Diterpenoids with a (9β-H)-pimarane nucleus having anti-colorectal cancer activity, their derivatives and preparation methods

Abstract

The present invention discloses a diterpenoid compound and its derivatives with a (9β-H)-pimarane nucleus having anti-colorectal cancer activity, and also discloses a preparation method and use of the above-mentioned compound. The present invention discovers that the new skeleton compounds 1 and 2 have a certain inhibitory effect on colorectal cancer cells. Among them, compound 2 has significant anti-proliferative effects on two colorectal cancer HT-29 and SW620 cell lines, and its effects are stronger than those of the positive drug 5-fluorouracil, and it has the potential to be developed into a new anti-colorectal cancer drug.

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and particularly relates to a derivative of (9β-H)-pimarane nucleus with anti-colon cancer activity, and a preparation method and application thereof. Background Art

[0002] Colorectal cancer is the third most common malignant tumor, second only to lung cancer and gastric cancer. Its incidence rate ranks the third among male malignant tumors and the second among female malignant tumors, and the incidence rate of colon cancer has increased particularly significantly. Colon cancer is one of the most prominent health killers in the world, and it is characterized by high incidence, easy recurrence, poor prognosis, and high mortality.

[0003] The common clinical treatment methods for colon cancer mainly include surgical resection, radiotherapy, chemotherapy, immunotherapy, molecular targeted therapy, etc. Surgical resection is mainly applicable to tumor patients with early limited metastasis, such as stage I colon cancer, while stage II and III colon cancer are treated with a combination of surgery and chemotherapy, and the treatment of advanced colon cancer mainly relies on chemotherapy. It can be seen that chemotherapy occupies an important position in the treatment of colon cancer at all stages and has become the main treatment method for colon cancer. Fluorouracil (5-fluorouracil, 5-FU), Oxaliplatin, and Irinotecan are commonly used chemotherapy drugs for the clinical treatment of colon cancer. Among them, 5-FU, as the basic drug for colon cancer, has been an important chemotherapy drug for more than 40 years. The drug has significant curative effects and a relatively broad anti-cancer spectrum. However, 5-FU will cause strong toxic and side effects to patients and irreversible drug resistance. In view of the strong toxic and side effects of colon cancer chemotherapy drugs such as 5-FU, which affect the quality of life of patients, it is urgent to research and discover new anti-cancer drugs with good curative effects and low toxic and side effects. Therefore, it is of great significance to isolate and screen non-toxic or low-toxic natural anti-tumor active ingredients from plants.

[0004] Icacina oliviformis (Poir.) J.Raynal is a perennial drought-resistant shrub of the genus Iodes in the family Icacinaceae, mainly distributed in the central and western regions of Africa. The tubers of I. oliviformis are rich in starch and can also be used as food to relieve hunger for the people during famine. In addition, the plant also has rich medicinal value. The local people use its tubers as medicine for the treatment of various diseases, such as poisoning, constipation, malaria, etc.; the alcohol extract of its tubers is also used by the local people as a first-aid drug for dealing with some emergencies such as food poisoning. Modern pharmacological studies have shown that I. oliviformis has various activities such as anti-tumor, anti-convulsant, sedative, analgesic, hypoglycemic, antibacterial, and seed germination inhibition. At present, the research on I. oliviformis mostly focuses on its tuber part, and the chemical composition research of its leaves is still lacking. In addition, compared with the tuber part, the leaves of the plant have strong regeneration ability and rich sources, and are an environmentally friendly natural resource. Summary of the Invention

[0005] The object of the present invention is to conduct in-depth research on the active ingredients of the leaves of I. oliviformis. Two new skeleton compounds of the 3,4-seco-17-nor-pimarane type were isolated from the dried leaves of the plant I. oliviformis and named secoicacinlivitholide F (1) and secoicacinlivitholide G (2). Another object of the present invention is to screen out a preparation method for this type of new skeleton compound.

[0006] Through activity evaluation, the present invention found that the compounds secoicacinlivitholide F and secoicacinlivitholide G have certain activities in inhibiting the proliferation of colon cancer cells. In particular, the compound secoicacinlivitholide G has significant anti-proliferation effects on two colon cancer cell lines, HT-29 and SW620, and its effects are stronger than those of the positive drug 5-FU. In addition, the inhibitory effect of the compound secoicacinlivitholide G on the colon cancer cell line HT-29 is about 3 times that of the positive drug 5-FU, and it can be used for the preparation of anti-colon cancer drugs or as a lead compound for the development of anti-colon cancer drugs.

[0007] The present invention provides the following technical solution: A derivative of the (9β-H)-pimarane nucleus with anti-colon cancer activity, which includes a compound with the following structural general formula:

[0008]

[0009] Wherein, R 1 and R 2Each may be hydrogen, acyl, glycosyl, alkyl, cycloalkyl, alkylaryl, aryl, arylalkyl, arylalkenyl, arylalkynyl, or heterocyclic group.

[0010] As a preferred embodiment, the above-mentioned (9β-H)-pimarane diterpene is characterized in that R 1 and R 2 are hydrogen or alkyl.

[0011] As a more preferred embodiment, R 1 is hydrogen or methyl, R 2 is hydrogen, and it is selected from the following compounds:

[0012]

[0013] The preparation method of two novel skeleton compounds of (9β-H)-pimarane diterpenes with anti-colorectal cancer activity according to the present invention is characterized by including the following steps:

[0014] (1) Weigh dry I. oliviformis leaves, crush them, soak the medicinal materials with ethanol or aqueous methanol solution at room temperature, filter and collect the filtrate, and concentrate it under reduced pressure until the alcohol smell disappears to obtain a concentrated solution;

[0015] (2) Extract the concentrated solution obtained in step (1) with petroleum ether, ethyl acetate and n-butanol respectively, and concentrate under reduced pressure to obtain a petroleum ether fraction, an ethyl acetate fraction and an n-butanol fraction;

[0016] (3) Take the n-butanol fraction obtained in step (2), use MCI reverse medium-pressure preparative chromatography, and elute with a mixed solvent of methanol and water with different volume ratios;

[0017] (4) The eluate fraction Bu-1 at 47.00–55.00 min obtained in step (3) is subjected to MCI reverse medium-pressure preparative gradient elution, and the effluent Bu-1-7 at 55.00–65.00 min is collected; The fraction Bu-1-7 is prepared into secoicacinlivitholide F (1) by preparative high-performance liquid chromatography.

[0018] (5) The eluate fraction Bu-2 at 55.00–63.00 min obtained in step (3) is subjected to MCI reverse medium-pressure preparative gradient elution, and the effluent Bu-2-10 at 65.00–74.00 min is collected; The fraction Bu-2-10 is prepared into secoicacinlivitholide G (2) by preparative high-performance liquid chromatography.

[0019] As a preferred embodiment, the preparation method of the present invention includes the following steps:

[0020] (1) Weigh 1.3 kg of dry I. oliviformis leaves, crush them, add 13 L of ethanol with a volume concentration of 95% and soak the medicinal materials at room temperature. Soak three times, 12 hours each time. After filtration, collect the filtrate and concentrate it under reduced pressure until the alcohol smell disappears to obtain an extract.

[0021] (2) Make the medicinal liquid obtained in step (1) into a suspension with an appropriate amount of water, and then extract it with equal volumes of petroleum ether, ethyl acetate, and n-butanol, 4 extractions each, to obtain the petroleum ether fraction, ethyl acetate fraction, n-butanol fraction, and residual water fraction respectively.

[0022] (3) For the n-butanol fraction obtained in step (2), mix the sample with MCI at a ratio of 1:1.2, and perform medium-pressure preparative - MCI column chromatography (MCI particle size is 75–150 μm, 3.0 cm × 60.0 cm, column volume is 400 mL) with gradient elution. Use methanol–water as the mobile phase for elution, where A is pure water and B is methanol; elution gradient: 0.01–10.00 min, 5% - 5% B; 10.00–20.00 min, 15% - 15% B; 20.00–30.00 min, 25% - 25% B; 30.00–50.00 min, 35% - 35% B; 50.00–70.00 min, 45% - 45% B; 70.00–90.00 min, 55% - 55% B; 90.00–110.00 min, 65% - 65% B; 110.00~130.00 min, 75% - 75% B; 130.00~160.00 min, 75% - 100% B. The elution flow rate is 20 mL / min, and the detection wavelengths are 250 nm and 310 nm; after high-performance liquid chromatography analysis, concentrate and combine using a rotary evaporator to obtain 4 fractions Bu-1 to Bu-4.

[0023] (4) For the eluate fraction Bu-1 from 47.00–55.00 min obtained in step (3), perform gradient elution by MCI reversed-phase medium-pressure preparation, using methanol–water as the mobile phase, where A is pure water and B is methanol; elution gradient: 0.01–10.00 min, 5% - 5% B; 10.00–20.00 min, 15% - 15% B; 20.00–30.00 min, 25% - 25% B; 30.00–50.00 min, 35% - 35% B; 50.00–70.00 min, 45% - 45% B; 70.00–120.00 min, 45% - 95% B. The elution flow rate is 20 mL / min, and the detection wavelengths are 250 nm and 310 nm. Collect the effluent Bu-1-7 from 55.00–65.00 min; subject the fraction Bu-1-7 to preparative high-performance liquid chromatography and perform isocratic elution with pure water (A) - acetonitrile (B) as the mobile phase (CH3 CN-H 2 (O, v / v, 41:59), collect the stock solution of Compound 1 in the time period of 53 - 57 min; the stock solution of Compound 1 is further subjected to preparative high performance liquid chromatography to obtain the monomer compound secoicacinlivitholide F. The column pressure is 9.8 MPa, the column temperature is 22 - 26 °C, the mobile phase is pure water - acetonitrile with a volume ratio of 60:40, the injection volume is 100 μL, the flow rate is 3 mL / min, and the detection wavelength is 250 nm.

[0024] (5) Subject the eluate fraction Bu-2 obtained in step (3) in the range of 55.00–63.00 min to MCI reversed-phase medium pressure preparative gradient elution, elute with methanol–water as the mobile phase, where A is pure water and B is methanol; elution gradient: 0.01–10.00 min, 5% - 5% B; 10.00–20.00 min, 15% - 15% B; 20.00–30.00 min, 25% - 25% B; 30.00–50.00 min, 35% - 35% B; 50.00–70.00 min, 45% - 45% B; 70.00–90.00 min, 55% - 55% B; 90.00–130.00 min, 55% - 95% B. The elution flow rate is 20 mL / min, and the detection wavelengths are 250 nm and 310 nm. Collect the effluent Bu-2-10 in the range of 65.00–74.00 min; subject the fraction Bu-2-10 to preparative high performance liquid chromatography, and perform isocratic elution with pure water (A) - acetonitrile (B) as the mobile phase (CH 3 CN-H 2 (O, v / v, 40:60), collect the stock solution of Compound 2 in the time period of 50 - 54 min; the stock solution is further subjected to preparative high performance liquid chromatography to obtain the monomer compound secoicacinlivitholide G.

[0025] The activity experiment was carried out by performing a proliferation inhibition experiment on HT-29 colon cancer cells, selecting clinically used 5-FU as the positive control drug, and the MTT method was used to test the IC of the compound against HT-29 colon cancer cells 50 (half inhibitory concentration), and finally proved that the above 2 compounds have good anti-colon cancer effects. The use of the above compounds in the preparation of anti-colon cancer drugs.

[0026] Beneficial effects:

[0027] Two new 3,4-seco-17-nor-pimarane-type skeletal compounds, secoicacinlivitholide F and secoicacinlivitholide G, were discovered from the West African plant I. oliviformis. The invention also provides an extraction and separation technique for the compounds, a structural identification method, and their use in anti-colon cancer cell proliferation. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1 1H NMR spectrum of Compound 1 1 (500 MHz, Methanol-d 4 )

[0029] Figure 2 13C NMR spectrum of Compound 1 13 (125 MHz, Methanol-d 4 )

[0030] Figure 3 DEPT 135 spectrum of Compound 1 (125 MHz, Methanol-d 4 )

[0031] Figure 4 1H- 1 1H COSY spectrum of Compound 1 (500 MHz, Methanol-d 1 (500 MHz, Methanol-d 4 )

[0032] Figure 5 HSQC spectrum of Compound 1 1 1H: 500 MHz, 13 13C: 125 MHz)

[0033] Figure 6 HMBC spectrum of Compound 1 1 1H: 500 MHz, 13 13C: 125 MHz)

[0034] Figure 7 NOESY spectrum of Compound 1 (500 MHz, Methanol-d 4 )

[0035] Figure 8 Mass spectrum of Compound 1

[0036] Figure 9 UV spectrum of Compound 1

[0037] Figure 10 1H NMR spectrum of Compound 2 1 (500 MHz, Methanol-d4 )。

[0038] Figure 11 Of Compound 2 13 13C NMR Spectrum (125 MHz, Methanol-d 4 )。

[0039] Figure 12 DEPT 135 Spectrum of Compound 2 (125 MHz, Methanol-d 4 )。

[0040] Figure 13 Of Compound 2 1 H- 1 H COSY Spectrum (500 MHz, Methanol-d 4 )。

[0041] Figure 14 HSQC Spectrum of Compound 2( 1 H: 500 MHz, 13 C: 125 MHz)。

[0042] Figure 15 HMBC Spectrum of Compound 2( 1 H: 500 MHz, 13 C: 125 MHz)。

[0043] Figure 16 NOSEY Spectrum of Compound 2 (500 MHz, Methanol-d 4 )。

[0044] Figure 17 Mass Spectrum of Compound 2.

[0045] Figure 18 UV Spectrum of Compound 2. Detailed Implementation Modes

[0046] Example 1

[0047] 1. Instruments and Materials

[0048] 1.1 Instruments

[0049]

[0050] 1.2. Experimental Materials

[0051] Hedera ODS preparative chromatography column (10 nm, 5 μm, 10 × 250 mm); Waters ACQUITY UPLC BEH C 18Chromatographic column (2.1 mm×100 mm, 1.7 μm); MCI GEL (CHP20, 75 - 150 μm), the gel is Sephadex LH-20, column chromatography silica gel (200 - 300 mesh); Chromatographic grade acetonitrile, methanol and formic acid were purchased from Merck, USA; Analytical grade petroleum ether, ethyl acetate, etc. were purchased from Nanjing Wanqing Chemical Reagent Co., Ltd.

[0052] The preparation method of the compound is carried out according to the following steps:

[0053] (1) Weigh 1.3 kg of dry leaves of I. oliviformis, crush them, add 13 L of ethanol with a volume concentration of 95%, soak the medicinal materials at room temperature, soak and extract three times, each time for 12 hours, filter and collect the filtrate, and concentrate it under reduced pressure until there is no alcohol smell to obtain an extract.

[0054] (2) Make the extract obtained in step (1) into a suspension with 1000 mL of water, then extract with equal volumes of petroleum ether, ethyl acetate and n-butanol, extract 4 times each, and obtain the petroleum ether fraction, ethyl acetate fraction, n-butanol fraction and residual water fraction respectively.

[0055] (3) For the n-butanol fraction obtained in step (2), mix the sample with MCI at a ratio of 1:1.2, and use medium-pressure preparative - MCI column chromatography (MCI particle size is 75–150 μm, 3.0 cm×60.0 cm, column volume is 400 mL) for gradient elution. Use methanol–water as the mobile phase for elution, where A is pure water and B is methanol; Elution gradient: 0.01–10.00 min, 5% - 5% B; 10.00–20.00 min, 15% - 15% B; 20.00–30.00 min, 25% - 25% B; 30.00–50.00 min, 35% - 35% B; 50.00–70.00 min, 45% - 45% B; 70.00–90.00 min, 55% - 55% B; 90.00–110.00 min, 65% - 65% B; 110.00~130.00 min, 75% - 75% B; 130.00~160.00 min, 75% - 100% B. The elution flow rate is 20 mL / min, and the detection wavelengths are 250 nm and 310 nm; After high-performance liquid chromatography analysis, concentrate and combine with a rotary evaporator to obtain 4 fractions Bu-1 to Bu-4.

[0056] (4) The fraction Bu-1 obtained in step (3) was subjected to gradient elution using medium-pressure preparative-MCI column chromatography (MCI particle size 75–150 μm, 3.0 cm × 60.0 cm, column volume 400 mL), with methanol–water as the mobile phase for elution, where A was pure water and B was methanol; elution gradient: 0.01–10.00 min, 5%-5% B; 10.00–20.00 min, 15%-15% B; 20.00–30.00 min, 25%-25% B; 30.00–50.00 min, 35%-35% B; 50.00–70.00 min, 45%-45% B; 70.00–120.00 min, 45%-95% B. The elution flow rate was 20 mL / min, and the detection wavelengths were 250 nm and 310 nm; after high-performance liquid chromatography analysis, it was concentrated and combined using a rotary evaporator to obtain 13 fractions Bu-1-1 to Bu-1-13.

[0057] (5) The fraction Bu-1-7 obtained in step (4) was purified by reversed-phase C 18 Preparative high-performance liquid chromatography (Hedera ODS, 5 μm, 2.1×250 mm) was used for purification. Isocratic elution was carried out with pure water (A)-acetonitrile (B) as the mobile phase (CH3CN-H2O, v / v, 41:59), the detection wavelengths were 250 nm and 310 nm, the flow rate was 3 mL / min, the column temperature was 30 °C, and the stock solution of compound 1 was collected in the time period of 53-57 min; the stock solution of compound 1 was further subjected to preparative high-performance liquid chromatography to obtain monomeric compound 1 (2.6 mg). The high-performance liquid chromatography column pressure was 9.8 MPa, the chromatographic column was Hedera ODS, the column temperature was 22-26 °C, the mobile phase was pure water-acetonitrile with a volume ratio of 60:40, the injection volume was 100 μL, the flow rate was 3 mL / min, and the detection wavelength was 250 nm.

[0058] (6) The fraction Bu-2 obtained in step (3) was subjected to gradient elution using medium-pressure preparative-MCI column chromatography (MCI particle size 75–150 μm, 3.0 cm × 60.0 cm, column volume 400 mL), with methanol–water as the mobile phase for elution, where A was pure water and B was methanol; elution gradient: 0.01–10.00 min, 5% - 5% B; 10.00–20.00 min, 15% - 15% B; 20.00–30.00 min, 25% - 25% B; 30.00–50.00 min, 35% - 35% B; 50.00–70.00 min, 45% - 45% B; 70.00–90.00 min, 55% - 55% B; 90.00–130.00 min, 55% - 95% B. The elution flow rate was 20 mL / min, and the detection wavelengths were 250 nm and 310 nm; after high-performance liquid chromatography analysis, it was concentrated and combined using a rotary evaporator to obtain 11 fractions Bu-2-1 to Bu-2-11.

[0059] (7) The fraction Bu-2-10 obtained in step (6) was purified by preparative reversed-phase C 18 preparative high-performance liquid chromatography (Hedera ODS, 5 μm, 1.5 × 250 mm). Isocratic elution was carried out with pure water (A) - acetonitrile (B) as the mobile phase (CH 3 CN - H 2 O, v / v, 40:60), the detection wavelengths were 250 nm and 310 nm, the flow rate was 3 mL / min, the column temperature was 30 °C, and the stock solution of compound 2 was collected in the time period of 50 - 54 min; the stock solution of compound 2 was further subjected to preparative high-performance liquid chromatography to obtain monomeric compound 2 (4.41 mg). The high-performance liquid chromatography column pressure was 9.8 MPa, the chromatographic column was Hedera ODS, the column temperature was 22 - 26 °C, the mobile phase was pure water - acetonitrile with a volume ratio of 60:40, the injection volume was 80 μL, the flow rate was 3 mL / min, and the detection wavelength was 250 nm.

[0060] 3. Structure elucidation of the compounds:

[0061] 3.1 Structure identification of Secoicacinlivitholide F (1)

[0062]

[0063] Compound 1 was a white powder, Combined with 13 13C NMR data and the quasi-molecular ion peak m / z 363.1426 ([M + H] + obtained from high-resolution electrospray ionization mass spectrometry (HRESIMS), C 19 H 23 O7 + The calculated value is: 363.1444), and the molecular formula of the compound can be deduced as C 19 H 22 O 7 , and the degree of unsaturation is 9. In the hydrogen spectrum of compound 1 (Table 1), there is a signal of one methyl proton at δ H (1.29, d, CH 3 -18) and two vinylic proton signals at δ H (6.67, d, H-15; 7.54, d, H-16). Combining with 13 the C NMR spectrum and DEPT spectrum show that the compound has 19 carbon signals, including one methyl carbon, five methylene carbons (including one oxygenated methylene), seven methine carbons (including one oxygenated methine), three quaternary carbons (including one quaternary carbon connected to another quaternary carbon) and three carbonyl carbons. Analyzing the 1 H and 13 C NMR data of compound 1, in compound 1, there are two methylene groups at δ C (30.7, C-1; 26.8, C-2), one carboxyl group at δ C (177.0, C-3), two methine groups at δ C (38.7, C-4; 46.9, C-5) and one quaternary carbon at δ C (50.8, C-10). The above evidence indicates that in compound 1, ring opening occurs between C-3 and C-4. In addition, in the HMBC spectrum, it shows that H 2 -20 is correlated with C-6 (δ C 77.6), indicating the presence of a 6,20-epoxy bridge. The planar structure of compound 1 is further confirmed by 1 H- 1 H COSY, HSQC and HMBC spectra.

[0064] The relative configuration of compound 1 was determined by analyzing the NOESY spectrum ( Figure 7 ). So far, the diterpenoids isolated from the genus Iodes are all (9β-H)-pimarane and its derivatives. Therefore, H-5 in compound 1 is assigned an α-orientation. In the NOESY spectrum, H-5 (δ H 2.48) and H-6 (δ H 4.46), CH 3 -18 (δ H 1.29) are correlated, indicating that H-5 / H-6 / CH 3 -18 are all α-oriented. H-4 (δ H 2.54) and H-20β (δ H 3.83) are correlated, H-20α (δH 4.02) is related to H-8 (δ H 3.48) and H-9 (δ H 2.65), indicating that

[0065]

[0066] H-8 / H-9 / H 2 -20 are all β-oriented. Therefore, the structure of compound 1 was determined and named secoicacinlivitholide F. Compound 1 is the first 3,4-seco-17-nor-pimarane new skeleton compound with a 6,20-epoxy bridge found in nature.

[0067] 3.2 Structure identification of secoicacinlivitholide G (2)

[0068] Compound 2 is a white powder, Combined with 13 the C-NMR data and the quasi-molecular ion peak m / z 377.1607 ([M + H] + , C 20 H 25 O 7 + calculated value: 377.1600) given by high-resolution mass spectrometry (HRESIMS), the molecular formula of the compound can be deduced as C 20 H 24 O 7 , and the degree of unsaturation is 9. Comparing the 1 H-NMR and 13 C-NMR spectra of compound 2 and 1, it was found that compound 2 has one more methoxy group than compound 1. The HMBC spectrum shows that OCH 3 (δ H 3.64, δ C 52.2) is related to C-3 (δ C 175.2), indicating that the methoxy group is connected to C-3

[0069]

[0070] connected. Therefore, it can be determined that compound 2 is a 3-methoxy derivative of compound 1. The planar structure of compound 2 was further confirmed by 1 H- 1 H COSY, HSQC and HMBC spectra, and it was named secoicacinlivitholide G. The relative configuration of compound 2 is the same as that of compound 1.

[0071]

[0072] Table 1. For Compounds 1 and 2 1 H and 13 C NMR data ( 1 H, 500 MHz; 13 C, 125 MHz)

[0073]

[0074] Example 2

[0075] The anti - colon cancer activity test research of the present invention was carried out according to the following steps:

[0076] 1. Tumor cell culture

[0077] The colon cancer cell lines HT - 29 and SW620 (Cell Bank of the Chinese Academy of Sciences) were cultured in DMEM medium (Gibco, USA) containing 10% fetal bovine serum at 37 °C and 5% CO 2 condition. When sub - culturing, first take out a plate of tumor cells, remove the culture medium, add 3 mL of PBS, wash twice; add 1 mL of trypsin for digestion, after 2 min add 1 mL of culture medium to terminate digestion, centrifuge at room temperature for 5 min at 1000 r / min; remove the supernatant, add 1 mL of DMEM medium containing 10% fetal bovine serum, and resuspend to obtain a cell suspension.

[0078] 2. Preparation of experimental drugs

[0079] Weigh an appropriate amount of Compounds 1 and 2 prepared in Example 1 above and dissolve them in DMSO to make the final concentration of the stock solution 4 mM, and store it in a 4 °C refrigerator. Before the experiment, dilute the stock solution with DMEM medium to make the drug concentration 20 μM, and ensure that the final concentration of DMSO is less than 0.1%. Add different volumes of DMEM medium to dilute the compound into different concentrations. At the same time, use DMEM medium containing 0.1% DMSO as the negative control.

[0080] 3. Toxicity of drugs to tumor cell lines

[0081] Suspend the tumor cells with the medium and inoculate them into a 96 - well plate (100 μL / well) at a cell density of 6×10 3 and culture them at 37 °C and 5% CO 2 for 24 h. During the logarithmic growth phase of the tumor cell line, add compounds with different concentrations and culture them at 37 °C and 5% CO 2 for 24 h.

[0082] 4. Detection of cell viability by MTT method

[0083] After the drug has acted on tumor cells for 24 h, add 20 μL of MTT solution (5 mg / mL) to each well and incubate in a sterile incubator for 3 h. Completely aspirate the supernatant, add 150 μL of DMSO to each well, and react on a shaker for 30 min to dissolve the blue-violet formazan crystals. Measure the OD value using an enzyme-linked immunosorbent assay (ELISA) reader at a wavelength of 570 nm. Calculate the IC 50 (half maximal inhibitory concentration) of the compound using GraphPad Prism 8 software, with 5-FU as the positive control.

[0084] 5. The experimental results are shown in Table 2:

[0085] Table 2. Inhibitory effects of Compounds 1 and 2 on colon cancer cell lines (IC 50 : μM)

[0086]

[0087] Experimental conclusion: Through the evaluation of the anti-colon cancer cell activities of Compounds 1 and 2, it was found that Compound 2 has significant anti-proliferative effects on two colon cancer cell lines, HT-29 and SW620, and its effects are stronger than those of the positive drug 5-FU. Among them, the inhibitory effect of Compound 2 on the colon cancer cell line HT-29 is about 3 times that of the positive drug 5-FU, indicating its potential for development into a new anti-colon cancer drug.

Claims

1. A diterpenoid compound with an (9 β -H)-pimarane nucleus and anti-colorectal cancer activity, It is characterized in that It is a compound of the following structural general formula: ； Among them, R 1 is hydrogen or methyl, and R 2 is hydrogen.

2. The preparation method of the diterpenoid compound with a (9 β -H)-pimarane nucleus having anti-colorectal cancer activity according to claim 1 It is characterized in that It includes the following steps: (1) Weigh out the dried I. oliviformis leaves (1.3 kg), crush them, add 13 L of ethanol with a volume concentration of 95% and soak the medicinal materials at room temperature for extraction. Soak three times, 12 hours each time. After filtration, collect the filtrate and concentrate it under reduced pressure until the alcohol smell disappears to obtain an extract. (2) The extract obtained in step (1) is made into a suspension with an appropriate amount of water, and then extracted 4 times with equal volumes of petroleum ether, ethyl acetate, and n-butanol to obtain a petroleum ether fraction, an ethyl acetate fraction, an n-butanol fraction, and a residual water fraction respectively; (3) For the n-butanol fraction obtained in step (2), the sample is mixed with MCI at a ratio of 1:1.2, and medium-pressure preparative-MCI column chromatography gradient elution is used, with methanol–water as the mobile phase for elution, where A is pure water and B is methanol; elution gradient: 0.01–10.00 min, 5%-5% B; 10.00–20.00 min, 15%-15% B; 20.00–30.00 min, 25%-25% B; 30.00–50.00 min, 35%-35% B; 50.00–70.00 min, 45%-45% B; 70.00–90.00 min, 55%-55% B; 90.00–110.00 min, 65%-65% B; 110.00~130.00 min, 75%-75% B; 130.00~160.00 min, 75%-100% B; the elution flow rate is 20 mL / min, and the detection wavelengths are 250 nm and 310 nm; after high-performance liquid chromatography analysis, it is concentrated and combined to obtain 4 fractions Bu-1~Bu-4; (4) The eluate fraction Bu-1 obtained in step (3) within 47.00–55.00 min was subjected to gradient elution by medium-pressure MCI reverse-phase preparation, using methanol–water as the mobile phase, where A was pure water and B was methanol; elution gradient: 0.01–10.00 min, 5%–5% B; 10.00–20.00 min, 15%–15% B; 20.00–30.00 min, 25%–25% B; 30.00–50.00 min, 35%–35% B; 50.00–70.00 min, 45%–45% B; 70.00–120.00 min, 45%–95% B; elution flow rate was 20 mL / min, detection wavelengths were 250 nm and 310 nm; the effluent Bu-1-7 within 55.00–65.00 min was collected; fraction Bu-1-7 was subjected to preparative high-performance liquid chromatography, using pure water A and acetonitrile B with a volume ratio of 59:41 as the mobile phase for isocratic elution, and the stock solution of compound 1 was collected in the time period of 53–57 min; the stock solution of compound 1 was further purified by preparative high-performance liquid chromatography, the column pressure was 9.8 MPa, the column temperature was 22–26 °C, the mobile phase was pure water–acetonitrile with a volume ratio of 60:40, the injection volume was 100 μL, the flow rate was 3 mL / min, and the detection wavelength was 250 nm to obtain the monomeric compound secoicacinlivitholide F, whose structural formula is: ； (5) The eluate fraction Bu-2 obtained in step (3) at 55.00–63.00 min was subjected to gradient elution by medium-pressure preparative MCI in the reverse phase, with methanol–water as the mobile phase for elution, where A is pure water and B is methanol; elution gradient: 0.01–10.00 min, 5%–5% B; 10.00–20.00 min, 15%–15% B; 20.00–30.00 min, 25%–25% B; 30.00–50.00 min, 35%–35% B; 50.00–70.00 min, 45%–45% B; 70.00–90.00 min, 55%–55% B; 90.00–130.00 min, 55%–95% B; elution flow rate 20 mL / min, detection wavelengths 250 nm and 310 nm; the effluent Bu-2-10 at 65.00–74.00 min was collected; fraction Bu-2-10 was subjected to preparative high-performance liquid chromatography, with pure water A–acetonitrile B at a volume ratio of 60:40 as the mobile phase for isocratic elution, and the stock solution of compound 2 was collected in the time period of 50–54 min; the stock solution of compound 2 was further purified by preparative high-performance liquid chromatography, with a column pressure of 9.8 MPa, column temperature 22–26 °C, mobile phase of pure water–acetonitrile at a volume ratio of 60:40, injection volume 80 μL, flow rate 3 mL / min, and detection wavelength 250 nm, to obtain the monomeric compound secoicacinlivitholide G; its structural formula is: 。 3. Use of the diterpenoid compound with a (9 β -H)-pimarane nucleus in the preparation of an anti-colorectal cancer drug.

4. A pharmaceutical composition comprising the diterpenoid compound of the (9 β -H)-pimarane nucleus as claimed in claim 1 and a pharmaceutically acceptable excipient.

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