Compound with ursane type triterpenes-enantiokaurane type diterpenes dimer new skeleton as well as preparation method and application thereof

Abstract

The invention discloses two compounds with ursane type triterpenes-enantiokaurane type diterpenes dimer new frameworks and a preparation method thereof, chemical components of a Western African plant I.trichantha tuber are deeply researched, two compounds Bisicacinol B (1) and Bisicacinol C (2) are obtained through separation, and the two compounds are used for preparing the novel framework of the ursane type triterpenes-enantiokaurane type diterpenes dimer new frameworks. The compound is a natural hybrid dimer which is formed by carrying out a DielsAlder addition reaction or a Michael addition reaction on ursane type triterpenes with a unique spiro structure and enantiokaurene type diterpenes. According to the present invention, the pharmaceutical experiment results show that the Bisicacinol B (1) and the Bisicacinol C (2) have significant antitumor activity (such as pancreatic cancer, colon cancer, lung cancer or liver cancer), and can be combined with the amphotericin B to provide the synergistic anti-Candida albicans activity;

Description

Technical Field

[0001] The present invention belongs to the field of medicine, and particularly relates to two new skeleton compounds of ursane-type triterpenoid-ent-kaurane-type diterpenoid dimers, a preparation method thereof, and an application thereof in anti-tumor treatment. Background Art

[0002] I. trichantha is a plant endemic to Central and West Africa in the genus Iodes, and is a medicinal plant used by indigenous tribes in Nigeria and surrounding countries. This plant has played an important role in traditional medicine in Nigeria, where local people use its tubers as medicine and widely apply them to the treatment of various diseases, such as poisoning, constipation, inducing vomiting, and treating malaria. The tubers of I. trichantha are rich in starch. During famine years, local people wash the tubers with water and then eat them. Modern research shows that I. trichantha has various physiological activities such as hypoglycemic, anticonvulsant, sedative, analgesic, and antibacterial effects. Currently, there is little research on the chemical constituents of I. trichantha. Summary of the Invention

[0003] The purpose of the present invention is to conduct in-depth research on the active ingredients of the tubers of I. trichantha, extract and isolate the potentially active ingredients, and provide a scientific basis for their clinical application.

[0004] Technical Solution: The present invention has isolated two new skeleton compounds of ursane-type triterpenoid-ent-kaurane-type diterpenoid heterodimers from the tubers of the plant I. trichantha, and the compounds are named Bisicacinol B (1) and Bisicacinol C (2).

[0005] The structural formulas of Compounds 1 and 2 of the present invention are:

[0006]

[0007] The preparation method of the new skeleton compound of ursane-type triterpenoid-ent-kaurane-type diterpenoid dimer described in the present invention includes the following steps:

[0008] (1) Weigh the dried tubers of I. trichantha, crush them, add ethanol-water for reflux extraction of the medicinal materials, filter and collect the filtrate, and concentrate it under reduced pressure until there is no alcohol smell to obtain a concentrated solution;

[0009] (2) Use petroleum ether, dichloromethane, ethyl acetate, and n-butanol to extract the concentrated solution obtained in step (1) respectively, and concentrate under reduced pressure to obtain a petroleum ether fraction, a dichloromethane fraction, an ethyl acetate fraction, an n-butanol fraction, and a residual water fraction respectively;

[0010] (3) Take the dichloromethane fraction obtained in step (2), and perform normal pressure column chromatography on AB-8 macroporous resin, eluting with ethanol-water to obtain 3 sub-fractions DCM-1 - DCM-3;

[0011] (4) Take the eluate fraction DCM-3 obtained in step (3), and perform normal pressure column chromatography on silica gel, eluting with dichloromethane-methanol to obtain 7 sub-fractions DCM-3-1 - DCM-3-7;

[0012] (5) Take the fraction DCM-3-5 obtained in step (4), and perform medium pressure preparative - MCI column chromatography with gradient elution, using methanol-water as the mobile phase to obtain 11 fractions DCM-3-5-1 - DCM-3-5-11; Subject the fraction DCM-3-5-9 to semi-preparative high performance liquid chromatography to obtain compounds 1 and 2.

[0013] As a preferred embodiment, the preparation method of the present invention comprises the following steps:

[0014] (1) Weigh dry I. trichantha tubers, crush them, add ethanol with a volume concentration of 80 - 95% to reflux and extract the medicinal materials, with a material-liquid ratio of 1:6 - 20, extract 1 - 3 times, each time for 1 - 3 h, filter and collect the filtrate, combine the filtrates, and concentrate under reduced pressure until the alcohol smell disappears to obtain an extract;

[0015] (2) Make the extract obtained in step (1) into a suspension with an appropriate amount of water, then extract with equal volumes of petroleum ether, dichloromethane, ethyl acetate, and n-butanol, extract 3 - 6 times each, to obtain petroleum ether fraction, dichloromethane fraction, ethyl acetate fraction, n-butanol fraction, and residual water fraction respectively.

[0016] (3) Mix the dichloromethane fraction obtained in step (2) with macroporous resin, perform normal pressure column chromatography on macroporous resin, and use ethanol-water with volume ratios of 30:70, 60:40, 90:10, 100:0 as the mobile phase for gradient elution in sequence, eluting 4 - 6 column volumes for each gradient; After analysis by thin layer and ultra-high performance liquid chromatography, concentrate and combine using a rotary evaporator to obtain 3 sub-fractions DCM-1 - DCM-3;

[0017] (4) Mix the eluate fraction DCM-3 obtained in step (3) with silica gel, then perform normal pressure column chromatography on silica gel, and use dichloromethane-methanol with volume ratios of 100:0, 50:1, 30:1, 20:1, 10:1, 7:1, 5:1, 3:1, 2:1, 1:1, 1:3, 1:7, 0:100 as the mobile phase for gradient elution in sequence, eluting 3 - 6 column volumes for each gradient; After analysis by thin layer and ultra-high performance liquid chromatography, concentrate and combine using a rotary evaporator to obtain 7 sub-fractions DCM-3-1 - DCM-3-7;

[0018] (5) The fraction DCM-3-5 obtained in step (4) was mixed with MCI, and medium-pressure preparative MCI column chromatography was used for gradient elution with methanol-water as the mobile phase, where A was water and B was methanol; the elution gradient program was as follows: 0.01 - 20.00 min, 30% - 30% B; 20.00 - 50.00 min, 40% - 40% B; 50.00 - 80.00 min, 45% - 45% B; 80.00 - 110.00 min, 50% - 50% B; 110.00 - 140.00 min, 55% - 55% B; 140.00 - 170.00 min, 60% - 60% B; 170.00 - 200.00 min, 70% - 70% B; 200.00 - 230.00 min, 85% - 85% B; 230.00 - 270.00 min, 100% - 100% B. The elution flow rate was 20 mL / min, and the detection wavelengths were 256 nm and 310 nm; after ultra-high performance liquid chromatography analysis, it was concentrated and combined to obtain 11 fractions DCM-3-5-1 - DCM-3-5-11;

[0019] (6) The eluate fraction DCM-3-5-9 eluted at 200 - 220 min obtained in step (5) was separated by semi-preparative high-performance liquid chromatography with pure water A - methanol B as the mobile phase for isocratic elution to obtain compound 1 and compound 2.

[0020] As a preferred scheme, the chromatographic conditions of the above-mentioned semi-preparative high-performance liquid chromatography were as follows: the chromatographic column model was Hedera ODS preparative chromatographic column (10 nm, 5 μm, 10×250 mm), the high-performance liquid chromatography column pressure was 9.8 MPa, the column temperature was 22 - 26 °C, the injection volume was 100 μL, and the mobile phase was MeOH-H 2 O (v / v, 80:20), the flow rate was 3 mL / min, and the detection wavelength was 310 nm. The monomeric compounds Bisicacinol B (1) and Bisicacinol C (2) were obtained.

[0021] Beneficial effects:

[0022] In the present invention, the chemical constituents of the tubers of the West African plant I. trichantha were deeply studied, and two new skeleton compounds Bisicacinol B (1) and Bisicacinol C (2) with ursane-type triterpenoid-ent-kaurane-type diterpenoid dimer were isolated. Through pharmacological experiments, it was found that Bisicacinol B (1) and Bisicacinol C (2) prepared in the present invention have significant anti-tumor activities (including pancreatic cancer, colon cancer, lung cancer or liver cancer), and also have good antibacterial activities (including Helicobacter pylori and Candida albicans).

[0023] The compounds Bisicacinol B (1) and Bisicacinol C (2) isolated by extraction of the present invention can be prepared into various dosage forms with pharmaceutically acceptable carriers. Brief Description of the Drawings

[0024] Figure 1 (+)-HR-ESI-MS spectrum of Compound 1;

[0025] Figure 2 UV spectrum of Compound 1;

[0026] Figure 3 For Compound 1 1 1H NMR spectrum (500 MHz, Methanol-d 4 );

[0027] Figure 4 For Compound 1 13 13C NMR spectrum (125 MHz, Methanol-d 4 );

[0028] Figure 5 DEPT 135 spectrum of Compound 1 (125 MHz, Methanol-d 4 );

[0029] Figure 6 For Compound 1 1 1H- 1 1H COSY spectrum (500 MHz, Methanol-d 4 );

[0030] Figure 7 HSQC spectrum of Compound 1 ( 1 1H: 500 MHz, 13 13C: 125 MHz, Methanol-d 4 );

[0031] Figure 8 HMBC spectrum of Compound 1 ( 1 1H: 500 MHz, 13 13C: 125 MHz, Methanol-d 4 );

[0032] Figure 9 NOESY spectrum of Compound 1 (500 MHz, Methanol-d 4 );

[0033] Figure 10 (+)-HR-ESI-MS spectrum of Compound 2;

[0034] Figure 11 UV spectrum of Compound 2;

[0035] Figure 12 of Compound 2 1 H NMR spectrum (500 MHz, Methanol-d 4 );

[0036] Figure 13 of Compound 2 13 C NMR spectrum (125 MHz, Methanol-d 4 );

[0037] Figure 14 DEPT 135 spectrum of Compound 2 (125 MHz, Methanol-d 4 );

[0038] Figure 15 of Compound 2 1 H- 1 H COSY spectrum (500 MHz, Methanol-d 4 );

[0039] Figure 16 HSQC spectrum of Compound 2 ( 1 H: 500 MHz, 13 C: 125 MHz, Methanol-d 4 );

[0040] Figure 17 HMBC spectrum of Compound 2 ( 1 H: 500 MHz, 13 C: 125 MHz, Methanol-d 4 );

[0041] Figure 18 NOSEY spectrum of Compound 2 (500 MHz, Methanol-d 4 );

[0042] Figure 19 of Compound 1 1 H- 1 H COSY, key HMBC and NOESY correlation diagrams;

[0043] Figure 20 of Compound 2 1 H- 1 H COSY, key HMBC and NOESY correlation diagrams.

[0044] Figure 21 Structural formulas of Compounds 1 and 2. Detailed implementation manners

[0045] Example 1

[0046] 1. Instruments and materials

[0047] 1.1 Instruments

[0048]

[0049]

[0050] 1.2 Experimental materials

[0051] Hedera ODS preparative chromatography column (10 nm, 5 μm, 10×250 mm); Waters ACQUITY UPLC BEH C 18 (2.1 mm×100 mm, 1.7 μm) chromatography column; MCI GEL (CHP20, 75 - 150 μm), 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] 2. The preparation method of the compound is carried out according to the following steps

[0053] (1) Weigh the dried tubers of I. trichantha, crush them, add ethanol with a volume concentration of 95% to reflux and extract the medicinal materials, with a material - liquid ratio of 1:10, extract 3 times, 2.5 h each time. After filtration, collect the filtrate, combine the filtrates, and concentrate under reduced pressure until the alcohol smell disappears to obtain an extract

[0054] (2) Make the extract obtained in step (1) into a suspension with an appropriate amount of water, and then extract with equal volumes of petroleum ether, dichloromethane, ethyl acetate and n - butanol, 5 extractions each, to obtain the petroleum ether fraction, dichloromethane fraction, ethyl acetate fraction, n - butanol fraction and residual water fraction respectively

[0055] (3) For the dichloromethane fraction obtained in step (2), mix the sample with macroporous resin at a ratio of 1:1.5, and use normal - pressure column chromatography on macroporous resin. Gradient elute successively with ethanol - water with a volume ratio of 30:70, 60:40, 90:10, 100:0 as the mobile phase, 5 column volumes for each gradient elution; After analysis by thin - layer and ultra - high - performance liquid chromatography, concentrate and combine using a rotary evaporator to obtain 3 sub - fractions DCM - 1 - DCM - 3

[0056] (4) The eluate fraction DCM-3 obtained in step (3) was mixed with the sample and silica gel at a ratio of 1:1.5, and silica gel normal pressure column chromatography was used. Dichloromethane-methanol with volume ratios of 100:0, 50:1, 30:1, 20:1, 10:1, 7:1, 5:1, 3:1, 2:1, 1:1, 1:3, 1:7, 0:100 was used as the mobile phase for gradient elution, and each gradient was eluted with 4 column volumes; after analysis by thin layer and ultra-high performance liquid chromatography, it was concentrated and combined using a rotary evaporator to obtain 7 sub-fractions DCM-3-1 - DCM-3-7;

[0057] (5) The fraction DCM-3-5 obtained in step (4) was mixed with the sample and MCI at a ratio of 1:1.2, and medium pressure preparative-MCI column chromatography gradient elution was used, with methanol-water as the mobile phase, where A is water and B is methanol; the elution gradient program was: 0.01 - 20.00 min, 30% - 30% B; 20.00 - 50.00 min, 40% - 40% B; 50.00 - 80.00 min, 45% - 45% B; 80.00 - 110.00 min, 50% - 50% B; 110.00 - 140.00 min, 55% - 55% B; 140.00 - 170.00 min, 60% - 60% B; 170.00 - 200.00 min, 70% - 70% B; 200.00 - 230.00 min, 85% - 85% B; 230.00 - 270.00 min, 100% - 100% B, the elution flow rate was 20 mL / min, and the detection wavelengths were 256 nm and 310 nm; after analysis by ultra-high performance liquid chromatography, it was concentrated and combined to obtain 11 fractions DCM-3-5-1 - DCM-3-5-11;

[0058] (6) The eluate fraction DCM-3-5-9 from 200 - 220 min obtained in step (5) was subjected to semi-preparative high performance liquid chromatography, and isocratic elution was carried out with pure water (A) - methanol (B) as the mobile phase (MeOH - H 2 O, v / v, 80:20). Monomeric compound 1 was collected in the time period of 52 - 57 min, and monomeric compound 2 was collected in the time period of 58 - 61 min.

[0059] 3. Structure analysis of the compound

[0060] 3.1 Structure identification of Bisicacinol B(1)

[0061]

[0062] Compound 1 is a white amorphous powder, (c 0.01, MeOH). Combined with 13The \(^{13}\)C NMR data and the quasi-molecular ion peak \(m / z\) 849.5523 ([M + Na]\(^{+}\), \(C\) 39 H 52 O 9 Na calculated to be 849.5517) led to the derivation of the molecular formula \(C\) 51 H 76 O 10 , with an unsaturation degree of 14. In the \(^{1}\)H NMR spectrum of compound 1 (Table 1), signals of 9 methyl protons were shown at \(\delta\) H (1.10, s, \(CH\) 3 -18; 1.04, s, \(CH\) 3 -19; 1.02, s, \(CH\) 3 -23′; 0.81, s, \(CH\) 3 -24′; 1.06, s, \(CH\) 3 -25′; 0.90, s, \(CH\) 3 -26′; 0.99, s, \(CH\) 3 -27′; 1.73, s, \(CH\) 3 -29′; 1.04, s, \(CH\) 3 -30′); 1 signal of a methoxy proton at \(\delta\) H (3.62, s, \(OCH\) 3 -28′). Combining the 13 \(^{13}\)C NMR spectrum and the DEPT spectrum showed that the compound had 51 carbon signals, including 9 methyl carbons, 14 methylene carbons (including 1 oxygenated methylene), 12 methine carbons (including 5 oxygenated methines), 13 quaternary carbons (including 1 oxygenated carbon) and 2 carbonyl carbons. Combining the carbon spectrum (Table 1), the DEPT spectrum and the HSQC spectrum of the compound indicated that there were 51 carbon signals, which could be attributed respectively to: 9 methyl carbons at \(\delta\) C 21.8 (\(CH\) 3 -18), 32.9 (\(CH\) 3 -19), 29.4 (\(CH\) 3 -23′), 17.6 (\(CH\) 3 -24′), 17.8 (\(CH\) 3 -25′), 18.6 (\(CH\) 3 -26′), 22.5 (\(CH\) 3 -27′), 16.3 (\(CH\) 3 -29′), 27.7 (\(CH\) 3 -30′); 1 methoxy carbon at \(\delta\) C 52.3 (\(OCH\) 3 -28); 14 methylene carbons (including 1 oxygenated methylene) at \(\delta\) C30.3 (C-2), 39.8 (C-3), 19.9 (C-11), 22.0 (C-12), 33.7 (C-17), 64.7 (C-20), 48.7 (C-1′), 19.4 (C-6′), 35.7 (C-7′), 39.1 (C-11′), 29.6 (C-15′), 32.6 (C-16′), 32.8 (C-21′) and 35.5 (C-22′); 12 methine carbons (including 5 oxygen-bonded methines) δ C 73.7 (C-1), 61.9 (C-5), 74.8 (C-6), 55.1 (C-9), 40.9 (C-13), 75.0 (C-14), 50.1 (C-16), 69.6 (C-2′), 84.4 (C-3′), 56.8 (C-5′), 49.4 (C-9′) and 127.8 (C-12′); 13 non-hydrogen-bonded carbons (including 1 oxygen-bonded carbon) δ C 34.6 (C-4), 98.0 (C-7), 49.6 (C-8), 42.1 (C-10), 40.2 (C-4′), 39.7 (C-8′), 39.1 (C-10′), 140.4 (C-13′), 46.0 (C-14′), 51.1 (C-17′), 136.0 (C-18′), 139.1 (C-19′) and 40.5 (C-20′); 2 carbonyl carbons δ C 225.7 (C-15) and 178.7 (C-28′).

[0063] The NMR data of Unit I in Compound 1 is highly similar to oridonin, except for one methine (δ C 50.1, C-16) and one methylene (δ C 33.7, C-17) in Unit I, instead of an exocyclic double bond (δ C 153.4, C-16; 120.6, C-17); the NMR data of Unit II in 1 is highly similar to goreishic acid, except for the presence of a methoxy group (δ H 3.62, δ C 52.3, OCH 3 -28′), and C-20 is a non-hydrogen-bonded carbon (δ C 40.5, C-20′), instead of a methine (δ H 2.28, m; δ C 24.8, C-20′). In the HMBC spectrum, CH-16 and C-20 as well as CH 2 -17 and C-19′ / C-20′ / CH 2 -21′ / CH3 The related signal proof units I and II of -30' are connected by a single bond via C-17 and C-20'.

[0064] The relative configuration of Compound 1 was determined by analyzing the NOESY spectrum ( Figure 9 ), and so far, the relevant reports have designated H-5 and H-5' as β-orientations. The NOESY related signals prove that CH 3 -19 / H-5β / H-1 / H-9 is in the β-orientation; according to the H-6 / CH 3 -18 / H-20a and H-20b / H-14 / H-13 / H-16 cross peaks indicate that H-6 / H-13 / H-14 / H-16 / CH 3 -18 is in the α-orientation; the correlation of H-5' / CH 3 -23' and H-3' / H-5' / H-9' / CH 3 -27' / H-16'α indicates that H-3' / H-5' / H-9' / CH 3 -23' / CH 3 -27' is in the α-orientation, and 28-COOCH 3 is in the β-orientation; due to the correlation between 28-COOCH 3 and CH 3 -30', it indicates that CH 3 -30' is also in the β-orientation; from the correlation of CH 3 -24' / H-2' / CH 3 -25' / CH 3 -26', it is inferred that H-2', CH 3 -24', CH 3 -25' and CH 3 -26' are in the β-orientation; through the measurement and calculation of ECD, the absolute configuration of C-16 is determined to be the R configuration, and finally the absolute configuration of Compound 1 is determined to be 1S,5R,6S,7S,8R,9S,10S,13S,14R,16R,2'R,3'R,5'R,8'R,9'R,10'R,14'S,17'S,20'S. Therefore, the complete structure of the new skeleton compound 1 is determined and named Bisicacinol B.

[0065] 3.2 Structure Identification of Bisicacinol C(2)

[0066]

[0067] Compound 2 is a white amorphous powder, (c 0.01,MeOH). Combining 13C NMR data and high resolution mass spectrometry (HRESIMS) gave a quasi-molecular ion peak of m / z 849.5522 ([M+Na], C 39 H 52 O 9 The calculated value of Na is 849.5517), and the molecular formula of the compound can be deduced to be C 51 H 76 O 10 , the unsaturation degree is 14. Its NMR measurement data are highly similar to those of 1 (Table 1), except that H-16 (δ H 2.20), C-16(δ C 54.4) and 1 in H-16 (δ H 2.93), C-16(δ C 50.1), it is speculated that 2 is the 16-diamer of 1; in addition, the coupling constant of H-16 in 2 (J=9.7Hz, d) is different from that in 1 (J=7.2Hz t), which also supports the above speculation. Through ECD measurement and calculation, it was determined that C-16 of 2 was S-configuration, and the absolute configuration of compound 2 was finally determined to be 1S, 5R, 6S, 7S, 8R, 9S, 10S, 13S, 14R, 16S, 2′R, 3′R, 5′R, 8′R, 9′R, 10′R, 14′S, 17′S, 20′S. Therefore, the complete structure of the new skeleton compound 2 was determined and named Bisicacinol C. Compounds 1 and 2 are the first heterodimers of enantiomeric kaurane-type diterpenes and ursane-type triterpenes.

[0068] Table 1 Compounds 1 and 2 1 H and 13 C NMR data ( 1 H, 500MHz; 13 C,125MHz)

[0069]

[0070]

[0071]

[0072] Example 2

[0073] The anti-tumor cell activity test study of the present invention is carried out according to the following steps:

[0074] 1. Tumor Cell Culture

[0075] The pancreatic cancer cell line MIA PaCa-2, colon cancer cell line HT-29, lung cancer cell line A549, and liver cancer cell line HepG2 (Cell Bank of the Chinese Academy of Sciences) were cultured in DMEM medium (Gibco) containing 10% fetal bovine serum at 37 °C and 5% CO 2 conditions. The media of HT-29, A549, and HepG2 cells were supplemented with 10% FBS and 1% PSN, and the medium of MIA PaCa-2 cells was supplemented with 10% FBS, 2.5% HS, and 1% PSN.

[0076] 2. Preparation of experimental drugs

[0077] Weighed appropriate amounts of Compound 1 and 2 prepared in Example 1 above and dissolved them in DMSO to make the final concentration of the stock solution 40 mM, and stored it in a refrigerator at 4 °C. Before the experiment, the stock solution was diluted with DMEM medium to make the drug concentration 20 μM, and ensure that the final concentration of DMSO was less than 0.1%. Different concentrations of the compound were diluted by adding different volumes of DMEM medium. At the same time, DMEM medium containing 0.1% DMSO was used as a negative control.

[0078] 3. Toxicity of drugs to tumor cell lines

[0079] The tumor cells were suspended with the medium. MIA PaCa-2, HT-29, and HepG2 were seeded in 96-well plates at a cell density of 8000, and A549 was seeded in 96-well plates at a cell density of 3000 (100 μL / well), and cultured at 37 °C and 5% CO 2 conditions for 24 h. At the logarithmic growth phase of the tumor cell lines, different concentrations of the compound (0, 0.004, 0.04, 0.4, 4, 40 μM) were added and cultured at 37 °C and 5% CO 2 conditions for 72 h.

[0080] 4. Detection of cell viability by CCK-8 method

[0081] After the drug acted on the tumor cells for 72 h, 10 μL of CCK-8 solution was added to each well and incubated in a sterile incubator for 1 h. Then, it was taken out and the OD value was measured at a wavelength of 450 nm using an enzyme-linked immunosorbent assay reader. The IC 50 (half maximal inhibitory concentration) of the compound was calculated using GraphPad Prism 8 software, with 5-FU and Carboplatin as positive controls.

[0082] 5. The experimental results are shown in Table 2

[0083] Table 2 Cytotoxic activities of Compound 1 and 2 (IC 50 : μM)

[0084]

[0085] Experimental conclusion: Through the evaluation of the cytotoxic activities of Compounds 1 and 2, it was found that Compound 1 had strong activities against adenocarcinoma cell line MIAPaCa-2, colon cancer cell line HT-29, lung cancer cell line A549, and liver cancer cell line HepG2, which were superior to Compound 2 and the positive control drug. Compound 2 also showed strong inhibitory activities against colon cancer cell line HT-29 and lung cancer cell line A549. In particular, it had a significant anti-proliferation effect on the A549 cell line, and its effects were stronger than those of the positive drugs 5-FU and Carboplatin, showing the potential to be developed into a new anti-lung cancer drug.

[0086] Example 3

[0087] The antibacterial activity test research of the present invention was carried out according to the following steps:

[0088] 1. Experimental materials and reagents

[0089] Helicobacter pylori (G27 and HP129) and Candida albicans (SC5314 and C5) were all from the laboratory of Professor Bihongkai of Nanjing Medical University. Main culture media and main reagents: Columbia medium, LB medium, selective antibiotics (metronidazole and amphotericin B), serum, etc.

[0090] 2. Preparation of culture media

[0091] Columbia liquid medium: Accurately weigh 29.0 g of Columbia liquid medium, heat and dissolve it in 1000 mL of double-distilled water. After the medium is completely dissolved, autoclave it at 121 °C for 15 min and set aside.

[0092] Columbia blood agar solid medium: Accurately weigh 39.0 g of Columbia blood agar solid medium, heat and dissolve it in 1000 mL of double-distilled water. After the medium is completely dissolved, autoclave it at 121 °C for 15 min. When it is naturally cooled to about 50 °C after autoclaving, quickly add 5% sterile defibrinated sheep blood, mix well, and pour it into a sterile petri dish while it is still hot.

[0093] LB medium: Glucose 15 g / L, yeast extract 10 g / L, peptone 5 g / L, sodium chloride 10 g / L.

[0094] 3. Resuscitation and culture of test strains

[0095] Take out the standard strain of Helicobacter pylori from the -80°C refrigerator and place it at room temperature. Accurately pipette 200 μL of the standard strain and transfer it onto the solid medium. Then use an L-shaped glass rod to spread the strain evenly. Place the culture dish containing the strain into a culture bag, and then put a microaerophilic gas-generating bag into the culture bag. Quickly seal the culture bag and place it in a 37°C constant temperature incubator for 72 hours. After the culture is completed, first identify Helicobacter pylori, and then scrape off the bacterial lawn on the solid medium and transfer it to 50 mL of liquid medium, which is used as the original bacterial solution.

[0096] 4. Determination of MIC

[0097] (1) Prepare the sample solutions of Compound 1 and 2 obtained in Example 1 with a concentration of 2 mg / mL.

[0098] (2) Preparation of the MIC plate: First, add 173.6 μL of the medium to the first well, and then add 6.4 μL of the antibacterial agent. Dilute it serially to the 7th well; do not add the drug to the 8th well and keep 90 μL of the medium as the control for adding bacteria without adding the drug.

[0099] (3) Preparation of the bacterial solution: Take the Helicobacter pylori growing in the logarithmic phase on the solid plate and make a bacterial suspension with BHI medium, and adjust the concentration to OD 600 to 0.3 (1×10 8 CFU / mL), and dilute it 10 times to 1×10 7 CFU / mL for standby.

[0100] (4) Inoculation of the bacterial solution: Add 10 μL to wells 1-8 (the concentration of the bacterial solution in each well is about 1.0×10 6 CFU / mL). Incubate for 72 hours to judge the results. The drug concentrations in wells 1 to 7 are 64, 32, 16, 8, 4, 2, and 1 μg / mL respectively.

[0101] (5) Result judgment: Take the lowest drug concentration that completely inhibits bacterial growth in the small well as the MIC. When the bacteria grow significantly in the positive control well 8 (i.e., without antibiotics), the test is meaningful. When there is a single skipped well in the microdilution method, the highest drug concentration that inhibits bacterial growth should be recorded. If there are multiple skipped wells, the result should not be reported and the test needs to be repeated. Each drug is tested 3 times.

[0102] 5. The experimental results are shown in Tables 3 and 4

[0103] Table 3 Inhibitory effects of Compound 1 and 2 on different strains (MIC: μg / mL)

[0104]

[0105]

[0106] MTZ, metronidazole; R, drug-resistant strain

[0107] Table 4 Synergistic inhibitory effects of Compound 1 and amphotericin B (AMB) on different strains (MIC: μg / mL)

[0108]

[0109] The above experimental results show that Compounds 1 and 2 have good antibacterial activities against one sensitive and one drug-resistant Helicobacter pylori strain, with MICs of 16 - 32 μg / mL, indicating that Compounds 1 and 2 have strong selective inhibitory effects on Helicobacter pylori. When used alone, Compound 1 has no significant inhibitory effect on the growth of two Candida albicans strains. However, when used in combination with AMB, it can significantly reduce its own MCI and lower the MCI of AMB to below 0.25 μg / mL, and can be used to prepare anti-Candida albicans drugs or as a lead compound for the development of anti-Candida albicans drugs.

Claims

1. A compound having a novel skeleton of ursane-type triterpene-enantiokaurane-type diterpene dimer, characterized in that: Including compounds 1 and 2 with the following structural formulas:

2. The method for preparing the compound according to claim 1, characterized in that: The following steps are involved: (1) Weigh the dried I. trichantha tubers, crush them, add ethanol water to reflux and extract the medicinal materials, collect the filtrate after filtering, and concentrate it under reduced pressure until there is no alcohol taste to obtain a concentrated solution; (2) extracting the concentrated solution obtained in step (1) with petroleum ether, dichloromethane, ethyl acetate and n-butanol, respectively, and concentrating under reduced pressure to obtain a petroleum ether fraction, a dichloromethane fraction, an ethyl acetate fraction, an n-butanol fraction and a raffinate fraction, respectively; (3) taking the dichloromethane fraction obtained in step (2), applying AB-8 macroporous resin to atmospheric pressure column chromatography, and eluting with ethanol-water to obtain three sub-fractions DCM-1 to DCM-3; (4) The eluate fraction DCM-3 obtained in step (3) was subjected to silica gel atmospheric pressure column chromatography and eluted with dichloromethane-methanol to obtain 7 sub-fractions DCM-3-1-DCM-3-7; (5) The fraction DCM-3-5 obtained in step (4) was subjected to medium pressure preparative MCI column chromatography with gradient elution and methanol-water as the mobile phase to obtain 11 fractions DCM-3-5-1-DCM-3-5-11; the fraction DCM-3-5-9 was subjected to semi-preparative HPLC to obtain compounds 1 and 2.

3. The method for preparing the compound according to claim 2, characterized in that: The following steps are involved: (1) Weigh the dried I. trichantha tubers, crush them, add ethanol with a volume concentration of 80-95% to reflux and extract the medicinal materials, with a solid-liquid ratio of 1:6-20, extract 1-3 times, each time for 1-3 hours, filter and collect the filtrate, combine the filtrates, and concentrate under reduced pressure until there is no alcohol taste 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 with equal volumes of petroleum ether, dichloromethane, ethyl acetate and n-butanol, each extracted 3 to 6 times, to obtain a petroleum ether fraction, a dichloromethane fraction, an ethyl acetate fraction, an n-butanol fraction and a raffinate fraction, respectively. (3) The dichloromethane fraction obtained in step (2) was mixed with a macroporous resin, and subjected to macroporous resin atmospheric pressure column chromatography, with ethanol-water as the mobile phase in a volume ratio of 30:70, 60:40, 90:10, and 100:0, respectively, and each gradient elution was 4 to 6 column volumes; after thin layer and ultra-high performance liquid chromatography analysis, the fractions were concentrated and combined using a rotary evaporator to obtain three sub-fractions DCM-1 to DCM-3; (4) The eluate fraction DCM-3 obtained in step (3) was mixed with silica gel, and then subjected to silica gel atmospheric pressure column chromatography, with dichloromethane-methanol as the mobile phase in a volume ratio of 100:0, 50:1, 30:1, 20:1, 10:1, 7:1, 5:1, 3:1, 2:1, 1:1, 1:3, 1:7, 0:100 as gradient elution, each gradient elution being 3 to 6 column volumes; after thin layer and ultra-performance liquid chromatography analysis, the fractions were concentrated and combined using a rotary evaporator to obtain 7 sub-fractions DCM-3-1-DCM-3-7; (5) The fraction DCM-3-5 obtained in step (4) was mixed with MCI and subjected to gradient elution by medium pressure preparative MCI column chromatography, with methanol-water as the mobile phase, wherein A is water and B is methanol; the elution gradient program is: 0.01-20.00 min, 30%-30% B; 20.00-50.00 min, 40%-40% B; 50.00-80.00 min, 45%-45% B; 80.00-110.00 min, 50%-50% B; 110.00-140.00 min, 55%-55% B; 140.00-170.00min, 60%-60% B; 170.00-200.00min, 70%-70% B; 200.00-230.00min, 85%-85% B; 230.00-270.00min, 100%-100% B, elution flow rate 20mL / min, detection wavelengths of 256nm and 310nm; after ultra-performance liquid chromatography analysis, concentrated and combined to obtain 11 fractions DCM-3-5-1-DCM-3-5-11; (6) The 200-220 min eluate fraction DCM-3-5-9 obtained in step (5) was subjected to semi-preparative high performance liquid chromatography using pure water A-methanol B as the mobile phase for isocratic elution to separate compound 1 and compound 2.

4. The method for preparing the compound according to claim 3, characterized in that: The chromatographic conditions of semi-preparative HPLC were as follows: the chromatographic column model was Hedera ODS preparative chromatographic column, with specifications of 10 nm, 5 μm, 10×250 mm, HPLC column pressure was 9.8 MPa, column temperature was 22-26°C, injection volume was 100 μL, mobile phase was MeOH and H2O in a volume ratio of 80:20, flow rate was 3 mL / min, and detection wavelength was 310 nm.

5. Use of the compound according to claim 1 in the preparation of anticancer drugs.

6. Use of the compound according to claim 1 in the preparation of drugs for treating pancreatic cancer, colon cancer, lung cancer or liver cancer.

7. Use of the compound according to claim 1 in the preparation of antibacterial and antifungal drugs.

8. Use of the compound according to claim 1 in the preparation of anti-Helicobacter pylori drugs.

9. Use of the compound according to claim 1 in combination with amphotericin B in the preparation of an anti-Candida albicans drug.

10. A pharmaceutical preparation, characterized in that The compound according to claim 1 and a pharmaceutically acceptable carrier are prepared into a pharmaceutical preparation.

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