An enantiomeric atisane diterpenoid compound, its preparation method and application

Abstract

The present invention belongs to the technical field of phytochemistry, and particularly relates to the isolation and identification of an ent-atisan diterpenoid compound from Isodon xerophilus, and naming it as xerophilsin K. The molecular formula of the compound is: C 20 H 30 O 3， The chemical structural formula is as follows: #imgabs0# The present invention also provides the ent-atisan diterpenoid compound, its preparation method and its application in cancer.

Description

Technical Field

[0001] The present invention belongs to the field of pharmaceutical technology, and particularly relates to an enantiomeric-atisanane diterpenoid compound, a preparation method thereof, and an application thereof. Background Art

[0002] Cancer has plagued humanity for centuries and is a leading cause of death in countries at all income levels around the world. In addition to the existing burden, with the growth of the population and the cancer-risk lifestyles that people have now, future cancer cases and deaths are expected to increase rapidly. Among them, breast cancer and cervical cancer are malignant diseases that seriously threaten women's health. Breast cancer is the second leading cause of cancer death in women worldwide after lung cancer. Therefore, it is very urgent to find drugs that can treat cancer and develop means for treating cancer, especially breast cancer and cervical cancer.

[0003] In recent years, more and more evidence has shown that natural products have broad application prospects in cancer prevention and treatment. Plants of the genus Isodon have been widely concerned as an anticancer medicinal plant. It has been found that many plants of the genus Isodon are used as drugs for treating cancer, infectious diseases, acute cholecystitis, hepatitis, sore throat, etc. in Chinese folk. Currently, more than 1200 diterpenoid compounds have been reported from the genus Isodon, and these diterpenoid compounds have important medicinal values in inflammation and anti-tumor aspects.

[0004] Isodon xerophilus is a plant of the genus Isodon unique to Yunnan Province, China, and is widely used by local people as an anti-tumor, antibacterial, and anti-inflammatory drug. Several enantiomeric-kaurane diterpenoids with significant biological activities, including xerophilusin A, xerophilusin B, and xerophilusin F, have been identified from this plant before.

[0005] The purpose of the present invention is to provide a new enantiomeric-atisanane diterpenoid compound xerophilsin K with anti-cancer activity, a preparation method thereof, and its application in anti-cancer activity, especially in breast cancer and cervical cancer. Summary of the Invention

[0006] The first aspect of the present invention provides a new enantiomeric-atisanane diterpenoid compound that has an inhibitory effect on human cancer cells and has not been reported relatedly so far.

[0007] The first aspect of the present invention provides an enantiomeric-atisanane diterpenoid compound, and the molecular formula of the compound is: C 20 H 30 O 3 , and the chemical structural formula is as follows:

[0008]

[0009] The compound is a colorless massive crystal (methanol) and is named xerophilsin K.

[0010] The second aspect of the present invention provides a method for preparing the enantiomeric-atisan diterpenoid compound described in the first aspect, which specifically includes the following steps:

[0011] (1) Extracting the extract: extracting the underground part of Rabdosia xerophila with an acetone / water solvent system to obtain an extract, concentrating the extract to remove acetone therein, and extracting with ethyl acetate, and concentrating the ethyl acetate phase under reduced pressure to obtain a first extract;

[0012] (2) Silica gel column chromatography: dry-packing a column with silica gel of 80 - 100 mesh for the first extract obtained in step (1) and performing silica gel column chromatography; performing gradient elution with a chloroform / acetone solvent system with volume ratios of 10:0, 9:1, 8:2, 7:3, 6:4, 5:5 respectively, combining the parts with the same polarity, collecting the eluates of each part and concentrating; collecting the eluate obtained when eluting with a chloroform / acetone solvent system with a volume ratio of 9:1, which is called the first eluate; concentrating the first eluate to obtain a second extract, and decolorizing the second extract with MCI using a first methanol / water solvent system to obtain a decolorized third extract; separating the third extract with an RP-18 chromatography column using a methanol / water solvent system with a volume ratio of 30:60 - 100:0, and collecting the eluate obtained when eluting with a methanol / water solvent system with a volume ratio of 90:10, which is called the second eluate; further separating the second eluate with a silica gel chromatography column, performing gradient elution with a petroleum ether / acetone solvent system with volume ratios of 9:1, 8:2, 7:3, 6:4, 5:5 respectively, and collecting the eluate obtained when eluting with a petroleum ether / acetone solvent with a volume ratio of 9:1, which is called the third eluate; performing Sephadex LH-20 on the third eluate and eluting with a chloroform / methanol solvent, dividing it into 5 parts, and obtaining the part with an Rf value of 0.49 when detected by TLC with a petroleum ether / acetone with a volume ratio of 8:2, which is called the fourth eluate; separating the fourth eluate with an RP-18 chromatography column using a second methanol / water system, dividing it into 9 parts, and obtaining the part with an Rf value of 0.49 when detected by TLC with a petroleum ether / acetone with a volume ratio of 8:2, which is called the fifth eluate; f =0.49, which is called the fourth eluate; separating the fourth eluate with an RP-18 chromatography column using a second methanol / water system, dividing it into 9 parts, and obtaining the part with an Rf value of 0.49 when detected by TLC with a petroleum ether / acetone with a volume ratio of 8:2, f =0.49, which is called the fifth eluate;

[0013] (3) High performance liquid chromatography separation: The fifth eluate obtained in step (2) is introduced into high performance liquid chromatography for separation and purification. The flow rate is 3.0 mL / min, the mobile phase is an acetonitrile / water solvent system with a volume ratio of 83:17, the detection wavelength of the ultraviolet detector is 202 nm, 30.0 μL of the fifth eluate is injected each time, and the eluate corresponding to the retention time of 26.7 min of the chromatographic peak after each injection is collected, which is called the sixth eluate. The enantiomeric atisane diterpenoid compound is obtained after removing the solvent from the sixth eluate.

[0014] Preferably, in step (1), the volume ratio of the acetone / water solvent system is 70:30, and extraction is carried out three times with ethyl acetate;

[0015] In step (2), the volume ratio of the first methanol / water solvent system is 90:10, the volume ratio of the chloroform / methanol solvent system is 5:5, and the volume ratio of the second methanol / water solvent system is 80:20.

[0016] The third aspect of the present invention provides a pharmaceutical composition, which mainly features including an active ingredient and a pharmaceutically acceptable carrier, and the active ingredient is the enantiomeric atisane diterpenoid compound described in the first aspect or a pharmaceutically acceptable salt.

[0017] The fourth aspect of the present invention provides the use of the compound or a pharmaceutically acceptable salt, or the pharmaceutical composition in the preparation of drugs for preventing and treating cancer.

[0018] Preferably, the cancer is cervical cancer or breast cancer. Description of the Drawings

[0019] Figure 1 It is the 1H NMR spectrum of the compound xerophilsin K(1) of the present invention at 500 MHz in CDN. 5 D 5 N, 500 MHz 1 1H NMR spectrum.

[0020] Figure 2 It is the 13C NMR spectrum of the compound xerophilsin K(1) of the present invention at 125 MHz in CDN. 5 D 5 N, 125 MHz 13 13C NMR spectrum.

[0021] Figure 3 It is the single crystal X-ray diffraction structure diagram of the compound xerophilsin K(1) of the present invention.

[0022] Figure 4 It is the graph of the inhibitory effect of the compound xerophilsin K(1) of the present invention on human cancer cells.

[0023] Figure 5 Figure showing that xerophilsin K induces apoptosis in HeLa cells.

[0024] Figure 6 Figure showing that xerophilsin K induces apoptosis in MDA-MB-231 cells. Detailed implementation manners

[0025] In order to make the technical means, creative features, achieved purposes and functions of the present invention easy to understand, the present invention will be further described below in conjunction with specific embodiments.

[0026] Example 1

[0027] Obtaining xerophilsin K(1)

[0028]

[0029] The present invention provides a method for extracting and isolating xerophilsin K(1) from the underground part of Isodon xerophilus. The specific steps are as follows:

[0030] Prepare a dry sample (8.0 kg) of the underground part of Isodon xerophilus. After pulverizing the sample, use an acetone / water solvent system (the volume ratio of the system is 70:30) to cold soak the sample four times at room temperature, about 25 L of the system solvent each time, 3 days per time, to obtain a combined extract;

[0031] Perform vacuum concentration on the extract to remove the acetone therein to obtain an aqueous suspension;

[0032] Extract the aqueous suspension three times with an equal volume of ethyl acetate, combine the ethyl acetate and perform vacuum concentration to obtain a first extract (120.0 g).

[0033] The ethyl acetate extract was dry-packed into a silica gel column with 80–100 mesh silica gel for silica gel column chromatography; gradient elution was carried out with chloroform / acetone solvent systems with volume ratios of 10:0, 9:1, 8:2, 7:3, 6:4, and 5:5 respectively. The parts with the same polarity were combined, and the eluents of each part were collected and concentrated to obtain seven fractions: Fractions A–G. The eluent obtained when eluting with a chloroform / acetone solvent system with a volume ratio of 9:1 was collected, Fraction C, also known as the first eluent. The eluent of Fraction C was concentrated to obtain the second extract. The second extract was decolorized by MCI using a methanol / water system (90:10, v / v) to obtain 26.0 g of the third extract; this 26.0 g of extract was further separated using an RP-18 chromatography column with a methanol / water system (30:60–100:0, v / v) and divided into 3 parts: C1–C3; the eluent C3 (12.0 g) obtained when eluting with a methanol / water solvent system (90:10, v / v) was collected, also known as the second eluent. C3 was further separated using a silica gel chromatography column, and gradient elution was carried out with a petroleum ether / acetone solvent system (9:1, 8:2, 7:3, 6:4, 5:5, v / v) and divided into 5 parts: C3-1–C3-5; the eluent C3-3 (3.6 g) obtained when eluting with a petroleum ether / acetone solvent system with a volume ratio of 9:1 was collected, also known as the third eluent; C3-3 was eluted with Sephadex LH-20 (chloroform / methanol, 5:5, v / v) and divided into 5 parts: C3-3-1–C3-3-5; for C3-3-2 (also known as the fourth eluent, when detected by TLC with a petroleum ether / acetone volume ratio of 8:2, R f = 0.49), it was further separated using an RP-18 chromatography column with a methanol / water system (80:20, v / v). When the solvent was eluted, the eluent was divided into 9 parts: C3-3-2-1–C3-3-2-9; among them, C3-3-2-1 (680.0 mg, also known as the fifth eluent. When detected by TLC with a petroleum ether / acetone volume ratio of 8:2, R f = 0.49), by semi-preparative HPLC, the flow rate was 3.0 mL / min, the mobile phase was an acetonitrile / water solvent system (83:17, v / v), detector UVλmax = 202 nm, and 30.0 μL of the C3-3-2-1 eluate was injected each time. The eluent corresponding to the retention time of 26.7 min of the chromatographic peak after each injection was collected to obtain Compound 1 (50.0 mg).

[0034] The structure characterization data of Compound 1 are as Figures 1 to 3 shown.

[0035] Xerophilsin K(1): Colorless massive crystals (methanol); m.p. 291.1 °C; [α] 19 D –76.9 (c 0.1, MeOH); UV (MeOH) λ max (logε) 324 (1.89), 230 (3.88), 204 (3.67), 195 (3.75) nm; IR (KBr) ν max 3559, 3527, 2948, 2870, 1684, 1617, 1482, 1438, 1390, 1229, 1162, 1075, 1032 cm –1 ; 1 H and 13 CNMR data; positive HRESIMS [M+Na] + m / z 341.2093 (calcd. for C 20 H 30 O 3 Na, 341.2087).

[0036] Among them, the 1 H NMR and 13 C NMR data (CDCl 3 ) of compound 1 are shown in Table 1 specifically.

[0037] Table 1

[0038]

[0039] X-ray crystal structure analysis. Crystals of compound 1 were obtained in MeOH. Intensity data were collected at 100 K using Cu Kα radiation on a Bruker APEX DUO diffractometer equipped with an APEX II CCD. Cell refinement and data reduction were performed using Bruker SAINT. The structure was solved by the direct method using SHELXS-97 and refined by full-matrix least-squares using anisotropic displacement parameters for non-hydrogen atoms. Hydrogen atoms were placed at calculated positions and refined using a riding model. The crystallographic data (excluding structure factor tables) for the structure of compound 1 have been deposited with the Cambridge Crystallographic Data Centre (CCDC) as Supplementary Publication No. CCDC 1999372. Copies of the data can be obtained free of charge from the CCDC, 12 Union Road, Cambridge CB 1EZ, UK [fax: International +44(0)(1223)336 033]; e-mail: deposit@ccdc.cam.ac.uk].[[]]

[0040] Xerophilsin K(1) crystal data: C 20 H30 O 3 , M = 652.92, α = 90°, β = 90°, γ = 90°, T = 100(2) K, space group P212121, Z = 4, μ(CuKα) = 0.624 mm -1 , measuring 17388 reflections, 6014 independent reflections (R int = 0.1114). The final R 1 value is 0.0703 (I > 2σ(I)). The final wR(F 2 ) value is 0.1482 (I > 2σ(I)). The final R 1 value is 0.1559 (all data). The final wR(F 2 ) value is 0.1885 (all data). The goodness of fit of F 2 is 1.102. The Flack parameter = 0.02(14).

[0041] The structure of the enantiomeric-atisanane diterpenoid compound prepared by the above method was determined by the following method:

[0042] Visual observation found that: The compound of the present invention is a colorless block crystal (methanol);

[0043] Infrared spectrum (KBr tablet) showed that there were hydroxyl groups (3559 cm –1 ), α,β-unsaturated ketone (1684 and 1617 cm –1 ) characteristic functional groups in the compound; High-resolution mass spectrometry (HRESIMS) gave the quasi-molecular ion peak 341.2093 [M+Na] + (calcd. 341.2087), which can determine that the molecular formula of the compound is C 20 H 30 O 3 . In the 1 H NMR spectrum of the compound, there were 2 singlet methyl signals δ H 1.03 and 1.30; 1 oxidized methine signal δ H 4.11 (d, J = 3.4 Hz); and 2 olefinic proton signals δ H 5.21 (d, J = 1.8 Hz) and 6.06 (d, J = 1.8 Hz). In the 13In the \(^{13}\)C NMR and DEPT spectra, it was found that the compound had 20 carbon atoms, including 2 methyl groups, 9 methylene groups (one oxidized methylene group and one terminal alkene), 4 methine groups (one oxidized methine group), 3 quaternary carbons, 1 protonless olefinic carbon atom, and 1 ketone carbonyl carbon atom. By analyzing the 1D NMR spectral data of the compound, it was found that the compound was very similar to the alboatisins C compound.

[0044] Comparing the 1D NMR spectral data of this compound with those of alboatisins C, it was found that their data were very similar. However, in alboatisins C, the C-13 position was an oxidized methine group, and its C-18 position was an oxidized methylene group. In the compound of the present invention, the corresponding positions were a methylene signal (δ c 28.7, t) and a methyl group (δ c 28.1, q), respectively. The C-19 methyl group of the compound alboatisins C was an oxidized methylene group (δ c 65.0, t) in the compound of the present invention. Further, through 1 H– 1 H COSY and HMBC and other two-dimensional NMR correlation signals, the planar structure of the compound could be determined. Single crystals of the compound were obtained by various single crystal cultivation methods, and the crystal data of the compound were obtained by single crystal X-ray diffraction with a copper target, and the Flack parameter was [0.02(14)], confirming that the absolute configuration of the compound was 4R, 5S, 7S, 8R, 9S, 10R, 12S.

[0045] The 1 H– 1 H COSY and HMBC key correlations of compound 1 were as follows:

[0046]

[0047] The single crystal X-ray diffraction structure of compound 1 was as Figure 3 shown.

[0048] In summary, the structure of the compound was confirmed to be 7β,19-dihydroxyent-atis-16-en-15-one, and it was named xerophilsin K.

[0049] Example 2

[0050] Cytotoxicity of xerophilsin K against various human cancer cell lines

[0051] The cytotoxicity of xerophilsin K obtained in Example 1 was tested by the Cell Counting Kit 8 (CCK8) method.

[0052] Cytotoxicity screening of xerophilsin K at different concentrations (5.0, 10.0, and 20.0 μM) was performed on a variety of human cancer cell lines, including HeLa (human cervical cancer cells), MDA-MB-231 (human triple-negative breast cancer cells), MCF7 (human Luminal A breast cancer cells), A549 (human lung cancer cells), SMMC-7721 (human liver cancer cells), U87 (human glioblastoma cells), HepG2 (human liver cancer cells), and SKOV3 (human ovarian cancer cells).

[0053] Specifically, the above various cell lines were seeded in 96-well plates and then returned to the incubator for 6.0 - 8.0 hours until the cells adhered. The corresponding concentration of xerophilsin K was added to the culture plates, and the same volume of DMSO was added to the control group. Then, it was returned to the incubator for incubation for 24.0 hours, and 3 replicate wells were set for each experimental concentration condition. 10.0 μL of CCK8 reagent was added to each well of the 96-well culture plate. After adding, the 96-well plate was gently shaken and then returned to the incubator for incubation. After incubation for 1.0 - 2.0 hours, when the control group showed orange-yellow color, the absorbance at 450 nm was detected using an enzyme-linked immunosorbent assay (ELISA) reader to obtain the experimental results.

[0054] The following formula was used to calculate the cell viability: Cell viability = (absorbance of the experimental group / absorbance of the control group) × 100%. The cytotoxicity results of xerophilsin K on each cell line are specifically shown in Table 2 and Figure 4 as shown.

[0055] Table 2

[0056]

[0057] The IC 50 values of xerophilsin K on Hela cells and MDA-MB-231 cells were calculated using GraphPad Prism 5.0 software. The specific IC 50 values are shown in Table 3.

[0058] Table 3

[0059]

[0060] As Figure 4As shown, xerophilsin K has strong cytotoxicity against HeLa (human cervical cancer cells), MDA-MB-231 (human triple-negative breast cancer cells), and MCF7 (human breast cancer cells). When the concentration is 5.0 μM, compared with the control group, the cell survival rate can be reduced to less than 50%. Among them, the toxicity to HeLa (human cervical cancer cells) and MDA-MB-231 (human triple-negative breast cancer cells) is more significant.

[0061] Example 3

[0062] Effect of xerophilsin K on apoptosis of HeLa cells and MDA-MB-231 cells

[0063] The experimental method is as follows: Seed HeLa and MDA-MB-231 cells in 6-well plates. Use a cell counting chamber to prepare a cell density of 4×10 4 cells / well, with a cell suspension volume of 2.0 mL / well, and allow the cells to adhere for approximately 6.0–8.0 hours. Add the corresponding concentrations of xerophilsin K (2.5 mM, 5.0 mM, 7.5 mM, 10.0 mM) to the culture plates. Add the same volume of DMSO to the control group. Then, place the plates back in the incubator and incubate for 24.0 hours. Collect the supernatant medium into 5.0 mL centrifuge tubes. After rinsing the cell surface with PBS, collect the PBS into the above 5.0 mL centrifuge tubes. Then, digest the cells with trypsin without EDTA and collect the cells into the above 5.0 mL centrifuge tubes, and wash the cells twice with PBS. Add 100.0 μL of binding buffer from the cell apoptosis detection kit to each centrifuge tube and resuspend the cells. Then, add 2.5 μL of Annexin V and 2.5 μL of PI to each centrifuge tube and mix well. Incubate the above samples at 37.0 °C for 15.0 min, gently invert the centrifuge tubes up and down every 5.0 min during this period to ensure uniform staining. After incubation, add 400.0 μL of binding buffer to each centrifuge tube and mix well. Detect the fluorescence intensity of the cells using a flow cytometer at excitation wavelengths of 488 nm and 561 nm.

[0064] The results are as Figure 5 and Figure 6 shown. As the concentration of xerophilsin K increases, the cell count in the Q2 and Q3 regions of HeLa and MDA-MB-231 cells shows a concentration-dependent increase, and obvious apoptosis occurs in HeLa cells and MDA-MB-231 cells. Among them, the cells in the Q1 region in the figure are mechanically damaged cells; the Q2 region is late apoptotic cells; the Q3 region is early apoptotic cells; the Q4 region is live cells.

[0065] In this specification, the invention has been described with reference to specific embodiments thereof. However, it will be apparent that various modifications and variations can be made without departing from the spirit and scope of the invention. Accordingly, the specification and drawings are to be regarded as illustrative rather than restrictive.

Claims

1. An enantiomeric atisane diterpenoid compound, characterized in that, The molecular formula of the described compound is: C 20 H 30 O 3 , and the described compound is named xerophilsin K, and its chemical structural formula is as follows:

2. A method for preparing the enantiomeric atisane diterpenoid compound according to claim 1, characterized in that, the method comprises the steps of: (1) Extracting the extract: extracting the underground part of Isodon xerophilus with an acetone / water solvent system to obtain an extract, concentrating the extract to remove acetone therein, and extracting with ethyl acetate, and concentrating the ethyl acetate phase under reduced pressure to obtain a first extract; (2) Silica gel column chromatography: The first extract obtained in step (1) is dry-packed into a column with 80–100 mesh silica gel for silica gel column chromatography; gradient elution is carried out with chloroform / acetone solvent systems with volume ratios of 10:0, 9:1, 8:2, 7:3, 6:4, and 5:5 respectively. The parts with the same polarity are combined, and the eluates of each part are collected and concentrated; the eluate obtained when eluting with a chloroform / acetone solvent system with a volume ratio of 9:1 is collected and called the first eluate; the first eluate is concentrated to obtain a second extract, and the second extract is decolorized by MCI using a first methanol / water solvent system to obtain a third extract; the third extract is separated continuously with an RP-18 chromatography column using a methanol / water solvent system with a volume ratio of 30:60–100:0, and the eluate obtained when eluting with a methanol / water solvent system with a volume ratio of 90:10 is collected and called the second eluate; the second eluate is separated continuously with a silica gel chromatography column, and gradient elution is carried out with petroleum ether / acetone solvent systems with volume ratios of 9:1, 8:2, 7:3, 6:4, and 5:5 respectively. The eluate obtained when eluting with a petroleum ether / acetone solvent system with a volume ratio of 9:1 is collected and called the third eluate; the third eluate is subjected to Sephadex LH-20 and eluted with a chloroform / methanol solvent system, and it is divided into 5 parts, and the part with an R f value of 0.49 when detected by TLC with a petroleum ether / acetone with a volume ratio of 8:2 is obtained and called the fourth eluate; the fourth eluate is separated continuously with an RP-18 chromatography column using a second methanol / water solvent system, and it is divided into 9 parts, and the part with an R f value of 0.49 when detected by TLC with a petroleum ether / acetone with a volume ratio of 8:2 is obtained and called the fifth eluate; (3) High performance liquid chromatography separation: passing the fifth eluate finally obtained in step (2) through high performance liquid chromatography for separation and purification, with a flow rate of 3.0 mL / min, a mobile phase of an acetonitrile / water solvent system with a volume ratio of 83:17, a detection wavelength of 202 nm for the ultraviolet detector, injecting 30.0 μL of the fifth eluate each time, collecting the eluate corresponding to a retention time of 26.7 min of the chromatographic peak after each injection, called the sixth eluate, and removing the solvent from the sixth eluate to obtain the enantiomeric atisane diterpenoid compound.

3. According to the preparation method described in claim 2, characterized in that, in the step (1), the volume ratio of the acetone / water solvent system is 70:30, and extracting with ethyl acetate three times; in the step (2), the volume ratio of the first methanol / water solvent system is 90:10, the volume ratio of the chloroform / methanol solvent system is 5:5, and the volume ratio of the second methanol / water solvent system is 80:

20.

4. A pharmaceutical composition, characterized in that, it comprises an active ingredient and a pharmaceutically acceptable carrier, and the active ingredient is the compound according to claim 1 or a pharmaceutically acceptable salt.

5. Use of the compound according to claim 1 or a pharmaceutically acceptable salt, or the pharmaceutical composition according to claim 4 in the preparation of a drug for preventing and treating cancer, wherein the cancer is cervical cancer or triple negative breast cancer.

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