Preparation method of diterpenoid and application thereof

Abstract

The application discloses a preparation method of diterpenoid compounds and application thereof. Ten diterpenoid compounds are separated from Euphorbia fischeriana, including six kinds of atisine type diterpenoid compounds, two kinds of gansuine type diterpenoid compounds, one kind of taxodine type diterpenoid compound and one kind of sequirine type diterpenoid compound. The diterpenoid compounds have the effect of promoting apoptosis of SW1990 pancreatic cancer cells, wherein the anticancer activity of compounds 1, 3 and 9 is better than that of positive drug paclitaxel, and the compounds can be used as leading compounds of anti-pancreatic cancer drugs, and also provide a basis and a new idea for extraction and medicinal research of the diterpenoid compounds.

Description

Technical Field

[0001] This invention belongs to the field of compound extraction technology, and relates to a method for preparing diterpenoid compounds and their applications. Background Technology

[0002] Pancreatic cancer is one of the most common highly malignant tumors of the digestive system. With changes in the environment and dietary structure, the global incidence and mortality rates of pancreatic cancer are increasing year by year, seriously threatening human life and health. Due to its location, pancreatic cancer has an insidious onset, lacks specific early symptoms, and is easily missed, often resulting in advanced stages when patients are diagnosed, making surgical treatment difficult. Therefore, conventional chemotherapy is often the first-line treatment strategy for pancreatic cancer patients. However, current drugs are not very effective in treating pancreatic cancer, leading to poor prognosis. There is an urgent need to develop new drugs for effective treatment of pancreatic cancer. Natural products play an important role in the development of anti-tumor drugs. Many natural products and their derivatives, such as paclitaxel, vincristine, etoposide, and camptothecin, have been successfully used in clinical anti-tumor treatment. Of the anti-tumor drugs marketed in the past 40 years, 45% are new drugs developed using natural products as lead compounds or with pharmacophores derived from natural products.

[0003] Big Wolfsbane ( Euphorbia jolkinii *Euphorbia pekinensis* (Boiss) is the rhizome of a plant in the genus *Euphorbia* of the family Euphorbiaceae, native to Sichuan and Yunnan provinces. In Yunnan, *Euphorbia pekinensis* is used as a traditional Chinese medicine, with the root used medicinally for its hemostatic, anti-inflammatory, and anti-swelling effects. It is also used to treat conditions such as ascites due to liver cirrhosis, pruritus, and scabies. This invention aims to isolate diterpenoid compounds with anti-pancreatic cancer activity from *Euphorbia pekinensis*, providing new pathways and ideas for the preparation of diterpenoid compounds and the development of anti-pancreatic cancer drugs. Summary of the Invention

[0004] The first objective of this invention is to provide an application of a diterpenoid compound or a pharmaceutical composition containing the diterpenoid compound, and the second objective of this invention is to provide a method for preparing the diterpenoid compound.

[0005] The first objective of this invention is achieved by using a diterpenoid compound or a pharmaceutical composition containing such a diterpenoid compound in the preparation of an anti-pancreatic cancer drug, wherein the diterpenoid compound has the structure shown in any one of the following 1 to 10:

[0006] The second objective of this invention is achieved by the method for preparing the diterpenoid compound, which comprises the following steps: 1) Extract the dried roots of *Euphorbia fischeriana* by cold soaking in 80% ethanol 3-4 times, each time for 6-8 days, to obtain an extract; 2) The extract was extracted with ethyl acetate, and the resulting extract was separated with D101 macroporous resin. The fractions Fr.1 to Fr.3 were obtained by elution with methanol / water at volume ratios of 50:50, 70:30, and 90:10. The fractions were analyzed by TLC. Then, the fraction Fr.2 containing the target compound was subjected to silica gel column chromatography and eluted with petroleum ether / ethyl acetate at volume ratios of 95:10, 80:20, 70:30, and 60:40 to obtain subfractions Fr.2-1 to Fr.2-4. 3) The subfluidic fraction Fr.2-1 was subjected to silica gel column chromatography, and eluted with petroleum ether-ethyl acetate at volume ratios of 80:20 and 70:30. Then, it was purified by Sephadex LH-20 chromatography and HPLC to obtain compound 4. 4) The subfluidic fraction Fr.2-2 was subjected to silica gel column chromatography with gradient elution using petroleum ether-acetone at volume ratios of 90:10, 70:30, and 60:40; followed by Sephadex LH-20 chromatography and HPLC purification to obtain compounds 2, 3, 5, and 8. 5) The subfluid fraction Fr.2-3 was subjected to silica gel column chromatography with gradient elution using petroleum ether-acetone at volume ratios of 90:10, 80:20 and 60:40; then it was purified by Sephadex LH-20 chromatography and HPLC to obtain compounds 1 and 9. 6) The subfluidic fraction Fr.2-4 was subjected to silica gel column chromatography with gradient elution using dichloromethane-acetone at volume ratios of 95:5, 90:10 and 80:20; then it was purified by Sephadex LH-20 chromatography and HPLC to obtain compounds 6, 7 and 10.

[0007] The beneficial effects of this invention are as follows: This invention isolates 10 diterpenoid compounds from *Euphorbia fischeriana*, including 6 atescenane-type diterpenoids, 2 euphorbia-type diterpenoids, 1 kauriane-type diterpenoid, and 1 sucralose-type diterpenoid. These diterpenoid compounds promote apoptosis in SW1990 pancreatic cancer cells. Among them, compounds 1, 3, and 9 exhibit superior anticancer activity compared to the positive control drug paclitaxel, and can serve as lead compounds for anti-pancreatic cancer drugs. This invention also provides a foundation and new ideas for the extraction and pharmaceutical research of such diterpenoid compounds. Attached Figure Description

[0008] Figure 1 Compound 1 of the present invention 1 H NMR spectrum; Figure 2 The DEPT spectrum of compound 1 of this invention is shown below. Figure 3 Compound 2 of the present invention1 H NMR spectrum; Figure 4 The DEPT spectrum of compound 2 of this invention is shown below. Figure 5 Compound 3 of the present invention 1 H NMR spectrum; Figure 6 The DEPT spectrum of compound 3 of this invention is shown below. Figure 7 Compound 4 of the present invention 1 H NMR spectrum; Figure 8 The DEPT spectrum of compound 4 of this invention is shown below. Figure 9 Compound 5 of the present invention 1 H NMR spectrum; Figure 10 The DEPT spectrum of compound 5 of this invention is shown below. Figure 11 Compound 6 of the present invention 1 H NMR spectrum; Figure 12 The DEPT spectrum of compound 6 of this invention is shown below. Figure 13 Compound 7 of the present invention 1 H NMR spectrum; Figure 14 The DEPT spectrum of compound 7 of this invention is shown below. Figure 15 Compound 8 of the present invention 1 H NMR spectrum; Figure 16 The DEPT spectrum of compound 8 of this invention; Figure 17 Compound 9 of the present invention 1 H NMR spectrum; Figure 18 The DEPT spectrum of compound 9 of this invention; Figure 19 Compound 10 of the present invention 1 H NMR spectrum; Figure 20 The DEPT spectrum of compound 10 of this invention; Figure 21 Cell viability (mean ± SEM, n = 3) after direct application of compounds 1–10 to SW1990 cells. p <0.01 vs Blank control (DMSO)). Detailed Implementation

[0009] The present invention will be further described below, but this is not intended to limit the invention in any way. Any modifications made based on the present invention are within the scope of protection of the present invention.

[0010] This invention provides the use of diterpenoid compounds or pharmaceutical compositions containing such diterpenoid compounds in the preparation of anti-pancreatic cancer drugs, wherein the diterpenoid compounds have the structures shown in any one of claims 1 to 10 below: .

[0011] The present invention also provides a pharmaceutical composition comprising at least one of the diterpenoid compounds and a pharmaceutically acceptable carrier or excipient.

[0012] The preparation method of the diterpenoid compound is carried out according to the following steps: 1) Extract the dried roots of *Euphorbia fischeriana* by cold soaking in 80% ethanol 3-4 times, each time for 6-8 days, to obtain an extract; 2) The extract was extracted with ethyl acetate, and the resulting extract was separated with D101 macroporous resin. The fractions Fr.1 to Fr.3 were obtained by elution with methanol / water at volume ratios of 50:50, 70:30, and 90:10. The fractions were analyzed by TLC. Then, the fraction Fr.2 containing the target compound was subjected to silica gel column chromatography and eluted with petroleum ether / ethyl acetate at volume ratios of 95:10, 80:20, 70:30, and 60:40 to obtain subfractions Fr.2-1 to Fr.2-4. 3) The subfluidic fraction Fr.2-1 was subjected to silica gel column chromatography, and eluted with petroleum ether-ethyl acetate at volume ratios of 80:20 and 70:30. Then, it was purified by Sephadex LH-20 chromatography and HPLC to obtain compound 4. 4) The subfluidic fraction Fr.2-2 was subjected to silica gel column chromatography with gradient elution using petroleum ether-acetone at volume ratios of 90:10, 70:30, and 60:40; followed by Sephadex LH-20 chromatography and HPLC purification to obtain compounds 2, 3, 5, and 8. 5) The subfluid fraction Fr.2-3 was subjected to silica gel column chromatography with gradient elution using petroleum ether-acetone at volume ratios of 90:10, 80:20 and 60:40; then it was purified by Sephadex LH-20 chromatography and HPLC to obtain compounds 1 and 9. 6) The subfluidic fraction Fr.2-4 was subjected to silica gel column chromatography with gradient elution using dichloromethane-acetone at volume ratios of 95:5, 90:10 and 80:20; then it was purified by Sephadex LH-20 chromatography and HPLC to obtain compounds 6, 7 and 10.

[0013] In each step, HPLC purification was performed using Rp-C 18 Chromatographic column.

[0014] In step 3), HPLC purification was performed using acetonitrile / water at a volume ratio of 75:25 for isocratic elution.

[0015] In step 4), HPLC purification was performed using acetonitrile / water at a volume ratio of 60:40 for isocratic elution.

[0016] In step 5), HPLC purification was performed using acetonitrile / water at a volume ratio of 50:50 for isocratic elution.

[0017] In step 6), HPLC purification was performed using acetonitrile / water at a volume ratio of 50:50 for isocratic elution.

[0018] Methanol was used as the eluent in each step of the Sephadex LH-20 chromatography process. Example 1: Preparation of compounds 1-10 Take 20.0 kg of dried roots of Euphorbia fischeriana, crush them, and extract them three times with 80% ethanol, each extraction lasting one week. Combine the ethanol extracts, and recover the ethanol under reduced pressure to obtain the extract. The extract was dispersed in water and then extracted with ethyl acetate. The ethyl acetate extract fraction was concentrated. The fraction was then passed through a D101 macroporous resin with methanol-water (50:50, 70:30, 90:10). v / v The eluent gradient yielded three fractions, denoted as Fr.1 to Fr.3. The three fractions were analyzed by TLC. Based on the results, fraction Fr.2 was eluted by silica gel column chromatography (eluent was petroleum ether-ethyl acetate, with volume ratios of petroleum ether and ethyl acetate of 95:10, 80:20, 70:30 and 60:40, respectively) to obtain four subfractions, which were designated as Fr.2-1 to Fr.2-4. The four subfractions were analyzed by TLC. Based on the results, subfraction Fr.2-1 was first subjected to silica gel column chromatography with petroleum ether-ethyl acetate (80:20 and 70:30). v / v Gradient elution, followed by Sephadex LH-20 (methanol elution), thin-layer chromatography (petroleum ether-ethyl acetate (50:50)), and HPLC purification, using Rp-C 18 The column was eluted isocratically with acetonitrile-water (75:25 volume ratio of acetonitrile to water) to give compound 4; Subfluid fraction Fr.2-2 was first subjected to silica gel column chromatography with petroleum ether-acetone (90:10, 70:30, and 60:40). v / v Gradient elution followed by Sephadex LH-20 column chromatography (methanol elution), then HPLC purification, using Rp-C 18 The column was eluted isocratically with acetonitrile-water (60:40 volume ratio of acetonitrile to water) to give compounds 2, 3, 5, and 8. Subfluid fraction Fr.2-3 was first subjected to silica gel column chromatography with petroleum ether-acetone (90:10, 80:20, and 60:40). v / v Gradient elution was performed, followed by Sephadex LH-20 column chromatography (methanol elution), and then HPLC purification was carried out using an Rp-C18 column with isocratic elution using acetonitrile-water (acetonitrile and water volume ratio of 50:50) to obtain compounds 1 and 9. The subfluid fraction Fr.2-4 was first subjected to silica gel column chromatography with dichloromethane-acetone (95:5, 90:10, and 80:20). v / v Gradient elution was performed, followed by Sephadex LH-20 column chromatography (methanol elution), and then HPLC purification was carried out using an Rp-C18 column with isocratic elution using acetonitrile-water (acetonitrile and water volume ratio of 50:50) to obtain compounds 6, 7, and 10.

[0019] The structures of compounds 1–10 were characterized, and the instrument information and NMR data of the compounds obtained are as follows: HRESI-MS was performed using an Agilent LC-MS-Q-TOF G6530 (Agilent Technologies, Santa Clara, USA) and a Shimadzu LC-MS-IT-TOF (Shimadzu, Kyoto, Japan); 1D NMR and 2D NMR were performed using an Avance III HD 400, AV 600 (Bruker, Bremerhaven, Germany) NMR spectrometer (TMS as internal standard); the semi-preparative high-performance liquid chromatograph was an LC-52 series product from Beijing Cypress Technology Co., Ltd., and the column model used was Basic C18 (5 μm, 10 × 250 mm); MCI CHP-20P GEL was purchased from Mitsubishi Chemical Corporation, Japan; Sephadex LH-20 was purchased from Pharmacia; commonly used organic reagents were domestic AR-grade reagents purchased from Yunnan Liyan Technology Co., Ltd.; thin-layer chromatography silica gel plates and column chromatography silica gel were purchased from Shanghai Haohong Biomedical Technology Co., Ltd.

[0020] The NMR data for compounds 1–10 are as follows: Compound 1: ent -(5 β 8 α 9 β 10 α ,11 α ,12 α 11-hydroxyatis-16-one-3,14-dione: White needle-like crystals, molecular formula C 20 H 28 O3ESI-MS m / z. 317.2109 [M+H] + (Calculated value: 317.2111); 1 H NMR (600 MHz, CDCl3): δ H 0.85-0.90 (1H, m, H-7), 1.02 (3H, s , 4 β -Me), 1.06 (3H, s , 4 α - Me ), 1.17 (3H, s, 10 β -Me), 1.26 (1H, dd , J = 1.6 / 12.4 Hz, H-5), 1.42-1.48 (1H, m ,H-6), 1.66-1.70 (1H, m , H-6′), 1.76 (1H, d , J = 10.0 Hz, H-9), 2.11-2.17 (1H, m ,H-15), 2.17-2.21 (1H, m , H-13), 2.28-2.32 (1H, m , H-2), 2.31-2.34 (1H, m , H-15′), 2.32-2.37 (1H, d , H-7), 2.56-2.59 (1H, m , H-1), 2.60-2.62 (1H, m , H-2′), 2.65-2.67 (1H, m , H-12), 2.69-2.71 (1H, m , H-1′), 2.93 (1H, dd , J= 1.6 / 14.2 Hz,H-13′), 4.52 (1H, dd , J = 4.0 / 10.0 Hz, H-11′), 4.52 (1H, br . s , H-17), 4.78 (1H, br . s (, H-17′); 13 C NMR (150 MHz, CDCl3): δ C 38.6 ( t , C-1), 34.5 ( t , C-2), 217.5( s , C-3), 47.9 ( s , C-4), 56.7 ( d , C-5), 20.3 ( t , C-6), 31.0 ( t , C-7), 48.8( s , C-8), 57.2 ( d , C-9), 39.0 ( s , C-10), 70.6 ( d , C-11), 48.4 ( d , C-12), 38.3 ( t , C-13), 216.5 ( s (C-14), 43.0 ( t , C-15), 144.3 ( s (, C-16), 109.5 ( t (C-17), 26.5 ( q , C-18), 21.9 ( q , C-19), 15.6 ( q (C-20) For example Figure 1-2 As shown.

[0021] Compound 2: ent -(3 α 5 β 8 α 9 β 10 α ,11 α ,12 α )-3,11-dihydroxyatis-16-en-14-one: White needle-like crystals, molecular formula C20 H 30 O3. 1 H NMR (600 MHz, CDCl3): δ H 0.70-0.78 (1H, m, H-5),0.76 (3H, s , 10 β -Me), 0.79-0.86 (1H, m , H-7), 0.95 (3H, s , 4 α - Me ), 0.98(3H, s , 4 β -Me), 1.18-1.20 (1H, m , H-1), 1.49-1.51 (1H, m , H-6), 1.50-1.52 (1H, m , H-6),1.53-1.56 (1H, m , H-2), 1.54-1.57 (1H, m , H-2′), 1.64 (1H, d , J =9.6Hz, H-9),2.07-2.11 (1H, m , H-15), 2.12-2.13 (1H, m , H-13), 2.28-2.31 (1H, m , H-15′),2.29-2.32 (1H, m , H-7), 2.44-2.46 (1H, m , H-1′), 2.59-2.61 (1H, m , H-12), 2.90(1H, dd , J = 2.0 / 18.8 Hz, H-13), 3.20 (1H, dd , J = 3.6 / 11.2 Hz, H-11), 4.63-1.65(1H, m, H-11′), 4.72(1H, br . s , H-17), 4.94 (1H, br . s , H-17′); 13 C NMR (150 MHz,CDCl3): δ C 38.1 (t, C-1), 23.7 (t, C-2), 80.9 (d, C-3), 38.1 (s, C-4), 55.9 (d,C-5), 19.1 (t, C-6), 31.4 (t, C-7), 48.9 (s, C-8), 58.2 (d, C-9), 39.2 (s, C-10), 71.0 (d, C-11), 48.4 (d, C-12), 38.0 (t, C-13), 212.2 (s, C-14), 43.3 (t, C-15), 144.6 (s, C-16), 109.4 (t, C-17), 28.7 (q, C-18), 16.8 (q, C-19), 16.2(q, C-20). For example... Figure 3-4 As shown.

[0022] Compound 3: ent -16,17dihydroxyatisan-3-one: White needle-like crystals, molecular formula C 20 H 32 O3ESI-MS m / z 343.2244 [M+Na] + (Calculated value: 343.2244); 1 H NMR (600 MHz, CDCl3): δ H 0.82 (1H, m ,H-14), 1.04 (3H, s , 4 β -Me), 1.07 (3H, s , 4 α -Me), 1.10 (1H, m , H-15), 1.11 (3H, s 10 β -Me), 1.18 (1H, m H-7 α ), 1.24 (2H, m , H-11 / H-15′), 1.30 (1H, m , H-5), 1.32(1H, m , H-9), 1.38 (1H, m H-1 α ), 1.42 (1H, m, H-7 β ), 1.45 (2H, m , H-6 α / H-6 β ),1.50 (1H, m , H-13),1.63 (1H, m , H-13′), 1.82 (1H, qn , J =3.0, H-12), 1.85 (1H, m ,H-1 β ), 1.87 (1H, m , H-14′), 2.03 (1H, m , H-11′), 2.33 (1H, ddd , J = 3.2 / 6.0 / 16.0Hz, H-2 α ), 2.58 (1H, ddd , J = 6.9 / 12.3 / 16.0 Hz, H-2 β ), 3.43-3.57 (2H, Abq , J = 10.9Hz, H-17 / H-17′); 13 C NMR (150 MHz, CDCl3): δ C 38.0 ( t , C-1), 34.1 ( t ,C-2), 217.8( s , C-3), 47.7 ( s , C-4), 55.7 ( d , C-5), 19.7 ( t , C-6), 38.8 ( t , C-7), 37.2( s , C-8), 50.9 ( d , C-9), 32.9 ( s , C-10), 23.0 ( t , C-11), 32.1 ( d , C-12), 23.3( t , C-13), 27.4 ( t , C-14), 52.4 ( t , C-15), 74.2 ( s, C-16), 68.9 ( t , C-17), 26.2 ( q , C-18), 21.7 ( q , C-19), 13.5 ( q (C-20). Figure 5-6 As shown.

[0023] Compound 4: ent -(3 α 5 β 8 α 9 β 10 α ,12 α )-3-hydroxyatis-16-en-14-one: White needle-like crystals, molecular formula C 20 H 30 O2ESI-MS m / z 303.2319[M+H] + (Calculated value: 303.2319); 1H NMR (600 MHz, CDCl3): δ H 0.67 (3H, s , 10 β -Me), 0.75 (3H, s , 4 β -Me), 0.79 (1H, dd , J = 3.6 / 10.8 Hz,H-5), 0.88-0.91 (1H, m , H-7), 0.97 (3H, s , 4 α -Me), 1.03-1.06 (1H, m , H-1), 1.47-1.49 (1H, m , H-2), 1.49-1.51 (1H, m , H-1), 1.55-1.58 (1H, m , H-6), 1.57-1.59(1H, m , H-6), 1.59 (1H, d , H-9), 1.60-1.64 (1H, m , H-2), 1.60-1.62 (1H, m , H-11), 1.80-1.84 (1H, m, H-11), 2.13-2.16 (1H, m , H-15), 2.19-2.22 (1H, m , H-15), 2.25-2.26 (1H, m , H-13), 2.28-2.32 (1H, m , H-7), 2.67-2.69 (1H, m , H-12), 3.19 (1H, dd , J = 3.6 / 11.2 Hz, H-3), 4.65 (1H, br.s , H-17), 4.85 (1H, br.s , H-17′); 13C NMR (150 MHz, CDCl3): δ C 36.8 ( t , C-1), 26.9 ( t , C-2), 79.0 ( d , C-3), 38.8 ( s , C-4), 54.7 ( d (C-5), 19.0 ( t , C-6), 31.6 ( t ,C-7), 47.9 ( s , C-8), 52.7 ( d , C-9), 37.9( s , C-10), 27.9 ( t , C-11), 38.5 ( d , C-12), 44.6 ( t , C-13), 216.9 ( s , C-14), 42.9 ( t , C-15), 147.6 ( s , C-16), 106.9 ( t , C-17), 28.4 ( q , C-18), 15.6 ( q , C-19), 13.3 ( q (C-20). Figure 7-8 As shown.

[0024] Compound 5:16 β ,17-dihydroxy- ent-atisan-1-ene-3-one: White solid, molecular formula: C 20 H 30 O3, HR-ESI-MS [M+H] + m / z 319.2268 (Calculated value: 319.2268); 1 H NMR (600 MHz, CDCl3): δ H 0.89-0.84 (1H, m H-14 α ), 1.08 (3H, s , 4 β -Me), 1.12 (3H, s , 4 α -Me), 1.23 (3H, s , 10 β -Me), 1.25 (2H, s , H-15 / 15′), 1.26-1.24 (1H, m , 7 β -Me), 1.42-1.39 (1H, m H-11 β ), 1.50 (2H, o , H-6 / 6′), 1.52-1.49 (2H, m H-13 β / H-9), 1.60-1.57 (1H, m H-13 α ), 1.65-1.61 (1H, m , H-5), 1.88 (1H, o H-12 α ), 1.89-1.87 (1H, m H-14 α ),3.45 (1H, d , J = 11.4 Hz, H-17 β ), 3.59 (1H, d , J = 11.4 Hz, H-17 α ), 5.82 (1H, d , J =10.1 Hz, H-2), 6.93 (1H, d , J= 10.1 Hz, H-1); 13 C NMR (150 MHz, CDCl3): δ C 158.7 ( d , C-1), 125.0 ( d , C-2), 205.3 ( s , C-3), 44.6 ( s , C-4), 53.6 ( d , C-5), 19.0( t , C-6), 38.7 ( t , C-7), 33.5( s ,C-8), 45.9 ( d , C-9), 40.1 ( s , C-10), 23.0 ( t ,C-11), 32.0 ( d , C-12), 23.1 ( t , C-13), 27.6 ( t , C-14), 52.3 ( t , C-15), 74.0 ( s , C-16), 68.8 ( t, C-17), 27.5 ( q , C-18), 21.6 ( q , C-19), 17.5 ( q (C-20). Figure 9-10 As shown.

[0025] Compound 6: ent -16 α -hydroxy-17-acetoxyatisan-3-one: White needle-like crystals, molecular formula C 22 H 34 O4ESI-MS m / z. 397.2151[M+Cl] − (Calculated value: 397.2198); 1 H NMR (600 MHz, CDCl3): δ H 0.84 (lH, m , H-14), 1.04 (3H, s , 4 β -Me), 1.08 (3H, s , 4α -Me), 1.11 (3H, s , 10 β -Me), 1.18 (1H, m , H-15), 1.20 (lH, m , H-7 α ), 1.22 (lH, m , H-11), 1.23 (lH, m , H-15), 1.30 (lH, dd , J = 2.8 / 11.2 Hz, H-5), 1.34 (lH, dd , J = 6.0 / 12.8 Hz, H-9), 1.38(lH, m , H-l α ), 1.40 (lH, m , H-7 β ), 1.45 (2H, m , H-6 α / H-6 β ), 1.48 (1H, m , H-13),1.59 (1H, m , H-13), 1.77 (1H, qn , J = 3.0 Hz, H-12), 1.83 (lH, ddd , J = 3.2 / 6.9 / 13.5Hz, H-l β ), 1.88 (lH, dddd , J = 3.1 / 3.2 / 11.4 / 14.0 Hz, H-l β ), 2.04 (1H, m , H-11),2.10 (3H, s , OAc), 2.33 (1H, ddd , J = 4.2 / 6.1 / 16.0 Hz, H-2 α ),2,56 (lH, ddd , J = 6.9 / 12.3 / 16.0 Hz, H-2 β ), 3.95-4.10 (2H, ABq , J = 11.4 Hz, H-17 / H-17′);13 C NMR (150MHz, CDCl3): δ C 38.0 ( t , C-1), 34.1 ( t , C-2), 217.3 ( s , C-3), 47.6 ( s , C-4), 55.6 ( d , C-5), 19.6 ( t , C-6), 38.7 ( t , C-7), 32.7 ( s , C-8), 50.6 ( d , C-9), 37.2 ( s , C-10), 22.9 ( t , C-11), 32.8 ( d , C-12), 23.2 ( t , C-13), 27.1 ( t , C-14), 52.2 ( t , C-15), 72.7 ( s , C-16), 70.3 ( t , C-17), 26.2 ( q , C-18), 21.6 ( q ,C-19), 13.5 ( q , C-20), 171.2 ( s , C-21), 21.0 ( q (C-22). For example Figure 11-12 As shown.

[0026] Compound 7: Euphoractine A: White needle-like crystals, molecular formula C 29 H 38 O6ESI-MS m / z. 517.2363[M+Cl] − (Calculated value: 517.2362); 1 H NMR (600 MHz, CDCl3): δ H 0.76 (3H, s , 6 α -Me), 0.81(3H, s , 12 α -Me), 0.99 (3H, s, 12 β -Me), 1.08(1H, d , J = 7.4 Hz, H-16), 1.13 (1H, m ,H-9), 1.16 (3H, s , 13 α -Me) 1.20 (1H, m , H-7), 1.50 (1H, m , H-8), 2.01 (1H, m , H-1), 2.07 (1H, m , H-4), 2.22 (1H, m , H-7), 2.51 (1H, m , H-2), 2.55 (1H, d , H-12),2.61 (1H, m , H-1), 3.42 (1H, d , J = 8.7 Hz, H-11), 4.46 (1H, t , J = 5.8 Hz, H-3),4.97(1H, t , J = 11.5 Hz, H-5), Cinnamoyl 6.60 (1H, d , J = 15.8Hz, H-3′), 7.30 (1H, m , H-7′), 7.36 (1H, m , H-5′ / H-9′), 7.53 (1H, m , H-6′ / H-8′), 7.76 (1H, d , J = 15.8Hz, H-3′); 13 C NMR (150 MHz, CDCl3): δ C 38.9 ( t , C-1), 35.3 ( d , C-2), 75.4 ( d , C-3), 55.4 ( d , C-4), 63.4 ( d , C-5), 47.5 ( s , C-6), 32.7 (t , C-7), 22.0 ( t, C-8), 39.5 ( d , C-9), 41.7 ( s , C-10), 73.6 ( d , C-11), 48.8 ( d , C-12), 56.8 ( s , C-13), 205.2 ( s , C-14), 94.2 ( s , C-15), 16.4 ( q (C-16), 18.0 ( q , C-17), 15.2( q (C-18), 28.0 ( q , C-19), 13.7 ( q (C-20), Cinnamoyl 166.9 ( s , C-1′), 147.4( d , C-2′), 117.8 ( d , C-3′), 134.1 ( s , C-4′), 129.0 ( d , C-5′ / C-9′), 128.5 ( d ,C-6′ / C-8′), 130.9 ( d (, C-7′). For example Figure 13-14 As shown.

[0027] Compound 8: cis -euphactins A: White solid, molecular formula: C 29 H 38 O6, HR-ESI-MS [M+H] − m / z 483.2791 (Calculated value: 483.2741). 1 H-NMR (600 MHz, CDCl3): δ H 7.66 (1H, d , J =7.7 Hz, H-9′), 7.53-7.51 (1H, m , H-5′), 7.42-7.39 (1H, m , H-6), 7.34-7.33 (2H,m , H-7′ / 8′), 7.04 (1H, d , J = 12.7 Hz, H-3′), 6.16 (1H, d , J = 16.5 Hz, H-12), 5.91 (1H, d , J = 12.7 Hz, H-2′), 5.38 (1H, d , J = 16.6 Hz, H-11), 4.87 (1H, d , J =11.6 Hz, H-5),4.37 (1H, br.s , H-3), 3.29 (1H, t , J = 9.6 Hz, H-9), 2.82 (1H, dd , J =15.4 / 11.4 Hz,H-1 α ), 2.48-2.43 (1H, m , H-2), 1.99 (1H, dd , J = 11.6 / 5.0 Hz, H-4), 1.81-1.77(2H, m , H-7 α / 8 α ), 1.72 (1H, dd , J = 15.5 / 4.0 Hz, H-1 β ), 1.59 (1H, o , H-8 β ), 1.16(3H, s , H-18), 1.15 (3H, s , H-16), 1.13 (3H, s , H-20), 1.09 (1H, d , J = 7.4 Hz, H-7 β ), 0.81 (3H, s, H-19), 0.69 (3H, s , H-17); 13 C-NMR (150 MHz, CDCl3): δ C 39.1 ( t,C-1), 35.7 ( d , C-2), 75.8 ( d , C-3), 55.0 ( d , C-4), 64.7 ( d , C-5), 43.1 ( s , C-6), 33.6 ( t , C-7), 29.2 ( t , C-8), 84.0 ( d , C-9), 53.4 ( s , C-10), 136.6 ( d , C-11), 130.0 ( d (, C-12), 62.0 ( s , C-13), 203.0 ( s , C-14), 94.2 ( s , C-15), 15.6( q , C-16), 17.7 ( q (C-17), 26.5 ( q , C-18), 16.1 ( q , C-19), 14.1 ( q (C-20), 163.7 ( s , C-1′), 116.6 ( d , C-2′), 147.8 ( d , C-3′), 136.6 ( s , C-4′), 128.3 ( d ,C-5′ / 9′), 130.2 ( d , C-6′ / 8′), 130.6 ( d (, C-7′). For example Figure 15-16 As shown.

[0028] Compound 9:3 α ,12 α -dihydroxy- ent -beyerane-15-ene-2-one: White solid. Molecular formula: C 20 H 30 O3, HR-ESI-MS [M+H] + m / z 319.2180 (Calculated value: 319.2268). 1H-NMR (150 MHz, CDCl3): δ H 5.77 (1H, d , J =5.7, H-15), 5.62 (1H, d , J =5.7 Hz, H-16), 3.89 (1H, d , J = 5.7 Hz,H-3), 3.50 (1H, dd , J = 9.2 / 6.2 Hz, H-12), 3.41 (1H, d , J = 4.8 Hz, 3 β -OH), 2.44(1H, d , J = 12.3 Hz, H-11), 2.12 (1H, d , J = 12.2 Hz, H-1 β ), 1.18 (3H, s , 4 α -Me),1.12 (3H, s , 10 β -Me), 0.73 (3H, s , 13-Me), 0.69 (3H, s, 4 β -Me); 13 C NMR (150MHz,CDCl3): δ C 51.5 ( t , C-1), 210.7 ( s , C-2), 82.7 ( d , C-3), 45.7 ( s , C-4), 52.0 ( d ,C-5), 19.9 ( t , C-6), 35.8 ( t , C-7), 48.5 ( s , C-8), 54.1 ( d , C-9), 43.6 ( s , C-10), 30.4 ( t , C-11), 73.9 ( d , C-12), 48.7 (s , C-13), 58.2 ( t , C-14), 136.4 ( d , C-15), 134.7 ( t , C-16), 20.5 (t, C-17), 29.4 (q, C-18), 16.6 (q, C-19), 15.7 (q, C-20). like Figure 17-18 As shown.

[0029] Compound 10:3 β 5 α ,20-trihydroxy-15 β -cinnamoyloxy-14-oxolathyra-6 Z ,12 E -diene, white needle-like crystals, molecular formula C 29 H 36 O6ESI-MS m / z. 481.2588 [M+H] + (Calculated value: 481.2585); 1 HNMR (600MHz, CDCl3): δ H 1.06 (3H, s , H-19), 1.07 (1H, d , J =6.7Hz, H-16), 1.09 (3H, s , H-18), 1.42 (1H, ddd , J = 4.2 / 7.9 / 12.5 Hz, H-9), 1.66 (1H, dd , J = 7.9 / 11.8 Hz,H-11), 1.70 (3H, t , J = 1.3 Hz, H-17), 1.75 (1H, t , J = 13.0 Hz, H-1 β ), 2.0 (1H, m ,H-2), 2.17 (1H, dd , J = 3.6 / 5.4 Hz, H-4), 2.34 (1H, m H-8 α ), 2.52 (1H, m, H-8 β ),3.29 (1H, dd , J = 7.1 / 13.0 Hz, H-1 α ), 4.23-4.27 (2H, q , J = 11.7 Hz), 4.35 (1H, t , J =3.6 Hz, H-3), 5.28 (1H, dd , J = 3.3 / 12.3 Hz, H-7), 5.54 (1H, d , J = 5.4 Hz, H-5),6.66 (1H, d , J = 16.0 Hz, H-2′), 6.79 (1H, d , J = 11.8 Hz, H-12), 7.43 (3H, m , H-6′ / 7′ / 8′), 7.61 (2H, m , H-5′ / 9′), 7.72 (1H, d , J = 16.0 Hz, H-3′); 13 C NMR (150 MHz,CDCl3): δ C 47.1 ( t , C-1), 37.8 ( d , C-2), 79.7 ( d , C-3), 54.8 ( d , C-4), 65.6 ( d ,C-5), 135.8 ( s , C-6), 127.2 ( d , C-7), 23.8 ( t , C-8), 33.4 ( d , C-9), 27.2 ( s ,C-10), 27.8 ( d , C-11), 147.2 ( d , C-12), 135.4 ( s , C-13), 199.3 ( s , C-14),92.5 ( s, C-15), 13.5 ( q , C-16), 16.9 ( q , C-17), 27.2( q , C-18), 16.9 ( q , C-19), 55.9 ( q (C-20), 166.0 ( s , C-1′), 117.2 ( d , C-2′), 146.8 ( d , C-3′), 134.0( s , C-4′), 128.3 ( d , C-5′), 129.1 ( d , C-6′), 130.9 ( d , C-7′), 129.1 ( d , C-8′), 128.3 ( d (, C-9′). For example Figures 19-20 As shown.

[0030] Application Examples 1-6 In the following application examples, conventional reagents are selected and formulations are prepared according to existing conventional methods. These application examples only demonstrate that at least one of compounds 1 to 10 described in this invention can be prepared into different formulations, and no specific limitations are made on the specific reagents and operations: Application Example 1 At least one of compounds 1 to 10 prepared in Example 1 was made into an oral liquid using conventional oral liquid preparation methods.

[0031] Application Example 2 At least one of compounds 1 to 10 prepared in Example 1 is dissolved in DMSO, and then water for injection is added according to conventional methods. The solution is then filtered, filled, and sterilized to prepare an injection solution with a concentration of 1.0 to 5.0 mg / mL.

[0032] Application Example 3 At least one of compounds 1 to 10 prepared in Example 1 was dissolved in DMSO, then dissolved in sterile water for injection, stirred until dissolved, filtered through a sterile vacuum funnel, then sterilely filtered, dispensed into ampoules, freeze-dried at low temperature, and then sterilely sealed to obtain a powder for injection.

[0033] Application Example 4 At least one of compounds 1 to 10 prepared in Example 1 was added to the excipient at a mass ratio of 8:2 to the excipient to prepare granules.

[0034] Application Example 5 At least one of compounds 1 to 10 prepared in Example 1 was added to the excipient at a mass ratio of 8:2, and then granulated and compressed into tablets.

[0035] Application Example 6 At least one of compounds 1 to 10 prepared in Example 1 was added to an excipient at a mass ratio of 8:2 to the excipient to form capsules.

[0036] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

[0037] Detection of the anti-pancreatic cancer SW1990 cell activity of compounds 1-10 1. Materials and Instruments 1) Materials: SW1990 cells were purchased from Wuhan Pronosai Co., Ltd.

[0038] 2) Instruments: Multi-functional microplate reader (Thermo Scientific Multiskan FC, USA); CO2 incubator (Thermo Forma 3310, USA).

[0039] 2. Detection Method The cytotoxicity of the compound was detected using the CCK-8 assay. SW1990 cells were cultured in a 37 ℃, 5% CO2 incubator in a medium containing 89% DMEM (basic 1×), 10% fetal bovine serum, and 1% penicillin-dextrose antibody. When the cell density on the culture dish reached 85%, the cells were digested with trypsin, centrifuged, and diluted to 5×10⁶ cells / mL. 5 Cells / mL were seeded into 96-well cell culture plates, 100 μL per well. After 24 h, the following groups were administered: blank group (0.1% DMSO to eliminate the influence of DMSO solvent), positive control group (paclitaxel 40 μM), and compound-administered groups (2 μM, 10 μM, 50 μM).

[0040] Control group: Normally cultured SW1990 cells (0.1% DMSO solvent); Positive control group: treated with paclitaxel 40 μM for 24 h; Drug group: treated with different concentrations (2 μM, 10 μM and 50 μM) of drug for 24 h; 24 h after drug administration, the cell culture medium was replaced with DMEM containing 10% CCK-8 in each well. After 2 hours of incubation, the absorbance of each well was read at 450 nm using a microplate reader, and the cell viability and IC50 of each group were calculated. 50 Values. The experiment was repeated 3 times. Cell viability results are as follows. Figure 21 Data are presented as mean ± SEM. Static analysis was performed using Prism 8.0 software. One-way analysis was used to analyze the data. p <0.05 is considered statistically significant), IC 50 The results are shown in Table 1.

[0041] Formula for calculating cell viability: Cell viability (%) = [A (加药) -A (空白) ] / [ A (对照) -A (空白) ]×100%.

[0042] Results analysis: From Figure 21 It was found that compounds 1-10 all possessed certain apoptosis-inducing activity in pancreatic cancer SW1990 cells. Among them, compounds 1, 3, and 9 exhibited strong anti-pancreatic cancer cell activity, with an IC50 value of [missing value]. 50 The values ​​were 26.50±6.36, 21.07±5.98, and 44.97±9.16, respectively. μ M is superior to the positive control drug paclitaxel, as shown in Table 1.

[0043] Table 1 IC50 of compounds 1-10 50 Calculation results Note: a Data are expressed as mean ± SD (n = 3).

Claims

1. The use of a diterpenoid compound or a pharmaceutical composition containing such diterpenoid compound in the preparation of an anti-pancreatic cancer drug, characterized in that, The diterpenoid compound is compound 1 with the following structural formula: 。 2. The application according to claim 1, characterized in that, The pharmaceutical composition comprises the diterpenoid compound of claim 1 and a pharmaceutically acceptable excipient.

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