Cadinane type sesquiterpene dimer in artemisia annua and pharmaceutical composition, preparation method and application thereof

Abstract

The invention provides arteannuin A1A4 (artemannuins A1A4, 14), arteannuin B1B4 (artemannuins B1B4, 57), arteannuin C1C2 (artemannuins C1, C2, 8 and 9) and arteannuin DH (artemannuins DH, 1014), a pharmaceutical composition of the arteannuin A1A4, the arteannuin B1B4, the arteannuin C1C2, the arteannuins C1, C2, 8 and 9) and the arteannuin DH, and a preparation method and application of the arteannuin A1A4, the arteannuin B1B4, the arteannuin C1C2, the arteannuin C1C2, the arteannuin DH and the arteannuin DH, and belongs to the technical field of medicine. The 14 cadinane type sesquiterpene dimers shown in the structural formula (I) have an inhibiting effect on human hepatoma cell lines HepG2, Huh7 and SK-Hep-1, can form a pharmaceutical composition with a pharmaceutically acceptable carrier or excipient, and can be used for preparing anti-hepatoma drugs.

Description

Technical fields:

[0001] This invention belongs to the field of pharmaceutical technology. Specifically, it relates to 14 juniper sesquiterpene dimers, artemannuins A1-A4 (1-4), B1-B4 (5-7), C1-C2 (8 and 9), and DH (10-14), their preparation methods and applications, pharmaceutical compositions using compounds 1-14 as active ingredients, and the application of these compounds and their pharmaceutical compositions in the preparation of drugs for treating liver cancer. Background technology:

[0002] Due to the traditional medicinal effects of plants from which natural products are derived, as well as their unique chemical structures, multiple targets, high efficiency, and low toxicity, natural products have significant advantages in innovative drug development. Statistics show that nearly 75% of clinically used drugs are derived from natural products and their derivatives. For example, plants in the Asteraceae family, rich in sesquiterpene lactones, have historically been used to treat inflammation, cancer, and infections. Sesquiterpene dimers are a class of rare natural hybrids with a C30 skeleton, biogenically formed from two sesquiterpene monomers through reactions such as Diels-Alder, [2+2] cycloaddition, Michael addition, free radical coupling, or esterification. The complex structure and rich pharmacological activities of sesquiterpene dimers make them highly promising drug candidates, attracting widespread attention from chemists and biologists.

[0003] Sesquiterpene dimers are mainly derived from Artemisia species, with over 260 reported to date. Most sesquiterpene dimers are guaiacolane-type dimers, exhibiting activities such as anti-hepatocellular carcinoma, antitumor, and anti-inflammatory effects. Furthermore, our team previously reported for the first time 40 sesquiterpene dimers containing juniperane units from Artemisia acutum, including juniperane-juniperane and juniperane with other types of monoterpenes (monocyclic farnesane, bisabolane, eucalyptane, 12-nor-juniperane, 4,5-fractured-juniperane, 4,5-fractured-12-nor-juniperane, 5,6-fractured-12-nor-juniperane, 5(4→3)-migratory-juniperane, 5(4→15)-migratory-juniperane, 5(4→3)-migratory-12-nor-juniperane, and 5(4→3)-migratory-4,15-dinor-juniperane).

[0004] Artemisia annua is a well-known traditional Chinese medicine used to treat diseases such as malaria. Reported chemical components in Artemisia annua include sesquiterpenes, sesquiterpene dimers, diterpenes, flavonoids, phenylpropanoids, coumarins, and volatile oils, exhibiting biological activities such as antimalarial, antibacterial, antiparasitic, antipyretic, and immune-enhancing effects. Juniperane sesquiterpenes are the main active components in Artemisia annua, with artemisinin exhibiting significant antimalarial activity. Two derivatives developed to address the insufficient solubility of artemisinin, artemether and artesunate, have been applied clinically. Literature also reports the anti-hepatocellular carcinoma activity of Artemisia annua, and network pharmacology predicts that its main active components are flavonoids.

[0005] To date, there are no reports in the prior art of artemannuins A1-A4 (1-4), B1-B4 (5-7), C1-C2 (8 and 9), and DH (10-14), nor are there any reports of pharmaceutical compositions containing them as active ingredients, nor are there any reports of their use in the preparation or treatment of drugs for liver cancer. Summary of the Invention:

[0006] The purpose of this invention is to address the aforementioned shortcomings of existing technologies by providing a new class of medicinally valuable artemisinins A1-A4 (1-4), B1-B4 (5-7), C1-C2 (8 and 9), and DH (10-14) as shown in formula (I), including preparation methods, pharmaceutical compositions, and applications. This invention also found that the ethyl acetate extract of Artemisia annua ethanol extract exhibits good inhibitory activity against three types of liver cancer cells (HepG2, Huh7, and SK-Hep-1), with inhibition rates of 53.2%, 52.1%, and 59.6% at 200 μg / mL, respectively. For the first time, activity-tracking isolation revealed 14 sesquiterpene dimers containing juniperane units, namely artemannuins A1-A4 (1-4), B1-B4 (5-7), C1-C2 (8 and 9), and DH (10-14). These compounds exhibit inhibitory activity against human liver cancer cell lines HepG2, Huh7, and SK-Hep-1, and could 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 14 juniperane sesquiterpene dimers, artemannuins A1-A4 (1-4), B1-B4 (5-7), C1-C2 (8 and 9), and DH (10-14), with structures shown in formula (I):

[0009]

[0010] The present invention also provides a method for preparing compounds 1-14 of Formula I. 48 kg of dried Artemisia annua aerial parts are 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 3.1 kg of the ethyl acetate extract. Subsequently, the ethyl acetate extract is subjected to silica gel column chromatography and eluted with a gradient of acetone-petroleum ether at volume ratios of 10:90, 20:80, and 30:70 and ethyl acetate to obtain four fractions, Frs.AD. Fractions A and B were combined and then subjected to silica gel column chromatography, eluted with MeOH-CHCl3 at volume ratios of 1:99, 2:98, 5:95, 10:90, 20:80, and 40:60 and a MeOH gradient, yielding five fractions Frs.B-1-B-5. 51.6 g of Fr.B-2 was eluted on an MCI gel CHP 20P column with MeOH-H2O and MeOH at ratios of 50:50, 70:30, and 90:10, yielding four subfractions Frs.B-2a-B-2d. 9.0 g of Fr.B-2b was subjected to Rp-C... 18 Column chromatography was performed, with MeOH-H2O gradients of 50:50, 60:40, 70:30, and 80:20 to obtain subfractions Frs.B-2b-1-B-2b-5. 3 g of Fr.B-2b-3 was subjected to silica gel column chromatography, with MeOH-CHCl3 eluents of 2:98, 5:95, and 10:90 to obtain five subfractions Frs.B-2b-3a-B-2b-3e. 560 mg of Fr.B-2b-3d was subjected to silica gel column chromatography, with MeOH-CHCl3 eluents of 5:95 and 10:90, followed by semi-preparative HPLC on an Agilent XDB-C... 18 Compounds 10 (38 mg) and 11 (4 mg) were purified on a column using acetonitrile-water at a ratio of 40:60. 10.3 g of Fr. B-2c was purified by reverse-phase Rp-C... 18Column chromatography was performed, eluting with MeOH-H2O at ratios of 50:50, 60:40, 70:30, and 80:20 to obtain five fractions: Frs.B-2c-1-B-2c-5. 4.2 g of Fr.B-2c-3 was subjected to silica gel column chromatography, eluting with MeOH-CHCl3 at ratios of 2:98, 5:95, and 10:90 to obtain five fractions: Frs.B-2c-3a-B-2c-3e. 1.7 g of Fr.B-2c-3a was subjected to Sephadex LH-20 column chromatography, eluting with MeOH-CHCl3 at a ratio of 50:50, followed by semi-preparative HPLC on an Agilent XDB-C... 18 Compounds 1 (700 mg), 2 (16 mg), 3 (34 mg), and 4 (10 mg) were purified by column chromatography with acetonitrile-water at a ratio of 40:60. 1.5 g of Fr. B-2c-3b was eluted with 50:50 MeOH-CHCl3 on a Sephadex LH-20 column and then subjected to semi-preparative HPLC on an Agilent XDB-C10 column. 18 Compounds 5 (53 mg), 6 (10 mg), and 7 (591 mg) were obtained by repeated purification on a column using acetonitrile-water (48:52) and methanol-water (75:25). 910 mg of Fr. B-2c-3c was purified by Sephadex LH-20 column chromatography, eluted with 50:50 MeOH-CHCl3, and then subjected to semi-preparative HPLC on an Agilent XDB-C... 18 Compounds 8 (591 mg) and 9 (15 mg) were purified by methanol-water at a ratio of 78:22 on a column. 8 g of Fr.B-4 was purified by MCI gel CHP 20P column chromatography, eluting with MeOH-H2O and MeOH at ratios of 50:50, 70:30, and 90:10 to give four subfractions Frs.B-4a-B-4d. 1.1 g of Fr.B-4c was purified by silica gel column chromatography, eluting with MeOH-CHCl3 at ratios of 5:95 and 10:90, followed by Sephadex LH-20 column chromatography with a 50:50 MeOH-CHCl3 treatment, and finally semi-preparative HPLC was performed on an Agilent XDB-C... 18 Compounds 12 (13 mg), 13 (11 mg), and 14 (17 mg) were purified on a column using methanol-water in a ratio of 65:35 and acetonitrile-water in a ratio of 42:58.

[0011] This invention provides the use of compounds 1-14 of Formula I in the preparation of anti-liver cancer drugs. This invention does not have any particular limitation on the method of the application, and 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 1–14 of formula (I) and a pharmaceutically acceptable carrier.

[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 1-14 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 compounds 1–14 is used to prepare an anti-liver cancer drug, the present invention preferably uses compounds 1–14 directly or in the form of a pharmaceutical composition.

[0015] The pharmaceutical composition provided by this invention comprises at least one of compounds 1-14 above and a pharmaceutically acceptable carrier or excipient. In this invention, the pharmaceutically acceptable carrier 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; any pharmaceutically acceptable carrier well-known in the art that is non-toxic to humans and animals and inert 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 1-14 can be directly mixed with a pharmaceutically acceptable carrier. 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 1-14 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 14 juniperine sesquiterpene dimers, artemannuins A1-A4 (1-4), B1-B4 (5-7), C1-C2 (8 and 9), and DH (10-14).

[0021] 2. This invention provides a novel method for preparing new compounds 1–14, which uses readily available raw materials, is easy to operate, and is suitable for industrial production.

[0022] 3. This invention provides pharmaceutical compositions with novel compounds 1–14 as active ingredients, providing new drugs with good pharmaceutical effects for novel anti-liver cancer drugs.

[0023] 4. Screening results of the anti-hepatocellular carcinoma activity of compounds 1–14 of the present invention showed that, at a concentration of 200 μg / mL, compound 13 exhibited a certain inhibitory effect on three hepatocellular carcinoma cell lines: HepG2, Huh7, and SK-Hep-1, with inhibition rates of 52.0%, 61.3%, and 61.7%, respectively. The esterified products 1a, 3a, 6a, and 8a, obtained after methyl esterification of compounds 1, 3, 6, and 8, all showed significant inhibitory activity against HepG2, Huh7, and SK-Hep-1 cells. At a concentration of 200 μM, their inhibition rates ranged from 74.6% to 96.5%, which was 10–18 times that of the original compounds. When the concentration was reduced to 100 μM, 1a, 3a, 6a, and 8a still showed inhibitory effects on the three hepatocellular carcinoma cell lines, with inhibition rates between 65.9% and 86.1%, which was 30–64 times that of the original compounds. The dose-response relationship for 1a, 3a, 6a, and 8a was further investigated, and the IC50 was obtained. 50 The values ​​were 45.9, 41.0, 41.2, and 38.4 μM (HepG2), 30.5, 35.4, 57.2, and 42.3 μM (Huh7), and 30.9, 30.1, 28.6, and 32.1 μM (SK-Hep-1). These results indicate that compounds 1–14 isolated from Artemisia annua can be used as drugs to treat liver cancer-related diseases. Attached image description:

[0024] Figure 1 The structural formulas of compounds 1–14 of this invention are shown below;

[0025] Figure 2 This is a schematic diagram of the X-ray single crystal structure of compound 1a of the present invention;

[0026] Figure 3 To investigate the inhibitory activity of compound 1-14 isolated from Artemisia annua on the proliferation of HepG2, Huh7, and SK-Hep-1 hepatocellular carcinoma cells at concentrations of 200 and 100 μM, sorafenib was used as a positive control. Detailed implementation method:

[0027] To better understand the essence of the present invention, the following, in conjunction with the accompanying drawings, uses experimental examples and embodiments of the present invention to further illustrate the preparation methods, structural identification, pharmacological effects, and preparation methods and drug compositions of artemannuins A1-A4 (1-4), B1-B4 (5-7), C1-C2 (8 and 9), and DH (10-14), but these experimental examples and embodiments are not intended to limit the present invention.

[0028] 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.

[0029] Example 1:

[0030] The present invention relates to the preparation of 14 juniperine sesquiterpene dimers, and artemannuins A1-A4 (1-4), B1-B4 (5-7), C1-C2 (8 and 9), and DH (10-14).

[0031] 48 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 3.1 kg of ethyl acetate extract. Subsequently, the ethyl acetate extract was subjected to silica gel column chromatography and eluted with gradients of acetone-petroleum ether at volume ratios of 10:90, 20:80, and 30:70 and ethyl acetate to obtain four fractions, Frs.AD. Fractions A and B were combined and then subjected to silica gel column chromatography, eluted with MeOH-CHCl3 at volume ratios of 1:99, 2:98, 5:95, 10:90, 20:80, and 40:60, yielding five fractions Frs.B-1-B-5. 51.6 g of Fr.B-2 was eluted on an MCI gel CHP 20P column with MeOH-H2O and MeOH at ratios of 50:50, 70:30, and 90:10, yielding four subfractions Frs.B-2a-B-2d. 9.0 g of Fr.B-2b was subjected to Rp-C... 18Column chromatography was performed, with MeOH-H2O gradients of 50:50, 60:40, 70:30, and 80:20 to obtain subfractions Frs.B-2b-1-B-2b-5. 3 g of Fr.B-2b-3 was subjected to silica gel column chromatography, with MeOH-CHCl3 eluents of 2:98, 5:95, and 10:90 to obtain five subfractions Frs.B-2b-3a-B-2b-3e. 560 mg of Fr.B-2b-3d was subjected to silica gel column chromatography, with MeOH-CHCl3 eluents of 5:95 and 10:90, followed by semi-preparative HPLC on an Agilent XDB-C... 18 Compounds 10 (38 mg) and 11 (4 mg) were purified on a column using acetonitrile-water at a ratio of 40:60. 10.3 g of Fr. B-2c was purified by reverse-phase Rp-C... 18 Column chromatography was performed, eluting with MeOH-H2O at ratios of 50:50, 60:40, 70:30, and 80:20 to obtain five fractions: Frs.B-2c-1-B-2c-5. 4.2 g of Fr.B-2c-3 was subjected to silica gel column chromatography, eluting with MeOH-CHCl3 at ratios of 2:98, 5:95, and 10:90 to obtain five fractions: Frs.B-2c-3a-B-2c-3e. 1.7 g of Fr.B-2c-3a was subjected to Sephadex LH-20 column chromatography, eluting with MeOH-CHCl3 at a ratio of 50:50, followed by semi-preparative HPLC on an Agilent XDB-C... 18 Compounds 1 (700 mg), 2 (16 mg), 3 (34 mg), and 4 (10 mg) were purified by column chromatography with acetonitrile-water at a ratio of 40:60. 1.5 g of Fr. B-2c-3b was chromatographically analyzed on a Sephadex LH-20 column with 50:50 MeOH-CHCl3 as elution, followed by semi-preparative HPLC on an Agilent XDB-C... 18 Compounds 5 (53 mg), 6 (10 mg), and 7 (591 mg) were obtained by repeated purification on a column using acetonitrile-water (48:52) and methanol-water (75:25). 910 mg of Fr. B-2c-3c was purified by Sephadex LH-20 column chromatography, eluted with 50:50 MeOH-CHCl3, and then subjected to semi-preparative HPLC on an Agilent XDB-C... 18Compounds 8 (591 mg) and 9 (15 mg) were purified by methanol-water at a ratio of 78:22 on a column. 8 g of Fr.B-4 was purified by MCI gel CHP 20P column chromatography, eluting with MeOH-H2O and MeOH at ratios of 50:50, 70:30, and 90:10 to give four subfractions Frs.B-4a-B-4d. 1.1 g of Fr.B-4c was purified by silica gel column chromatography, eluting with MeOH-CHCl3 at ratios of 5:95 and 10:90, followed by Sephadex LH-20 column chromatography with a 50:50 MeOH-CHCl3 treatment, and finally semi-preparative HPLC was performed on an Agilent XDB-C... 18 Compounds 12 (13 mg), 13 (11 mg), and 14 (17 mg) were purified by column chromatography using methanol-water in a 65:35 ratio and acetonitrile-water in a 42:58 ratio. Structural data for compounds 1-14 are as follows:

[0032] Optical rotation was measured using an Autopol VI polarimeter (Rudolph Research Analytical, Hackettstown, USA); infrared (IR) spectroscopy was performed using the KBr pellet method on a Bio-Rad FTS-135 infrared spectrometer (Hercules, California, USA); ultraviolet (UV) spectroscopy was performed using a UV-2401PC UV spectrometer (Shimadzu, Kyoto, Japan); ECD spectroscopy was performed using an Applied Photophysics circular dichroism spectrometer (Agilent, Santa Clara, United States); nuclear magnetic resonance (1D and 2D NMR) spectroscopy was performed using an Avance spectrometer. The analysis was performed using a Type III-600 superconducting nuclear magnetic resonance spectrometer (Bruker, Bremerhaven, Germany) with deuterated chloroform as the solvent; high-resolution mass spectrometry (HRESIMS) was performed using a Shimadzu LCMS-IT-TOF mass spectrometer (Shimadzu, Kyoto, 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; 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 column model. XDB-C18 (5μm, 9.4×250mm); chromatographic grade acetonitrile was purchased from Mitsubishi Chemical Corporation; MCI gel CHP20P (75~150μm) was purchased from Mitsubishi Chemical Corporation (Tokyo, Japan); the colorimetric reagent was 10% H2SO4-EtOH solution.

[0033]

[0034] Artemannuin A1 (1)

[0035] Molecular formula: C 30 H 38 O6

[0036] Molecular weight: 494

[0037] Appearance: White powder;

[0038] HRESIMS m / z 495.2752 [M+H]+ (calcd.for C 30 H 39 O6,495.2741);

[0039] IR(KBr)ν max :3435,1699,1629,1455,1417,1353,1256,1173,1096cm –1 ;

[0040] ECD(c0.29,MeOH)λ max (Δε):209(–26.11),287(–1.45)nm;

[0041] (c0.12,MeOH);

[0042] 1 H NMR and 13 C NMR (DEPT) data are shown in Tables 1 and 2.

[0043]

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

[0045] Crystal data for 1a:C 32 H 42 O6,M=522.65, α=90°, β=107.9550(10)°, γ=90°, T=150.(2)K,space group P1211,Z=2,μ(Cu Kα)=0.659mm -1 ,28511measured reflections,5427independent reflections(R int =0.0368).The finalR1 values ​​were 0.0289(I>2σ(I)).The final wR(F 2 )values ​​were 0.0735(I>2σ(I)).Thefinal R1 values ​​were 0.0298(all data).The final wR(F 2 )values ​​were 0.0743(alldata).The goodness of fit on F 2was 1.042.Flack parameter=0.03(5).CCDC2299801.

[0046]

[0047] Artemannuin A2,2

[0048] Molecular formula: C 30 H 38 O6

[0049] Molecular weight: 494

[0050] Appearance: White powder;

[0051] HRESIMS m / z 495.2739 [M+H] + (calcd.for C 30 H 39 O6,495.2741);

[0052] IR(KBr)ν max :3435,1670,1629,1455,1416,1354,1255,1174,1097cm –1 ;

[0053] ECD(c 0.22,MeOH)λ max (Δε):209(–19.30),284(–0.32)nm;

[0054] (c 0.10,MeOH);

[0055] 1 H NMR and 13 C NMR (DEPT) data are shown in Tables 1 and 2.

[0056]

[0057] Artemannuin A3,3

[0058] Molecular formula: C 30 H 38 O6

[0059] Molecular weight: 494

[0060] Appearance: White powder;

[0061] HRESIMS m / z 495.2746 [M+H] + (calcd.for C30 H 39 O6,495.2741);

[0062] IR(KBr)ν max :3434,1699,1628,1454,1417,1353,1256,1173,1096cm –1 ;

[0063] ECD(c 0.21,MeOH)λ max (Δε):209(–24.94),284(–1.34)nm;

[0064] (c 0.13,MeOH);

[0065] 1 H NMR and 13 C NMR (DEPT) data are shown in Tables 1 and 2.

[0066]

[0067] Artemannuin A4 (4)

[0068] Molecular formula: C 30 H 38 O6

[0069] Molecular weight: 494

[0070] Appearance: White powder;

[0071] HRESIMS m / z 495.2742 [M+H] + (calcd.for C 30 H 39 O6,495.2741);

[0072] IR(KBr)ν max :3435,1700,1629,1455,1416,1354,1254,1172,1096cm –1 ;

[0073] ECD(c0.23,MeOH)λ max (Δε):209(–16.83),284(–0.84)nm;;

[0074] (c0.16,MeOH);

[0075] 1 H NMR and 13C NMR (DEPT) data are shown in Tables 1 and 2.

[0076]

[0077] Artemannuin B1 (5)

[0078] Molecular formula: C 30 H 40 O6

[0079] Molecular weight: 496

[0080] Appearance: White powder;

[0081] HRESIMS m / z 495.2759 [M–H] - (calcd.for C 30 H 39 O6,495.2752);

[0082] IR(KBr)ν max :3432,1700,1630,1456,1415,1355,1258,1176,1099cm –1 ;

[0083] ECD(c 0.25,MeOH)λ max (Δε):210(–42.78),294(+10.10)nm;

[0084] (c 0.09,MeOH);

[0085] 1 H NMR and 13 C NMR (DEPT) data are shown in Tables 1 and 2.

[0086]

[0087] Artemannuin B2 (6)

[0088] Molecular formula: C 30 H 40 O6

[0089] Molecular weight: 496

[0090] Appearance: White powder;

[0091] HRESIMS m / z 495.2758 [M–H] - (calcd.for C 30 H 39O6,495.2752);

[0092] IR(KBr)ν max :3436,1700,1629,1456,1415,1383,1255,1174cm –1 ;

[0093] ECD(c0.21,MeOH)λ max (Δε):209(–24.80),302(+1.62)nm;

[0094] (c0.16,MeOH);

[0095] 1 H NMR and 13 C NMR (DEPT) data are shown in Tables 1 and 2.

[0096]

[0097] Artemannuin B3 (7)

[0098] Molecular formula: C 30 H 38 O6

[0099] Molecular weight: 494

[0100] Appearance: White powder;

[0101] HRESIMS m / z 495.2756[M–H]-(calcd.for C 30 H 39 O6,495.2752);

[0102] IR(KBr)ν max :3429,1701,1630,1457,1416,1354,1252,1174cm –1 ;

[0103] ECD(c0.23,MeOH)λ max (Δε):209(–16.84),304(+1.90)nm;

[0104] (c0.08,MeOH);

[0105] 1 H NMR and 13 C NMR (DEPT) data are shown in Tables 1 and 2.

[0106]

[0107] Artemannuin C1, 8

[0108] Molecular formula: C 30 H 38 O6

[0109] Molecular weight: 494

[0110] Appearance: White powder;

[0111] HRESIMS m / z 493.2586[M–H]-(calcd.for C 30 H 37 O6,493.2596);

[0112] IR(KBr)ν max :3436,1698,1629,1455,1413,1355,1256,1177cm –1 ;

[0113] ECD(c0.24,MeOH)λ max (Δε):209(–56.96),294(+9.33)nm;

[0114] (c0.11,MeOH);

[0115] 1 H NMR and 13 C NMR (DEPT) data are shown in Tables 1 and 2.

[0116]

[0117] Artemannuin C2, 9

[0118] Molecular formula: C 30 H 38 O6

[0119] Molecular weight: 494

[0120] Appearance: White powder;

[0121] HRESIMS m / z 493.2588 [M–H] - (calcd.for C 30 H 37 O6,493.2596);

[0122] IR(KBr)ν max:3436,1698,1629,1454,1412,1383,1258,1179cm –1 ;

[0123] ECD(c0.22,MeOH)λ max (Δε):209(–48.71),294(+10.19)nm;

[0124] (c0.11,MeOH);

[0125] 1 H NMR and 13 C NMR (DEPT) data are shown in Tables 1 and 2.

[0126]

[0127] Artemannuin D (10)

[0128] Molecular formula: C 30 H 42 O7

[0129] Molecular weight: 514

[0130] Appearance: White powder;

[0131] HRESIMS m / z 537.2819 [M+Na] + (calcd.for C 30 H 42 O7Na,537.2823);

[0132] IR(KBr)ν max :3433,1710,1628,1454,1383,1175cm –1 ;

[0133] ECD(c0.25,MeOH)λ max (Δε):224(+3.96),290(+2.10)nm;

[0134] (c0.10,MeOH);

[0135] 1 H NMR and 13 C NMR (DEPT) data are shown in Tables 3 and 4.

[0136]

[0137] Artemannuin E (11)

[0138] Molecular formula: C 30 H 40 O7

[0139] Molecular weight: 528

[0140] Appearance: White powder;

[0141] HRESIMS m / z 529.2800 [M+H] + (calcd.for C 30 H 41 O7,529.2796);

[0142] IR(KBr)ν max :3436,1784,1717,1631,1596,1458,1385,1260,1112cm –1 ;

[0143] UV(MeOH)λ max (logε): 237(2.77)nm;

[0144] ECD(c0.27,MeOH)λ max (Δε):208(-3.02),229(-6.08),280(+0.18),322(-0.33)nm;

[0145] (c0.10,MeOH);

[0146] 1 H NMR and 13 C NMR (DEPT) data are shown in Tables 3 and 4.

[0147]

[0148] Artemannuin F (12)

[0149] Molecular formula: C 30 H 42 O8

[0150] Molecular weight: 530

[0151] Appearance: White powder;

[0152] HRESIMS m / z 531.2951 [M+H] + (calcd.for C 30 H 43 O8,531.2951);

[0153] IR(KBr)ν max :3436,1776,1708,1625,1451,1420,1381,1170cm –1 ;

[0154] ECD(c0.36,MeOH)λ max (Δε):203(-2.74),222(-0.93),232(-1.16),261(+0.17),290(-0.19)nm;

[0155] (c0.13,MeOH);

[0156] 1 H NMR and 13 C NMR (DEPT) data are shown in Tables 3 and 4.

[0157]

[0158] Artemannuin G (13)

[0159] Molecular formula: C 30 H 46 O7

[0160] Molecular weight: 518

[0161] Appearance: White powder;

[0162] HRESIMS m / z 517.3167[MH] - (calcd.for C 30 H 45 O7,517.3171);

[0163] IR(KBr)ν max :3432,1715,1629,1455,1383,1265,1172cm –1 ;

[0164] ECD(c0.27,MeOH)λ max (Δε):221(-0.15),246(+0.26)nm;

[0165] (c0.12,MeOH);

[0166] 1 H NMR and 13 C NMR (DEPT) data are shown in Tables 3 and 4.

[0167]

[0168] Artemannuin H (14)

[0169] Molecular formula: C 30 H 46 O7

[0170] Molecular weight: 518

[0171] Appearance: White powder;

[0172] HRESIMS m / z 517.3170[MH] - (calcd.for C 30 H 45 O7,517.3171);

[0173] IR(KBr)ν max :3435,1714,1630,1455,1383,1262,1175cm –1 ;

[0174] ECD(c0.25,MeOH)λ max (Δε):216(+0.46),248(+0.60)nm;

[0175] (c0.08,MeOH);

[0176] 1 H NMR and 13 C NMR (DEPT) data are shown in Tables 3 and 4.

[0177] Table 1. Compounds 1-9 13 C NMR (150MHz, δin ppm) data

[0178]

[0179]

[0180] a Deuterated methanol was used as the solvent; b Deuterated chloroform was used as the solvent.

[0181] Table 2. Compounds 1-9 1 ¹H NMR (600MHz, J in Hz, δ in ppm) data 1-9.

[0182]

[0183] aDeuterated methanol was used as the solvent; b Deuterated chloroform was used as the solvent.

[0184] Table 3 Compounds 10-14 13 C NMR (150MHz, δin ppm) data (CD3OD)

[0185]

[0186] Table 4. Compounds 10-14 1 1H NMR (600MHz, J in Hz, δ in ppm) data (CD3OD).

[0187]

[0188] Inhibitory activity of compounds 1–14 against three hepatocellular carcinoma cell lines.

[0189] 1. Materials and Methods

[0190] 1.1 Materials

[0191] The HepG2 cell line was donated by the Viable Screening Center of Kunming Institute of Botany, Chinese Academy of Sciences; SK-Hep-1 and Huh7 cell lines were purchased from Shanghai Jining Biotechnology Co., Ltd.; the culture medium (Dulbecco's Modified Eagle Medium, DMEM) was purchased from Thermo Fisher Scientific (Suzhou, China); the serum (fetal bovine serum, FBS) was purchased from Life Technologies (NY, USA); and RPMI-1640 was purchased from ThermoFisher Biochemical Products (Beijing, China).

[0192] 1.2 Instruments

[0193] Flex Station 3 benchtop multi-functional microplate reader (Bio-RAD 680, USA); analytical balance (AG135, Metler Toledo, China); incubator (DHP-9082, Shanghai).

[0194] 1.3 Experimental Procedure

[0195] 1) Take liver cancer cells in the logarithmic growth phase, discard the old culture medium, wash twice with PBS, and discard the PBS;

[0196] 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.

[0197] 3) Stop digestion with DMEM complete medium containing 10% FBS 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;

[0198] 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;

[0199] 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;

[0200] 6) Remove the MTT solution, add 100 μL of DMSO to each well, and incubate in an incubator for 10 min;

[0201] 7) Measure the absorbance at 490 nm using a microplate reader. Calculate the cell inhibition rate using the formula: Inhibition rate = (Negative group - Experimental group) / (Negative group - Blank group) × 100%. Calculate the IC50 using the statistical software GraphPad Prism 5. 50 The experiment was repeated 3 times.

[0202] 2. Results

[0203] The anti-hepatocellular carcinoma activity of all compounds was evaluated, and the results are as follows: Figure 3 As shown, at a concentration of 200 μg / mL, only compound 13 exhibited some inhibitory activity against three hepatocellular carcinoma cell lines: HepG2, Huh7, and SK-Hep-1, with inhibition rates of 52.0%, 61.3%, and 61.7%, respectively. The other compounds showed weaker inhibitory effects, with inhibition rates below 50%. These results indicate that although compounds 13 and 14 are composed of the same sesquiterpene monomers, compound 13, with its C-12 / C-15' ester bond, exhibited 2-5 times greater inhibitory activity against the three hepatocellular carcinoma cell lines than compound 14 (with its C-5 / C-1' ester bond). This suggests that the linkage mode of the two sesquiterpene monomers affects their activity.

[0204] According to literature reports, compounds containing α-methylene-γ-esters are widely considered as active drug carriers in biology due to their ability to react with many sites on proteins, and generally possess biological activity. Based on this fact, compounds 1, 3, 6, and 8 were selected for methyl esterification to obtain their esterified products 1a, 3a, 6a, and 8a. Anti-hepatocellular carcinoma activity results showed that, at the same concentration, all four derivatives exhibited significant inhibitory activity against HepG2, Huh7, and SK-Hep-1 cells, with inhibition rates ranging from 74.6% to 96.5%, which was 10-18 times that of the original compounds. When the concentration was reduced to 100 μM, 1a, 3a, 6a, and 8a still showed inhibitory effects on the three hepatocellular carcinoma cell lines, with inhibition rates between 65.9% and 86.1%, which was 30-64 times that of the original compounds. Further studies investigated the dose-response relationship of 1a, 3a, 6a, and 8a, obtaining the IC50 values. 50 The values ​​were 45.9, 41.0, 41.2 and 38.4 μM (HepG2), 30.5, 35.4, 57.2 and 42.3 μM (Huh7), and 30.9, 30.1, 28.6 and 32.1 μM (SK-Hep-1), respectively, as shown in Table 5.

[0205] Table 5. Anti-hepatocellular carcinoma activity of compounds 1a, 4a, 6a and 8a

[0206]

[0207] 3. Conclusion

[0208] Screening results for anti-hepatocellular carcinoma activity showed that at a concentration of 200 μg / mL, only compound 13 exhibited inhibitory activity against three hepatocellular carcinoma cell lines: HepG2, Huh7, and SK-Hep-1, with inhibition rates of 52.0%, 61.3%, and 61.7%, respectively. Other compounds showed weaker inhibitory effects, with inhibition rates below 50%. Methyl esterification of compounds 1, 3, 6, and 8 yielded esterified products 1a, 3a, 6a, and 8a, all of which showed significant inhibitory activity against HepG2, Huh7, and SK-Hep-1 cells. At a concentration of 200 μM, their inhibition rates ranged from 74.6% to 96.5%, which was 10-18 times higher than the original compounds. Even at a concentration reduced to 100 μM, 1a, 3a, 6a, and 8a still showed inhibitory activity against the three hepatocellular carcinoma cell lines, with inhibition rates between 65.9% and 86.1%, which was 30-64 times higher than the original compounds. The dose-response relationship for 1a, 3a, 6a, and 8a was further investigated, and the IC50 was obtained. 50The values ​​were 45.9, 41.0, 41.2, and 38.4 μM (HepG2), 30.5, 35.4, 57.2, and 42.3 μM (Huh7), and 30.9, 30.1, 28.6, and 32.1 μM (SK-Hep-1). These results indicate that compounds 1-14 isolated from Artemisia annua can be used as drugs to treat liver cancer-related diseases.

[0209] Formulation Examples

[0210] In the following formulation examples, conventional reagents were selected and the formulations were prepared according to existing conventional methods. This application example only demonstrates that at least one of the compounds 1–14 described in this invention can be prepared into different formulations, and no specific limitations are made on the specific reagents and operations:

[0211] 1. Dissolve at least one of the compounds 1–14 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.

[0212] 2. Dissolve at least one of the compounds 1–14 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.

[0213] 3. At least one of the compounds 1–14 of the present invention is added to an excipient at a weight ratio of 9:1 to prepare a powder.

[0214] 4. Add at least one of the compounds 1–14 of the present invention to the excipient at a weight ratio of 5:1, and granulate and compress the mixture into tablets.

[0215] 5. Prepare an oral liquid from at least one of the compounds 1–14 of the present invention using conventional oral liquid preparation methods.

[0216] 6. At least one of the compounds 1–14 of the present invention is added to an excipient at a weight ratio of 5:1 to the excipient to form a capsule.

[0217] 7. Add at least one of the compounds 1–14 of the present invention to the excipient at a weight ratio of 5:1 to prepare granules.

[0218] As can be seen from the above embodiments, the present invention provides a class of juniperane sesquiterpene dimers, their preparation methods and applications, pharmaceutical compositions and their applications. The 14 compounds provided by the present invention exhibit varying degrees of inhibitory effects on 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.

[0219] 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. Artemisinin A1-A4, B1-B4, C1-C2 and DH, as shown in structural formula (I), i.e., compounds 1-14, 2. The method for preparing artemisinin A1-A4, B1-B4, C1-C2 and DH, i.e., compounds 1-14, as shown in structural formula (I) of claim 1, is characterized in that... The method includes the following steps: The dried aerial parts of Artemisia annua are 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. The ethyl acetate extract is then subjected to silica gel column chromatography and eluted with a gradient of acetone-petroleum ether at volume ratios of 10:90, 20:80, and 30:70 and ethyl acetate to obtain four fractions, Frs.AD. Fractions A and B are combined and then subjected to silica gel column chromatography, eluted with a MeOH-CHCl3 gradient at volume ratios of 1:99, 2:98, 5:95, 10:90, 20:80, and 40:60 and MeOH to obtain five fractions, Frs.B-1-B-5. Fr.B-2 is subjected to MCI gel CHP chromatography. 20P column chromatography, eluted with MeOH-H2O and MeOH at ratios of 50:50, 70:30, and 90:10, yielded four subfractions Frs.B-2a-B-2d; Fr.B-2b was purified by Rp-C... 18 Column chromatography was performed, with MeOH-H2O gradients of 50:50, 60:40, 70:30, and 80:20 to obtain subfractions Frs.B-2b-1-B-2b-5; Fr.B-2b-3 was further eluted by silica gel column chromatography with MeOH-CHCl3 at ratios of 2:98, 5:95, and 10:90 to obtain five subfractions Frs.B-2b-3a-B-2b-3e; Fr.B-2b-3d was further eluted by silica gel column chromatography with MeOH-CHCl3 at ratios of 5:95 and 10:90, and then subjected to semi-preparative HPLC at an Agilent XDB-C... 18 Compounds 10 and 11 were purified by column chromatography with acetonitrile-water at a ratio of 40:60; Fr.B-2c was purified by reverse-phase Rp-C 18 Column chromatography was performed, eluting with MeOH-H2O at ratios of 50:50, 60:40, 70:30, and 80:20 to obtain five fractions: Frs.B-2c-1-B-2c-5. Fr.B-2c-3 was then subjected to silica gel column chromatography, eluting with MeOH-CHCl3 at ratios of 2:98, 5:95, and 10:90 to obtain five fractions: Frs.B-2c-3a-B-2c-3e. Fr.B-2c-3a was subjected to Sephadex LH-20 column chromatography, eluting with MeOH-CHCl3 at a ratio of 50:50, followed by semi-preparative HPLC on an Agilent XDB-C... 18 Compounds 1, 2, 3, and 4 were purified by column chromatography with acetonitrile-water at a ratio of 40:

60. Fr.B-2c-3b was purified by Sephadex LH-20 column chromatography with elution of MeOH-CHCl3 at a ratio of 50:50, followed by semi-preparative HPLC on an Agilent XDB-C10 column. 18 Compounds 5, 6, and 7 were obtained by repeated purification on a column using acetonitrile-water (48:52) and methanol-water (75:25). Fr.B-2c-3c was purified by Sephadex LH-20 column chromatography, eluted with 50:50 MeOH-CHCl3, and then subjected to semi-preparative HPLC on an Agilent XDB-C10 column. 18 Compounds 8 and 9 were purified by methanol-water at a ratio of 78:22 on a column. Fr.B-4 was purified by MCI gel CHP 20P column chromatography, eluted with MeOH-H2O and MeOH at ratios of 50:50, 70:30, and 90:10, to obtain four subfractions Frs.B-4a-B-4d. Fr.B-4c was purified by silica gel column chromatography, eluted with MeOH-CHCl3 at ratios of 5:95 and 10:90, then purified by Sephadex LH-20 column chromatography, treated with MeOH-CHCl3 at a ratio of 50:50, and finally purified by semi-preparative HPLC on an Agilent XDB-C10 column. 18 Compounds 12, 13, and 14 were purified on a column using methanol-water in a ratio of 65:35 and acetonitrile-water in a ratio of 42:

58.

3. The use of artemisinin A1-A4, B1-B4, C1-C2 and DH, i.e. compounds 1-14, as shown in structural formula (I) of claim 1 in the preparation of anti-hepatocellular carcinoma drugs.

4. A pharmaceutical composition comprising at least one of artemisinin A1-A4, B1-B4, C1-C2 and DH, i.e., compounds 1-14, as shown in structural formula (I) 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: The dried aerial parts of Artemisia annua are 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. The ethyl acetate extract is then subjected to silica gel column chromatography and eluted with a gradient of acetone-petroleum ether at volume ratios of 10:90, 20:80, and 30:70 and ethyl acetate to obtain four fractions, Frs.AD. Fractions A and B are combined and then subjected to silica gel column chromatography, eluted with a MeOH-CHCl3 gradient at volume ratios of 1:99, 2:98, 5:95, 10:90, 20:80, and 40:60 and MeOH to obtain five fractions, Frs.B-1-B-5. Fr.B-2 is subjected to MCI gel CHP chromatography. 20P column chromatography, eluted with MeOH-H2O and MeOH at ratios of 50:50, 70:30, and 90:10, yielded four subfractions Frs.B-2a-B-2d; Fr.B-2b was purified by Rp-C... 18 Column chromatography was performed, with MeOH-H2O gradients of 50:50, 60:40, 70:30, and 80:20 to obtain subfractions Frs.B-2b-1-B-2b-5; Fr.B-2b-3 was further eluted by silica gel column chromatography with MeOH-CHCl3 at ratios of 2:98, 5:95, and 10:90 to obtain five subfractions Frs.B-2b-3a-B-2b-3e; Fr.B-2b-3d was further eluted by silica gel column chromatography with MeOH-CHCl3 at ratios of 5:95 and 10:90, and then subjected to semi-preparative HPLC at an Agilent XDB-C... 18 Compounds 10 and 11 were purified by column chromatography with acetonitrile-water at a ratio of 40:60; Fr.B-2c was purified by reverse-phase Rp-C 18 Column chromatography was performed, eluting with MeOH-H2O at ratios of 50:50, 60:40, 70:30, and 80:20 to obtain five fractions: Frs.B-2c-1-B-2c-5. Fr.B-2c-3 was then subjected to silica gel column chromatography, eluting with MeOH-CHCl3 at ratios of 2:98, 5:95, and 10:90 to obtain five fractions: Frs.B-2c-3a-B-2c-3e. Fr.B-2c-3a was subjected to Sephadex LH-20 column chromatography, eluting with MeOH-CHCl3 at a ratio of 50:50, followed by semi-preparative HPLC on an Agilent XDB-C... 18 Compounds 1, 2, 3, and 4 were purified by column chromatography with acetonitrile-water at a ratio of 40:

60. Fr.B-2c-3b was purified by Sephadex LH-20 column chromatography with elution of MeOH-CHCl3 at a ratio of 50:50, followed by semi-preparative HPLC on an Agilent XDB-C10 column. 18 Compounds 5, 6, and 7 were obtained by repeated purification on a column using acetonitrile-water (48:52) and methanol-water (75:25). Fr.B-2c-3c was purified by Sephadex LH-20 column chromatography, eluted with 50:50 MeOH-CHCl3, and then subjected to semi-preparative HPLC on an Agilent XDB-C10 column. 18 Compounds 8 and 9 were purified by methanol-water at a ratio of 78:22 on a column. Fr.B-4 was purified by MCI gel CHP 20P column chromatography, eluted with MeOH-H2O and MeOH at ratios of 50:50, 70:30, and 90:10, to obtain four subfractions Frs.B-4a-B-4d. Fr.B-4c was purified by silica gel column chromatography, eluted with MeOH-CHCl3 at ratios of 5:95 and 10:90, then purified by Sephadex LH-20 column chromatography, treated with MeOH-CHCl3 at a ratio of 50:50, and finally purified by semi-preparative HPLC on an Agilent XDB-C10 column. 18 Compounds 12, 13 and 14 were purified on a column using methanol-water in a ratio of 65:35 and acetonitrile-water in a ratio of 42:58; then one of compounds 1-14 or any combination thereof was added to a pharmaceutically acceptable carrier.

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