Vibsane type diterpenoid compound with seven-membered ring skeleton characteristic in Japanese coral tree or salt of vibsane type diterpenoid compound as well as preparation method and application of vibsane type diterpenoid compound

Abstract

The invention belongs to the technical field of natural medicinal chemistry, and relates to vibsane type diterpenoid compounds with seven-membered ring skeleton characteristics in Japanese coral trees or salts of the vibsane type diterpenoid compounds as well as a preparation method and application of the vibsane type diterpenoid compounds. The invention particularly relates to 15 vibane type diterpenoid compounds which are extracted and separated from dried leaves of viburnum awabuki of a viburnaceae linden plant, wherein the vibane type diterpenoid compounds have a seven-membered ring skeleton structure. According to the present invention, the vibsane type diterpenoid compounds are obtained through silica gel column chromatography, HP20 macroporous resin column chromatography, ODS column chromatography and HPLC separation, and the antitumor activity of the compounds is investigated by testing the inhibition effects of the vibsane type diterpenoid compounds on three tumor cell lines HepG2, A549 and MCF-7. # imgabs0 #

Description

Technical Field

[0001] The present invention belongs to the technical field of natural drug chemistry, and specifically relates to a vibsane-type diterpenoid compound or its salt having a seven-membered ring skeleton characteristic from a Japanese coral tree and a preparation method thereof, and also relates to the application of the compound in the preparation of an anti-tumor drug. Background Art

[0002] Japanese coral tree (Viburnum awabuki): Viburnum genus, Viburnaceae, evergreen shrub or small tree. Distributed in China, Japan and Korea, it is often cultivated in the lower reaches of the Yangtze River in my country. Because of its strong resistance and absorption capacity to soot and toxic gases, it is often used as a garden plant and courtyard green plant. Its main chemical components include characteristic vibsane-type diterpenes, triterpenes, lignans, etc. It has anti-tumor, antioxidant, and nerve factor growth promotion activities.

[0003] Cancer: According to the World Health Organization (WHO), the number of cancer patients worldwide may increase by 60% in the next 20 years. As the number of cancer patients increases, cancer has become a global public health issue as the second leading cause of death in the world. Natural products are considered to be an important source of drug research and development due to their wide variety of skeletons. At present, a variety of small molecule compounds from natural sources, such as paclitaxel, have been approved for marketing and used in the treatment of cancer. my country is rich in natural product resources, so continuing to search for natural products with anti-tumor activity is one of the important ways to develop anti-tumor drugs. Summary of the invention

[0004] The present invention aims to provide 15 vibsane-type diterpenoid compounds or their salts separated from the dried leaves of Viburnum awabuki, a plant of the genus Viburnum of the Viburnaceae family, and their preparation methods, as well as the use of such compounds or their salts in the preparation of antitumor drugs.

[0005] The structure of the vibsane type diterpenoid compound is shown in the figure:

[0006]

[0007] The preparation technology scheme of the present invention comprises the following steps:

[0008] (1) The dried leaves of the Japanese coral tree were extracted with ethanol, and the extract was concentrated to obtain an extract, which was extracted with n-butanol. The n-butanol layer extract was subjected to silica gel column chromatography with a CH2Cl2 / CH3OH gradient elution (50:1-1:1) (v / v) to obtain four fractions Fr.A-Fr.D;

[0009] (2) Fractions Fr.A and Fr.B were subjected to HP20 column chromatography with an ethanol-water gradient elution to obtain a total of 6 fractions Fr.A1-Fr.A3 and Fr.B1-Fr.B3;

[0010] (3) Fractions Fr.A1-Fr.A3 and Fr.B1-Fr.B3 were subjected to ODS column chromatography with an ethanol-water gradient elution, respectively, to obtain a total of 7 fractions Fr.1-Fr.4 and Fr.5-Fr.7;

[0011] (4) Fraction Fr.2 was eluted by silica gel column chromatography with petroleum ether:ethyl acetate (50:1-1:1) (v / v) to obtain four subfractions Fr.2-1 to Fr.2-4. Fr.2-4 was purified by semi-preparative liquid chromatography with acetonitrile-water to obtain compounds 5, 6, and 12; Fr.2-3 was purified by semi-preparative liquid chromatography with acetonitrile-water to obtain compound 15.

[0012] (5) Fraction Fr.1 was eluted by silica gel column chromatography with petroleum ether:ethyl acetate (50:1-1:1) (v / v) to obtain 7 subfractions Fr.1-1 to Fr.1-7. Fr.1-6 was purified by semi-preparative liquid phase with acetonitrile-water to obtain compound 7; Fr.1-4 was purified by semi-preparative liquid phase with acetonitrile-water to obtain compounds 2 and 9. Fr.1-3 was purified by semi-preparative liquid phase with acetonitrile-water to obtain compounds 10 and 11; Fr.1-2 was purified by semi-preparative liquid phase with acetonitrile-water to obtain compounds 3, 4 and 8.

[0013] (6) Fraction Fr.6 was eluted by silica gel column chromatography with petroleum ether:ethyl acetate (50:1-1:1) (v / v) to obtain 6 subfractions Fr.6-1 to Fr.6-6. Fr.6-4 was purified by semi-preparative liquid chromatography with acetonitrile-water to obtain compounds 13 and 14; Fr.6-2 was purified by semi-preparative liquid chromatography with acetonitrile-water to obtain compound 1.

[0014] The above preparation method, wherein:

[0015] In the step (1), the Japanese coral tree refers to the Japanese coral tree (Viburnumawabuki K. Koch) of the genus Viburnum of the Viburnum family. The dried leaves of the Japanese coral tree are extracted by reflux with 70%-80% industrial ethanol.

[0016] In the step (2), the fractions Fr.A and Fr.B are eluted by HP20 column chromatography with a gradient of ethanol-water (70:30-100:0) (v / v).

[0017] In the step (3), the fractions Fr.A1-Fr.A3 and Fr.B1-Fr.B3 are respectively eluted by ODS column chromatography with a gradient of ethanol-water (60:40-100:0 and 20:80-100:0) (v / v).

[0018] In the step (4), Fr.2-4 was purified by semi-preparative liquid phase elution with acetonitrile-water (48:52, v / v) to obtain compounds 5, 6, and 12; Fr.2-3 was purified by semi-preparative liquid phase elution with acetonitrile-water (57:43, v / v) to obtain compound 15.

[0019] In the step (5), Fr.1-6 was purified by eluting with acetonitrile-water (75:25, v / v) via semi-preparative liquid phase to obtain compound 7; Fr.1-4 was purified by eluting with acetonitrile-water (62:38, v / v) via semi-preparative liquid phase to obtain compounds 2 and 9. Fr.1-3 was purified by eluting with acetonitrile-water (60:40, v / v) via semi-preparative liquid phase to obtain compounds 10 and 11; Fr.1-2 was purified by eluting with acetonitrile-water (48:52, v / v) via semi-preparative liquid phase to obtain compounds 3, 4 and 8.

[0020] In the step (6), Fr.6-4 was purified by semi-preparative liquid phase elution with acetonitrile-water (55:45, v / v) to obtain compounds 13 and 14; Fr.6-2 was purified by semi-preparative liquid phase elution with acetonitrile-water (40:60, v / v) to obtain compound 1.

[0021] The obtained compounds were systematically identified and the results are as follows:

[0022] The structures of compounds 1-15 were identified by high-resolution mass spectrometry, one-dimensional NMR, two-dimensional NMR, induced CD, quantum chemical calculation NMR and ECD.

[0023] vibsawabinA(1): colorless oil. UV(MeOH)λ max (logε)204(2.34)nm. HRESIMS gives the quasi-molecular ion peak m / z417.26364[M+H] + (calcd for C 25 H 37 O5,417.26355), combined 1 H-NMR and 13 C-NMR confirmed that the molecular formula of the compound is C 25 H 36 O5, calculated unsaturation is 8. 1 In the H-NMR spectrum (600 MHz, DMSO-d6), the characteristic signal δ H5.58 (1H, p, J = 1.3 Hz), 2.03 (3H, d, J = 1.3 Hz), 1.84 (3H, d, J = 1.3 Hz) suggest the presence of a senecioyloxy fragment. H 4.90 (1H, dd, J = 18.2, 1.0 Hz), 4.99 (1H, dd, J = 11.4, 1.0 Hz), 5.88 (1H, dd, J = 18.2, 11.4 Hz), 4.65 (1H, o.), 5.01 (1H, m) are olefinic hydrogen signals. 13 C-NMR spectrum (150 MHz, DMSO-d6) combined with DEPT spectrum showed that there were 25 carbon signals in the molecule, including 1 keto carbonyl carbon signal, 1 ester carbonyl carbon signal, 8 olefin carbon signals, 2 sp 3 Hybridized quaternary carbon signal, 3 sp 3 Hybridized secondary carbon signal, 4 sp 3 Hybridized subcarbon signals and 6 methyl carbon signals. The planar structure of the compound was further analyzed by combining the two-dimensional NMR spectrum.

[0024] exist 1 H- 1 In the H COSY spectrum, the correlations of H-2 / H2-1, H-5 / H-6, H-8 / H-9 / H-10, H2-12 / H2-13 / H-14 indicate the existence of four spin coupling systems I-IV in the molecule. In the HBMC spectrum, H-2 is correlated with C-4 / C-9 / C-18, H2-1 is correlated with C-3, H2-18 is correlated with C-4 / C-9, H-5 / H-6 is correlated with C-4, and H3-19 is correlated with C-6 / C-7 / C-8, indicating the existence of a seven-membered ring structure fragment in the molecule, with the vinyl group attached to the C-3 position and the methyl group attached to the C-7 position. In addition, H-8 is correlated with C-1' by HMBC, indicating that the senesilyl group is located at the C-8 position of the seven-membered ring. In addition, the HMBC correlation between H3-20 and C-10 / C-11 / C-12 connects the two spin coupling systems III and IV. Combined with the HMBC correlation between H3-16 / H3-17 and C-14 / C-15, and the HMBC correlation between H-8 and C-10, the connection mode and connection position of the side chain at C-9 of the seven-membered ring are confirmed. Considering the chemical shift values ​​of C-6 and C-18 (δ C-6 76.4,δ C-18 68.9) and the unsaturation of the molecule, combined with the HMBC correlation between H2-18 and C-6, it is inferred that C-18 and C-6 are connected by an ether bond. In summary, the planar structure of the molecule was determined.

[0025] The relative configuration of the compound was determined by NOESY spectrum. In the NOESY spectrum, it can be observed that H3-19 is correlated with H-9, and H-9 is correlated with H-18β, indicating that CH3-19, H-9 and the oxygen bridge are all located on the plane and are β-oriented. Due to ring strain, although H-6 is α-oriented, a strong NOESY correlation with H3-19 is still observed. The NOESY correlation between H-5β / H-18α and H-8 / H-5α indicates that H-8 is α-oriented. The large coupling constant between H-8 and H-9 (J=11.7Hz) further proves that H-8 and H-9 are in opposite directions. Therefore, the relative configuration of the compound was determined to be (3R*, 6S*, 7R*, 8R*, 9S*). The absolute configuration of the compound was determined by comparing the measured and calculated ECD curves. The ECD curve of the (3R,6S,7R,8R,9S)-1 configuration was calculated at the B3LYP / 6-311++G(2d,p) / / B3LYP / 6-31G(d) level, and it was found that the measured curve fit the calculated curve well, thus determining the absolute configuration of the compound to be (3R,6S,7R,8R,9S).

[0026] Vibsawabin B(2): colorless oil. UV(MeOH)λ max (logε)230(2.76). HRESIMS gives the quasi-molecular ion peak 465.2844[M+H] + (calcd for C 26 H 41 O7,465.2847), combined 1 H-NMR and 13 C-NMR confirmed that the molecular formula of the compound is C 26 H 40 O7, the calculated unsaturation is 7. 1 In the H-NMR spectrum (600 MHz, CD3OD), the characteristic signal δ H 5.74 (1H, p, J = 1.3 Hz), 2.21 (3H, d, J = 1.3 Hz), 1.98 (3H, d, J = 1.3 Hz) suggest the presence of a senecioyloxy fragment. H 7.23 (1H, d, J = 6.8 Hz), 4.95 (1H, dd, J = 11.2, 6.8 Hz) are a group of cis double bond hydrogen signals, δ H 5.66 (1H, ddd, J = 15.1, 7.9, 6.8 Hz), 5.51 (1H, m), 6.64 (1H, ddt, J = 9.0, 5.0, 1.8 Hz) indicate the presence of three additional olefinic hydrogen signals. H 3.16 (3H, s) is the methoxy signal.13 C-NMR spectrum (150 MHz, CD3OD) combined with HSQC spectrum showed that there were 26 carbon signals in the molecule, including 2 keto carbonyl carbon signals, 1 ester carbonyl carbon signal, 8 olefin carbon signals, 2 sp 3 Hybridized quaternary carbon signal, 2 methine carbon signals, 4 methylene carbon signals, 6 methyl carbon signals, and 1 methoxy carbon signal. The above NMR signals indicate that the compound is a seven-membered ring vibsane type diterpene.

[0027] 1 H- 1 In the H COSY spectrum, the correlations of H2-1 / H-2 / H-3 / H2-18, H2-6 / H-5 / H-10 / H-9 / H-8, and H2-12 / H2-13 / H-14 indicate that there are three spin coupling systems I-III in the molecule. In the HBMC spectrum, H2-18 is correlated with C-2 / C-4, OCH3-18 is correlated with C-18, H2-6 is correlated with C-4 / C-10, and H3-20 is correlated with C-10 / C-11 / C-1. Combining the spin coupling systems, a seven-membered ring fragment was constructed. The HMBC correlation of OCH3-18 with C-18 indicates that the methoxy group is connected to the C-18 position. The HMBC correlation of H3-19 with C-6 / C-7 indicates that the acetyl group is connected to the C-6 position. The HMBC correlation of H-8 with C-1' indicates that the sedum acyl group is connected to the C-8 position. Calculation of the unsaturation of the compound showed the presence of another ring fragment in the structure. Considering the chemical shift values ​​of C-2 and C-14, combined with the key HMBC correlation of H-14 with C-2 and the HMBC correlation of H3-20 with C-12, it was proved that C-2 and C-14 were connected by an ether bond to form another seven-membered ring. The HMBC correlation of H3-16 / H3-17 with C-14 and C-15 indicated the structure of the substituent attached to C-15. Thus, the planar structure of the compound was determined.

[0028] The relative configuration of the compound was determined by NOESY spectrum. In the NOESY spectrum, correlation signals were observed for H-5 / H-1β, H-3 / H-1β, H-5 / H3-20, H-9 / H3-20, H-1α / H-14, and H-2 / H-14, indicating that these hydrogens were oriented in the same direction, β. NOESY correlation signals of H-10 and H-8 indicate that H-10 was oriented in the α direction. The relative configuration of the compound was determined to be (2S*, 3S*, 5S*, 10S*, 11S*, 14R*). By comparing the measured ECD and calculated ECD spectra, the absolute configuration of the compound was determined to be (2S, 3S, 5S, 10S, 11S, 14R).

[0029] Vibsawabin C(3): colorless oil. UV(MeOH)λ max (logε)232(2.68). HRESIMS gave the quasi-molecular ion peak m / z 447.27225[M+H] + (calcd for C 26 H 39 O6,447.27412), combined 1 H-NMR and 13 C-NMR confirmed that the molecular formula of the compound is C 26 H 38 O6, calculated unsaturation is 8. 1 In the H-NMR spectrum (600 MHz, CD3OD), the characteristic signal δ H 5.74 (1H, p, J = 1.3 Hz), 2.21 (3H, d, J = 1.3 Hz), 1.98 (3H, d, J = 1.3 Hz) suggest the presence of a senecioyloxy fragment. H 7.23 (1H, d, J = 6.8 Hz), 4.95 (1H, dd, J = 11.2, 6.8 Hz) are a group of cis double bond hydrogen signals, δ H 5.66 (1H, ddd, J = 15.1, 7.9, 6.8 Hz), 5.51 (1H, m), 6.64 (1H, ddt, J = 9.0, 5.0, 1.8 Hz) indicate the presence of three additional olefinic hydrogen signals. H 3.16 (3H, s) is the methoxy signal. 13 C-NMR spectrum (150 MHz, CD3OD) combined with HSQC spectrum showed that there were 26 carbon signals in the molecule, including 2 keto carbonyl carbon signals, 1 ester carbonyl carbon signal, 8 olefin carbon signals, 2 sp 3 Hybridized quaternary carbon signal (δ C 41.2,76.5), 2 methine carbon signals, 4 methylene carbon signals, 6 methyl carbon signals, and 1 methoxy carbon signal. The above NMR signals indicate that the compound is a seven-membered ring vibsane-type diterpene.

[0030] 1 H- 1In the H COSY spectrum, the correlations of H-2 / H2-1, H2-6 / H-5 / H-10 / H-9 / H-8, and H2-12 / H-13 / H-14 indicate that there are three spin coupling systems in the molecule. In the HBMC spectrum, H-2 is correlated with C-4 / C-11, H-5 is correlated with C-11, H2-18 is correlated with C-2 / C-4, H2-6 is correlated with C-4 / C-10, H3-20 is correlated with C-10 / C-1, and H3-19 is correlated with C-6 / C-7, proving that there is a seven-membered ring in the structure, and the hydroxymethyl group is connected to C-3 and the acetyl group is connected to C-6. In addition, H-8 is correlated with C-1' by HMBC, indicating that the acyloxy group of the senecioyl group is located at the C-8 position. H3-16 / H3-17 and C-15 / C-14 have HMBC correlation, OCH3-15 and C-15 have HMBC correlation, H3-20 and C-12 have HMBC correlation, and combined with the spin coupling system, the connection order of the side chain connected to the C-11 position was determined. Thus, the planar structure of the compound was determined.

[0031] The relative configuration of the compound was determined by NOESY spectrum and quantum chemical calculation. In the NOESY spectrum, a strong correlation was observed between H-10 / H3-20 and H3-20 / H-1β, indicating that H-10 and CH3-20 were in the same orientation, both in the β orientation. The NOESY correlation between H-5 and H-1α and the larger coupling constant (9.7Hz) between H-5 and H-10 indicated that H-5 and H-10 were in the opposite orientation, in the α orientation. The smaller coupling constant (6.8Hz) between H-8 and H-9 and the NOESY correlation of H-8 / H-9 indicated that there was a cis double bond at that location. Since this compound is the first natural vibsane diterpene compound with H-10 in β orientation, in order to further verify the relative configuration of C-10 and C-5, the quantum chemical coupling constants of four possible configurations (5R*,10R*,11S*)-A, (5R*,10S*,11S*)-B, (5S*,10R*,11S*)-C, (5S*,10S*,11S*)-D were calculated at the B97-2 / pcJ-1_2006 theoretical level. The results show that when H-5 and H-10 are in opposite directions, the coupling constant between the two is large, about 9 to 10 Hz (A and D); when H-5 and H-10 are in the same direction, the coupling constant between the two is small, about 3 to 4 Hz (B and C). According to the measured coupling constant J 5,10=9.7Hz, indicating that the H-5 and H-10 of the compound are in opposite directions. Then, quantum chemical NMR calculations were performed on two possible configurations A and D at the mPW1PW91 / 6-311+G(d,p) / / B3LYP / 6-31G(d) level, and the calculation results were analyzed using statistical analysis methods. The results showed that the calculated value of (5R*,10R*,11S*)-A had the highest fit with the measured NMR (correlation coefficient R 2 =0.9969), and its confidence level custom DP4+ probability analysis is 100%, so the relative configuration of the compound is determined to be (5R*, 10R*, 11S*). By comparing the measured ECD and calculated ECD spectra, the absolute configuration of the compound is determined to be (5R, 10R, 11S).

[0032] Vibsawabin D(4): colorless oil. UV(MeOH)λ max (logε)231(1.87). HRESIMS gave a quasi-molecular ion peak m / z 447.27255[M+H] + (calcd for C 26 H 39 O6,447.27412), combined 1 H-NMR and 13 C-NMR confirmed that the molecular formula of the compound is C 26 H 38 O6, the calculated unsaturation is 8. By comparison, it was found that the NMR signal of this compound was similar to the NMR data of 3. Combined with HMBC and 1 H- 1 The H COSY spectrum confirmed that the compound has the same planar structure as 3, except that the chemical shift value of H-5 and the smaller coupling constant (3.4 Hz) between H-5 and H-10 indicate that H-5 and H-10 are facing the same side of the molecule. The strong correlation signals of H-5 / H3-20 and H-10 / H3-20 in the NOESY spectrum indicate that H-5, H-10 and CH3-20 are facing in a β direction. The relative configuration of the compound was determined to be (5S*, 10R*, 11S*). The absolute configuration of the compound was determined to be (5S, 10R, 11S) by comparing the calculated ECD and measured ECD spectra.

[0033] Vibsawabin E(5): colorless oil. UV(MeOH)λ max (logε)231(2.32). HRESIMS gives the quasi-molecular ion peak m / z 433.25723[M+H] + (calcd for C25 H 37 O6,433.25847), combined 1 H-NMR and 13 C-NMR confirmed that the molecular formula of the compound is C 25 H 36 O6, the calculated unsaturation is 8. By comparison, it is found that the NMR signal of this compound is similar to the NMR data of 4, the difference is that in the C6 side chain fragment connected to C-11, this compound has one less methyl and methoxy signal than 4. In the HMBC spectrum, H2-16 / H3-17 is correlated with C-14 / C-15, H3-20 is correlated with C-12, OH-14 is correlated with C-14, and H2-12 is correlated with C-14. Combined with the coupling system C-12-C-13-C-14, the side chain fragment connected to C-11 is determined. In the NOESY spectrum, the correlation signals of H-5 / H3-20 and H-10 / H3-20 indicate that H-5, CH3-20 and H-10 are located on the same side of the molecule, in the β direction. The smaller coupling constant (3.5Hz) between H-5 and H-10 further indicates that H-5 and H-10 are in the same direction. The small coupling constant (6.5Hz) between H-8 and H-9 indicates that there is a cis double bond there. Therefore, the relative configuration of part of the compound was determined to be (5S*, 10R*, 11S*). The chiral center C-14 is located on the flexible chain and far away from the main chromophore of the molecule, so it has no significant effect on the ECD curve of the compound. Therefore, by comparing the calculated ECD and the measured ECD, the absolute configuration of part of the compound was determined to be (5S, 10R, 11S). The absolute configuration of C-14 was determined by the Rh2(OCOCF3)4 induced ECD method. The results showed that its induced ECD spectrum showed a positive Cotton effect at 336-347nm, so the absolute configuration of C-14 was S. Therefore, the absolute configuration of the compound was determined to be (5S, 10R, 11S, 14S).

[0034] Vibsawabin F(6): colorless oil. UV(MeOH)λ max (logε)231(1.89). HRESIMS gave the quasi-molecular ion peak m / z 433.25845[M+H] + (calcd for C 25 H 37 O6,433.25847), combined 1 H-NMR and 13 C-NMR confirmed that the molecular formula of the compound is C 25 H 36 O6, calculated unsaturation is 8. Analysis 1 H-NMR spectrum and 13C-NMR spectrum showed that the NMR signal of this compound was very similar to that of 5, except for the chemical shift value of H-5 and the coupling constant between H-5 and H-10 (δ H-5 3.42,J 5,10 =3.5Hz; 6 in delta H-5 2.89,J 5,10 =9.6 Hz), indicating that 6 may be a diastereomer of 5 about C-5. 1 H- 1 H COSY and HMBC are correlated, proving that the compound has the same planar structure as 5. In the NOESY spectrum, there are related signals for H-10 / H3-20, while the related signals for H-5 / H3-20 are missing. Combined with the large coupling constant (9.6Hz) between H-5 and H-10, it indicates that H-10 and CH3-20 are in the same orientation, β orientation, while H-5 is in the opposite orientation, α orientation. The correct structure was further confirmed by calculating the NMR method. The chiral center C-14 has little effect on the NMR data because its configuration difference is located on the flexible chain. The NMR calculations were performed for its two possible configurations (5R*,10R*,11S*,14S*)-A and (5S*,10S*,11S*,14S*)-B. The calculation results were analyzed by statistical methods. The structure shows that the calculated value of (5R*,10R*,11S*,14S*)-A has a small deviation from the measured value (R 2 =0.9967), and the custom DP4+ probability analysis is 99.99%, so the relative configuration of part of the compound is determined to be (5R*, 10R*, 11S*). Similar to 5, by comparing the calculated ECD and the measured ECD, the absolute configuration of part of the compound is determined to be (5R, 10R, 11S). The absolute configuration of C-14 is determined by the Rh2(OCOCF3)4 induced ECD method. The results show that its induced ECD spectrum shows a positive Cotton effect at 336-347nm, so the absolute configuration of C-14 is S. Therefore, the absolute configuration of the compound is determined to be (5R, 10R, 11S, 14S).

[0035] Vibsawabin G(7): colorless oil. UV(MeOH)λ max (logε)231(2.29). HRESIMS gave a quasi-molecular ion peak m / z 447.27377[M+H] + (calcd for C 26 H 39 O6,447.27412), combined 1 H-NMR and 13 C-NMR confirmed that the molecular formula of the compound is C26 H 38 O6, and the calculated unsaturation degree is 8. By analyzing the one-dimensional and two-dimensional NMR data of the compound, it was found that the compound has a planar structure that is highly similar to 3. The difference is that in the HMBC spectrum, correlation signals between OCH3-18 and C-18, and OH-15 and C-15 were observed, indicating that the methoxy and hydroxyl groups are connected to the C-18 and C-15 positions, respectively. Thus, the planar structure of the compound was determined. In the NOESY spectrum, H-5 / H3-20 and H-9 / H3-20 are correlated, indicating that H-5, H-9, and CH3-20 are located on the same side of the molecule, in the β direction; H-8 / H-10 is correlated, indicating that H-10 is in the α direction. The smaller chemical shift value (δ H-5 2.97) further proves the orientation of H-5. The large coupling constant (12.3Hz) between H-8 and H-9 indicates that the double bond there is a trans double bond. Therefore, the relative configuration of the compound is determined to be (5S*, 10S*, 11S*). By comparing the calculated ECD and the measured ECD, the absolute configuration of the compound is determined to be (5S, 10S, 11S).

[0036] Vibsawabin H (8): colorless oil. UV(MeOH)λ max (logε)231(1.71). HRESIMS gave the quasi-molecular ion peak m / z 447.27249[M+H] + (calcd for C 26 H 39 O6,447.27412), combined 1 H-NMR and 13 C-NMR confirmed that the molecular formula of the compound is C 26 H 38 O6, the calculated unsaturation is 8. By comparison, it is found that the NMR signal of this compound is highly similar to the NMR data of 8, indicating that the two have similar skeletons. In the HMBC spectrum, H2-12 and C-14 are correlated, H2-16 / H3-17 and C-14 are correlated, OH-14 and C-14 are correlated, and H3-20 and C-12 are correlated. 1 H- 1 The structure of the C6 side chain connected to C-11 was confirmed by H COSY spectrum. H-5(3.39) The relative configuration of the compound was determined to be (5R*, 10S*, 11S*). By comparing the calculated ECD and the measured ECD, the absolute configuration of the compound was determined to be (5R, 10S, 11S). Similar to compound 5, the absolute configuration of C-14 was determined by the Rh2(OCOCF3)4 salt induced ECD method. The results showed that its induced ECD spectrum showed a negative Cotton effect at 336-347nm, so the absolute configuration of C-14 was R. Thus, the absolute configuration of the compound was determined to be (5R, 10S, 11S, 14R).

[0037] Vibsawabin I (9): colorless oil. UV(MeOH)λ max (logε)230(1.90). HRESIMS gave the quasi-molecular ion peak m / z 465.28497[M+H] + (calcd for C 26 H 41 O7,465.28468), combined 1 H-NMR and 13 C-NMR confirmed that the molecular formula of the compound is C 26 H 40 O7, the calculated unsaturation is 7. By comparison, it is found that the NMR signal of this compound is highly similar to the NMR data of 8, the difference is that the C6 side chain connected to C-11 is replaced by the hydroxyl group in 9 instead of the methoxy group in 8. In the HMBC spectrum, H3-16 / H3-17 is correlated with C-14 / C-15, H2-12 is present with C-14, and H3-20 is correlated with C-12. Combined with the chemical shift values ​​of C-14 and C-15, it shows that there is a hydroxyl group at each position of C-14 and C-15, and the structure of the C6 side chain connected to C-11 is determined. In the NOESY spectrum, H-5 / H3-20 and H-9 / H3-20 are correlated, indicating that H-5, H-9, and CH3-20 are located on the same side of the molecule, which is β-oriented; H-10 / H2-12 and H-8 / H-10 are correlated, indicating that H-10 is α-oriented. Thus, the relative configuration of the compound was determined to be (5S*, 10S*, 11S*). Similar to 8, the absolute configuration of the compound was determined to be (5S, 10S, 11S) by comparing the calculated ECD and the measured ECD. The absolute configuration of C-14 was determined by the Mo2(OAc)4 salt induced ECD method. The results showed that the positive Cotton effect displayed at 310nm in its induced ECD spectrum indicated that the absolute configuration of C-14 was S. Thus, the absolute configuration of the compound was determined to be (5S, 10S, 11S, 14S).

[0038] Vibsawabin G (10): colorless oil. UV(MeOH)λ max (logε)232(2.46). HRESIMS gave the quasi-molecular ion peak m / z 465.28323[M+H] + (calcd for C 26 H 41 O7,465.28468), combined 1 H-NMR and 13 C-NMR confirmed that the molecular formula of the compound is C 26 H 40 O7, the calculated unsaturation is 7. By comparison, it is found that the NMR signal of this compound is similar to the NMR data of 9, except that the methoxy and hydroxyl substitution positions in the structure are different. In the HMBC spectrum, H3-16 / H3-17 are correlated with C-14 / C-15, OCH3-15 is correlated with C-15, H2-12 is present with C-14, and H3-20 is correlated with C-12. Combined with the coupling system, it shows that the methoxy group is connected to the C-15 position, and the structure of the C6 side chain connected to C-11 is determined. H2-18 is HMBC correlated with C-2 / C-4, and combined with the chemical shift value of C-18, it shows that the hydroxyl group is connected to the C-18 position. The relative configuration of the compound C-5, C-10 and C-11 positions was determined by NOESY spectrum, which is the same as 9. By comparing the calculated ECD and the measured ECD, the absolute configuration of the compound part was determined to be (5S, 10S, 11S). The absolute configuration of the chiral center C-14 was determined by the Mo2(OAc)4 induced ECD method, and the positive Cotton effect shown in its induced ECD spectrum at 310 nm indicated that the absolute configuration of C-14 was S. Thus, the absolute configuration of the compound was determined to be (5S, 10S, 11S, 14S).

[0039] Vibsawabin K(11): colorless oil. UV(MeOH)λ max (logε)232(1.99). HRESIMS gave a quasi-molecular ion peak m / z 465.28378[M+H] + (calcd for C 26 H 41 O7,465.28468), combined 1 H-NMR and 13 C-NMR confirmed that the molecular formula of the compound is C 26 H 40 O7, the calculated unsaturation is 7. By comparison, it was found that the NMR signal of this compound was highly similar to the NMR data of 10. Combined with HMBC and 1 H- 1The H COSY spectrum confirmed that the compound has the same planar structure as 10. The difference is that the chemical shift values ​​of the two compounds H2-12 / H2-13 are different [δ H 0.99, 1.40, 10 in delta H (1.07, 1.40) / (1.05, 1.40)], it is inferred that 11 may be the diastereomer of 10 about C-14. The relative configurations of C-5, C-10 and C-11 of the compound were determined by NOESY spectrum, which were the same as 10. The absolute configuration of the compound part was determined to be (5S, 10S, 11S) by comparing the calculated ECD and the measured ECD. The absolute configuration of the chiral center C-14 was determined by the Mo2(OAc)4 induced ECD method, and the negative Cotton effect shown in its induced ECD spectrum at 310nm indicated that the absolute configuration of C-14 was R. Thus, the absolute configuration of the compound was determined to be (5S, 10S, 11S, 14R).

[0040] Vibsawabin L(12): colorless oil. UV(MeOH)λ max (logε)231(2.26). HRESIMS gave a quasi-molecular ion peak m / z 433.25674[M+H] + (calcd for C 25 H 37 O6,433.25847), combined 1 H-NMR and 13 C-NMR confirmed that the molecular formula of the compound is C 25 H 36 O6, the calculated unsaturation degree is 8. The NMR data of this compound are similar to those of the known compound vibsanin C, except that the characteristic methyl signal of the senecioyl group is not observed in 12, and there is an additional oxygen-carbon signal. In the HMBC spectrum, H3-4' / H2-5' is correlated with C-2' / C-3', H2-5' is correlated with C-4', H-2' is correlated with C-1', and H-8 is correlated with C-1'. Combined with the chemical shift value of C-5' and the mass spectrometry data of the compound, it is determined that one of the methyl groups in the characteristic senecioyl group side chain connected to C-8 has become a hydroxymethyl group. In the NOESY spectrum, correlation signals of H-5 / H3-20 and H-9 / H3-20 can be observed, indicating that H-5 and CH3-20 are in the same orientation, which is β orientation. The NOESY correlation of H-10 / H-8 shows that H-10 is in the opposite orientation, which is α orientation. Thus, the relative configuration of the compound was determined to be (5S*, 10S*, 11S*). By comparing the calculated ECD with the measured ECD, the absolute configuration of the compound was determined to be (5S, 10S, 11S).

[0041] Vibsawabin M(13): colorless oil. UV(MeOH)λ max (logε)231(2.43). HRESIMS gave a quasi-molecular ion peak m / z 437.2537[M+H] + (calcd for C 24 H 37 O7,437.2534), combined 1 H-NMR and 13 C-NMR confirmed that the molecular formula of the compound is C 24 H 36 O7, the calculated unsaturation is 7. By comparison, it is found that the NMR signal of this compound is similar to that of compound 10, except that the C-11 of 13 is connected to the C6 side chain. In the HMBC spectrum, OCH3-14 is correlated with C-14, H3-20 is correlated with C-12, and H2-12 is correlated with C-14. 1 H- 1 The H2-12-H2-13-H-14 coupling system observed in the H COSY spectrum indicates that there are two methoxy groups attached to the C-14 position, and the structure of the side chain attached to C-11 of the compound is determined. The NOESY correlation of H-5 / H3-20, H-9 / H3-20 and H-8 / H-10 shows that H-5 and CH3-20 are β-oriented, and H-10 is α-oriented. The relative configuration of the compound is determined to be (5S*, 10S*, 11S*). By comparing the measured ECD and calculated ECD spectra, the absolute configuration of the compound is determined to be (5S, 10S, 11S).

[0042] Vibsawabin N(14): colorless oil. UV(MeOH)λ max (logε)224(2.27). HRESIMS gave a quasi-molecular ion peak m / z 415.2470[M+H] + (calcd for C 25 H 35 O5,415.2479), combined 1 H-NMR and 13 C-NMR confirmed that the molecular formula of the compound is C 25 H 34O5, the calculated unsaturation is 9. By comparison, it was found that the NMR signal of this compound was similar to that of 12, indicating that the two have similar structures. Characteristic sedum acyloxy signals were observed in the one-dimensional NMR spectrum. In the HMBC spectrum, H3-19 is correlated with C-7 / C-6, H-6 is correlated with C-4, and H-9 is correlated with C-5, indicating that C-5-C-6 is a carbon-carbon double bond. In the NOESY spectrum, the correlation signals of H-8 / H-10, H-10 / H-12 and H-9 / H3-20 indicate that H-10 is α-oriented and CH3-20 is β-oriented. The absence of the NOESY correlation signal between H-6 and H-10 indicates that Δ 5 The relative configuration of the compound was determined to be (10R*, 11S*). The absolute configuration of the compound was determined to be (10R, 11S) by comparing the measured ECD and calculated ECD spectra.

[0043] Vibsawabin O(15): colorless oil. UV(MeOH)λ max (logε)226(2.21). HRESIMS gave the quasi-molecular ion peak m / z 399.25183[M+H] + (calcd for C 25 H 35 O4,399.25299), combined 1 H-NMR and 13 C-NMR confirmed that the molecular formula of the compound is C 25 H 34 O4, the calculated unsaturation is 9. 1 In the H-NMR spectrum (600 MHz, DMSO-d6), the characteristic signal δ H 5.71 (1H, p, J = 1.3 Hz), 2.12 (3H, d, J = 1.3 Hz), 1.91 (3H, d, J = 1.3 Hz) suggest the presence of a senecioyloxy fragment. H 7.01 (1H, d, J = 12.6 Hz), 5.36 (1H, dd, J = 12.6, 9.4 Hz) are a group of trans-olefin hydrogen signals, δ H 5.56 (1H, dd, J = 7.4, 3.7 Hz), 5.96 (1H, d, J = 1.1 Hz), 4.96 (1H, m) indicate the presence of three additional olefinic hydrogen signals. H 2.20 (3H, d, J = 1.1 Hz), 0.91 (3H, s), 1.57 (3H, d, J = 1.3 Hz), and 1.46 (3H, d, J = 1.3 Hz) are the four methyl proton signals. 13C-NMR spectrum (150 MHz, DMSO-d6) combined with HSQC spectrum showed that there were 25 carbon signals in the structure, including 1 ester carbonyl carbon signal, 12 olefin carbon signals, and 1 sp 3 Hybridized quaternary carbon signal, 1 methine carbon signal, 4 methylene carbon signals, and 6 methyl carbon signals. The planar structure of the compound was analyzed in combination with the two-dimensional spectrum.

[0044] 1 H- 1 In the H COSY spectrum, the correlation signals of H2-1 / H-2, H-8 / H-9 / H-10, H2-12 / H2-13 / H-14 were observed, indicating that there are three spin coupling systems in the structure. In the HMBC spectrum, H3-20 is correlated with C-10 / C-1, H2-1 is correlated with C-3 / C-10, H-2 is correlated with C-4 / C-11, H-6 is correlated with C-4 / C-5 / C-10, and H-10 is correlated with C-4, proving that there is a seven-membered ring structure fragment in the compound. H3-19 is HMBC correlated with C-6 / C-7, combined with the remaining unsaturation in the structure and the chemical shift values ​​of C-4 and C-7 in the low field region (δ C 145.9,149.3), indicating that C-4 and C-7 are connected by an ether bond to form a furan ring and fused with a seven-membered ring. The HMBC correlation between H-2 and C-18 indicates that there is a hydroxymethyl group connected to the C-3 position. The HMBC correlation between H-8 and C-1' indicates that the senecioyl group is connected to the C-1' position. H3-16 / H3-17 have HMBC correlations with C-14 / C-15, and H3-20 has HMBC correlations with C-12. Combined with the spin coupling fragment C-12-C-13-C-14, it indicates the structure of the C6 side chain connected to C-11. The planar structure of the compound was thus determined.

[0045] The relative configuration of the compound was determined by NOESY spectrum. In the NOESY spectrum, the correlation of H-9 / H3-20 showed that CH3-20 and H-9 were located on the same side of the molecule. The correlation of H-10 / H2-13 and H-10 / H-8 showed that H-10 was oriented in the opposite direction, α. Therefore, the relative configuration of the compound was determined to be (10R*, 11S*). By comparing the calculated ECD and the measured ECD, the absolute configuration of the compound was determined to be (10R, 11S).

[0046] The NMR data of compounds 1-15 are shown in Tables 1-4.

[0047] Table 1: Compound 1-4 (a: DMSO-d6 and b: CD3OD, c: CDCl3) 1 H(600MHz) and 13C (150 MHz) NMR data

[0048]

[0049]

[0050]

[0051] Table 2: Compound 5-8 (a: DMSO-d6 and b: CD3OD, c: CDCl3) 1 H(600MHz) and 13 C (150 MHz) NMR data

[0052]

[0053]

[0054] Table 3: Compounds 9-12 (a: DMSO-d6 and b: CD3OD, c: CDCl3) 1 H(600MHz) and 13 C (150 MHz) NMR data

[0055]

[0056]

[0057] Table 4: Compounds 13-15 (a: DMSO-d6 and b: CD3OD, c: CDCl3) 1 H(600MHz) and 13 C (150 MHz) NMR data

[0058]

[0059]

[0060]

[0061] The cytotoxic activities of the 15 vibsane-type diterpenoid compounds of the present invention on human liver cancer cells HepG2, lung cancer cells A549 and breast cancer cells MCF-7 were investigated. The experimental results showed that compound 3 showed significant anti-tumor activity against MCF-7; compound 13 showed moderate anti-tumor activity against MCF-7. Compounds 1 and 13 had good anti-tumor activity against HepG2 and A549. Therefore, the vibsane-type diterpenoid compounds of the present invention have new medical uses with anti-tumor effects.

[0062] The present invention also provides a pharmaceutical composition, which comprises the vibsane diterpenoid compound or a salt thereof of the present invention and a pharmaceutically acceptable carrier or excipient.

[0063] The advantages of the present invention are that the vibsane-type diterpenoid compounds of the present invention have novel structures and are all optically pure compounds with a definite stereoconfiguration. At the same time, they have strong anti-tumor activity and are worth further development. BRIEF DESCRIPTION OF THE DRAWINGS

[0064] Figure 1 Key HMBC correlation and COSY correlation of compounds 1-15;

[0065] Figure 2 Key NOESY correlations of compounds 1-15;

[0066] Figure 3 Comparison of measured and calculated ECDs of compounds 1-15;

[0067] Figure 4 Rhodium salts of compounds 5-6, 8 induced CD;

[0068] Figure 5 Molybdenum salts of compounds 9-11 induced CD. DETAILED DESCRIPTION

[0069] The following examples are intended to help those skilled in the art to better understand the present invention, but are not intended to limit the present invention in any way.

[0070] Example 1

[0071] 40 kg of dried leaves of Japanese coral tree of Viburnum genus of Viburnum family were extracted with 75% ethanol by reflux for 3 times, each time for 2 hours, and the extract was concentrated to obtain an extract, which was extracted with n-butanol, and the n-butanol layer extract (3000 g) was subjected to silica gel column chromatography, and eluted with CH2Cl2-CH3OH system gradient (50:1-1:1) (v / v) to obtain 4 fractions Fr.A-Fr.D. Fr.A (150 g) and Fr.B (160 g) were subjected to HP20 macroporous resin with ethanol-water gradient elution (70:30-100:0) (v / v), and a total of 6 fractions Fr.A1-Fr.A3 and Fr.B1-Fr.B3 were obtained. Fractions Fr.A1-Fr.A3 and Fr.B1-Fr.B3 were subjected to ODS column chromatography with ethanol-water gradient elution (60:40-100:0 and 20:80-100:0) (v / v), respectively, to obtain 7 fractions Fr.1-Fr.4 and Fr.5-Fr.7. Fraction Fr.2 (4 g) was subjected to silica gel column chromatography with petroleum ether: ethyl acetate (50:1-1:1) (v / v) to obtain 4 subfractions Fr.2-1 to Fr.2-4. Fr.2-4 was purified by semi-preparative liquid phase with acetonitrile-water (48:52, v / v) to obtain compounds 5 (2.8 mg), 6 (3.7 mg), and 12 (3.0 mg); Fr.2-3 was purified by semi-preparative liquid phase with acetonitrile-water (57:43, v / v) to obtain compound 15 (15.0 mg). Fraction Fr.1 was eluted by silica gel column chromatography with petroleum ether: ethyl acetate (50:1-1:1) (v / v) to obtain 7 subfractions Fr.1-1 to Fr.1-7. Fr.1-6 was purified by semi-preparative liquid phase with acetonitrile-water (75:25, v / v) to obtain compound 7 (2.9 mg); Fr.1-4 was purified by semi-preparative liquid phase with acetonitrile-water (62:38, v / v) to obtain compounds 2 (10.6 mg) and 9 (4.1 mg). Fr.1-3 was purified by semi-preparative liquid chromatography with acetonitrile-water (60:40, v / v) to obtain compounds 10 (9.6 mg) and 11 (6.2 mg); Fr.1-2 was purified by semi-preparative liquid chromatography with acetonitrile-water (48:52, v / v) to obtain compounds 3 (6.0 mg), 4 (3.5 mg) and 8 (3.1 mg). Fraction Fr.6 was eluted by silica gel column chromatography with petroleum ether: ethyl acetate (50:1-1:1) (v / v) to obtain 6 subfractions Fr.6-1 to Fr.6-6. Fr.6-4 was purified by semi-preparative liquid phase with acetonitrile-water (55:45, v / v) to obtain compounds 13 (3.8 mg) and 14 (12.5 mg); Fr.6-2 was purified by semi-preparative liquid phase with acetonitrile-water (40:60, v / v) to obtain compound 1 (5.0 mg).

[0072] Example 2

[0073] The antitumor activity of compounds 1-15 against liver cancer cells HepG2, lung cancer cells A549, and breast cancer cells MCF-7 was investigated in vitro.

[0074] The MTT method was used to investigate the cytotoxic activity of compounds 1-15 against tumor cells A549, HepG2, and MCF-7. The cells were placed in a 96-well plate and cultured with culture medium for 12 hours. The three tumor cells were treated with compounds of different concentrations (50, 25, 12.5, 6.25, and 3.125 μM), and cisplatin (DDP), sorafenib (SORA), and doxorubicin (DOX) were used as positive control groups. After 48 hours of action, 20 μL MTT reagent was added and placed at 37°C for 4 hours. The cells treated with different concentrations were detected using an enzyme reader at a wavelength of 490 nm. The results show (Table 5) that compound 3 has significant cytotoxic activity against MCF-7 cells, IC 50 The value was 9.72±0.43μM; compound 13 had moderate cytotoxic activity against MCF-7 cells, IC 50 The value was 12.85±0.30μM. Compounds 1 and 13 had good cytotoxic activity against HepG2 and A549 cells, and the IC 50 The values ​​were 19.59±1.12μM, 11.79±0.19μM, 15.31±1.34μM, and 19.13±0.30μM, respectively.

[0075] Table 5: Cytotoxic activity results of compounds 1-15 against three tumor cells (IC 50 ,μM)

[0076] Compound HepG2 A549 MCF-7 1 19.59±1.12 11.79±0.19 28.40±1.07 2 >50 >50 >50 3 29.97±0.38 >50 9.72±0.43 4 30.37±0.54 >50 >50 5 >50 >50 >50 6 >50 >50 >50 7 >50 >50 >50 8 >50 >50 >50 9 >50 >50 >50 10 >50 >50 >50 11 >50 >50 >50 12 39.13±0.19 >50 >50 13 15.31±1.34 19.13±0.30 12.85±0.30 14 33.7±0.01 40.69±3.94 37.23±3.94 15 >50 >50 >50 SORA 2.11±0.01 DDP 6.76±0.17 DOX 0.02±0.001

[0077] Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalents, the present invention is also intended to include these modifications and variations.

Claims

1. A vibsane-type diterpenoid compound having a seven-membered ring skeleton characteristic from a Japanese coral tree or a salt thereof, characterized in that: The structural formula of the vibsane type diterpenoid compound is:

2. The vibsane-type diterpenoid compound having a seven-membered ring skeleton characteristic from Japanese coral tree according to claim 1 or a salt thereof, characterized in that: The vibsane type diterpenoid compound is isolated from the dried leaves of Viburnum awabuki K. Koch, a plant of the genus Viburnum of the Viburnaceae family.

3. A method for preparing a vibsane-type diterpenoid compound or a salt thereof having a seven-membered ring skeleton as claimed in claim 1 or claim 2, characterized in that: The preparation method of the vibsane-type diterpenoid compound comprises the following steps: (1) The dried leaves of Japanese coral tree were extracted with ethanol, and the extract was concentrated to obtain an extract, which was extracted with n-butanol. The n-butanol layer extract was subjected to silica gel column chromatography with a gradient elution of CH2Cl2:CH3OH volume ratio of 50:1-1:1 to obtain four fractions Fr.A-Fr.D; (2) Fractions Fr.A and Fr.B were subjected to HP20 column chromatography with gradient elution using ethanol and water to obtain a total of 6 fractions Fr.A1-Fr.A3 and Fr.B1-Fr.B3; (3) Fractions Fr.A1-Fr.A3 and Fr.B1-Fr.B3 were subjected to ODS column chromatography with a gradient elution of ethanol:water in a volume ratio of 60:40-100:0 and 20:80-100:0, respectively, to obtain a total of 7 fractions Fr.1-Fr.4 and Fr.5-Fr.7; (4) Fraction Fr.2 was subjected to silica gel column chromatography with a gradient elution of petroleum ether:ethyl acetate in a volume ratio of 50:1-1:1 to obtain 4 subfractions Fr.2-1 to Fr.2-4. Fr.2-4 was purified by semi-preparative liquid chromatography with acetonitrile-water to obtain compounds 5, 6, and 12; Fr.2-3 was purified by semi-preparative liquid chromatography with acetonitrile-water to obtain compound 15; (5) Fraction Fr.1 was subjected to silica gel column chromatography with a gradient elution of petroleum ether:ethyl acetate in a volume ratio of 50:1-1:1 to obtain 7 subfractions Fr.1-1 to Fr.1-7. Fr.1-6 was purified by semi-preparative liquid phase with acetonitrile-water to obtain compound 7; Fr.1-4 was purified by semi-preparative liquid phase with acetonitrile-water to obtain compounds 2 and 9; Fr.1-3 was purified by semi-preparative liquid phase with acetonitrile-water to obtain compounds 10 and 11; Fr.1-2 was purified by semi-preparative liquid phase with acetonitrile-water to obtain compounds 3, 4 and 8; (6) Fraction Fr.6 was purified by silica gel column chromatography with a gradient elution of petroleum ether:ethyl acetate in a volume ratio of 50:1-1:1 to obtain 6 subfractions Fr.6-1 to Fr.6-6. Fr.6-4 was purified by semi-preparative liquid chromatography with acetonitrile-water to obtain compounds 13 and 14; Fr.6-2 was purified by semi-preparative liquid chromatography with acetonitrile-water to obtain compound 1.

4. The method for preparing a vibsane-type diterpenoid compound having a seven-membered ring skeleton according to claim 3, characterized in that: In the step (1), the Japanese coral tree refers to the Japanese coral tree (Viburnumawabuki K. Koch) of the genus Viburnum of the Viburnum family; the dried leaves of the Japanese coral tree are extracted by reflux with 70%-80% industrial ethanol.

5. The method for preparing a vibsane-type diterpenoid compound having a seven-membered ring skeleton according to claim 3, characterized in that: In the step (2), the fractions Fr.A and Fr.B are subjected to gradient elution using an HP20 column chromatography with an ethanol:water ratio of 70:30-100:0 by volume.

6. The method for preparing a vibsane-type diterpenoid compound having a seven-membered ring skeleton according to claim 3, characterized in that: In the step (4), Fr.2-4 is purified by semi-preparative liquid phase elution with acetonitrile:water in a volume ratio of 48:52 to obtain compounds 5, 6, and 12; Fr.2-3 is purified by semi-preparative liquid phase elution with acetonitrile:water in a volume ratio of 57:43 to obtain compound 15.

7. The method for preparing a vibsane-type diterpenoid compound having a seven-membered ring skeleton according to claim 3, characterized in that: In the step (5), Fr.1-6 is purified by semi-preparative liquid phase with acetonitrile:water in a volume ratio of 75:25 to obtain compound 7; Fr.1-4 is purified by semi-preparative liquid phase with acetonitrile:water in a volume ratio of 62:38 to obtain compounds 2 and 9, and Fr.1-3 is purified by semi-preparative liquid phase with acetonitrile:water in a volume ratio of 60:40 to obtain compounds 10 and 11; Fr.1-2 is purified by semi-preparative liquid phase with acetonitrile-water in a volume ratio of 48:52 to obtain compounds 3, 4, and 8.

8. The method for preparing a vibsane-type diterpenoid compound having a seven-membered ring skeleton according to claim 3, characterized in that: In the step (6), Fr.6-4 was purified by semi-preparative liquid phase elution with acetonitrile:water in a volume ratio of 55:45 to obtain compounds 13 and 14; Fr.6-2 was purified by semi-preparative liquid phase elution with acetonitrile:water in a volume ratio of 40:60 to obtain compound 1.

9. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the vibsane-type diterpenoid compound having a seven-membered ring skeleton characteristic as claimed in claim 1 or claim 2 or a salt thereof and a pharmaceutically acceptable carrier or excipient.

10. Use of the vibsane-type diterpenoid compound having a seven-membered ring skeleton characteristic according to claim 1 or claim 2 or its salt or the pharmaceutical composition according to claim 9 in the preparation of anti-tumor drugs.

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