Heterocyclic compounds, pharmaceutical compositions comprising the same, and methods of use thereof

Abstract

The application provides artemordins A-S (compounds 1-19), a pharmaceutical composition thereof, and a preparation method and application thereof, and belongs to the technical field of drugs. The 19 sesquiterpene dimers shown in structural formula (I) of the application have cytotoxic activity on human hepatoma cell strains HepG2, Huh7 and SK-Hep-1, can be combined with a pharmaceutically acceptable carrier or excipient to form a pharmaceutical composition, and can be used for preparing an anti-hepatoma drug.

Description

Technical fields:

[0001] This invention belongs to the field of pharmaceutical technology. Specifically, it relates to 19 novel sesquiterpene dimers, artemordins A–S (compounds 1-19), their preparation methods and applications, and the application of pharmaceutical compositions using compounds 1-19 as active ingredients in the preparation of drugs for treating liver cancer. 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 progression, and difficulty in early diagnosis of hepatocellular carcinoma, most patients are already in locally advanced stages or have metastasized at the time of diagnosis, making them unsuitable for the above treatments and only able to receive drug therapy.

[0003] The genus *Artemisia* in the family Asteraceae comprises approximately 380 species, distributed worldwide, with 186 species and 44 varieties found in my country. Among them, some plants are well-known folk and traditional Chinese medicines, such as *Artemisia capillaris*, *Artemisia argyi*, and *Artemisia annua*, used to treat various diseases including malaria, hepatitis, cancer, eczema, diarrhea, bruises, and rheumatism. Phytochemical studies have shown that sesquiterpenes are among the most important chemical components in *Artemisia* plants, with common types including eucalyptane, guaiacolane, gemmaane, and juniperane. Literature reports that some of these sesquiterpenes possess significant antitumor, anti-inflammatory, antiviral, and immunomodulatory effects.

[0004] Black sand wormwood (A. ordosica Krasch.) is a perennial herb belonging to the genus Artemisia in the family Asteraceae, mainly distributed in northwestern and northern my country. Black sand wormwood has a bitter and slightly warm nature; the whole plant can be used to treat urinary retention; the root is used to stop bleeding; the stems, leaves, and flower buds can be used to treat rheumatoid arthritis and boils; the fruit has anti-inflammatory, swelling-reducing, chest-relieving, and insecticidal properties, and can be applied externally to treat mumps and boils, and taken internally to treat hernia. Mongolian medicine uses it as an anti-inflammatory, hemostatic, wind-dispelling, and heat-clearing medicine. Black sand wormwood is rich in chemical components, mainly phenols, coumarins, and enynes, as well as terpenes and sterols. However, to date, no reports have been found on sesquiterpenes or anti-liver cancer activity in black sand wormwood.

[0005] As a continuation of previous research, this invention isolated 19 novel sesquiterpene dimers from Artemisia aquilina, namely artemordins A–S (compounds 1-19). To date, there are no reports in the prior art of artemordins A–S (compounds 1-19), nor of pharmaceutical compositions containing them as active ingredients, nor of the application of these compounds and their pharmaceutical compositions in the preparation or treatment of liver cancer drugs. Summary of the Invention:

[0006] The purpose of this invention is to provide a new class of artemordins A–S (compounds 1-19) with medicinal value, as shown in formula (I), along with their preparation methods, pharmaceutical compositions, and applications. This invention has isolated and identified 19 novel sesquiterpene dimers from Artemisia annua, namely artemordins A–S (compounds 1-19). These compounds exhibit significant cytotoxic activity against human liver cancer cell lines HepG2, Huh7, and SK-Hep-1, and can be used to prepare anti-liver cancer drugs.

[0007] To achieve the above-mentioned objectives of the present invention, the present invention provides the following technical solution:

[0008] This invention provides a series of sesquiterpene dimer compounds, artemordins A–S (compounds 1-19), with the following structure (I):

[0009]

[0010] The present invention also provides a method for preparing compounds 1-19 of formula I.

[0011] The dried aerial parts of Artemisia annua were pulverized and extracted twice with three times the volume of 90% ethanol. The extracts were combined and concentrated under reduced pressure to obtain a crude extract, which was then dispersed in water and extracted with ethyl acetate to obtain the ethyl acetate extract. Subsequently, the ethyl acetate extract was subjected to silica gel column chromatography and eluted with acetone-petroleum ether gradients of 0:100, 5:95, 10:90, 20:80, 40:60, and 100:0 to obtain six fractions, Frs.AF. Fraction Fr.D was subjected to silica gel column chromatography and eluted with ethyl acetate-petroleum ether at ratios of 10:90, 20:80, 30:70, 50:50, and 0:100 to obtain four fractions, Fr.D1-Fr.D4. Fr.D2 was subjected to MCI gel chromatography. CHP20P column chromatography was performed using methanol-water elution at ratios of 40:60, 50:50, 70:30, 90:10, and 100:0 to obtain four subfractions Frs.D2.1-D2.4; Fr.D2.3 was then eluted with Rp-C 18 Column chromatography was performed with methanol-water at ratios of 50:50, 70:30, 90:10, and 100:0 to obtain five fractions Fr.D2.3.1-D2.3.4. Fr.D2.3.2 was then subjected to silica gel column chromatography with methanol-chloroform at ratios of 2:98-15:85 to obtain four fractions Fr.D2.3.2a-D2.3.2d. Fr.D2.3.2b was finally subjected to semi-preparative high-performance liquid chromatography at an Agilent XDB-C100000 column. 18The compounds were purified by methanol-water elution at a ratio of 77:23 to obtain compounds 1 (artemisinin A), 2 (artemisinin B), 3 (artemisinin C), and 4 (artemisinin D); Fr.D2.3.2c. Finally, the compounds were purified by preparative high-performance liquid chromatography (HPLC) at an Agilent XDB-C10000 HPLC system. 18 Compounds 5 (artemisinin E) and 6 (artemisinin F) were purified by column chromatography with acetonitrile-water at a ratio of 65:35. Fr.D2.3.3 was then purified by silica gel column chromatography with methanol-chloroform at a ratio of 2:98–15:85, followed by Sephadex LH-20 column chromatography with methanol-chloroform at a ratio of 50:50. Finally, the purified fractions were purified by semi-preparative high-performance liquid chromatography (HPLC) at an Agilent XDB-C10 HPLC system. 18 Purification with methanol-water at a ratio of 80:20 yielded compounds 7 (artemisinin G), 8 (artemisinin H), 9 (artemisinin I), and 10 (artemisinin J). Fr.D3 was further purified by MCI gel CHP 20P column chromatography, eluting with methanol-water at ratios of 40:60, 50:50, 70:30, 90:10, and 100:0 to obtain four subfractions Frs.D3.1–D3.4. Fr.D3.3 was then purified by Rp-C... 18 Column chromatography was performed with methanol-water mixtures of 40:60, 50:50, 60:40, 70:30, and 100:0 to obtain four fractions, Fr.D3.3.1–D3.3.4. Fr.D3.3.1 was further eluted with silica gel column chromatography using ethyl acetate-petroleum ether at ratios of 10:90–50:50, followed by Sephadex LH-20 column chromatography with methanol-chloroform at a ratio of 50:50. Finally, the fractions were purified by semi-preparative high-performance liquid chromatography (HPLC) at an Agilent XDB-C10 column. 18 Compound 11, artemisinin K, was purified by column chromatography with methanol-water at a ratio of 77:23 to obtain Fr.D3.3.2. The compound was then subjected to silica gel column chromatography with methanol-chloroform at a ratio of 2:98-15:85, followed by Sephadex LH-20 column chromatography with methanol-chloroform at a ratio of 50:50. Finally, it was purified by semi-preparative high-performance liquid chromatography (HPLC) at an Agilent XDB-C10. 18 Compounds 17 (artemisinin Q), 18 (artemisinin R), and 19 (artemisinin S) were purified by column chromatography with acetonitrile-water at a ratio of 67:33 and methanol-water at a ratio of 80:20. Following purification with silica gel column chromatography using methanol-chloroform at a ratio of 2:98–15:85, followed by Sephadex LH-20 column chromatography with methanol-chloroform at a ratio of 50:50, the final product was purified by semi-preparative high-performance liquid chromatography (HPLC) at an Agilent XDB-C10. 18The compounds 12 (artemisinin L), 13 (artemisinin M), 14 (artemisinin N), 15 (artemisinin O), and 16 (artemisinin P) were purified by column elution with acetonitrile-water in a ratio of 72:28 and methanol-water in a ratio of 84:16. This invention provides the application of compounds 1-19 of Formula I in the preparation of anti-hepatocellular carcinoma drugs. This invention does not impose any particular limitation on the method of application; any method well-known in the art can be used.

[0012] The present invention also provides a pharmaceutical composition comprising at least one of compounds 1-19 of formula (I) and a pharmaceutically acceptable carrier or excipient.

[0013] Furthermore, the application of the aforementioned pharmaceutical composition in the preparation of anti-liver cancer drugs is also provided. A method for preparing the aforementioned pharmaceutical composition is also provided: compounds 1-19 of the present invention are prepared using the above-described method for preparing compounds, and then a pharmaceutically acceptable carrier is added.

[0014] When at least one of the compounds 1-19 is used to prepare an anti-liver cancer drug, the present invention preferably uses the compounds 1-19 directly or in the form of a pharmaceutical composition.

[0015] The pharmaceutical composition provided by this invention comprises at least one of compounds 1-19 described above and a pharmaceutically acceptable carrier or excipient. In this invention, the pharmaceutically acceptable carrier or excipient is preferably a solid, semi-solid, or liquid diluent, filler, or pharmaceutical excipient. This invention does not impose any particular limitation on the pharmaceutically acceptable carrier or excipient; any pharmaceutically acceptable carrier and / or excipient well-known in the art that is non-toxic and inert to humans and animals may be selected.

[0016] The present invention does not impose any particular limitation on the preparation method of the pharmaceutical composition. At least one of compounds 1-19 can be directly mixed with a pharmaceutically acceptable carrier or excipient. The present invention does not impose any particular limitation on the mixing process. Any process well known in the art that can obtain the pharmaceutical composition can be selected.

[0017] This invention provides the application of the pharmaceutical composition described in the above technical solution in the preparation of anti-liver cancer drugs. There are no special limitations on the method of application, and any method well known in the art can be selected.

[0018] In this invention, when the pharmaceutical composition is used to prepare an anti-liver cancer drug, the content of the composition in the drug is preferably 0.1% to 99%; in the pharmaceutical composition, the content of at least one of compounds 1-19 is preferably 0.5% to 90%. The pharmaceutical composition of this invention is preferably used in the form of a dose per unit body weight. In this invention, the prepared drug is preferably administered by both injection (intravenous injection, intramuscular injection) and oral administration.

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

[0020] 1. This invention provides 19 novel sesquiterpene dimer compounds, artemordins A–S (compounds 1-19).

[0021] 2. This invention provides a new method for preparing new compounds 1-19, which uses readily available raw materials and is easy to operate.

[0022] 3. This invention provides a pharmaceutical composition with novel compounds 1-19 as active ingredients, providing a new drug with good medicinal effects for new anti-liver cancer drugs.

[0023] 4. Compounds 1-19 of the present invention exhibit cytotoxic activity against three liver cancer cell lines, HepG2, Huh7, and SK-Hep-1, at 200 μM. Compounds 2, 8, 10, 11, and 17 show inhibitory activity against HepG2 cells, with an IC50 value of [missing value]. 50 The values ​​were 26.9, 25.1, 25.5, 23.1, and 30.7 μM; compounds 2, 8, 10, 16, and 17 had inhibitory effects on Huh7 cells, with IC50 values ​​of 26.9, 25.1, 25.5, 23.1, and 30.7 μM. 50 The concentrations were 29.5, 18.3, 26.5, 30.3, and 27.4 μM; compounds 2, 8, and 15 had inhibitory effects on SK-Hep-1 cells, with IC50 values ​​of 29.5, 18.3, 26.5, 30.3, and 27.4 μM. 50 The values ​​were 19.7, 15.7, and 30.7 μM. These results indicate that compound 1-19 isolated from Artemisia annua can be used as a drug to treat liver cancer-related diseases. Attached image description:

[0024] Figure 1 The structural formulas of compounds 1-19 of this invention are shown below;

[0025] Figure 2 The diagram shows the X-ray single crystal structures of compounds 1, 3, 5, and 7. Detailed implementation method:

[0026] To better understand the essence of the present invention, the following description, in conjunction with the accompanying drawings, uses experimental examples and embodiments of the present invention to further illustrate the preparation method, structural identification, pharmacological effects, preparation method, and drug composition of artemordins A–S (compounds 1-19) of the present invention, but does not limit the present invention to these experimental examples and embodiments.

[0027] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. All other embodiments obtained by those skilled in the art based on the embodiments of this invention without creative effort are within the scope of protection of this invention.

[0028] Example 1:

[0029] Preparation of the sesquiterpene dimer, artemordins A–S (compound 1-19), according to the present invention:

[0030] 51.5 kg of dried Artemisia annua aerial parts were pulverized and extracted twice with three times the volume of 90% ethanol. The extracts were combined and concentrated under reduced pressure to obtain a crude extract, which was dispersed in water and extracted with ethyl acetate to obtain the ethyl acetate extract. Subsequently, the ethyl acetate extract was subjected to silica gel column chromatography and eluted with acetone-petroleum ether gradients 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 ratios 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 chromatography. 20P column chromatography was performed, eluting with methanol-water at ratios of 40:60, 50:50, 70:30, 90:10, and 100:0 to obtain four subfractions Frs.D2.1-D2.4; Fr.D2.3 (25 g) was then eluted with Rp-C 18 Column chromatography was performed with methanol-water at ratios of 50:50, 70:30, 90:10, and 100:0 to obtain five fractions Fr.D2.3.1-D2.3.4. Fr.D2.3.2 (1.2 g) was then subjected to silica gel column chromatography with methanol-chloroform at ratios of 2:98-15:85 to obtain four fractions Fr.D2.3.2a-D2.3.2d. Fr.D2.3.2b (98 mg) was finally subjected to semi-preparative high-performance liquid chromatography at an Agilent XDB-C1000000 column. 18The compounds were purified by methanol-water elution at a ratio of 77:23 to obtain compounds 1 (artemisinin A, 16.1 mg), 2 (artemisinin B, 12.5 mg), 3 (artemisinin C, 7.6 mg), and 4 (artemisinin D, 5.8 mg); Fr.D2.3.2c (78 mg) was then purified by semi-preparative high-performance liquid chromatography (HPLC) at an Agilent XDB-C1000 column. 18 Compound 5, artemisinin E (19.0 mg), and compound 6, artemisinin F (33.7 mg), were purified by column chromatography with acetonitrile-water at a ratio of 65:35. Fr.D2.3.3 (4 g) was purified by silica gel column chromatography with methanol-chloroform at a ratio of 2:98-15:85, followed by Sephadex LH-20 column chromatography with methanol-chloroform at a ratio of 50:50. Finally, the precipitate was purified by semi-preparative high-performance liquid chromatography (HPLC) at an Agilent XDB-C10. 18 The compounds were purified on a column using methanol-water at a ratio of 80:20 to obtain compounds 7 (artemisinin G, 34 mg), 8 (artemisinin H, 16.1 mg), 9 (artemisinin I, 22.6 mg), and 10 (artemisinin J, 18.0 mg). Fr.D3 (70 g) was purified by MCI gel CHP 20P column chromatography with methanol-water at ratios of 40:60, 50:50, 70:30, 90:10, and 100:0 to obtain four subfractions Frs.D3.1-D3.4. Fr.D3.3 (15 g) was then purified by Rp-C... 18 Column chromatography was performed with methanol-water mixtures of 40:60, 50:50, 60:40, 70:30, and 100:0 to obtain four fractions, Fr.D3.3.1-D3.3.4. Fr.D3.3.1 (3 g) was subjected to silica gel column chromatography, eluted with ethyl acetate-petroleum ether at 10:90-50:50, followed by Sephadex LH-20 column chromatography, eluted with methanol-chloroform at 50:50, and finally purified by semi-preparative high-performance liquid chromatography (HPLC) at an Agilent XDB-C10. 18 Compound 11, artemisinin K (16.1 mg), was purified by methanol-water chromatography with a 77:23 ratio on a column to obtain compound 11. Fr.D3.3.2 (5 g) was then purified by silica gel column chromatography with methanol-chloroform at a ratio of 2:98-15:85, followed by Sephadex LH-20 column chromatography with methanol-chloroform at a ratio of 50:50, and finally purified by semi-preparative high-performance liquid chromatography at an Agilent XDB-C10. 18 The compounds 17 (artemisinin Q, 18 mg), 18 (artemisinin R, 14 mg), and 19 (artemisinin S, 3 mg) were purified by column chromatography with acetonitrile-water at a ratio of 67:33 and methanol-water at a ratio of 80:20. Fr.D3.3.4 (3 g) was then purified by silica gel column chromatography with methanol-chloroform at a ratio of 2:98-15:85, followed by Sephadex LH-20 column chromatography with methanol-chloroform at a ratio of 50:50. Finally, the purified product was purified by semi-preparative high-performance liquid chromatography (HPLC) at an Agilent XDB-C10 HPLC system. 18The compounds were purified by elution on the column with acetonitrile-water in a ratio of 72:28 and methanol-water in a ratio of 84:16 to obtain compounds 12 (9 mg), 13 (4 mg), 14 (7 mg), 15 (12 mg), and 16 (2 mg).

[0031] Structural data of compounds 1-19:

[0032] Optical rotation was measured using an Autopol VI polarimeter (Rudolf, USA); infrared spectroscopy was performed using the KBr pellet method on a Bio-Rad FTS-135 infrared spectrometer (Bio-Rad Laboratories, USA); ultraviolet spectroscopy was performed using an ultraviolet-2401PC ultraviolet spectrometer (Shimadzu Corporation, Japan); circular dichroism spectroscopy was performed using an Applied Photophysics circular dichroism spectrometer (Agilent Technologies, USA); nuclear magnetic resonance spectroscopy (one-dimensional and two-dimensional) was performed using an Avance III-600 superconducting nuclear magnetic resonance spectrometer (Bruker, Germany) with CDCl3 as solvent; high-resolution mass spectrometry was performed using a Shimadzu LCMS-IT-TOF mass spectrometer (Shimadzu Corporation, Japan); thin-layer chromatography silica gel plates HSGF254 were products of Yantai Jiangyou Silica Gel Development Co., Ltd.; column chromatography silica gel (200-300 mesh) was produced by Linyi Haixiang Chemical Co., Ltd.; dextran gel LH-20 (Sephadex LH-20) was purchased from GE Healthcare Bio-Sciences. AB Company; the high-performance liquid chromatograph was manufactured by Shimadzu Corporation, with a controller model of CBM-20A, a pump model of LC-20AR, a detector model of SPD-M20A, a column oven model of AT-350, and an Agilent-EClipse XDB-C18 column (5μm, 9.4×250mm); chromatographic grade acetonitrile was purchased from Meredith Corporation; MCI gel CHP20P (75~150μm) was purchased from Mitsubishi Chemical Corporation, Japan; the colorimetric reagent was 10% H2SO4-EtOH solution.

[0033]

[0034] Artemordin A (1)

[0035] Molecular formula: C 30 H 40 O6

[0036] Molecular weight: 496

[0037] Properties: Orthorhombic crystals;

[0038] High-resolution mass spectrometry in positive ion mode: m / z 497.2899 [M+H] +(Calculated as C) 30 H 41 O6,497.2898).

[0039] Infrared (KBr)v max 1748,1665,1632,1454,1384,1138,1020cm -1

[0040] Circular dichroism (MeOH)λ max (Δε)221(-3.2)nm;

[0041]

[0042] The proton and carbon NMR spectra are shown in Tables 1 and 5.

[0043]

[0044] X-ray single-crystal diffraction structure of compound 1

[0045] Crystal data for compound 1: C 30 H 40 O6,M=496.62, α=90°, β=90°, γ=90°, T = 100.(2)K, space group P212121, Z = 4, μ(Cu Kα) = 0.702mm -1 ,23946measured reflections,4864independent reflections(R int =0.1875).The final R1 values ​​were 0.1366(I>2σ(I)).The final wR(F 2 )valueswere 0.3639(I>2σ(I)).The final R1 values ​​were 0.1520(all data).The final wR(F 2 )values ​​were 0.3743(all data).The goodness of fit on F 2 was 1.619.Flack parameter = 0.19(13).

[0046]

[0047] Artemordin B (2)

[0048] Molecular formula: C 30 H 40 O6

[0049] Molecular weight: 496

[0050] Appearance: White amorphous powder;

[0051] High-resolution mass spectrometry in positive ion mode: m / z 237.1487 [M+H] + (Calculated as C) 14 H 21 O3,237.1485).

[0052] Infrared (KBr)v max 1751,1666,1630,1454,1384,1155,1019cm -1

[0053] Circular dichroism (MeOH)λ max (Δε)220.3(-6.4)nm;

[0054]

[0055] The proton and carbon NMR spectra are shown in Tables 1 and 5.

[0056]

[0057] Artemordin C, 3

[0058] Molecular formula: C 30 H 42 O6

[0059] Molecular weight: 498

[0060] Properties: Monoclinic crystals;

[0061] High-resolution mass spectrometry in positive ion mode: m / z 499.3061 [M+H] + (Calculated as C) 30 H 43 O6,499.3054).

[0062] Infrared (KBr)v max 1750,1667,1454,1384,1154,1025cm -1

[0063] Circular dichroism (MeOH)λ max (Δε)222(-3.4)nm;

[0064]

[0065] The proton and carbon NMR spectra are shown in Tables 1 and 5.

[0066]

[0067] X-ray single-crystal diffraction structure of compound 3

[0068] Crystallographic data for compound 3: C 30 H 42 O6,M=498.63, α=90°, β=95.856(2)°, γ=90°, T = 150.(2)K, space group P1 21 1, Z = 2, μ(Cu Kα) = 0.664mm -1 ,22626measured reflections,5032independent reflections(R int =0.1383).The finalR1 values ​​were 0.0464(I>2σ(I)).The final wR(F 2 )values ​​were 0.0984(I>2σ(I)).Thefinal R1 values ​​were 0.0662(all data).The final wR(F 2 )values ​​were 0.1087(alldata).The goodness of fit on F 2 was 1.037.Flack parameter = -0.06(19).

[0069]

[0070] Artemordin D, 4

[0071] Molecular formula: C 30 H 42 O6

[0072] Molecular weight: 498

[0073] Appearance: White amorphous powder;

[0074] High-resolution mass spectrometry in positive ion mode: m / z 499.3057 [M+H] + (Calculated as C) 30 H 43 O6,499.3054).

[0075] Infrared (KBr)v max 1750,1668,1454,1384,1139,1022cm -1

[0076] Circular dichroism (MeOH)λ max (Δε)222(-0.3)nm;

[0077]

[0078] The proton and carbon NMR spectra are shown in Tables 1 and 5.

[0079]

[0080] Artemordin E (5)

[0081] Molecular formula: C 30 H 40 O6

[0082] Molecular weight: 496

[0083] Properties: Orthorhombic crystals;

[0084] High-resolution mass spectrometry in positive ion mode: m / z 497.2904 [M+H] + (Calculated as C) 30 H 41 O6,497.2898).

[0085] Infrared (KBr)v max 1777,1711,1651,1455,1384,1259,1172,1138cm -1

[0086] Ultraviolet (MeOH)λ max (logε)220(0.40)nm

[0087] Circular dichroism (MeOH)λ max (Δε)202(-1.2),215(-2.6),230(+1.0),246(-2.0)nm;

[0088]

[0089] The proton and carbon NMR spectra are shown in Tables 1 and 5.

[0090]

[0091] X-ray single-crystal diffraction structure of compound 5

[0092] Crystallographic data for compound 5: C 30 H 40 O6,M=496.62, α=90°, β=90°, γ=90°, T = 150.(2)K, space group P212121, Z = 4, μ(Cu Kα) = 0.680mm -1 ,40874measured reflections,5058independent reflections(R int =0.1637).The final R1 values ​​were 0.0349(I>2σ(I)).The final wR(F 2 )valueswere 0.0707(I>2σ(I)).The final R1 values ​​were 0.0524(all data).The final wR(F 2 )values ​​were 0.0768(all data).The goodness of fit on F 2 was 1.015.Flack parameter = 0.01(9).

[0093]

[0094] Artemordin F (6)

[0095] Molecular formula: C 30 H 40 O6

[0096] Molecular weight: 496

[0097] Appearance: White amorphous powder;

[0098] High-resolution mass spectrometry in positive ion mode: m / z 497.2897 [M+H] + (Calculated as C) 30 H 41 O6,497.2898)

[0099] Infrared (KBr)v max 1748,1685,1449,1385,1152,1018cm -1

[0100] Ultraviolet (MeOH)λ max (logε)220(0.46)nm

[0101] Circular dichroism (MeOH)λ max (Δε)214(-4.5),230(+0.8),246(-2.8)nm;

[0102]

[0103] The proton and carbon NMR spectra are shown in Tables 1 and 5.

[0104]

[0105] Artemordin G (7)

[0106] Molecular formula: C 30 H 40 O6

[0107] Molecular weight: 496

[0108] Properties: Orthorhombic crystals;

[0109] High-resolution mass spectrometry in positive ion mode m / z 497.2898 ([M+H]) + Calculated as C 30 H 41 O6,497.2898).

[0110] Infrared (KBr)v max 1766,1711,1675,1453,1384,1353,1171cm -1

[0111] Circular dichroism (MeOH)λ max (Δε)207(+9.2),227(-2.6),256(+1.2),282(+0.8),320(+2.5)nm;

[0112]

[0113] The proton and carbon NMR spectra are shown in Tables 2 and 5.

[0114]

[0115] X-ray single-crystal diffraction structure of compound 7

[0116] Crystallographic data for compound 7: C 30 H 40 O6,M=496.62, α=90°, β=90°, γ=90°, T = 100.(2)K, space group P212121, Z = 12, μ(Cu Kα) = 0.701mm -1 ,66082measured reflections,14798independentreflections(R int =0.0760).The final R1 values ​​were 0.0535(I>2σ(I)).The finalwR(F 2 )values ​​were 0.1287(I>2σ(I)).The final R1 values ​​were 0.0613(all data).The final wR(F 2 )values ​​were 0.1334(all data).The goodness of fit on F 2 was1.035.Flack parameter = 0.02(4).

[0117]

[0118] Artemordin H (8)

[0119] Molecular formula: C 30 H 36 O6

[0120] Molecular weight: 492

[0121] Appearance: White amorphous powder;

[0122] High-resolution mass spectrometry in positive ion mode, m / z 493.2585 ([M+H]). + Calculated as C 30 H 37 O6,493.2585)

[0123] Infrared (KBr)v max 1762, 1711, 1676, 1384, 1174cm -1

[0124] Ultraviolet (MeOH)λ max (logε)214(0.7)nm

[0125] Circular dichroism (MeOH)λ max (Δε)220(-10.5),254(+3.6),285(+0.9),320(+2.9)nm

[0126]

[0127] The proton and carbon NMR spectra are shown in Tables 2 and 5.

[0128]

[0129] Artemordin I (9)

[0130] Molecular formula: C 30 H 38 O6

[0131] Molecular weight: 494

[0132] Appearance: White amorphous powder;

[0133] High-resolution mass spectrometry in positive ion mode, m / z 495.2751 ([M+H]). + Calculated as C 30 H 39 O6,495.2741).

[0134] Infrared (KBr)v max 1776,1738,1712,1456,1371,1238,1175cm -1

[0135] Ultraviolet (MeOH)λ max (logε)210(0.4)nm

[0136] Circular dichroism (MeOH)λ max (Δε)201(+8.7),221(-8.1),266(+1.6),286(+1.1),320(+2.4)nm;

[0137]

[0138] The proton and carbon NMR spectra are shown in Tables 2 and 5.

[0139]

[0140] Artemordin J (10)

[0141] Molecular formula: C 30 H 38 O6

[0142] Molecular weight: 494

[0143] Appearance: White amorphous powder;

[0144] High-resolution mass spectrometry in positive ion mode m / z 495.2737 ([M+H]) + Calculated as C 30 H 39 O6,495.2741).

[0145] Infrared (KBr)v max 1776,1738,1710,1456,1369,1237,1175cm -1

[0146] Ultraviolet (MeOH)λ max (logε)214(0.4)nm

[0147] Circular dichroism (MeOH)λ max (Δε)224(-0.6),247(+2.8),281(+0.3),322(+2.1)nm

[0148]

[0149] The proton and carbon NMR spectra are shown in Tables 2 and 5.

[0150]

[0151] Artemordin K (11)

[0152] Molecular formula: C 30 H 42 O4

[0153] Molecular weight: 466

[0154] Appearance: White amorphous powder;

[0155] High-resolution mass spectrometry in positive ion mode: m / z 467.3163 [M+H] + (Calculated as C) 30 H 43 O4,467.3156).

[0156] Infrared (KBr)v max 3434,1707,1628,1446,1382,1266,1163cm -1

[0157] Circular dichroism (MeOH)λ max (Δε)231(+3.6),264(+1.5),297(+3.6)nm

[0158]

[0159] The proton and carbon NMR spectra are shown in Tables 2 and 5.

[0160]

[0161] Artemordin L, 12

[0162] Molecular formula: C 30 H 44 O7

[0163] Molecular weight: 516

[0164] Appearance: White amorphous powder;

[0165] High-resolution mass spectrometry in positive ion mode m / z 517.3165 ([M+H]) + Calculated as C 30 H 45 O7,517.3160).

[0166] Infrared (KBr)v max 3442,1756,1703,1631,1455,1384,1249,1160,1056cm -1

[0167] Ultraviolet (MeOH)λ max (logε): 220(2.94)nm;

[0168] Circular dichroism (c 0.20, MeOH)λ max (Δε): 232(+0.2), 282(+3.0)nm

[0169]

[0170] The proton and carbon NMR spectra are shown in Tables 3 and 6.

[0171]

[0172] Artemordin M (13)

[0173] Molecular formula: C 30 H 44 O7

[0174] Molecular weight: 516

[0175] Appearance: White amorphous powder;

[0176] High-resolution mass spectrometry in positive ion mode m / z 517.3164 ([M+H]) + Calculated as C 30 H 45O7,517.3160).

[0177] Infrared (KBr)v max 3434,1770,1709,1627,1454,1380,1268,1173,1158,1119,1058cm - 1. Ultraviolet (MeOH)λ max (logε): 220(2.85)nm;

[0178] Circular dichroism (c 0.21, MeOH)λ max (Δε):212(–11.1),245(+2.6)nm;

[0179]

[0180] The proton and carbon NMR spectra are shown in Tables 3 and 6.

[0181]

[0182] Artemordin N, 14

[0183] Molecular formula: C 30 H 46 O8

[0184] Molecular weight: 534

[0185] Appearance: White amorphous powder;

[0186] High-resolution mass spectrometry in positive ion mode m / z 557.3078 ([M+Na)) + The calculated value is 557.3085.

[0187] Ultraviolet (MeOH)λ max (logε):215(3.04);

[0188] Circular dichroism (MeOH)λ max (Δε)212(-6.8),253(+0.9)nm;

[0189]

[0190] The proton and carbon NMR spectra are shown in Tables 3 and 6.

[0191]

[0192] Artemordin O (15)

[0193] Molecular formula: C 30 H46 O5

[0194] Molecular weight: 486

[0195] Appearance: Colorless oily substance;

[0196] High-resolution mass spectrometry in positive ion mode, m / z 509.3234 ([M+Na)). + Calculated as C 30 H 46 O5Na, 509.3237). Infrared (KBr) v max 3438,1724,1629,1455,1381,1264,1177,1159cm -1

[0197] Circular dichroism (MeOH)λ max (Δε)204(-6.3),237(+3.3)nm;

[0198]

[0199] The proton and carbon NMR spectra are shown in Tables 3 and 6.

[0200]

[0201] Artemordin P, 16

[0202] Molecular formula: C 30 H 46 O5

[0203] Molecular weight: 486

[0204] Appearance: Colorless oily substance;

[0205] High-resolution mass spectrometry in positive ion mode m / z 509.3238 ([M+Na)) + Calculated as C 30 H 46 O5Na, 509.3237).

[0206] Infrared (KBr)v max 3437,1727,1629,1454,1380,1264,1176,1158cm -1

[0207] Circular dichroism (MeOH)λ max (Δε)206(-2.8),234(+3.2)nm;

[0208]

[0209] The proton and carbon NMR spectra are shown in Tables 3 and 6.

[0210]

[0211] Artemordin Q (17)

[0212] Molecular formula: C 30 H 46 O5

[0213] Molecular weight: 486

[0214] Appearance: Colorless oily substance;

[0215] High-resolution mass spectrometry in positive ion mode, m / z 509.3242 ([M+Na)). + Calculated as C 30 H 46 O5Na, 509.3237).

[0216] Infrared (KBr)v max 3434,1711,1624,1456,1380,1266,1175,1120cm -1

[0217] Circular dichroism (MeOH)λ max (Δε)216(+1.1),223(+0.2),242(+1.4),280(-3.5)nm;

[0218]

[0219] The proton and carbon NMR spectra are shown in Tables 4 and 6.

[0220]

[0221] Artemordin R (18)

[0222] Molecular formula: C 30 H 48 O5

[0223] Molecular weight: 488

[0224] Appearance: Colorless oily substance;

[0225] High-resolution mass spectrometry in positive ion mode m / z 511.3392 ([M+Na)) + Calculated as C 30 H 48 O5Na, 511.3394).

[0226] Infrared (KBr)vmax 3438,1724,1639,1629,1455,1381,1264,1177,1117,1059cm -1

[0227] Ultraviolet (MeOH)λ max (logε):216(3.00);

[0228] Circular dichroism (MeOH)λ max (Δε)212(-2.9),234(+3.9)nm

[0229]

[0230] The proton and carbon NMR spectra are shown in Tables 4 and 6.

[0231]

[0232] Artemordin S, 19

[0233] Molecular formula: C 30 H 50 O5

[0234] Molecular weight: 490

[0235] Appearance: Colorless oily substance;

[0236] High-resolution mass spectrometry in positive ion mode, m / z 513.3554 ([M+Na)). + C 30 H 50 O5Na, calculated as 513.3550).

[0237] Infrared (KBr)v max 3438,1724,1639,1628,1455,1381,1264,1177,1117,1059cm -1 Circular dichroism (MeOH)λ max (Δε)208(-1.2),224(+1.8)nm;

[0238]

[0239] The proton and carbon NMR spectra are shown in Tables 4 and 6.

[0240] Table 1. 1H NMR spectral data of compounds 1-6 (CDCl3, 600MHz)

[0241]

[0242] Note: "ol" indicates signal overlap, and the peak shape and coupling constant cannot be read.

[0243] Table 2. 1H NMR data of compound 7-11 (CDCl3, 600MHz)

[0244]

[0245] Table 3. 1H NMR spectra of compounds 12-16 (CDCl3, 600MHz)

[0246]

[0247] Table 4. 1H NMR spectra of compounds 17-19 (CDCl3, 600MHz)

[0248]

[0249]

[0250]

[0251] Example 2:

[0252] Cytotoxic activity of compounds 1-19 against three hepatocellular carcinoma cell lines.

[0253] 1. Materials and Methods

[0254] 1.1 Materials

[0255] 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 Co., Ltd. (Suzhou, China); serum (fetal bovine serum, FBS) was purchased from Thermo Fisher Scientific Co., Ltd.; and RPMI-1640 was purchased from Thermo Fisher Scientific Biochemicals (Beijing) Co., Ltd.

[0256] 1.2 Instruments

[0257] Flex Station 3 benchtop multi-functional microplate reader (Bio-RAD 680, USA); analytical balance (AG135, Metler Toledo, China); incubator (DHP-9082, Shanghai).

[0258] 1.3 Experimental Procedure

[0259] 1) Take liver cancer cells in the logarithmic growth phase, discard the old culture medium, wash twice with PBS, and discard the PBS;

[0260] 2) Digest the cells with 0.25% trypsin. When the cell outlines are observed to darken and become rounded under a microscope, quickly remove the trypsin.

[0261] 3) Stop digestion with DMEM complete medium containing 10% serum and resuspend the cells. Take 10 μL of the cell suspension, count the cells using a cell counter, and adjust the cell concentration to 1 × 10⁻⁶ cells with culture medium. 4 / mL, seeded into 96-well plates, add 100μL of cell suspension to each well, and incubate in a 37℃, 5% CO2 incubator for 24h to allow the cells to adhere;

[0262] 4) Remove the culture medium, add the diluted sample to the plate, 100 μL per well, set 3 replicates for each concentration, and continue incubation in the incubator for 48 h;

[0263] 5) Remove the culture medium, add the prepared MTT solution (1 mg / mL), add 100 μL to each well, and incubate in an incubator for 4 h;

[0264] 6) Remove the MTT solution, add 100 μL of DMSO to each well, and incubate in an incubator for 10 min;

[0265] 7) Measure the absorbance at 490nm using an ELISA reader. The inhibition rate is calculated using the formula: Inhibition rate = (Negative group - Experimental group).

[0266] The cell inhibition rate was calculated as (negative control group - blank group) × 100%, and the IC50 was calculated using the statistical software GraphPad Prism 5. 50 The experiment was repeated 3 times.

[0267] Table 7 Half-inhibitory concentrations of compounds on hepatocellular carcinoma cell cytotoxicity.

[0268]

[0269] Table 8. Hepatocellular carcinoma cell cytotoxicity of compounds 1-19

[0270]

[0271] Note: Sorafenib is a positive control, IC50 50 The concentrations were 8.65 (HepG2), 7.24 (Huh7), and 10.97 (SK-Hep-1) μg / mL. 2. Results

[0272] The anti-cytotoxic activity against hepatocellular carcinoma cells was evaluated for all isolated compounds, and the results are shown in Tables 7 and 8. Compounds 1-19 exhibited cytotoxic activity against three hepatocellular carcinoma cell lines (HepG2, Huh7, and SK-Hep-1) at 200 μM. Compounds 2, 8, 10, 11, and 17 showed inhibitory effects on HepG2 cells, with IC50 values ​​of [missing value].50 The values ​​were 26.9, 25.1, 25.5, 23.1, and 30.7 μM; compounds 2, 8, 10, 16, and 17 had inhibitory effects on Huh7 cells, with IC50 values ​​of 26.9, 25.1, 25.5, 23.1, and 30.7 μM. 50 The concentrations were 29.5, 18.3, 26.5, 30.3, and 27.4 μM; compounds 2, 8, and 15 had inhibitory effects on SK-Hep-1 cells, with IC50 values ​​of 29.5, 18.3, 26.5, 30.3, and 27.4 μM. 50 The values ​​were 19.7, 15.7, and 30.7 μM. In summary, compound 7 exhibited the best cytotoxic activity against the three hepatocellular carcinoma cell lines HepG2, Huh7, and SK-Hep-1, with an IC50 value of 19.7, 15.7, and 30.7 μM. 50 The values ​​were 35.1, 35.0, and 32.7 μM, respectively.

[0273] 3. Conclusion

[0274] Experimental results showed that compounds 1-19 exhibited cytotoxic activity against three liver cancer cell lines: HepG2, Huh7, and SK-Hep-1. Among them, compound 7 showed moderate cytotoxic activity against HepG2 cells, with an IC50 value of [missing information]. 50 The value was 35.1 μM; compound 7 exhibited moderate cytotoxic activity against Huh7 cells, with an IC50 value of 35.1 μM. 50 The value was 35.0 μM; compound 7 exhibited moderate cytotoxic activity against SK-Hep-1 cells, with an IC50 value of 35.0 μM. 50 The concentration value was 32.7 μM. The remaining compounds all showed some inhibitory activity against the three hepatocellular carcinoma cell lines at 200 μM. In summary, compound 7 exhibited the best cytotoxic activity against the three hepatocellular carcinoma cell lines HepG2, Huh7, and SK-Hep-1, with an IC50 value of 32.7 μM. 50 The concentrations were 35.1, 35.0, and 32.7 μM, respectively. These results indicate that compound 1-19 isolated from Artemisia annua can be used as a drug to treat liver cancer-related diseases.

[0275] Formulation Examples

[0276] In the following formulation examples, conventional reagents were selected and the formulations were prepared according to existing conventional methods. These formulation examples only demonstrate that at least one of the compounds 1-19 described in this invention can be prepared into different formulations, and no specific limitations are made on the specific reagents and operations:

[0277] 1. Dissolve at least one of the compounds 1-19 of the present invention in a small amount of DMSO, add water for injection as usual, filter, fill and sterilize to prepare an injection solution, wherein the concentration of the injection solution is 0.5-5 mg / mL.

[0278] 2. Dissolve at least one of the compounds 1-19 of the present invention in a small amount of DMSO, then dissolve it in sterile water for injection, stir to dissolve, filter with a sterile suction funnel, then filter aseptically, dispense into ampoules, freeze-dry at low temperature, and then seal aseptically to obtain a powder for injection.

[0279] 3. At least one of the compounds 1-19 of the present invention is added to the excipient at a weight ratio of 9:1 to prepare a powder.

[0280] 4. Add at least one of the compounds 1-19 of the present invention to the excipient at a weight ratio of 5:1, and granulate and compress the mixture into tablets.

[0281] 5. Prepare an oral liquid from at least one of the compounds 1-19 of the present invention using conventional oral liquid preparation methods.

[0282] 6. At least one of the compounds 1-19 of the present invention is added to an excipient at a weight ratio of 5:1 to the excipient to form a capsule.

[0283] 7. Add at least one of the compounds 1-19 of the present invention to the excipient at a weight ratio of 5:1 to prepare granules.

[0284] As can be seen from the above embodiments, the present invention provides a compound from Artemisia annua, its preparation method and application, and a pharmaceutical composition and its application. The Artemisinin provided by the present invention mainly comprises 19 novel sesquiterpene dimers. These compounds exhibit varying degrees of cytotoxic activity against liver cancer cells and can be combined with pharmaceutically acceptable carriers or excipients to form pharmaceutical compositions, enabling the preparation of anti-liver cancer drugs.

[0285] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. Compounds 2, 8, 10, 11, 15, 16, and 17, as shown in the following structural formulas, ； ； ； ； ； ； 。 2. The method for preparing compounds 2, 8, 10, 11, 15, 16, and 17 as shown in claim 1, characterized in that... The method includes the following steps: The dried aerial parts of *Artemisia annua* are taken, pulverized, and extracted twice with three times the volume of 90% ethanol. The extracts are combined and concentrated under reduced pressure to obtain a crude extract, which is then dispersed in water and extracted with ethyl acetate to obtain the ethyl acetate extract. Subsequently, the ethyl acetate extract is subjected to silica gel column chromatography and eluted with gradients of acetone-petroleum ether at volume ratios of 0:100, 5:95, 10:90, 20:80, 40:60, and 100:0 to obtain six fractions, Frs. AF. Fraction Fr. D is subjected to silica gel column chromatography and eluted with ethyl acetate-petroleum ether at ratios of 10:90, 20:80, 30:70, 50:50, and 0:100 to obtain four fractions, Fr. D1-Fr. D4. Fr. D2 is subjected to MCI gel CHP 20P column chromatography and eluted with methanol-water at ratios of 40:60, 50:50, 70:30, 90:10, and 100:0 to obtain four subfractions, Frs. D2.1-D2.4; Fr. D2.3 then use Rp-C 18 Column chromatography was performed with methanol-water at ratios of 50:50, 70:30, 90:10, and 100:0 to obtain five fractions Fr. D2.3.1–D2.3.

4. Fr. D2.3.2 was then subjected to silica gel column chromatography with methanol-chloroform at ratios of 2:98–15:85 to obtain four fractions Fr. D2.3.2a–D2.3.2d. Fr. D2.3.2b was finally subjected to semi-preparative high-performance liquid chromatography (HPLC) at an Agilent XDB-C1000000 column. 18 Compound 2, artemisinin B, was purified by column chromatography with methanol-water at a ratio of 77:

23. Fr. D2.3.3 was then purified by silica gel column chromatography with methanol-chloroform at a ratio of 2:98-15:85, followed by Sephadex LH-20 column chromatography with methanol-chloroform at a ratio of 50:

50. Finally, the purified compound was purified by semi-preparative high-performance liquid chromatography (HPLC) at an Agilent XDB-C10. 18 Compounds 8 (artemisinin H) and 10 (artemisinin J) were purified by column chromatography with methanol-water at a ratio of 80:

20. Fr. D3 was then purified by MCI gel CHP 20P column chromatography with methanol-water at ratios of 40:60, 50:50, 70:30, 90:10, and 100:0 to obtain four subfractions Frs. D3.1–D3.

4. Fr. D3.3 then Rp-C 18 Column chromatography was performed with methanol-water mixtures of 40:60, 50:50, 60:40, 70:30, and 100:0 to obtain four fractions, Fr. D3.3.1–D3.3.

4. Fr. D3.3.1 was subjected to silica gel column chromatography, eluted with ethyl acetate-petroleum ether 10:90-50:50, followed by Sephadex LH-20 column chromatography, eluted with methanol-chloroform 50:50, and finally settled by preparative high performance liquid chromatography on an Agilent XDB-C10 column. 18 Compound 11, artemisinin K, was purified by column chromatography with methanol-water at a ratio of 77:23 to obtain the compound. Following silica gel column chromatography with methanol-chloroform at a ratio of 2:98-15:85, the compound was further purified by Sephadex LH-20 column chromatography with methanol-chloroform at a ratio of 50:

50. Finally, the compound was purified by semi-preparative high-performance liquid chromatography (HPLC) at an Agilent XDB-C10 HPLC system. 18 Compound 17, artemisinin Q, was purified by column chromatography using acetonitrile-water (67:33) and methanol-water (80:20). Following silica gel column chromatography with methanol-chloroform (2:98-15:85), followed by Sephadex LH-20 column chromatography with methanol-chloroform (50:50), the final product was purified by semi-preparative high-performance liquid chromatography (HPLC) at an Agilent XDB-C10. 18 The compounds 15 (artemisinin O) and 16 (artemisinin P) were purified by column elution with acetonitrile-water in a ratio of 72:28 and methanol-water in a ratio of 84:

16.

3. The use of compounds 2, 8, 10, 11, 15, 16, and 17, as shown in the structural formula of claim 1, in the preparation of anti-hepatocellular carcinoma drugs.

4. A pharmaceutical composition comprising at least one of compounds 2, 8, 10, 11, 15, 16, 17 as shown in the structural formula of claim 1 and a pharmaceutically acceptable carrier.

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

6. The method for preparing the pharmaceutical composition according to claim 4, characterized in that... The method includes the following steps: first, compounds 2, 8, 10, 11, 15, 16, and 17 are obtained using the preparation method described in claim 2; then, any one of the above compounds or any combination thereof is added to a pharmaceutically acceptable carrier.

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