Compound with anti-tumor activity in Daphne koreana and its preparation method and application

Abstract

Compounds with anti-tumor activity in Daphne koreana and their preparation methods and applications belong to the field of medical technology, and specifically relate to the preparation methods and applications of compounds with anti-tumor activity extracted and separated from Daphne koreana. The extraction method comprises the following steps: Daphne koreana is extracted with ethanol under reflux, concentrated under reduced pressure to obtain a total extract, extracted and concentrated, and subjected to LH-20 column chromatography to obtain a crude fraction, and then the fraction is subjected to in-depth and systematic separation using silica gel, reverse HP-20, reverse phase ODS, high performance liquid chromatography and other methods to obtain compounds. The anti-lung cancer, liver cancer and breast cancer activities of these compounds were investigated by testing their inhibitory effects on A549 lung cancer cells, HepG2 liver cancer cells and MCF-7 breast cancer cells. The compounds are all optically pure compounds with a determined stereoconfiguration, and at the same time, they have strong anti-lung cancer activity and are worthy of further development. #imgabs0#

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and particularly relates to diterpenoids and sesquiterpenoids compounds with anti-tumor activity in Daphne koreana and their preparation and application in the preparation of drugs against liver cancer or lung cancer. Background Art

[0002] Cancer: Also known as a malignant tumor. The human body needs to continuously produce new cells to replace aging and dying cells to maintain normal function. Therefore, most cells can replicate. However, this replication is limited by telomere length, typically only 50 to 60 divisions. Cancer cells, the fundamental unit of cancer, possess telomerase, which can repair telomeres lost during replication, allowing them to replicate indefinitely. This depletes the body's nutrients and releases various toxins, causing weight loss, weakness, anemia, loss of appetite, fever, and organ damage.

[0003] Daphne gemmate is a deciduous shrub native to northwest and western Sichuan, my country. It is a traditional Chinese medicinal herb known for its heat-clearing, detoxifying, and wind-removing dampness properties. Currently, no research has been conducted on the chemical constituents of Daphne gemmate. Therefore, research on the active chemical components of Daphne gemmate and their potential anticancer activity is of great significance. Summary of the Invention

[0004] The present invention provides a compound with anti-tumor activity prepared from Daphne koreana, which is any one of the following compounds:

[0005]

[0006] The method for preparing the compound with anti-tumor activity from Daphne koreana of the present invention comprises the following steps:

[0007] (1) The dried whole plant of Daphne koreana was extracted with ethanol under reflux;

[0008] (2) concentrating the extract under reduced pressure to obtain an extract, and extracting the extract with ethyl acetate and n-butanol;

[0009] (3) The extract was concentrated to obtain an extract, which was separated by glucose gel LH-20 column chromatography to obtain two fractions Fr.A to Fr.B;

[0010] (4) Fr.A to Fr.B are separated by silica gel vacuum column chromatography to obtain Fr.A1 to Fr.A2 and Fr.B1 to Fr.B4 respectively;

[0011] (5) Fr.A1 was separated by reverse phase HP-20 column chromatography and ODS column chromatography to obtain Fr.A1-1 to Fr.A1-4;

[0012] (6) Fr.B1 was separated by reverse phase HP-20 column chromatography and ODS column chromatography to obtain Fr.B1-1 to Fr.B1-4;

[0013] (7) Fr.A1-3 was separated by semi-preparative HPLC to obtain compound 1;

[0014] (8) Fr.B1-1 was separated by semi-preparative HPLC to obtain compound 2-8.

[0015] The method for preparing the compound with anti-tumor activity from Daphne koreana mentioned above, wherein:

[0016] The ethanol used in step (1) is industrial ethanol, the mass concentration of ethanol in the industrial ethanol is 70-90%; the extraction times are 2-3 times.

[0017] The temperature for the reduced pressure concentration in step (2) is 55-65°C.

[0018] The gradient elution conditions of the glucose gel LH-20 column chromatography in step (3) are EtOH:H2O=20:80, 70:30, 100:0.

[0019] The gradient elution condition of silica gel vacuum column chromatography in step (4) is CH2Cl2:MeOH=1:0-0:1.

[0020] The gradient elution conditions of the reversed-phase HP-20 column chromatography and the ODS column chromatography in step (5) are EtOH:H2O=20:80-100:0.

[0021] The gradient elution conditions of the reversed-phase HP-20 column chromatography and the ODS column chromatography in step (6) are EtOH:H2O=20:80-100:0.

[0022] The gradient elution condition of the semi-preparative HPLC in step (7) is CH3CN:H2O=35:65.

[0023] The gradient elution condition of the semi-preparative HPLC in step (8) is CH3CN:H2O=30:70-50:50.

[0024] The obtained compounds 1-8 were systematically identified, as Figure 1-26 , the results are as follows:

[0025] Compound 1: white amorphous powder (methanol); -45.6(c 0.1,MeOH); UV(MeOH)λ max (logε):253nm(1.79); ECD(MeOH)196(Δε+8.26),207(Δε+2.30),226(Δε+35.31),248(Δε-12.46)nm; HRESIMS gives the quasi-molecular ion peak m / z 491.2007[M+Na] + (calcd for C 27 H 32 O7Na,491.204), combined 1 H-NMR, 13 C-NMR suggests that its molecular formula is C 27 H 32 O7, calculated unsaturation is 12.

[0026] exist 1 In the H-NMR (600MHz, CDCl3) spectrum, δ H 0.97 (1H, d, J = 5.5 Hz, H-14), 1.19 (3H, s, H-16), 1.06 (3H, s, H-17) suggest the presence of a geminal dimethylcyclopropane group, δ H 1.12 (3H, d, J = 6.6 Hz, H-18), 1.82 (3H, dd, J = 3.0, 1.4 Hz, H-19) suggest the presence of two methyl groups, which is a typical feature of the tigliane-type diterpene skeleton. H 7.61 (1H, m), 5.67 (1H, d, J = 5.5 Hz) are two olefin proton signals, δ H 8.02 (1H, dd, J = 8.3, 1.4 Hz), 7.46 (1H, m), 7.59 (1H, m), 7.46 (1H, m), 8.02 (1H, dd, J = 8.3, 1.4 Hz) are a group of proton signals on the monosubstituted aromatic ring, δ H 4.05 (2H, q, J = 12.9 Hz) is the signal of two oxymethylene protons, δ H 1.12 (3H, d, J = 6.6 Hz), 1.82 (3H, dd, J = 3.0, 1.4 Hz) indicate two methyl proton signals. 13 The C-NMR (150 MHz, CDCl3) spectrum showed a total of 27 carbon atoms, of which δ C 208.7 (C-3), 169.1 (C-1') suggest two ketone carbonyl carbon signals, δ C129.7 (C-2'), 129.9 (C-3'), 128.7 (C-4'), 133.6 (C-5'), 128.7 (C-6'), 129.9 (C-7') are a group of aromatic ring carbon signals, δ C 160.2(C-1),133.7(C-2);δ C 140.9 (C-6) and 129.3 (C-7) are two groups of double bond carbon signals, δ C 88.5 (C-12), 79.4 (C-9), 73.6 (C-4), 68.3 (C-20), and 61.0 (C-13) are five oxygen-linked carbon signals. C Four methyl carbon signals were identified: 22.3 (C-16), 17.3 (C-17), 16.4 (C-18), and 10.3 (C-19). These hydrocarbon data indicate that the compound is a tigliane-type diterpenoid. All signals were assigned using HSQC and further analyzed using the remaining two-dimensional spectra.

[0027] In the HMBC spectrum, H3-19 (δ H 1.82) and C-1(δ C 160.2) and C-3(δ C 208.7) is correlated, H-1(δ H 7.61) and C-3(δ C 208.7) and C-4(δ C 73.6), indicating the presence of an α,β unsaturated ketone unit and the attachment of H3-19 to C-2. H 1.12) and C-9(δ C 79.4) and C-12 (δ C 88.5) is correlated, indicating that H3-18 is linked to C-11. H 4.05) and C-5(δ C 39.0) and C-7 (δ C 129.3) is correlated, indicating that H-20 is attached to C-6. H 4.85) and C-12(δ C 88.5),C-14(δ C 35.6),C-15(δ C 28.2) is correlated, indicating that OH-13 is connected at position 13. H 8.02), H-7'(δ H 8.02) and C-1'(δ C169.1) showed a correlation, indicating the presence of benzoyl groups. H 5.08) and C-1'(δ C 169.1) showed a correlation, indicating that the benzoyl group was attached to C-12. Therefore, the planar structure of compound 1 was confirmed.

[0028] The relative configuration of compound 1 was determined by NOESY spectroscopy. In the NOESY spectrum, H-8 and H-11 showed correlation, suggesting that H-11 and H-8 were on the same side, defined as a β orientation. Correlations between H-10 / H-1, H-1 / H3-18, and H-12 / H3-18 indicated an α orientation for H-10, H-1, H3-18, and H-12. Furthermore, the NOESY correlations of H-11 / H3-17 and H-8 / H3-17 inferred a β orientation for the gem-dimethylcyclopropane at C-13 and C-14, while the NOESY correlation of H-14 / H3-16 indicated an α orientation for H-14 and OH-13. The relative configuration of the compound was determined to be 4R*, 8S*, 9S*, 10S*, 11R*, 12R*, 13S*, and 14R*.

[0029] The absolute configuration of compound 1 was determined by comparing the calculated and measured ECD curves. The measured ECD curves of the compound were in good agreement with the calculated ECD curves of the 4R, 8S, 9S, 10S, 11R, 12R, 13S, 14R configurations, and the absolute configuration of compound 1 was determined to be 4R, 8S, 9S, 10S, 11R, 12R, 13S, 14R.

[0030] Compound 2: white amorphous powder (methanol); -32.3(c 0.1,MeOH); UV(MeOH)λ max (logε):253(1.79); ECD(MeOH)206(Δε-4.77),223(Δε+5.00),236(Δε-2.05),245(Δε+0.7 3), 250 (Δε-0.67), 263 (Δε+43.95), 315 (Δε-9.26); HRESIMS gives the quasi-molecular ion peak m / z291.1544[M+Na] + (calcd for C 15 H 24 O4Na,291.157), combined 1 H-NMR, 13 C-NMR suggests that its molecular formula is C 15 H 24 O4, the calculated unsaturation is 4.

[0031] 1In the H NMR (600 MHz, DMSO-d6) spectrum, δ H 0.83 (3H, d, J = 7.0 Hz) indicates a methyl proton signal, δ H 4.68 (1H, t, J = 5.4 Hz), 4.43 (1H, t, J = 5.3 Hz), 4.15 (1H, d, J = 6.0 Hz), 4.05 (1H, d, J = 1.5 Hz) indicate four hydroxyl proton signals, δ H 5.09 (1H, d, J = 2.2 Hz) and 5.02 (1H, d, J = 2.2 Hz) indicated two olefin proton signals. 13 The C NMR (150 MHz, DMSO-d6) spectrum gave a total of 15 carbon signals, including 4 olefinic carbon signals (δ C 157.4, 145.8, 128.8, 107.3), 1 methyl carbon signal (δ C 15.5), 4 methylene carbon signals (δ C 38.1, 34.0, 32.8, 28.9), 4 oxygen-carbon signals (δ C 78.4, 71.6, 62.9, 61.2), 2 tertiary carbon signals (δ C 38.8, 37.0). The above hydrocarbon data indicate that the compound is a guaiacyl-type sesquiterpenoid. All signals were assigned using HSQC and further analyzed using the remaining two-dimensional spectra.

[0032] δ in HMBC spectrum H 2.12(H-2) and δ C 35.0 (C-4) and 128.8 (C-10) are correlated, δ H 0.83(H3-15) and δ C There is a correlation between 32.8 (C-3) and 38.8 (C-5), 1 H- 1 δ in the H COSY spectrum H 2.12(H-2) and δ H 1.23(H-3),δ H 1.97(H-4) and δ H 1.23(H-3),δ H 2.84(H-5) and δ H 0.83(H3-15),δ H 0.83(H3-15) and δ H 1.97 (H-4) is correlated, proving that C-15 is connected to C-4 and C-1, C-2, C-3, C-4, and C-5 form a five-membered ring. In addition, the HMBC spectrum shows that δH 3.89(H-14) and δ C There is a correlation between 145.8 (C-1) and 34.0 (C-9), δ H 2.00(H-9) and δ C 78.4 (C-7) and δ C 62.9 (C-14) is related, δ H 1.28(H-6) and δ C There is a correlation between 71.6 (C-8), 37.0 (C-4) and 145.8 (C-1). 1 H- 1 δ in the H COSY spectrum H 3.38 (H-8) and δ H 2.00(H-9) is correlated, δ H 2.84(H-5) and δ H 1.28 (H-6) is related, indicating that C-14 is connected to C-10 and there is a seven-membered ring fragment composed of C-1, C-5, C-6, C-7, C-8, C-9, and C-10. The seven-membered ring and the five-membered ring are connected through C-1 and C-5. According to the δ H 3.97(H-13) and δ C 78.4 (C-7) and δ C 107.3(C-12) is related, δ H 5.09(H-12) and δ C 78.4 (C-7) and δ C 157.4 (C-11) is correlated, confirming that the hydroxypropylene side chain is connected to C-7. H 4.15(OH-8) and δ C 78.4 (C-7) and δ C The presence of a correlation at 34.0 (C-9) confirmed that OH-8 was connected to C-8. Therefore, the planar structure of compound 2 was confirmed.

[0033] The relative configuration of this compound was determined by NOESY spectroscopy. In the NOESY spectrum, H-5 correlated with OH-7 and H-3α, and OH-8 correlated with OH-7, suggesting that H-5, OH-7, and OH-8 were on the same side, defined as an α orientation. H-8 correlated with H-13, and H3-15 correlated with H-3β, indicating that H3-15 and H-8 were in a β orientation. Therefore, the relative configuration of compound 2 was determined to be 4S*, 5S*, 7S*, and 8S*.

[0034] The compound was crystallized in methanol:water (9:1) and single crystal X-ray diffraction was performed using a Bruker D8 VENTURE PHOTON II diffractometer (Flack constant of 0.11), thereby determining its absolute configuration to be 4S, 5S, 7S, 8S.

[0035] Compound 3: white amorphous powder; -46.0(c 0.1,MeOH); UV(MeOH)λ max (logε): 253 (1.79); ECD (MeOH) 195 (Δε + 3.65), 202 (Δε + 0.35), 243 (Δε + 26.37); HRESIMS gave a quasi-molecular ion peak m / z 291.1532 [M + Na] + (calcd for C 15 H 24 O4Na,291.157), combined 1 H-NMR, 13 C-NMR suggests that its molecular formula is C 15 H 24 O4, the calculated unsaturation is 4.

[0036] 1 In the H NMR (600 MHz, DMSO-d6) spectrum, δ H 1.62 (3H, d, J = 1.9 Hz), 1.02 (3H, s), 0.68 (3H, d, J = 5.3 Hz) indicate three methyl proton signals, δ H 5.29 (1H, s), 4.54 (1H, s), 4.56 (1H, t, J = 5.5 Hz) indicated three hydroxyl proton signals. 13 The C NMR (150 MHz, DMSO-d6) spectrum gave 15 carbon signals, including one carbonyl carbon signal (δ C 208.2), two olefinic carbon signals (δ C 174.7,136.2), 3 methyl carbon signals (δ C 21.3, 16.5, 8.0), 3 methylene carbon signals (δ C 38.2, 21.6, 32.0), three oxygen-carbon signals (δ C 68.2, 74.8, 67.7), three tertiary carbon signals (δ C The above information suggests that compound 3 is a typical guaiacyl sesquiterpenoid compound.

[0037] δ in HMBC spectrum H 2.31(H-2) and δC 208.2 (C-3), 174.7 (C-5) and 31.4 (C-10) are correlated, δ H 1.62(H3-15) and δ C 208.2 (C-3) and 174.7 (C-5) are correlated, proving that C-15 is connected to C-4 and C-1, C-2, C-3, C-4, and C-5 form a five-membered ring. H 0.68(H3-14) and δ C There is a correlation between 42.9 (C-1) and 32.0 (C-9), δ H 1.44(H-9) and δ C 48.5 (C-7) and δ C 16.5(C-14) is related, δ H 4.84(H-6) and δ C There is a correlation between 21.6 (C-8), 136.2 (C-4) and 42.9 (C-1). 1 H- 1 The presence of a C6-C7-C8-C9-C10-C14 spin system was observed in the H COSY spectrum, indicating that C-14 is connected to C-10 and there is a seven-membered ring fragment composed of C-1, C-5, C-6, C-7, C-8, C-9, and C-10. The seven-membered ring and the five-membered ring are connected through C-1 and C-5. According to the δ H 1.02(H-13) and δ C 48.5 (C-7) and δ C 67.7 (C-12) is related, δ H 3.29(H-12) and δ C A correlation of 48.5 (C-7) confirmed that C-11 was attached to C-7. The HMBC spectrum also confirmed the position of the hydroxyl group. Therefore, the planar structure of compound 3 was confirmed.

[0038] In the NOESY spectrum, it can be observed that OH-6 and H-7 have NOESY correlations, and H-7 and H-10 have correlations, and H-7 and H-1 have correlations, thus confirming that OH-6, H-7, H-10 and H-1 are on the same side of the plane. Further, quantum chemical NMR calculations were performed using the mPW1PW91 / 6-311+G(d,p) basis set level for the two possible configurations (ab) at the 11th position of the flexible side chain of this structure. Statistical analysis methods (R 2,DP4+Probability Analysis), comparing the correlation coefficients and confidence probabilities between compound 3 and the two possible configurations, revealed that the compound best matched the calculated results for configuration b. The final relative configurations of compound 3 were determined to be 1R*, 6R*, 7S*, 10R*, and 11R*.

[0039] The absolute configuration of the compound was determined by comparing the calculated ECD with the measured ECD. The calculated configuration of compound 3 was consistent with that of 1S, 6S, 7R, 10S, 11S, so its absolute configuration was determined to be 1S, 6S, 7R, 10S, 11S.

[0040] Compound 4: white amorphous powder; -40.0(c 0.1,MeOH); UV(MeOH)λ max (logε):253(1.79); ECD(MeOH)195(Δε-5.42),203(Δε+3.73),214(Δε-0.35),222 (Δε+2.05), 233 (Δε-5.84), 251 (Δε+22.18), 319 (Δε-9.82); HRESIMS gives the quasi-molecular ion peak m / z 291.1546[M+Na] + (calcd for C 15 H 24 O4Na,291.157), combined 1 H-NMR, 13 C-NMR suggests that its molecular formula is C 15 H 24 O4, the calculated unsaturation is 4.

[0041] 1 In the H NMR (600 MHz, DMSO-d6) spectrum, δ H 0.95 (3H, d, J = 6.9 Hz), 1.59 (3H, s), 1.01 (3H, s) indicate three methyl proton signals, δ H 4.89 (1H, s), 4.07 (1H, s), 4.50 (1H, t, J = 5.3 Hz) indicated three hydroxyl proton signals. 13 The C NMR (150 MHz, DMSO-d6) spectrum gave 15 carbon signals, including one carbonyl carbon signal (δ C 205.5), two olefinic carbon signals (δ C 176.7,133.9), 3 methyl carbon signals (δ C 21.1, 18.1, 7.2), 4 methylene carbon signals (δ C49.9, 27.7, 26.1, 24.0), three oxygen-carbon signals (δ C 77.5,74.2,67.4), two tertiary carbon signals (δ C 45.1, 40.1). The above information suggests that compound 4 is a typical guaiacyl sesquiterpenoid compound.

[0042] δ in HMBC spectrum H 2.25(H-2) and δ C 133.9 (C-4), 176.7 (C-5) and 45.1 (C-10) are correlated, δ H 1.59(H3-15) and δ C 205.5 (C-3) and 176.7 (C-5) are correlated, proving that C-15 is connected to C-4 and C-1, C-2, C-3, C-4, and C-5 form a five-membered ring. H 0.95(H3-14) and δ C There is a correlation between 77.5 (C-1) and 27.7 (C-9), δ H 1.80(H-9) and δ C 40.1(C-7) and δ C 18.1(C-14) is related, δ H 2.47(H-6) and δ C There is a correlation between 26.1(C-8), 133.9(C-4) and 77.5(C-1). 1 H- 1 The presence of a C6-C7-C8-C9-C10-C14 spin system was observed in the H COSY spectrum, indicating that C-14 is connected to C-10 and there is a seven-membered ring fragment composed of C-1, C-5, C-6, C-7, C-8, C-9, and C-10. The seven-membered ring and the five-membered ring are connected through C-1 and C-5. According to the δ H 1.01(H-13) and δ C 40.1(C-7) and δ C 67.4 (C-12) is related, δ H 3.30(H-12) and δ C 67.4 (C-7) is correlated, confirming that C-11 is connected to C-7. H 4.50(OH-12) and δ C The presence of a correlation at 67.4 (C-12) confirmed the substitution position of the hydroxyl group. Combined with the HSQC spectrum, the compound was assigned to a direct carbon-hydrogen relationship, thereby confirming the planar structure of compound 4.

[0043] In the NOESY spectrum, it can be observed that OH-1 and CH3-14 have NOESY correlation, while OH-1 and H-10 do not have correlation, thus confirming that OH-1 and CH3-14 are on the same side of the plane. Due to the stacking of H-10 and H-7 signals, the relative configuration of position 7 determined by the NOESY spectrum is inaccurate. Further quantum chemical ECD and NMR calculations of the remaining four possible configurations (ad) of the structure were performed using the mPW1PW91 / 6-311+G(d,p) basis set level. Various statistical analysis methods (R 2 , DP4+ probability analysis), comparing the correlation coefficient and confidence probability between compound 4 and the four possible configurations, it was found that the calculated results of the compound and the a configuration were the best. The DP4+ probability analysis of the hydrocarbon data was 100%, and there was a small calculation deviation in the linear correlation analysis (R 2 =0.9993). Thus, the relative configuration of compound 4 was finally determined.

[0044] The absolute configuration at position 11 of compound 4 was determined using Snatzke's method. The complex formed by 4 and Mo2(OAc)4 induces a negative Cotton effect near 327 nm. Based on the empirical rule proposed by Snatzke et al., the absolute configuration at position 11 was determined to be 11S. The absolute configuration of compound 4 was further confirmed by comparing the calculated ECD with the measured ECD. The calculated configuration of compound 4 was in good agreement with the calculated 1S, 7R, 10S, and 11S configurations, and the absolute configuration was therefore determined to be 1S, 7R, 10S, and 11S.

[0045] Table 1 Compound 1-4 (a:CDCl3 and b:DMSO-d6) 1 H (600MHz) and 13 C (150 MHz) NMR data

[0046]

[0047]

[0048] Compound 5: white amorphous powder; -36.2(c 0.1,MeOH); UV(MeOH)λ max (logε):253(1.79); ECD(MeOH)200(Δε+3.20),207(Δε+6.11),217(Δε-1.30),225(Δε+1.5 1), 234 (Δε-2.55), 240 (Δε+1.70), 253 (Δε-52.87), 323 (Δε+15.26); HRESIMS gives the quasi-molecular ion peak m / z 269.1726[M+H]+ (calcd for C 15 H 25 O4,269.175), combined 1 H-NMR, 13 C-NMR suggests that its molecular formula is C 15 H 24 O4, the calculated unsaturation is 4.

[0049] 1 In the H NMR (600 MHz, DMSO-d6) spectrum, δ H 0.98 (3H, s), 1.57 (3H, s), 0.61 (3H, d, J = 7.2 Hz) indicate three methyl proton signals. 13 The C NMR (150 MHz, DMSO-d6) spectrum gave 15 carbon signals, including one carbonyl carbon signal (δ C 204.4), two olefinic carbon signals (δ C 173.7,136.3), 3 methyl carbon signals (δ C 20.8, 14.3, 7.6), 4 methylene carbon signals (δ C 50.6, 30.8, 30.1, 24.6), three oxygen-carbon signals (δ C 81.2, 73.8, 67.5), two tertiary carbon signals (δ C The above information suggests that compound 5 is a typical guaiacyl sesquiterpenoid compound.

[0050] δ in HMBC spectrum H 2.42(H-2) and δ C 136.3 (C-4), 173.7 (C-5) and 39.4 (C-10) are correlated, δ H 1.57(H3-15) and δ C 204.4 (C-3) and 173.7 (C-5) are correlated, proving that C-15 is connected to C-4 and C-1, C-2, C-3, C-4, and C-5 form a five-membered ring. H 0.61(H3-14) and δ C There is a correlation between 81.2 (C-1) and 30.8 (C-9), δ H 1.99(H-9) and δ C 40.8 (C-7) and δ C 14.3(C-14) is related, δ H 2.77(H-6) and δ CThere is a correlation between 24.6 (C-8), 136.3 (C-4) and 81.2 (C-1). 1 H- 1 The presence of a C6-C7-C8-C9-C10-C14 spin system was observed in the H COSY spectrum, indicating that C-14 is connected to C-10 and there is a seven-membered ring fragment composed of C-1, C-5, C-6, C-7, C-8, C-9, and C-10. The seven-membered ring and the five-membered ring are connected through C-1 and C-5. According to the δ H 0.98(H-13) and δ C 40.8 (C-7) and δ C 67.5(C-12) is related, δ H 3.28(H-12) and δ C A correlation of 40.8 (C-7) confirmed that C-11 was attached to C-7. The HMBC spectrum also confirmed the position of the hydroxyl group. Combined with the HSQC spectrum, direct carbon-hydrogen correlation was performed, confirming the planar structure of compound 5.

[0051] In the NOESY spectrum, it can be observed that H-9β has a NOESY correlation with CH3-14, while H-9α has no correlation with CH3-14. In addition, H-9α has a NOESY correlation with H-7, while H-9β has no correlation with H-7, thus confirming that H-7 and CH3-14 are on opposite sides of the plane. Due to the lack of OH-1 signal and the flexible side chain at position 11, quantum chemical NMR calculations were further performed on the remaining four possible configurations (ad) of the structure using the mPW1PW91 / 6-311+G(d,p) basis set level. Statistical analysis methods (R 2 ), comparing the correlation coefficients and confidence probabilities between compound 5 and the four possible configurations revealed good agreement with the calculated results for configurations c and d, with minimal calculation deviation in the linear correlation analysis. This confirmed the relative configurations of compound 5 at positions 1, 7, and 10.

[0052] The absolute configuration at position 11 of compound 5 was determined using Snatzke's method. The complex formed by compound 5 and Mo2(OAc)4 induces a negative Cotton effect near 323 nm. Based on the empirical rule proposed by Snatzke et al., the absolute configuration at position 11 was determined to be 11R. The absolute configuration of compound 5 was further confirmed by comparing the calculated ECD with the measured ECD. The calculated configuration of compound 5 was in good agreement with the 1R, 7R, 10S, and 11R configurations, and the absolute configuration was therefore determined to be 1R, 7R, 10S, and 11R.

[0053] Compound 6: white amorphous powder; -33.0(c 0.1,MeOH); UV(MeOH)λ max (logε): 253 (1.79); ECD (MeOH) 207 (Δε + 32.71), 250 (Δε -89.90), 326 (Δε + 30.92); HRESIMS gave a quasi-molecular ion peak m / z 273.1434 [M+Na] + (calcd for C 15 H 22 O3Na,273.146), combined 1 H-NMR, 13 C-NMR suggests that its molecular formula is C 15 H 22 O3, the calculated unsaturation is 4.

[0054] 1 In the H NMR (600 MHz, DMSO-d6) spectrum, δ H 1.76 (3H, s), 1.07 (3H, s), 0.97 (3H, d, J = 7.3) indicate three methyl proton signals, δ H 5.59 (1H, d, J = 6.0 Hz), 5.22 (1H, s) indicate two hydroxyl proton signals, δ H 4.77 (1H, d, J = 1.8 Hz) and 4.70 (1H, d, J = 1.8) indicate a group of terminal double bond proton signals. 13 The C NMR (150 MHz, DMSO-d6) spectrum gave 15 carbon signals, including one carbonyl carbon signal (δ C 202.2), 4 olefinic carbon signals (δ C 171.7, 150.7, 141.1, 109.0), 3 methyl carbon signals (δ C 22.4, 20.3, 16.7), 3 methylene carbon signals (δ C 32.0×2,30.0), 2 oxygen-carbon signals (δ C 81.1,78.0), two tertiary carbon signals (δ C The above information suggests that compound 6 is a typical guaiacyl-type sesquiterpenoid compound.

[0055] δ in HMBC spectrum H 3.92(H-3) and δ C There is a correlation between 202.2 (C-2) and 78.0 (C-4), δ H 1.07(H3-15) and δ C78.0 (C-4) and 171.7 (C-5) are correlated, δ H 5.59(OH-3) and δ C There is a correlation between 202.2 (C-2) and 78.0 (C-4), δ H 5.22(OH-4) and δ C 81.1 (C-3), 78.0 (C-4), 171.7 (C-5) and 22.4 (C-15) are correlated, proving that C-15, OH-4 are connected to C-4, OH-3 is connected to C-3 and C-1, C-2, C-3, C-4 and C-5 form a five-membered ring. In addition, the HMBC spectrum shows that δ H 0.97(H3-14) and δ C There is a correlation between 141.1 (C-1) and 32.0 (C-9), δ H 1.40(H-9) and δ C 141.1(C-1),45.9(C-7),26.0(C-10) and δ C 16.7 (C-14) is related, δ H 2.56(H-6) and δ C There is a correlation between 150.7 (C-11), 30.0 (C-8), 78.0 (C-4) and 141.1 (C-1). 1 H- 1 The presence of a C6-C7-C8-C9-C10-C14 spin system was observed in the H COSY spectrum, indicating that C-14 is connected to C-10 and there is a seven-membered ring fragment composed of C-1, C-5, C-6, C-7, C-8, C-9, and C-10. The seven-membered ring and the five-membered ring are connected through C-1 and C-5. According to the δ H 1.76(H-13) and δ C 45.9 (C-7) and δ C 109.0(C-12) is related, δ H 4.77(H-12) and δ C The presence of correlations at 45.9 (C-7) and 150.7 (C-11) confirmed that the side chain was an allyl group with C-11 attached to C-7. Combined with the HSQC spectrum, direct carbon-hydrogen correlations were performed on the compound, confirming the planar structure of compound 6.

[0056] The NOESY spectrum shows a NOESY correlation between CH3-14 and H-7, while H-6β is correlated with CH3-14 but not with CH3-15, suggesting that CH3-14 and H-7 are on the same side, defined as a β orientation. A NOESY correlation between CH3-15 and OH-3 indicates that CH3-15 and OH-3 are on the same side, defined as an α orientation.

[0057] The absolute configuration of the compound was determined by comparing the calculated ECD with the measured ECD. The calculated configuration of compound 6 was consistent with that of 3S, 4R, 7S, 10S, so the absolute configuration was determined to be 3S, 4R, 7S, 10S.

[0058] Compound 7: white amorphous powder; -53.0(c 0.1,MeOH); UV(MeOH)λ max (logε):253(1.79); ECD(MeOH)204(Δε+8.22),214(Δε+3.26),223(Δε+7.00),245(Δε-3.08); HRESIMS gives the quasi-molecular ion peak m / z 293.1698[M+Na] + (calcd for C 15 H 26 O4Na,293.172), combined 1 H-NMR, 13 C-NMR suggests that its molecular formula is C 15 H 26 O4, the calculated unsaturation is 3.

[0059] 1 In the H NMR (600 MHz, DMSO-d6) spectrum, δ H 0.98 (3H, s), 0.85 (3H, s) indicate two methyl proton signals, δ H 4.55 (1H, d, J = 4.9 Hz), 4.42 (1H, t, J = 5.7 Hz), 3.83 (1H, s), 4.37 (1H, t, J = 5.6 Hz) indicate four hydroxyl proton signals, δ H 5.64 (1H, dt, J = 8.6, 2.6 Hz) indicates an olefin proton signal. 13 The C NMR (150 MHz, DMSO-d6) spectrum gave a total of 15 carbon signals, including two olefinic carbon signals (δ C 143.7,123.8), 2 methyl carbon signals (δ C 22.0,18.2), 4 methylene carbon signals (δ C51.5, 42.0, 26.1, 25.6), 4 oxygen-carbon signals (δ C 67.6,74.6,69.8,64.6), 2 tertiary carbon signals (δ C 46.4,49.7), 1 quaternary carbon signal (δ C The above information suggests that compound 7 is a typical carotene-type sesquiterpenoid compound.

[0060] δ in HMBC spectrum H 1.38(H-2) and δ C 25.6 (C-3), 46.4 (C-4) 49.7 (C-5) and 18.2 (C-15) are correlated, δ H 0.85(H3-15) and δ C 42.0 (C-2) and 51.5 (C-10) are related. This proves that C-15 is connected to C-1 and C-1, C-2, C-3, C-4, and C-5 form a five-membered ring. In addition, the HMBC spectrum shows that δ H 0.98(H3-13) and δ C 46.4 (C-4), 74.6 (C-11) and 69.8 (C-12) are correlated, δ H 3.12 (H-12) and δ C 46.4 (C-4), 74.6 (C-11) and δ C 22.0(C-13) is related, δ H 3.83(OH-11) and δ C There is a correlation between 46.4 (C-4), 74.6 (C-11) and 22.0 (C-13), proving that the side chain group is connected to C-4 via C-11. 1 H- 1 The C2-C3-C4-C5-C6-C7 spin system was observed in the H COSY spectrum, and δ H 3.95(H-14) and δ C 123.8 (C-7), 143.7 (C-8) and 67.6 (C-9) are correlated, δ H 4.37(OH-14) and δ C There is a correlation between 143.7 (C-8) and 64.6 (C-14), which proves that C-14 is connected to C-8 and there is a seven-membered ring fragment composed of C-1, C-5, C-6, C-7, C-8, C-9, and C-10. The seven-membered ring and the five-membered ring are connected through C-1 and C-5. According to the δ H 4.55(OH-9) and δ C 143.7(C-8),δC 67.6 (C-9) and δ C The presence of a correlation at 51.5 (C-10) confirmed the position of the hydroxyl substitution. Combined with the HSQC spectrum, the compound was assigned to a direct carbon-hydrogen correlation, and the planar structure of compound 7 was confirmed.

[0061] In the NOESY spectrum, it can be observed that H-5 and H-2α have NOESY correlation, and H-2α has correlation with H-4, while H-15 has correlation with H-9. This confirms that H-5, H-4, and OH-9 are on the same side of the plane, which is defined as α orientation, while H-15 and H-9 are on the same side of the plane, which is defined as β orientation. Quantum chemical NMR calculations were further performed using the mPW1PW91 / 6-311+G(d,p) basis set level to calculate the two possible configurations (ab) at the 11th position of the flexible side chain of this structure. Statistical analysis methods (R 2 ,DP4+probabilistic analysis), comparing the correlation coefficients and confidence probabilities between compound 7 and the two possible configurations, revealed that the compound best matched the calculated results for configuration a. The relative configuration of compound 7 was ultimately determined to be 1R*, 4S*, 5S*, 9S*, 11R*.

[0062] The absolute configuration at position 11 of compound 7 was then determined using Snatzke's method. The complex formed by compound 7 and Mo2(OAc)4 induces a negative Cotton effect near 318 nm. Based on the empirical rule proposed by Snatzke et al., the absolute configuration at position 11 was determined to be 11R. The absolute configuration of compound 7 was further confirmed by comparing the calculated ECD with the measured ECD. The calculated configuration of compound 7, 1R, 4S, 5S, 9S, and 11R, showed good agreement, and the absolute configuration was therefore determined to be 1R, 4S, 5S, 9S, and 11R.

[0063] Compound 8: white amorphous powder; -50.0(c 0.1,MeOH); UV(MeOH)λ max (logε):253(1.79); ECD(MeOH)201(Δε-7.36),213(Δε+6.21),219(Δε+3.81),229 (Δε+6.86), 231 (Δε+6.79), 246 (Δε+11.66), 304 (Δε-1.83); HRESIMS gives the quasi-molecular ion peak m / z 293.1692[M+Na] + (calcd for C 15 H 26 O4Na,293.172), combined 1 H-NMR, 13C-NMR suggests that its molecular formula is C 15 H 26 O4, the calculated unsaturation is 3.

[0064] 1 In the H NMR (600 MHz, DMSO-d6) spectrum, δ H 1.14 (3H, s), 0.84 (3H, s) indicate two methyl proton signals, δ H 4.53 (1H, d, J = 5.9 Hz), 4.44 (1H, m), 3.85 (1H, s), 4.38 (1H, t, J = 5.7 Hz) indicate four hydroxyl proton signals, δ H 5.62 (1H, dt, J = 8.2, 2.6 Hz) indicates an olefin proton signal. 13 The C NMR (150 MHz, DMSO-d6) spectrum gave a total of 15 carbon signals, including two olefinic carbon signals (δ C 123.2, 143.2), two methyl carbon signals (δ C 25.8,17.7), 4 methylene carbon signals (δ C 51.3, 41.9, 27.1, 25.0), 4 oxygen-carbon signals (δ C 73.9, 68.5, 67.5, 64.7), two tertiary carbon signals (δ C 49.4, 48.2), 1 quaternary carbon signal (δ C The above information suggests that compound 8 is a typical carotene-type sesquiterpenoid compound.

[0065] δ in HMBC spectrum H 1.34(H-2) and δ C 25.0 (C-3), 48.2 (C-4), 49.4 (C-5) and 17.7 (C-15) are correlated, δ H 0.84(H3-15) and δ C 41.9 (C-2) and 51.3 (C-10) are related. This proves that C-15 is connected to C-1 and C-1, C-2, C-3, C-4, and C-5 form a five-membered ring. In addition, the HMBC spectrum shows that δ H 1.14(H3-13) and δ C 48.2 (C-4), 73.9 (C-11) and 68.5 (C-12) are correlated, δ H 3.26(H-12) and δ C 48.2 (C-4), 73.9 (C-11) and δ C 25.8 (C-13) is related, δH 3.85(OH-11) and δ C There is a correlation between 48.2 (C-4), 73.9 (C-11) and 25.8 (C-13), proving that the flexible side chain is connected to C-4 via C-11. 1 H- 1 The C2-C3-C4-C5-C6-C7 spin system was observed in the H COSY spectrum, and δ H 3.95(H-14) and δ C 123.3 (C-7), 143.2 (C-8) and 67.5 (C-9) are correlated, δ H 4.38(OH-14) and δ C 143.2 (C-8) and 64.7 (C-14) are correlated, proving that C-14 is connected to C-8 and there is a seven-membered ring fragment composed of C-1, C-5, C-6, C-7, C-8, C-9, and C-10. The seven-membered ring and the five-membered ring are connected through C-1 and C-5. According to the δ H 4.53(OH-9) and δ C 143.2(C-8),δ C 67.5 (C-9) and δ C The presence of a correlation at 51.3 (C-10) confirmed the position of the hydroxyl substitution. Combined with the HSQC spectrum, the compound was assigned to a direct carbon-hydrogen correlation, and the planar structure of compound 8 was confirmed.

[0066] In the NOESY spectrum, it can be observed that H-5 and H-2β have NOESY correlation, and H-2β has correlation with H-15, and H-15 has correlation with H-9, thus confirming that H-5, H-15 and H-9 are on the same side of the plane, which is defined as β orientation. H-4 has NOESY correlation with H-2α, so H-4 and OH-9 are on the same side of the plane, which is defined as α orientation. Further quantum chemical NMR calculations were performed using the mPW1PW91 / 6-311+G(d,p) basis set level for the two possible configurations (ab) at position 11 of the flexible side chain of this structure, and statistical analysis methods (R 2 ,DP4+probabilistic analysis), comparing the correlation coefficients and confidence probabilities between compound 8 and the two possible configurations, revealed that the compound best matched the calculated results for configuration b. The final relative configuration of compound 8 was determined to be 1R*, 4S*, 5R*, 9S*, 11S*.

[0067] The absolute configuration at position 11 of compound 8 was then determined using Snatzke's method. The complex formed by compound 8 and Mo2(OAc)4 induces a positive Cotton effect near 315 nm. Based on the empirical rule proposed by Snatzke et al., the absolute configuration at position 11 was determined to be 11S. The absolute configuration of compound 8 was further confirmed by comparing the calculated ECD with the measured ECD. The calculated configuration of compound 8 was in good agreement with the calculated 1R, 4S, 5R, 9S, and 11S configurations, and the absolute configuration was therefore determined to be 1R, 4S, 5R, 9S, and 11S.

[0068] Table 2 Compound 5-8 (a: CDCl3 and b: DMSO-d6) 1 H (600MHz) and 13 C (150 MHz) NMR data

[0069]

[0070] The obtained compounds 1-8 were evaluated for their cytotoxicity against three human cancer cell lines: HepG2 (liver cancer), A549 (lung cancer), and MCF-7 (breast cancer). The results showed that compound 1 exhibited the strongest cytotoxic activity against HepG2 and A549 cells (IC 50 The values were 4.062 μM and 6.353 μM, respectively. Therefore, the compound with anti-tumor activity in Daphne koreana of the present invention has the prospect of further development of anti-cancer drugs.

[0071] The present invention also provides a pharmaceutical composition, which is prepared by mixing the compound with anti-tumor activity or a pharmaceutically acceptable salt thereof in Daphne koreana as an active ingredient with a pharmaceutically acceptable excipient and preparing the composition into a clinically acceptable dosage form. The excipient refers to a diluent, adjuvant or carrier that can be used in the pharmaceutical field, and the dosage form is an injection, tablet or capsule.

[0072] The invention relates to the use of the compound with anti-tumor activity in Daphne koreana or its pharmaceutically acceptable salt or the pharmaceutical composition thereof in the preparation of an anti-tumor drug. The tumor is lung cancer or liver cancer.

[0073] The advantage of the present invention is that it is the first time that the compounds extracted from Daphne koreana are used in the technical field of preparing anti-tumor drugs. The compounds are all optically pure compounds with a determined stereoconfiguration. At the same time, they have strong anti-lung cancer activity and are worthy of further development. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 UV spectrum of compound 1;

[0075] Figure 2 (+)-HRESIMS of compound 1;

[0076] Figure 3 UV spectrum of compound 2;

[0077] Figure 4 (+)-HRESIMS of compound 2;

[0078] Figure 5 Single crystal image of compound 2;

[0079] Figure 6 UV spectrum of compound 3;

[0080] Figure 7 (+)-HRESIMS of compound 3;

[0081] Figure 8 Computed NMR analysis of compound 3;

[0082] Figure 9 UV spectrum of compound 4;

[0083] Figure 10 (+)-HRESIMS of compound 4;

[0084] Figure 11 Computed NMR analysis of compound 4;

[0085] Figure 12 UV spectrum of compound 5;

[0086] Figure 13 (+)-HRESIMS of compound 5;

[0087] Figure 14 Computed NMR analysis of compound 5;

[0088] Figure 15 UV spectrum of compound 6;

[0089] Figure 16 (+)-HRESIMS of compound 6;

[0090] Figure 17 UV spectrum of compound 7;

[0091] Figure 18 (+)-HRESIMS of compound 7;

[0092] Figure 19 Computational NMR analysis of compound 7;

[0093] Figure 20 UV spectrum of compound 8;

[0094] Figure 21(+)-HRESIMS of compound 8;

[0095] Figure 22 Computational NMR analysis of compound 8;

[0096] Figure 23 Key HMBC correlation and COSY correlation of compounds 1-8;

[0097] Figure 24 Key NOESY correlations for compounds 1-8;

[0098] Figure 25 Comparison of measured and calculated ECDs of compounds 1, 3–8;

[0099] Figure 26 ICD spectra of compounds 4, 5, 7, and 8;

[0100] Figure 27 The figure shows compound 1-induced apoptosis in HepG2 and A549 cells. Cell morphology changes were observed under phase contrast microscopy; nuclear morphology changes were observed by Hoechst 33324 staining; and apoptotic cells were observed by AO / EB staining. DETAILED DESCRIPTION

[0101] The following examples are intended to help those skilled in the art better understand the present invention, but are not intended to limit the present invention in any way.

[0102] Example 1

[0103] The preparation of compound 1-8 was carried out according to the following steps:

[0104] (1) 100.0 kg of dried Daphne koreana whole plant was extracted with 70% ethanol under reflux for 3 times, each time for 2 h;

[0105] (2) After vacuum concentration at 60°C, 5000 g of total extract was obtained, which was extracted with ethyl acetate and n-butanol;

[0106] (3) The extracts were concentrated to obtain 1492.3 g of extract in the ethyl acetate layer and 441 g of extract in the n-butanol layer. The samples from the two extract layers were then subjected to the next step of treatment. They were eluted with an ethanol:water gradient (20:80, 70:30, 100:0) and separated by glucose gel LH-20 column chromatography to quickly separate them into two crude fractions, Fr.A to Fr.B;

[0107] (4) Fr.A (266.3 g) to Fr.B (1305.1 g) were separated by silica gel column chromatography under reduced pressure using a gradient elution of dichloromethane:methanol (1:0-0:1) to obtain Fr.A1-Fr.A2 and Fr.B1-Fr.B4;

[0108] (5) Fr.A1 (46.8 g) was eluted with ethanol:water (20:80-100:0) gradient and separated by reverse phase HP-20 column chromatography and ODS column chromatography to obtain Fr.A1-1 to Fr.A1-4;

[0109] (6) Fr.B1 (68.7 g) was eluted with ethanol:water (20:80-100:0) gradient and separated by reverse phase HP-20 column chromatography and ODS column chromatography to obtain Fr.B1-1 to Fr.B1-4;

[0110] (7) Fr.A1-3 was separated by semi-preparative HPLC with acetonitrile:water (35:65) to obtain compound 1 (0.5 mg);

[0111] (8) Fr.B1-1 was separated by semi-preparative HPLC with acetonitrile:water (30:70-50:50) to obtain compounds 2 (3.3 mg), 3 (3.7 mg), 4 (1.8 mg), 5 (3.0 mg), 6 (1.3 mg), 7 (43.9 mg), and 8 (8.3 mg).

[0112] Example 2

[0113] The antitumor activity of compounds 1-8 against liver cancer cells HepG2, lung cancer cells A549 and breast cancer cells MCF-7 was investigated in vitro.

[0114] The cytotoxic activity of compounds 1-8 against tumor cells HepG2, A549 and MCF-7 was investigated using the MTT assay. The cells were placed in a 96-well plate and cultured with culture medium for 12 hours. HepG2, A549 and MCF-7 cells were treated with compounds at different concentrations, and sorafenib, cisplatin and doxorubicin were used as positive controls, respectively. 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 a microplate reader at a wavelength of 490 nm. The results showed (Table 3) that compound 1 exhibited good cytotoxic activity, with an IC of 0.05 for HepG2 and A549 cells. 50 The values were 4.062 μM and 6.353 μM, respectively.

[0115] In order to investigate whether compound 1 can induce apoptosis of HepG2 and A549 cells, we used Hoechst staining and AO / EB staining to conduct experiments on compound 1. HepG2 and A549 cells were placed in 24-well plates, treated with compound 1 for 48 hours, and the cell morphology changes were observed using a phase contrast microscope. After washing the cells three times with PBS, they were stained with Hoechst 33324 or AO / EB, and the morphological changes of the cells were observed using a fluorescence microscope after 15 minutes in a dark environment. The results showed that after the cells were treated with compound 1, characteristic morphological changes of cell apoptosis such as wrinkling and cell membrane blebbing appeared. Nuclear fragments and chromatin condensation appeared after staining with Hoechst. The AO / EB staining experiment found the appearance of apoptotic cells stained yellow-green. The above experimental data show that compound 1 can induce apoptosis of HepG2 and A549 cells (such as Figure 27 ).

[0116] Table 3 Cytotoxic activity IC 50 (μM)

[0117]

Claims

1. A compound with anti-tumor activity from Daphne koreana, characterized in that: It is any one of the compounds shown below: 。 2. A method for preparing the compound with anti-tumor activity from Daphne koreana according to claim 1, characterized in that: The steps include: (1) The dried whole plant of Daphne koreana was extracted with ethanol under reflux; (2) The extract is concentrated under reduced pressure to obtain an extract, and the extract is extracted with ethyl acetate and n-butanol; (3) The extract was concentrated to obtain an extract, which was separated by glucose gel LH-20 column chromatography to obtain two fractions, Fr. A to Fr. B; the gradient elution conditions of the glucose gel LH-20 column chromatography were EtOH:H2O = 20:80, 70:30, 100:0; (4) Fr. A to Fr. B were separated by silica gel vacuum column chromatography to obtain Fr. A1 to Fr. A2 and Fr. B1 to Fr. B4, respectively; the gradient elution conditions of silica gel vacuum column chromatography were CH2Cl2:MeOH = 1:0-0:1; (5) Fr. A1 was separated by reverse-phase HP-20 column chromatography and ODS column chromatography to obtain Fr. A1-1 to Fr. A1-4; the gradient elution conditions for reverse-phase HP-20 column chromatography and ODS column chromatography were EtOH:H2O = 20:80-100:0; (6) Fr. B1 was separated by reverse-phase HP-20 column chromatography and ODS column chromatography to obtain Fr. B1-1 to Fr. B1-4; the gradient elution conditions for reverse-phase HP-20 column chromatography and ODS column chromatography were EtOH:H2O = 20:80-100:0; (7) Fr. A1-3 was separated by semi-preparative HPLC to obtain compound 1; the gradient elution condition of semi-preparative HPLC was CH3CN:H2O = 35:65; (8) Fr. B1-1 was separated by semi-preparative HPLC to obtain compound 3; the gradient elution condition of semi-preparative HPLC was CH3CN:H2O = 30:70-50:

50.

3. The method for preparing the compound having anti-tumor activity from Daphne koreana according to claim 2, characterized in that: The ethanol used in step (1) is industrial ethanol, and the mass concentration of ethanol in the industrial ethanol is 70-90%; the number of extractions is 2-3 times.

4. A pharmaceutical composition, characterized in that A composition prepared by mixing the compound with anti-tumor activity in Daphne koreana of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient with a pharmaceutically acceptable excipient, and prepared into a clinically acceptable dosage form, wherein the excipient refers to a diluent, adjuvant or carrier that can be used in the pharmaceutical field, and the dosage form is an injection, tablet or capsule.

5. Use of the compound 1 or a pharmaceutically acceptable salt thereof according to claim 1, or a pharmaceutical composition comprising the compound 1, in the preparation of an anti-lung cancer or anti-liver cancer drug.

6. Use of the compound 3 or a pharmaceutically acceptable salt thereof or a pharmaceutical composition comprising the compound 3 according to claim 1 in the preparation of an anti-lung cancer drug.

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