Vibsane-type diterpenes from the leaves of euclea natalensis and their preparation and medical use as anti-tumor agents

Abstract

This invention belongs to the field of natural product chemistry, and relates to four Vibsane-type diterpenes extracted and enriched from the leaves of *Viburnum odoratissimum* Ker-Gawl. var. *odoratissimum*, their preparation methods, and applications. In particular, it relates to a method for preparing Vibsane-type diterpenes with significant antitumor activity from the leaves of the medicinal and horticultural crop *Viburnum odoratissimum*. The Vibsane-type diterpenes were obtained through repeated silica gel, ODS column chromatography, and HPLC separation. This invention also utilizes the MTT assay to test the cytotoxic activity of these four Vibsane-type diterpenes against HepG2 liver cancer cells.

Description

Technical Field

[0001] This invention belongs to the field of natural product chemistry technology, and relates to the extraction of natural products from coral trees (… Viburnum odoratissimum Vibsane-type diterpenes extracted and enriched from the leaves of *Ker-Gawl. var. odoratissimum*, their preparation methods and applications, especially a new method for preparing Vibsane-type diterpenes with significant antitumor activity from the leaves of the medicinal and garden crop *Ker-Gawl. var. odoratissimum*. Background Technology

[0002] Coral tree (Viburnum odoratissimum Ker-Gawl. var. odoratissimum) is a plant belonging to the genus Viburnum in the family Adoxaceae. Its branches and leaves are used medicinally, possessing properties that promote blood circulation and relieve pain, and are often used in traditional folk medicine for anti-inflammation, analgesia, and anti-rheumatism. Our research group studied the leaves of the coral tree and isolated various chemical components from its branches and leaves. Among them, Vibsane-type diterpenoids are characteristic compounds of the coral tree. These compounds have complex structures and exhibit excellent anti-hepatocellular carcinoma activity.

[0003] Liver cancer (HCC) is a common malignant tumor of the digestive system. Its most prominent pathological change is the abnormal proliferation and differentiation of hepatocytes, ultimately leading to dysfunction of liver metabolism, detoxification, and synthesis, seriously threatening patients' lives and health, and also causing a huge medical and economic burden. Current research on the treatment of liver cancer mainly focuses on surgical resection, local ablation, targeted therapy, and immunotherapy. The exact causes of liver cancer are not yet fully understood. Hepatitis B virus (HBV) or hepatitis C virus (HCV) infection, aflatoxin exposure, alcoholic liver disease, non-alcoholic fatty liver disease, and genetic factors may all be involved in the malignant transformation of hepatocytes. However, substantial evidence suggests that abnormal apoptosis induced by oxidative stress is closely related to the occurrence and development of liver cancer. Summary of the Invention

[0004] The purpose of this invention is to extract and enrich four Vibsane-type diterpenes from the leaves of *Viburnum odoratissimum* Ker-Gawl. var. *odoratissimum*, a plant belonging to the genus *Viburnum*. A method for preparing these diterpenes is provided, along with their use in antitumor applications.

[0005] The structures of the four novel vibsane-type diterpenes isolated from the leaves of *Viburnum odoratissimum* Ker-Gawl. var. *odoratissimum* (family Viburnaceae) described in this invention are as follows: .

[0006] The preparation technique for Vibsane-type diterpenes using coral tree leaves according to the present invention includes the following steps: (1) Take 40 kg of dried coral tree leaves and extract them three times by heating and refluxing with 70% ethanol.

[0007] (2) After solvent recovery, the total extract was concentrated and extracted with ethyl acetate. Subsequently, the obtained extract (600 g) was separated by silica gel column chromatography with a gradient elution of 100:0-5:1 using a dichloromethane-methanol system to obtain four fractions (Fr. A–Fr. D). Fr. B (180 g) was subjected to silica gel column chromatography (petroleum ether / ethyl acetate, gradient elution of 50:0-1:1), HP-20 macroporous adsorption resin column chromatography (gradient elution of 30%, 60%, and 90% ethanol aqueous solution), and ODS column chromatography (ethanol / water, gradient elution of 10:90-100:0) to obtain six subfractions (Fr. B2-1–Fr. B2-6).

[0008] (3) Fr. B2-1 (11 g) was purified by preparative and semi-preparative high performance liquid chromatography and eluted with an acetonitrile-water system of 70:30 to obtain compound 2 (3.2 mg). (4) Fr. B2-3 (9 g) was purified by preparative and semi-preparative high performance liquid chromatography. The compound 4 (6 mg) was obtained by elution with an acetonitrile-water system of 60:40 and the compound 1 (18.2 mg) was obtained by elution with an acetonitrile-water system of 57:43.

[0009] (5) Fr. B2-6 (7 g) was purified by preparative and semi-preparative high performance liquid chromatography to obtain compound 3 (5 mg). In step (1), the reflux extraction is performed 3 times, each time for 2 to 3 hours.

[0010] The preparation method described above used dried leaves of *Viburnum odoratissimum* Ker-Gawl. var. *odoratissimum*, collected in May 2020 from Guangxi Zhuang Autonomous Region, China (21°29'0.68"N, 109°06'56.89"E), and identified by Professor Lu Jincai of the School of Traditional Chinese Medicine, Shenyang Pharmaceutical University. The voucher specimen (No.: 20200517) is preserved in the Herbarium of Shenyang Pharmaceutical University.

[0011] The results of systematic structural identification of the obtained compounds are shown in Tables 1 and 2. Figure 1 to Figure 29 As shown: The structures of compounds 1-4 were identified using high-resolution mass spectrometry, one-dimensional NMR, two-dimensional NMR, and computational ECD techniques.

[0012] Anpluralvibsane B (1): a colorless oil. High-resolution mass spectrometry (HRESIMS) showed a quasi-molecular ion peak (C0) at m / z 469.2168 [M+Na]+. 25 H 34 The calculated value of O7Na is 469.2202, thus determining its molecular formula to be C. 25 H 34 O7 has 9 degrees of unsaturation. 1 1H NMR (CD3OD, 600 MHz) showed the presence of six methyl groups. d H 1.27 (s,3H, H-16), 1.28 (s, 3H, H-17), 2.17 (s, 3H, H-19), 1.08 (s, 3H, H-20), 2.19 (br s, 3H, H-4'), 1.95 (br s, 3H, H-5')], a oxymethylene group [ d H 4.32 (d, J = 13.2Hz, 1H, H-18a) / 4.10 (d, J = 13.2 Hz, 1H, H-18b)], two methylene groups [ d H 2.49 (m, 1H, H-1a) / 2.28 (br d, J = 13.9 Hz, 1H, H-1b), 3.10 (dd, J = 17.2, 2.9 Hz, 1H, H-13a) / 2.53, (overlap, 1H, H-13b)], four methyl groups [ d H 2.76 (dd, J = 8.1, 2.5 Hz, 1H, H-5),2.94 (dd, J = 7.8, 2.5 Hz, 1H, H-6), 2.67 (overlap, 1H, H-10), 2.66 (overlap, 1H, H-12)], and four alkene protons [ d H 6.59 (br d, J = 6.8 Hz, 1H, H-2), 7.32 (d, J =12.2 Hz, 1H, H-8), 5.20 (dd, J = 12.2, 10.6 Hz, 1H, H-9), 5.70 (br s, 1H, H-2')]. 13 C{ 1 ¹H-NMR (CD3OD, 150 MHz) and DEPT spectra resolved 25 carbon signals, including three ketone carbonyl groups ( d C 202.7, 211.1, 216.8), one ester carbonyl group ( d C 164.3), six olefin carbons ( d C 109.7, 115.2, 139.6, 140.1, 141.2, 162.2), six methyl carbons ( d C 20.6, 26.6, 26.7, 26.9, 27.6, 29.8), three aliphatic methylene carbons (including one oxygen-containing carbon) d C 37.2, 38.0, 63.6), four methylene carbons ( d C 48.5, 50.4, 60.6, 62.4) and two aliphatic quaternary carbons ( d C (49.3, 77.9). Although NMR data showed typical SMT-type VibD characteristics, the presence of two additional non-oxymethylene signals suggests that this scaffold derives from a novel scaffold. 1 H- 1Further analysis of the HCOSY spectrum revealed two isolated spin systems: H2-1 / H-2 and H-8 / H-9 / H-10 / H-5 / H-6 / H-12 / H-13. These substructures are clearly linked by HMBC correlations. Specifically, HMBC cross-peaks from H-8 to C-10 / C-1', H-10 to C-4, H2-18 to C-2 / C-4, and H3-20 to C-1 / C-10 / C-11 / C-12 establish the core SMT-type VibD framework. Crucially, an unprecedented C-6 / C-12 bond derived from a key HMBC correlation between H-12 and C-7 confirms the novel chemical structure of compound 2. Detailed analysis of the two-dimensional NMR spectral data is available in the supplementary information.

[0013] By combining NOESY correlation analysis, coupling constant analysis, and ring strain considerations, the relative configuration of compound 1 was determined. Due to the inherent constraints of the ring system, the C–C bonds at C-5 / C-6 and C-11 / C-12 are geometrically constrained to have the same orientation. Given that C-10 is located at the bridgehead vertex formed by C-5 / C-10 / C-11, its… β The configuration was confirmed by the NOE correlation between H-9 and H-1a. The key NOE cross-peaks between H-5 and H3-19, and H-9, along with the cross-peak between H-6 and H-13a, combined with the large coupling constant (J = 7.8 Hz) between H-6 and H-12, established the configuration of H-6. α Configuration and H-12 β Configuration. Furthermore, the large ortho-coupling constant (J = 12.3 Hz) between H-8 and H-9 indicates that the C-8 / C-9 double bond has an E configuration. The absolute configuration of 1 was determined by comparing the ECD spectrum of 1 with quantum chemical calculations. The ECD curves calculated for (8E, 5R, 6R, 10S, 11R, 12R)-2 showed excellent agreement with experimental data, thus confirming the absolute stereochemical configuration of 1.

[0014] Anpluralvibsane C(2): Colorless oily substance. Based on HRESIMS analysis (which gives [M+Na]... + Peak m / z 427.2117, calculated value C 23 H 32 O6Na, 427.2096), its molecular formula was determined to be C 23 H 32 O6 has an unsaturation degree of 8.

[0015] ¹H NMR spectrum (DMSO-d6, 600 MHz) shows the presence of four methyl groups. d H1.37 (3H, s, H-16), 0.74 (3H, s, H-17), 2.16 (3H, br s, H-4'), 1.93 (3H, br s, H-5')]; one methoxy group [ d H 3.36 (3H, s, 14-OCH3)]; one oxymethylene group [ d H 3.92 (2H, m, H2-15)]; three methylene groups [ d H 2.36 (1H, br d, J = 19.1 Hz, H-1 β ) / 1.72 (1H, m, H-1 α ), 2.20 (1H, t, J = 12.5Hz, H-6 β ) / 1.77 (1H, m, H-6 α ), 1.79 (1H, overlap, H-12a) / 1.26 (1H, m, H-12b)]; one oxymethyl group [ d H 4.73 (1H, d, J = 7.7 Hz, H-14)]; three methines [ d H 2.13 (1H, m, H-5), 2.46 (1H, t, J = 9.2 Hz, H-10), 1.79 (1H, overlap, H-13)]; four olefin proton signals [ d H 5.32 (1H, m, H-2), 7.25 (1H, d, J = 12.3 Hz, H-8), 5.45 (1H, dd, J = 12.3, 9.2 Hz, H-9), 5.75 (1H, br s, H-2')]; and a exchangeable proton signal [ d H 4.47 (1H, t, J =5.4 Hz, 15-OH).

[0016] 13 C{ 1 A total of 23 carbon signals were resolved by ¹H NMR spectroscopy (DMSO-d6, 150 MHz) and DEPT experiments, including one ester carbonyl group ( d C 162.8); six olefinic carbons ( d C 114.2, 115.0, 119.1, 136.0, 143.3, 160.4); four methyl carbons ( d C 20.2, 27.1, 30.0, 34.5); four aliphatic methylene carbons (including one oxygen-containing carbon) d C 36.5, 38.4, 42.8, 59.8); four methylene carbons (including one oxygen-containing carbon) d C 41.4, 46.7, 49.3, 102.2); three aliphatic quaternary carbons (including two oxygen-bound carbons) d C 34.4, 82.7, 103.6); and one methoxy carbon ( d C 55.9).

[0017] The spectral characteristics exhibit both SMT-type and RT-type VibDs, indicating that compound 2 possesses a novel framework. This conclusion is further supported by detailed two-dimensional NMR analysis. ¹H-¹H COSY spectroscopy identified four independent spin systems in the compound: H2-1 / H-2, H-8 / H-9 / H-10 / H-5 / H-6, H2-12 / H-13 / H-14, and H2-15 / 15-OH. These substructures were further linked and confirmed by HMBC spectroscopy. HMBC correlation signals (H-5 with C-3 / C-9, H-8 with C-10 / C-1', H-10 with C-4, H-2 with C-15 / C-4, H3-16 with C-6 / C-7, and H3-17 with C-1 / C-10 / C-11 / C-12) confirm that the compound possesses a typical SMT-type VibDs framework. The key HMBC correlations (H2-12 with C-4) revealed an unprecedented C-4 / C-13 connection. Furthermore, HMBC correlations with H2-6 and C-4, H2-12 and C-4, H-5 and C-3, 15-OH and C-3 / C-15, and 14-OCH3 and C-14 indicate that the remaining oxygen-containing carbons are interconnected via ether bonds, and that the methoxy group is attached to C-14. Therefore, compound 2 was identified as a novel VibD characterized by a 5 / 6 / 6 / 7 tetracyclic system and a previously unreported, highly oxidized bicyclic [4.2.1] skeleton.

[0018] Based on biosynthetic considerations, the C-12 / C-13 bridged ring system must be located below the seven-membered ring formed by C-1 / C-2 / C-3 / C-4 / C-5 / C-10 / C-11. Therefore, due to ring strain constraints, the C-4-OC-7 ether bridge can only be located above this same seven-membered ring. H-9 / H-5, H-9 / H-1 β The NOE correlation between H-5 and H-10 / H3-17 indicates that H-5 is... β Orientation, H-10 is α Orientation. Due to H-13, H-12b and H-6 α The chemical shifts of H-10 and H-13 are almost identical, making it impossible to definitively assign the configuration of H-13 using NOESY. Therefore, the correlation between H-10 and H-13 is unavailable. However, only H-13 is... α The orientation conforms to the ring tension constraint, thus confirming its... α Configuration. Furthermore, the coupling constant between H-8 and H-9 (J = 12.3 Hz) indicates that the C-8 / C-9 double bond is in the E configuration. Meanwhile, since no significant NOE correlation was observed at H-14, its relative configuration was determined based on the coupling constant between H-13 and H-14 (J = 7.7 Hz). Due to the presence of a dioxygen substituent at the H-14 position, the coupling constant at this position could not be estimated using the applicable Karplus equation. Therefore, the coupling constant was calculated using the "mixed" keyword at the B97-2 / pcJ-1 theoretical level. The results show that when H-14 is... β When oriented, the calculated value agrees very well with the experimental value of 7.7 Hz, thus confirming the relative configuration of H-14 as follows. β .

[0019] The absolute configuration of compound 2 was determined by comparing the calculated ECD spectrum with the experimentally measured ECD spectrum. The calculated ECD spectrum of (8E, 4S, 5S, 7S, 10S, 11R, 13S, 14S)-3 showed good agreement with the experimental ECD spectrum, thus confirming the absolute configuration of compound 2.

[0020] The NMR data of compounds 1-2 are assigned as shown in Table 1.

[0021] Table 11-2 (a: CD3OD and b: DMSO) H (600 MHz) and 13 NMR data at C (150 MHz) Compound 3 (Anpluralvibsane D): Colorless oil. Analysis according to HRESIMS (showing [M + Na)). + Peak m / z 473.2505, calculated value C25 H 38 O7Na, 473.2510), its molecular formula is determined to be C 25 H 38 O7 has an unsaturation degree of 7.

[0022] 1 H-NMR spectrum (CD3OD, 600 MHz) showed the presence of: six methyl groups [ d H 1.15 (3H, s, H-16), 1.18 (3H, s, H-17), 2.10 (3H, s, H-19), 1.05 (3H, s, H-20), 2.20 (3H, br s, H-4'), 1.96 (3H, br s, H-5')]; one oxymethylene group [ d H 4.17 (1H, dd, J = 11.7, 7.5 Hz, H-18a) / 3.73 (1H, dd, J = 11.7, 7.5 Hz, H-18b)]; three methylene groups [ d H 1.63(1H, dd, J =13.5, 6.7 Hz, H-1 β ) / 2.06 (1H, br d, J = 13.5 Hz, H-1 α ), d H 3.15 (1H, overlapped,H-6a) / 2.70 (1H, dd, J = 18.5, 2.7 Hz, H-6b), d H 1.74 (1H, overlapped, H-12a) / 1.51 (1H, dd, J = 15.1, 9.1 Hz, H-12b)]; six methines [ d H 2.75 (1H, dd, J = 6.7,2.5 Hz, H-2), 2.60 (1H, td, J = 7.0, 2.5 Hz, H-3), 2.97 (1H, td, J = 9.8, 2.7Hz, H-5), 1.72 (1H, br d, J= 11.0 Hz, H-10), 1.91 (1H, m, H-13), 3.17 (1H, d, J = 6.7 Hz, H-14)]; and three olefin proton signals [ d H 7.09 (1H, d, J = 12.3 Hz, H-8), 5.25 (1H, dd, J = 12.3, 11.0 Hz, H-9), 5.73 (1H, br s, H-2')].

[0023] 13 C{ 1 The H NMR spectrum (CD3OD, 150 MHz) and DEPT spectrum resolved a total of 25 carbon signals, including two ketone carbonyl groups ( d C 211.5, 216.5); one ester carbonyl group ( d C 164.8); four olefinic carbon signals ( d C 115.5, 117.7, 138.0, 161.7); six methyl carbons ( d C 20.5, 26.0, 27.3, 27.6, 29.9, 32.3); four aliphatic methylene carbons (including one oxygen-containing carbon signal) d C 36.0, 46.3, 48.0, 64.3); six methylene carbons (including one hydroxyl carbon signal) d C 42.1, 46.9, 48.1, 53.0, 65.6, 81.2); and two aliphatic quaternary carbons (including one oxygen-containing carbon signal) ( d C (45.6, 74.6). The data indicate that compound 3 is structurally highly similar to vibsatin A, but has four more carbon atoms, suggesting that it may be the first prototype compound of this type of skeleton before the side chain degrades.

[0024] Further analysis 1 H- 1¹H COSY spectroscopy identified two independent spin systems: H₂-6 / H-5 / H-10 / H-9 / H-8 and H₂-1 / H-2 / H-3 / H-18 / H-13 / H₂-12 / H-14, which were further linked and confirmed by HMBC spectroscopy. In the HMBC spectrum, correlations were observed between H₂-18 and C₂, C₃, and C₄, indicating that C₁₈ is attached to C₃. A correlation between H₃-19 and C₆ / C₇ suggested the presence of a C₆-C₇-C₁₈ fragment. HMBC correlations between H₃-20 and C₁₁, C₀, C₁, and C₁₂ confirmed that compound 3 possesses the skeletal characteristics of SMT-type VibDs. Furthermore, based on the HMBC correlation between H3-16 / H3-17 and C-14 / C-15, and the cross peak between H-14 and C-12 / C-2, it was further confirmed that compound 3 is the prototype compound of vibsatin A before the degradation of the C-4 side chain (C-14 / C-15 / C-16 / C-17).

[0025] Compound 4 (Anpluralvibsane K) is a colorless oil. According to HRESIMS analysis (which gives [M + H]),... + Peak m / z 415.2484, calculated value C 25 H 35 O5, 415.2484), its molecular formula was determined to be C 25 H 34 O5 has an unsaturation degree of 9.

[0026] 1 H-NMR spectroscopy (DMSO- d (6, 600 MHz) showed the presence of: six methyl groups [ d H 2.16 (3H, s, H-19), 1.07 (3H, s, H-17), 0.97 (3H, s, H-16), 0.81 (3H, s, H-20), 1.94 (3H, d, = 1.4 Hz,H-4'), 2.16 (3H, d, J = 1.4 Hz, H-5')]; one oxymethylene [ d H 4.06 (1H, dd, J = 12.0, 2.8 Hz, H-18 α ) / 3.64 (1H, dd, J = 12.0, 5.0 Hz, H-18 β )]; three methylene groups [ d H1.68 (1H,dd, J = 14.6, 9.5 Hz, H-1 α ) / 1.58 (1H, overlapped, H-1 β ), 1.62 (1H, m, H-12a) / 1.30 (1H, overlapped, H-12b), 1.44 (1H, overlap, H-13a) / 1.12 (1H, m, H-13b)]; four methines [ d H 3.01 (1H, d, J = 11.6 Hz, H-10), 2.73 (1H, m, H-3), 1.53 (1H, m, H-14), 2.22 (1H, m, H-2)]; and four aromatic / olefin proton signals [ d H 6.33 (1H, s, H-6), 7.29(1H, t, J = 11.6 Hz, H-8), 5.69 (1H, t, J = 11.6 Hz, H-9), 5.76 (1H, p, J = 1.4Hz, H-2').

[0027] 13 C{ 1 H-NMR spectroscopy (DMSO-) d A total of 22 carbon signals were resolved from the 6, 150 MHz and DEPT spectra. Based on the HSQC and HMBC spectra, four missing carbon signals in the carbon spectrum were identified. d C 26.4, 35.9, 52.2, 123.3). Furthermore, based on the molecular weight of compound 4, the carbon spectra... d C No HSQC or HMBC related signal was observed at 99.5, which was identified as an impurity peak. The aforementioned carbon signal included two ketone carbonyl groups (δ...). C 207.9, 197.0); one ester carbonyl group (δ C 162.7); six olefinic carbon signals (δ C 113.4, 114.0, 123.3, 136.1, 160.3, 160.8); six methyl carbon signals (δ C 18.0, 20.3, 26.4, 27.1, 27.5, 30.3); four aliphatic methylene carbon signals (including one oxygen-containing carbon signal) (δ C21.3, 35.1, 35.9, 58.8); four methylene carbon signals (δ C 28.6, 43.3, 49.2, 52.2); and two aliphatic quaternary carbon signals (including one oxygen-containing carbon signal) (δ C 36.3, 75.8).

[0028] analyze 1 H- 1 ¹H COSY spectroscopy identified two independent spin systems in the compound: H-8 / H-9 / H-10 and H2-1 / H-2 / H-3 / H2-18 / H-14 / H2-13 / H2-12. Observed HMBC correlations (H2-1 with C-10 / C-12, H-6 with C-4 / C-10, H-8 with C-1, H-10 with C-4, H2-18 with C-4, H3-19 with C-6 / C-7, H-4' with C-2' / C-3' / C-5', H-5' with C-3' / C-2' / C-4', and H3-20 with C-1 / C-10 / C-11 / C-12) confirmed that the compound possesses the skeletal characteristics of SMT-type VibDs. Furthermore, the HMBC correlations between H2-18 and C-15, and between H3-16 / H3-17 and C-14 / C-15, confirm that compound 4 possesses the essential VibDs characteristics of a 7 / 6 / 6 fused ring system.

[0029] The relative configuration of compound 4 was determined by NOESY spectroscopy and biosynthetic pathway analysis. From a biosynthetic perspective, based on the spatial orientation of the fixed 20-CH3 methyl group, C-12 was confirmed as the origin of the synthesis. α - Orientation. In the NOESY spectrum, H-10 / H-14, H-14 / H3-17, and H3-17 / H-18 were observed. α The NOE correlation between them confirmed that H-10, H-14, and H3-17 were... α - Orientation. Simultaneously, H-3 / H-1 was observed. β and H-18 β The NOE correlation between H-3 and H3-16 indicates that H-3 and H3-16 are β - Orientation. Furthermore, the coupling constant of H-8 / H-9 ( J = 11.6 Hz) indicates that the C-8 / C-9 double bond is E Configuration. Therefore, the relative configuration of this compound is determined to be 8. E , 2 S ,3 S , 10 R , 11 S , 14 S .

[0030] The absolute configuration of compound 4 was determined by comparing calculated and experimental ECD spectra. The experimental ECD curve of this compound was compared with the calculated one. E , 2 S , 3 S , 10 R , 11 S, 14 S The ECD curves for the configurations showed good consistency, thus confirming the absolute configuration of compound 4. The NMR data for compounds 3-4 are assigned as shown in Table 2.

[0031] Table 23-4 (a: CD3OD and b: DMSO) 1 H (600 MHz) and 13 NMR data at C (150 MHz) The in vitro antitumor activity of four novel Vibsane-type diterpenoid compounds isolated from coral tree leaves, as described in this invention, against the human hepatocellular carcinoma HepG2 cell line was tested using the MTT assay. The results showed that compound 1 exhibited significant inhibitory activity against this cell line in vitro. Therefore, the Vibsane-type diterpenoid compounds described in this invention show promise for further development into clinical drugs for the prevention and treatment of hepatocellular carcinoma.

[0032] The advantage of this invention is that the compounds are all optically pure compounds with defined stereoconfigurations, and they also have good anti-hepatocellular carcinoma activity, making them worthy of further development. Attached Figure Description

[0033] Figure 1 UV spectrum of compound 1; Figure 2 HRESIMS spectrum of compound 1; Figure 3 HSQC spectrum of compound 1 (600 MHz, CDCl3); Figure 4 HMBC spectrum of compound 1 (600 MHz, CDCl3); Figure 5 Compound 1 1 H- 1 H COSY spectrum (600 MHz, CDCl3); Figure 6 NOESY spectrum of compound 1 (600 MHz, CDCl3); Figure 7 DEPT spectrum of compound 1 (600 MHz, CDCl3); Figure 8 UV spectrum of compound 2; Figure 9 HRESIMS spectrum of compound 2; Figure 10 HSQC spectrum of compound 2 (600 MHz, DMSO); Figure 11 HMBC spectrum of compound 2 (600 MHz, DMSO); Figure 12 Compound 2 1 H- 1 H COSY spectrum (600 MHz, DMSO); Figure 13 NOESY spectrum of compound 2 (600 MHz, DMSO); Figure 14 DEPT spectrum of compound 2 (150 MHz, DMSO); Figure 15 UV spectrum of compound 3; Figure 16 HRESIMS spectrum of compound 3; Figure 17 HSQC spectrum of compound 3 (600 MHz, CDCl3); Figure 18 HMBC spectrum of compound 3 (600 MHz, CDCl3); Figure 19 Compound 3 1 H- 1 H COSY spectrum (600 MHz, CDCl3); Figure 20 NOESY spectrum of compound 3 (600 MHz, CDCl3); Figure 21 DEPT spectrum of compound 3 (600 MHz, CDCl3); Figure 22 UV spectrum of compound 4; Figure 23 HRESIMS spectrum of compound 4; Figure 24 HSQC spectrum of compound 4 (600 MHz, DMSO); Figure 25 HMBC spectrum of compound 4 (600 MHz, DMSO); Figure 26 Compound 4 1 H- 1 H COSY spectrum (600 MHz, DMSO); Figure 27 NOESY spectrum of compound 4 (600 MHz, DMSO); Figure 28 DEPT spectrum of compound 4 (600 MHz, DMSO); Figure 29 Compounds 1-4 1 H 1 H COSY, HMBC, NOESY and ECD spectra. Detailed Implementation

[0034] The embodiments listed below are intended to help those skilled in the art better understand the present invention, but do not limit the invention in any way.

[0035] Example 1: Preparation of compounds 1-4 40 kg of dried coral tree leaves were extracted three times by reflux with 70% ethanol. After solvent recovery, the extract was concentrated to obtain a total extract (3800 g), which was then extracted with ethyl acetate. Subsequently, the obtained extract (600 g) was separated by silica gel column chromatography using a dichloromethane-methanol system as the eluent at a gradient of 100:0–5:1 to obtain four fractions (Fr. A–Fr. D). Fr. B (180 g) was then subjected to silica gel column chromatography (petroleum ether / ethyl acetate, gradient elution 50:0–1:1), HP-20 macroporous adsorption resin column chromatography (30%, 60%, 90% ethanol / water gradient elution), and ODS column chromatography (ethanol / water, gradient elution 10:90–100:0) to obtain six subfractions (Fr. B2-1–Fr. B2-6). Fr. B2-1 (11 g) was purified by preparative and semi-preparative high-performance liquid chromatography (HPLC), and eluted with an acetonitrile-water system of 70:30 to obtain compound 2 (3.2 mg). Fr. B2-3 (9 g) was purified by preparative and semi-preparative HPLC, and eluted with an acetonitrile-water system of 60:40 to obtain compound 4 (6 mg) and with an acetonitrile-water system of 57:43 to obtain compound 1 (18.2 mg). Fr. B2-6 (7 g) was purified by preparative and semi-preparative HPLC to obtain compound 3 (5 mg). Example 2: In vitro cytotoxic activity of compounds 1-4 in the HepG2 tumor cell line The cytotoxic activity of compounds 1-4 against HepG2 liver cancer cells was investigated using the MTT assay. Cells were placed in 96-well plates and incubated statically with culture medium for 12 h. HepG2 cells were treated with different concentrations of the compounds, with sorafenib used as a positive control. IC50 was used. 50 The value was 3.58 ± 0.96 μM. After 48 h of treatment, 20 μL of MTT reagent was added and the mixture was incubated at 37℃ for 4 h. Cells treated with different concentrations were then analyzed using a microplate reader at a wavelength of 490 nm. The results are shown in Table 3. Compound 1 showed good cytotoxic activity against the human hepatocellular carcinoma HepG2 cell line, with an IC50 value of 3.58 ± 0.96 μM. 50 The value is 3.84 ± 0.50 μM.

[0036] Table 3. Anti-hepatocellular carcinoma activity of compounds 1-4 in HepG2 tumor cells (sorafenib 3.58±0.96) in vitro. Obviously, those skilled in the art can make various modifications and variations to this invention without departing from its spirit and scope. Therefore, if these modifications and variations fall within the scope of the claims of this invention and their equivalents, this invention also intends to include these modifications and variations.

Claims

1. Vibsane-type diterpenes in coral tree leaves, characterized by: The diterpene has the following structure: 。 2. The method for preparing diterpenes according to claim 1, characterized in that: The preparation method includes the following steps: (1) Take dried coral tree leaves and extract them by heating and reflux with 70% ethanol; (2) After recovering the solvent, the total extract was concentrated and extracted with ethyl acetate. Subsequently, the extract was separated by silica gel column chromatography with a gradient elution of 100:0-5:1 using a dichloromethane-methanol system as the eluent to obtain four fractions Fr. A–Fr. D. Fr. B was then subjected to silica gel column chromatography, HP-20 macroporous adsorption resin column chromatography, and ODS column chromatography to obtain six subfractions Fr. B2-1–Fr. B2-6. (3) Fr. B2-1 was purified by preparative and semi-preparative high performance liquid chromatography, and eluted with an acetonitrile-water system of 70:30 to obtain compound 2; (4) Fr. B2-3 was purified by preparative and semi-preparative high performance liquid chromatography, and compound 4 was obtained by elution with an acetonitrile-water system of 60:40 and compound 1 was obtained by elution with an acetonitrile-water system of 57:43; (5) Fr. B2-6 was purified by preparative and semi-preparative high performance liquid chromatography to obtain compound 3.

3. The preparation method according to claim 2, characterized in that: In step (1), reflux extraction is performed 3 times, each time for 2-3 hours.

4. The preparation method according to claim 2, characterized in that: In step (2), the elution conditions for the silica gel column chromatography are petroleum ether / ethyl acetate, gradient elution of 50:0-1:

1.

5. The preparation method according to claim 2, characterized in that: In step (2), the elution conditions for the HP-20 macroporous adsorption resin column chromatography are gradient elution with 30%, 60%, and 90% ethanol aqueous solution.

6. The preparation method according to claim 2, characterized in that: In step (2), the elution conditions for the ODS column chromatography are ethanol / water, gradient elution of 10:90-100:

0.

7. A pharmaceutical composition, characterized in that: It comprises the Vibsane-type diterpenoid as described in claim 1 and a pharmaceutically acceptable carrier.

8. The use of the Vibsane-type diterpenoid of claim 1 or the pharmaceutical composition of claim 7 in the preparation of an antitumor drug.

9. The application according to claim 8, characterized in that: The tumor in question is liver cancer.

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