge crystal data center, with the extraction numbers: CDCC 1999124. Website address: https:// www.ccdc.cam.ac.uk. The X-single crystal diffraction structure of Compound 15 is shown in FIG. 5.

¹H NMR and¹³the C NMR (DEPT) data are shown in tables 3 and 5.

Artemisia sphaerocephala lactone P (16)

The molecular formula is as follows: c₃₀H₃₈O₆

Molecular weight: 494.27

The characteristics are as follows: orthorhombic crystal

Melting point: 190-192 DEG C

And (3) optical rotation:

(c 0.03, methanol)

HRESIMS (+) M/z Experimental value 517.2556[ M + Na]⁺Calculated value 517.2561[ M + H]⁺。

IR(KBr)ν_max:3498,1783,1728,1655,1632,1383,1242,1037cm^-1。

ECD (methanol) lambda_max(Δε):198(-1.04),217(+1.30)nm。

Crystal data：C₃₀H₃₈O₆·CHCl₃,M＝613.97,

α＝90°,β＝90°,γ＝90°,

100.(2) K, lattice size P2 ₁2₁2₁,Z＝4,μ(Cu Kα)＝3.102mm^-1The crystalline data were measured using a D8 QUEST type crystallograph diffractometer (copper target) for a total number of diffractions of 54935 times, of which 5892 times were observed (R_int＝0.0346),I>2σ(I),R₁＝0.0285,wR(F²)＝0.0755,F²1.067, and 0.045 (3). The crystal parameters for compound 16 have been stored in the cambridge crystal data center, with the extraction numbers: CDCC 1999125. Website address: https:// www.ccdc.cam.ac.uk.

The X-ray single crystal diffraction structure of Compound 16 is shown in FIG. 6.

¹HNMR and¹³CNMR (DEPT) data are shown in tables 3 and 5.

Artemisia sphaerocephala lactone Q (17)

The molecular formula is as follows: c₃₀H₃₆O₆

Molecular weight: 492.25

The characteristics are as follows: orthorhombic crystal

Melting point: 190-191 deg.C

And (3) optical rotation:

(c 0.06, methanol)

HRESIMS (+) M/z Experimental value 493.2575[ M + H]⁺Calculated value 493.2585[ M + H]⁺。

IR(KBr)ν_max:3486,1762,1751,1664,1641,1386,1337,1271 cm^-1。

ECD (methanol) lambda_max(Δε):201(-3.18),219(-0.06),233(-0.54),263(+0.12)nm。

Crystal data: c₃₀H₃₆O₆,M＝492.59,

α＝90°,β＝90°,γ＝90°,

100.(2) K, lattice size P2 ₁2₁2₁,Z＝4,μ(Cu Kα)＝0.724 mm^-1The crystal data were measured using a D8 QUEST type crystal diffractometer (copper target) for a total number of diffraction cycles of 24692, of which 4932 cycles (R) were observed_int＝0.0367),I>2σ(I),R₁＝0.0362,wR(F²)＝0.0944,F²1.065, and 0.05 (3). The crystal parameters for compound 17 have been stored in the cambridge crystal data center, with the extraction numbers: CDCC 1999126. Website address: https:// www.ccdc.cam.ac.uk. The X-single crystal diffraction structure of Compound 17 is shown in FIG. 7.

¹HNMR and¹³CNMR (DEPT) data are shown in tables 3 and 5.

Artemisia sphaerocephala lactone R (18)

The molecular formula is as follows: c₃₀H₃₆O₆

Molecular weight: 492.25

The characteristics are as follows: white amorphous powder

And (3) optical rotation:

(c 0.06, methanol)

HRESIMS (+) m/z Experimental value 515.2405[M+Na]⁺Calculated value 515.2404[ M + H]⁺。

IR(KBr)ν_max:3499,1765,1733,1668,1630,1382,1294,1136cm^-1。

ECD (methanol) lambda_max(Δε):201(-4.05),223(+0.44),239(+0.08),261(+0.27)nm。

¹HNMR and¹³CNMR (DEPT) data are shown in tables 3 and 5.

TABLE 4 preparation of compounds 1 to 9¹³C NMR (DEPT) data (. delta.in ppm)

^aRecordedin CDCl₃.^bRecordedin CD₃OD.^cRecorded at 150MHz.^dRecorded at 100MHz.

TABLE 5 preparation of compounds 10-18¹³C NMR (DEPT) data (. delta.in ppm)

Example 2:

cytotoxic activity of compounds 1-18 against three hepatoma cell lines.

1. Materials and methods

1.1 materials

HepG2 cell line was awarded by Kunming plant research institute of Chinese academy of sciences, and SMMC-7721 and Huh7 cell lines were purchased from Shanghai Jinning Biotech, Inc.; medium (Dulbecco's Modified Eagle Medium, DMEM) was purchased from Thermo Fisher Scientific, Suzhou, China; serum (total bone 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 desktop multifunctional microplate reader (Bio-RAD 680, USA); analytical balance (AG135, Metler Toledo, china); incubator (DHP-9082, Shanghai).

1.3 Experimental procedures

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

2) digesting the cells by 0.25% of trypsin, and quickly absorbing the trypsin when the cell contour deepens and tends to become round under a microscope;

3) the digestion was stopped with 10% FBS-containing DMEM complete medium and the cells were resuspended, 10. mu.L of the cell suspension was taken, counted with a cell counter, and the cell concentration was adjusted to 1X 10 with the medium⁴Perml, seeded in 96-well plates, 100. mu.L of cell suspension per well, 5% CO at 37 ℃₂The culture box is incubated for 24 hours to ensure that the cells adhere to the wall;

4) the culture medium is aspirated, the diluted sample is added into the plate, 100 μ L of the diluted sample is added into each well, 3 multiple wells are set for each concentration, and the incubation is continued in the incubator for 48 h;

5) the culture medium is aspirated, the prepared MTT solution (1mg/mL) is added, 100 mu L of the MTT solution is added into each hole, and the mixture is incubated in an incubator for 4 hours;

6) the MTT solution is aspirated, DMSO is added, 100 mu L of DMSO is added into each hole, and the mixture is incubated in an incubator for 10 min;

7) measuring absorbance values using a microplate reader at 490nm wavelength by the formula: inhibition rate (negative-experimental group)/(negative-blank group) × 100% cells were calculatedInhibition rate, and calculating IC by using statistical software GraphPad prism 5₅₀The experiment was repeated 3 times.

2. Results

The cytotoxic activity of the compounds 1 to 18 against three hepatoma cells (HepG2, SMMC-7721 and Huh7) is shown in Table 6, which indicates that the compounds 1 to 18 have cytotoxicity against three hepatoma cells (HepG2, SMMC-7721 and Huh 7). For HepG2 cells, compounds 1,3 and 9 have strong cytotoxic activity and IC₅₀The values are respectively 4.4,3.3 and 5.3 mu M, and are better than the positive medicament sorafenib; compounds 5,10-12,15 and 18 also exhibited certain cytotoxic activity, their IC₅₀The values were 6.0,7.6,6.7,8.8,7.1 and 6.4 μ M, respectively, comparable to the positive drug sorafenib.

Compounds

10 and 11 have potent cytotoxic activity against SMMC-7721 cells, and their IC₅₀The values are respectively 6.6 and 6.0 mu M, which is better than that of the positive drug sorafenib; compounds 1,4,6,14 and 17 also have certain cytotoxic activity, their IC₅₀The values were 9.6,8.9,8.9,11.4 and 10.1 μ M, respectively, comparable to the positive drug sorafenib.

Compounds

3,4,6,9 and 11 have potent cytotoxic activity against Huh7 cells, and their IC' s₅₀The values are respectively 5.7,4.5,5.9,4.0 and 5.6 mu M, which are superior to the positive drug sorafenib; compounds 1,7,8,10,13 and 16 also have certain cytotoxic activity, their IC₅₀The values were 7.6,8.2,9.1,6.9,8.4 and 10.4 μ M, respectively, comparable to the positive drug sorafenib.

Compounds

5,15 and 18 showed significant cytotoxic activity against HepG2 cells only, with IC₅₀Values of 6.0,7.1, and 6.4. mu.M, respectively; compounds 4 and 6 showed significant cytotoxic activity against SMMC-7721 and Huh7 cell lines, and their IC₅₀A value of less than 10.0. mu.M; compounds 3 and 9 showed stronger cytotoxic activity than the yang drug sorafenib only against HepG2 and Huh7 cell lines; interestingly, compounds 1,10 and 11 all had strong cytotoxic activity against three hepatoma cells, with their IC' s₅₀The values were 4.4,7.6 and 6.7. mu.M (HepG2),9.6,6.6 and 6.0. mu.M (SMMC-7721),7.6,6.9 and 5.6. mu.M (Huh7), respectively.

TABLE 6 cytotoxic Activity of Compounds 1-18 against three hepatoma cells

^aThe numerical values being expressed as IC₅₀±SD

3. Conclusion

Experimental results show that the compounds 1-18 have cytotoxicity to three liver cancer cells (HepG2, SMMC-7721 and Huh 7). For HepG2 cells, 3 compounds (1,3 and 9) showed stronger cytotoxic activity than the positive drug Sorafenib, and IC thereof₅₀Values of 4.4,3.3 and 5.3. mu.M, respectively; compounds 10 and 11 have potent cytotoxic activity against SMMC-7721 cells, and their IC₅₀The values are respectively 6.6 and 6.0 mu M, which are superior to the positive drug sorafenib; for Huh7 cells, 5 compounds (3,4,6,9 and 11) showed stronger cytotoxic activity than the positive drug Sorafenib, and IC thereof₅₀Values of 5.7,4.5,5.9,4.0 and 5.6. mu.M, respectively; the other compounds also show cytotoxic activity of different degrees, especially, the

compounds

1,10 and 11 have strong cytotoxic activity on three hepatoma cells and IC thereof₅₀Values are respectively the IC thereof₅₀The values were 4.4,7.6 and 6.7. mu.M (HepG2),9.6,6.6 and 6.0. mu.M (SMMC-7721),7.6,6.9 and 5.6. mu.M (Huh7), respectively. The results show that the compounds 1-18 separated from artemisia scoparia can be used as medicines for treating liver cancer related diseases.

Formulation examples 1-7:

in the following application examples, conventional reagents were selected and the preparation was carried out according to the conventional methods, and this application example embodies that only at least one of the compounds 1 to 18 of the present invention can be prepared into various preparations, and the specific reagents and operations are not particularly limited:

1. dissolving at least one of the compounds 1-18 prepared in example 1 in DMSO, adding water for injection by a conventional method, finely filtering, filling 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 1-18 prepared in example 1 in 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 by aseptic melting to obtain powder for injection.

3. At least one of the compounds 1 to 18 prepared in example 1 was added to an excipient in a mass ratio of 9:1 to prepare a powder.

4. At least one of the compounds 1 to 18 prepared in example 1 was added with an excipient in a mass ratio of 5:1 to the excipient, and granulated and tabletted.

5. At least one of the compounds 1 to 18 prepared in example 1 was prepared into an oral liquid according to a conventional method for preparing an oral liquid.

6. At least one of the compounds 1 to 18 prepared in example 1 is added with an excipient according to the mass ratio of 5:1 to the excipient, and then the mixture is prepared into capsules.

7. At least one of the compounds 1 to 18 prepared in example 1 was added to an excipient at a ratio of 5:1 by mass to the excipient, and the mixture was made into granules.

From the above embodiments, the invention provides a compound in artemisia scoparia, a preparation method and application thereof, a pharmaceutical composition and application thereof. The artemisia anomala lactone mainly comprises 18 compounds with novel structures, the compounds have cytotoxic activities with different degrees 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 only a preferred embodiment of the present invention, and it should be noted that, for those skilled in the art, various modifications and decorations can be made without departing from the principle of the present invention, and these modifications and decorations should also be regarded as the protection scope of the present invention.

Claims

1. A compound of the structural formula (I) is incarvillea lactone A-R (artemimatrolides A-R,1-18),

2. rightsA process for the preparation of compounds 1 to 18 of formula (I) according to claim 1, characterized in that it comprises the following steps: crushing dry aerial parts of artemisia scoparia, extracting twice with 10 times of 90% ethanol, combining ethanol extracting solutions, concentrating under reduced pressure until no ethanol smell exists to obtain an ethanol extract, dispersing the extract in water, extracting for 3 times with ethyl acetate to obtain an ethyl acetate extracting part, performing silica gel column chromatography on the ethyl acetate extracting part, and performing gradient elution with acetone-petroleum ether (10:90,30:70,50:50, v/v) and acetone to obtain 3 fractions Fr.1-Fr.3; fr.2 is subjected to silica gel column chromatography (ethyl acetate-petroleum ether, 10:90,20:80,40:60,100:0, v/v) to obtain 4 components Fr.2-1-Fr.2-4; subjecting Fr.2-1 to medium pressure MCI CHP 20P column chromatography, and gradient eluting with water-methanol (50:50,30:70,10:90,0:100) to obtain 4 components Fr.2-1-4; fr.2-1-3 is subjected to silica gel column chromatography (ethyl acetate-petroleum ether, 10:90 to 40:60) to obtain 5 fractions Fr.2-1-3-1 to Fr.2-1-3-5; fr.2-1-3-4 is chromatographed by silica gel column (acetone-petroleum ether, 15:85 to 30:70) to obtain 3 fractions Fr.2-1-3-4 a-Fr.2-1-3-4 c; fr.2-1-3-4b by recrystallization (MeOH-CH)₂Cl₂10:90) to give compound 1, the remainder was purified by semi-preparative HPLC (water-acetonitrile, 44:56) to give compounds 2,10 and 17; fr.2-1-3-5 by silica gel column chromatography (ethyl acetate-chloroform, 2:98 to 10:90) to obtain 3 fractions Fr.2-1-3-5 a-Fr.2-1-3-5 c; fr.2-1-3-5a was purified by preparative HPLC (water-acetonitrile, 44:56) and semi-preparative HPLC (water-methanol, 20:80) to give compounds 5,15,16 and 18; fr.2-1-3-5b purification by semi-preparative HPLC (water-acetonitrile, 48:52) gave compound 11; performing medium-pressure MCI CHP 20P column chromatography on Fr.2-2, and performing gradient elution with water-methanol (50:50,30:70,10:90,0:100) to obtain 4 fractions Fr.2-2-1-Fr.2-2-4; fr.2-2-2 is subjected to silica gel column chromatography (ethyl acetate-petroleum ether, 20:80 to 50:50) to obtain 4 fractions Fr.2-2-2-1 to Fr.2-2-4; fr.2-2-2-2 is subjected to silica gel column chromatography (acetone-petroleum ether, 15:85,20:80,30:70) to obtain 3 fractions Fr.2-2-2 a-Fr.2-2-2 c; fr.2-2-2-2a is purified by preparative TLC (ethyl acetate-chloroform, 50:50) using Sephadex LH-20 gel column chromatography (methanol-chloroform, 50:50) to give compound 4; fr.2-2-2-2b was first purified by silica gel column chromatography (ethyl acetate-chloroform, 15:85) by preparative HPLC (water-acetonitrile, 52:48), semi-preparative HPLC (water-methanol, 37:63) to give the compoundSubstances 7,8 and 9; subjecting Fr.2-2-2-2c to Sephadex LH-20 gel column chromatography (methanol-chloroform, 50:50) to obtain two fractions Fr.2-2-2-2c-1-Fr.2-2-2 c-2; fr.2-2-2-2c-1 was purified by semi-preparative HPLC (water-methanol, 35:65) to give compounds 12 and 13; fr.2-2-2-2c-2 was purified by semi-preparative HPLC (water-methanol, 40:60) to give compounds 6 and 14; fr.2-2-2-3 by silica gel column chromatography (acetone-petroleum ether, 20:80), preparative HPLC (water-acetonitrile, 45:55), semi-preparative HPLC (water-acetonitrile, 53:47) to give Compound 3.

3. Use of compounds 1-18 of formula (I) according to claim 1 for the preparation of a medicament against liver cancer.

4. A pharmaceutical composition comprising at least one compound of formula (I) 1-18 according to claim 1 and a pharmaceutically acceptable carrier or excipient.

5. The use of the pharmaceutical composition of claim 4 for the preparation of a medicament against liver cancer.