A diterpene glycoside compound, preparation method and application

Abstract

The present application provides a diterpene glycoside compound, a preparation method and an application thereof, belonging to the field of pharmaceutical biotechnology. Lemnalia The diterpene glycosides isolated and extracted from soft corals of the genus genus have not been reported in existing journal articles regarding their molecular structure, isolation method, and pharmacological activity. The diterpene glycoside compounds isolated and purified in this application have anti-tumor effects and inhibitory activity against the drug-resistant human small cell lung cancer cell line NCI-H446 / EP. They can be used to develop candidate molecules for the treatment of this disease and can also serve as lead compounds for the chemical synthesis and structural modification of other drugs. The preparation method provided in this application is simple and rapid, and the extracted diterpene glycoside compounds are of high purity.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine and relates to a diterpene glycoside compound, a preparation method and an application thereof. Background Art

[0002] Compared with terrestrial organisms, the living environment of marine organisms is characterized by high salinity, high pressure, low oxygen, and low light, covering various ecological environments such as intertidal zones, polar regions, deep sea abysses, hydrothermal vents, and cold springs. This has shaped the particularity of marine organisms in metabolism, survival methods, information transmission, and adaptation mechanisms.

[0003] Soft corals are lower marine invertebrates with a large number and variety. The secondary metabolites they produce are also diverse, and they have great potential for marine medicinal development. Lemnalia Soft corals are widely distributed in marine ecosystems. Their primary secondary metabolites are terpenoids, including sesquiterpenes, diterpenes, and diterpenoid glycosides. These secondary metabolites possess diverse and promising biological activities, including antibacterial, antiviral, neuroprotective, and cytotoxic activities. Therefore, research on marine organisms, including soft corals, can yield structurally diverse natural products and discover monomeric compounds with excellent biological activity, providing valuable candidate molecules for drug development and synthesis, while also providing a scientific basis for the rational development and utilization of marine biological resources.

[0004] Lung cancer is a common malignant tumor that poses a serious threat to human health. It can be divided into non-small cell lung cancer and small cell lung cancer based on its histological characteristics. Small cell lung cancer is a fast-growing, easily metastatic neuroendocrine tumor of the lung with an extremely high mortality rate. Currently, the clinical treatment of small cell lung cancer generally uses a combination of chemotherapy and radiotherapy. This method usually responds well in the early stages of treatment, but patients quickly develop drug resistance and relapse. Clinical testing of early-stage targeted drugs has been less than ideal, and new targeted drugs and effective treatments for small cell lung cancer are urgently needed. Summary of the Invention

[0005] The purpose of the present invention is to provide a diterpene glycoside compound, a preparation method and an application thereof, so as to solve the problem that existing treatment methods for small cell lung cancer are prone to drug resistance and recurrence.

[0006] To achieve the above object, the present invention adopts the following technical solutions:

[0007] In the first aspect, the present application provides a diterpene glycoside compound, which is extracted from soft coral, especially Lemnalia Belongs to soft coral. The diterpene glycoside compounds in this application are named lemnaliside A , which is a colorless transparent oil with the molecular formula C 26 H36 O6, molecular structure formula is:

[0008] .

[0009] In the second aspect, the present application provides a diterpene glycoside compound lemnaliside A A preparation method comprising:

[0010] S01: The soft coral is chopped and soaked in methanol at room temperature to obtain an extract, which is then concentrated under reduced pressure, desalted with anhydrous methanol, and concentrated under reduced pressure again to obtain a crude extract.

[0011] Store frozen Lemnalia Soft corals were thawed at room temperature and chopped into fingernail-sized pieces. The chopped soft corals were then soaked in room-temperature methanol six times, each soaking for three days. After each soaking, all the soaking solutions were filtered and combined to obtain an extract. The extract was concentrated under reduced pressure at 37°C and 0.1 MPa to a paste. Anhydrous methanol was added to the concentrated paste to redissolve it. After filtration and desalination, the extract was concentrated under reduced pressure at 37°C and 0.1 MPa to obtain a crude extract.

[0012] S02: The crude extract was separated by silica gel vacuum column chromatography, gradient elution, and thin layer chromatography color detection, and the same fractions were combined and concentrated to obtain 16 primary fractions F1-F16.

[0013] The crude extract was chromatographed on a silica gel vacuum column with a column size of 160 mm * 500 mm and a silica gel particle size of 200-300 mesh. The separated products were gradient eluted with petroleum ether, petroleum ether / acetone with a volume ratio of 100 / 1, 50 / 1, 30 / 1, 20 / 1, 10 / 1, 5 / 1, 3 / 1, 2 / 1, and 1 / 1, dichloromethane, and dichloromethane / methanol with a volume ratio of 10 / 1, 5 / 1, 3 / 1, 2 / 1, 1 / 1, and 0 / 1. The eluted fractions were detected by thin layer chromatography. The same fractions were combined and concentrated to obtain 16 primary fractions F1-F16. The thin layer chromatography detection conditions were as follows: the developing solvent was petroleum ether and propanol in a volume ratio of 3:1, and the color developer was a 10% sulfuric acid ethanol solution.

[0014] S03: After the F16 in the first-level fractions F1-F16 is separated by silica gel column chromatography, gradient elution, and thin layer chromatography color detection, the same fractions are combined and concentrated to obtain 6 second-level fractions F161-F166.

[0015] Component F16 from the primary fractions F1-F16 was chromatographed using a 60 mm x 500 mm silica gel column with 300-400 mesh particles. The separated products were eluted using a gradient of dichloromethane / methanol in a volume ratio of 10 / 1, 5 / 1, 3 / 1, and 1 / 1, respectively. The eluted fractions were analyzed by thin-layer chromatography. Identical fractions were combined and concentrated to obtain six secondary fractions F161-F166. The thin-layer chromatography detection conditions were: a 3:1 volume ratio of petroleum ether and propanol as the developing solvent, and a 10% ethanolic sulfuric acid solution as the color developer.

[0016] S04: F165 in the secondary components F161-F166 is subjected to high performance liquid chromatography analysis, medium pressure preparative liquid chromatography separation, and isocratic elution to obtain four tertiary components F1651-F1654.

[0017] The component F165 in the secondary components F161-F166 was subjected to HPLC analysis to determine that the separation and preparation chromatographic conditions were methanol / water with a volume ratio of 80 / 20, wherein the HPLC analysis conditions were: the analytical column was 4.6 mm*250 mm, the particle size was 5 μm, and the ODS C 18 Column, flow rate 1ml / min. Component F165 was separated using a medium-pressure preparative liquid chromatograph. The separated product was isocratically eluted with methanol / water at a volume ratio of 80 / 20 to obtain four tertiary components F1651-F1654. According to the retention time in the HPLC chart of component F165, the following were determined: 0.00-7.60min for component F1651; 7.60-12.50min for component F1652; 12.50-17.50min for component F1653; and 17.50-25.00min for component F1654. The medium-pressure preparative liquid chromatography separation conditions were as follows: the preparative column was 20mm*250mm, and the particle size was 5μm. 18 column, with a flow rate of 10 ml / min.

[0018] S05: F1654 in the tertiary components F1651-F1654 is subjected to high performance liquid chromatography analysis, high performance preparative liquid chromatography separation, and isocratic elution to obtain a diterpene glycoside compound.

[0019] The component F1654 in the tertiary components F1651-F1654 was purified by high performance liquid chromatography to determine that the separation and preparation chromatography conditions were acetonitrile / water with a volume ratio of 35 / 65. Among them, the high performance liquid chromatography analysis conditions were: the analytical column was 4.6mm*250mm, the particle size was 5μm, and the ODS C 18Column, flow rate is 1ml / min. Component F1654 was separated and purified by high performance preparative liquid chromatography, and the separated product was isocratically eluted with acetonitrile / water with a volume ratio of 35 / 65 at a retention time of 127min to obtain diterpene glycoside compounds. lemnaliside A The separation conditions of HPLC were as follows: the chromatographic column was 10 mm*250 mm, the particle size was 5 μm, and the ODS C 18 column, with a flow rate of 2 ml / min.

[0020] In the third aspect, the diterpene glycoside compounds prepared in this application lemnaliside A Used to prepare drugs for treating drug-resistant human small cell lung cancer.

[0021] The present invention has the following beneficial effects:

[0022] (1) This application is the first Lemnalia Diterpenoid glycosides were isolated from soft corals of the genus lemnaliside A , the diterpene glycoside compound lemnaliside A Its molecular structure, isolation method and pharmacological activity research have not been reported in existing journal articles.

[0023] (2) The diterpene glycoside compound lemnaliside A It has anti-tumor effects and has inhibitory activity against the drug-resistant human small cell lung cancer cell line NCI-H446 / EP. It can be used to develop candidate molecules for the treatment of the disease and can also be used as a lead compound for the chemical synthesis and structural modification of other drugs.

[0024] (3) The diterpene glycoside compound lemnaliside A The preparation method is simple and rapid, and the extracted diterpene glycoside compounds are of high purity. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 The diterpene glycoside compound prepared in Example 1 of the present application lemnaliside A HPLC analysis chromatogram of F165 component during separation;

[0026] Figure 2 The diterpene glycoside compound prepared in Example 1 of the present application lemnaliside A UV (Ultraviolet Visible Absorption Spectroscopy) graph;

[0027] Figure 3 The diterpene glycoside compound prepared in Example 1 of the present application lemnaliside A IR (Infrared Spectroscopy) graph;

[0028] Figure 4 The diterpene glycoside compound prepared in Example 1 of the present application lemnaliside A HRESIMS (high resolution electrospray ionization mass spectroscopy) map;

[0029] Figure 5 The diterpene glycoside compound prepared in Example 1 of the present application lemnaliside A of 1 H-NMR (Nuclear Magnetic Resonance Spectroscopy of Hydrogen) spectrum;

[0030] Figure 6 The diterpene glycoside compound prepared in Example 1 of the present application lemnaliside A of 13 C-NMR (Carbon-13 nuclear magnetic resonance) spectrum;

[0031] Figure 7 The diterpene glycoside compound prepared in Example 1 of the present application lemnaliside A HSQC (heteronuclear singular quantum correlation) diagram;

[0032] Figure 8 The diterpene glycoside compound prepared in Example 1 of the present application lemnaliside A HMBC (1H detected heteronuclear multiple bond correlation) diagram;

[0033] Figure 9 The diterpene glycoside compound prepared in Example 1 of the present application lemnaliside A 1H-1H COSY (Homonuclear Chemical Shift Correlation Spectroscopy, proton correlation spectrum) diagram;

[0034] Figure 10 The diterpene glycoside compound prepared in Example 1 of the present application lemnaliside A Comparison of CD values ​​calculated by TDDFT-ECD and tested experimentally. DETAILED DESCRIPTION

[0035] The technical solution of the present invention is further explained and illustrated by means of specific embodiments below.

[0036] Example 1

[0037] The present invention provides a diterpene glycoside compound. lemnaliside A , the diterpene glycoside compound lemnaliside A It is a colorless, transparent oil with the molecular formula C 26 H 36 O6, molecular structure formula is:

[0038] .

[0039] Example 2

[0040] The present invention provides a diterpene glycoside compound. lemnaliside A A preparation method comprising:

[0041] S201: The frozen Lemnalia Soft corals were thawed at room temperature and chopped into fingernail-sized pieces. The chopped soft corals were then soaked in room-temperature methanol six times, each soaking for three days. After each soaking, all the soaking solutions were filtered and combined to obtain an extract. The extract was concentrated under reduced pressure at 37°C and 0.1 MPa to a paste. Anhydrous methanol was added to the concentrated paste to redissolve it. After filtration and desalination, the extract was concentrated under reduced pressure at 37°C and 0.1 MPa to obtain a crude extract.

[0042] S202: The crude extract was chromatographed on a silica gel vacuum column with a column size of 160 mm * 500 mm and a silica gel particle size of 200-300 mesh. The separated products were gradient eluted with petroleum ether, petroleum ether / acetone with a volume ratio of 100 / 1, 50 / 1, 30 / 1, 20 / 1, 10 / 1, 5 / 1, 3 / 1, 2 / 1, and 1 / 1, dichloromethane, and dichloromethane / methanol with a volume ratio of 10 / 1, 5 / 1, 3 / 1, 2 / 1, 1 / 1, and 0 / 1. The eluted fractions were subjected to thin layer chromatography for color detection. The fractions with the same fractions were combined and concentrated to obtain 16 primary fractions F1-F16. The thin layer chromatography color detection conditions were as follows: the developing solvent was petroleum ether and propanol with a volume ratio of 3:1, and the color developer was a sulfuric acid ethanol solution with a mass concentration of 10%.

[0043] S203: Component F16 from the first-stage fractions F1-F16 was chromatographed using a 60 mm x 500 mm silica gel column with 300-400 mesh silica particles. The separated products were eluted using a gradient elution of dichloromethane / methanol in a volume ratio of 10 / 1, 5 / 1, 3 / 1, and 1 / 1, respectively. The eluted fractions were analyzed by thin-layer chromatography. Identical fractions were combined and concentrated to obtain six second-stage fractions F161-F166. The thin-layer chromatography detection conditions were as follows: the developing solvent was petroleum ether and propanol in a volume ratio of 3:1, and the color developer was a 10% sulfuric acid-ethanol solution.

[0044] S204: Analyze the component F165 in the secondary components F161-F166 by high performance liquid chromatography to determine the separation and preparation chromatography conditions are methanol / water with a volume ratio of 80 / 20, and obtain the attached Figure 1 The HPLC analysis conditions are as follows: the analytical column is 4.6 mm*250 mm, the particle size is 5 μm, and the ODS C 18 Column, flow rate is 1ml / min. Figure 1 The HPLC chromatogram shown shows that component F165 was separated using a medium-pressure preparative liquid chromatograph. The separated product was isocratically eluted with methanol / water at a volume ratio of 80 / 20 to obtain four tertiary components F1651-F1654. Specifically, based on the retention times, the four tertiary components F1651-F1654 were determined as follows: 0.00-7.60 min for component F1651; 7.60-12.50 min for component F1652; 12.50-17.50 min for component F1653; and 17.50-25.00 min for component F1654. The medium-pressure preparative liquid chromatography separation conditions were: a 20 mm x 250 mm preparative column with a 5 μm particle size of Green Baicao ODS C 18 column, with a flow rate of 10 ml / min.

[0045] S205: Component F1654 in the tertiary components F1651-F1654 was subjected to HPLC purification analysis to determine that the separation and preparation chromatographic conditions were acetonitrile / water with a volume ratio of 35 / 65. The HPLC analysis conditions were: the analytical column was 4.6 mm*250 mm, the particle size was 5 μm, and the ODS C 18 Column, flow rate is 1ml / min. Component F1654 was separated and purified by high performance preparative liquid chromatography, and the separated product was isocratically eluted with acetonitrile / water with a volume ratio of 35 / 65 at a retention time of 127min to obtain diterpene glycoside compounds. lemnaliside A The separation conditions of HPLC were as follows: the chromatographic column was 10 mm*250 mm, the particle size was 5 μm, and the ODS C18 column, with a flow rate of 2 ml / min.

[0046] Example 3

[0047] To determine the diterpene glycoside compounds extracted by the preparation method provided in Example 2 lemnaliside A The structure of the diterpene glycoside compound extracted from Example 2 is lemnaliside A UV spectroscopy, infrared spectroscopy, high resolution mass spectrometry, 1 H-NMR, 13 C-NMR, HSQC, HMBC, 1 H- 1 The absolute configuration was determined by H COSY detection and ECD calculation. Figure 2-10 and the 1D and 2D NMR data shown in Table 1 .

[0048] By the attached Figure 2-4 It can be seen that diterpene glycosides lemnaliside A middle,[ α ] 25 D -30.1( c 1.0, MeOH);ECD ( c 0.5, MeOH) = Δ ε 195 -1.56, Δ ε 208 –0.81, Δ ε 224 +4.45; UV (MeOH) λ max (log ε )=231(1.78)nm; IR (KBr) ν max = 3421, 1631, 1592, 1369 cm -1 ; HRESIMS m / z 489.2499[M +CH2O2- H] - , the calculated value is C 27 H 37 O8, 489.2494, which suggests that the molecular formula of the compound is C 26 H 36 O6, unsaturation is 6.

[0049] Table 1: Diterpene glycosides lemnaliside A 1D and 2D NMR data (CDOD3, 500MHz)

[0050]

[0051] By the attached Figure 5 、 7 It can be seen that in the low field region δ H 7.94, m; δ H 7.93, m; δ H 7.36, d, J = 9.2 Hz; δ H 7.24, m; δ H 7.22, m total 5 benzene ring olefin hydrogen proton signals, in the high field region there are δ H 2.63, s; δ H 2.55, s total 2 singlet methyl signals and δ H 1.34, d, J = 6.9 Hz; δ H 0.84, d, J = 6.8 Hz, with two double-peak methyl signals. Figure 6 、 8 It can be seen that the compound has 26 carbon signals, 6 of which are a sugar unit structure, that is δ C / δ H 103.1 / 4.78,77.5 / 3.49, 78.1 / 3.47, 67.5 / 4.01, 82.0 / 3.70, 68.7 / 3.88 / 3.45. The remaining 20 carbon signals, combined with the characteristics of the number of methyl groups and olefinic hydrogen numbers, can be inferred to be diterpene structure fragments.

[0052] Furthermore, the planar structure was analyzed by two-dimensional nuclear magnetic resonance data. Figure 9 Continuous proton-related signals and Figure 8 Related signals can build compounds lemnaliside A The planar structure of the compound is compared with the known compounds of this type. lemnaliside A The difference is that the decahydronaphthalene ring of its diterpene part is changed to naphthalene.

[0053] For compounds lemnaliside A The configuration is determined by comparing the NMR data with the same type of compounds with single crystal structures and considering the biosynthesis. lemnaliside A The glycoside part is β -D-glucose, C-15 and C-16 positions are RConfiguration, determination of the configuration of the diterpene part, the compound only has the configuration of the C-11 position that needs to be determined, combined with the perspective of biosynthesis, it can be determined that the configuration of the diterpene part C-11 is 11 S . Finally passed Figure 10 The TDDFT-ECD calculations shown in the figure are compared with the experimental CD values ​​to verify the compound lemnaliside A The absolute configuration of .

[0054] Example 4

[0055] The diterpene glycoside compound provided in Example 1 of the present application lemnaliside A Can be used as candidate molecules for the preparation of anti-tumor drugs, i.e. compounds lemnaliside A It has significant inhibitory activity against drug-resistant human small cell lung cancer cell line NCI-H446 / EP. The details are as follows:

[0056] The diterpene glycoside compounds provided in Example 1 were tested by MTT method lemnaliside A The inhibitory effect on human chronic myeloid leukemia cells K562 was also tested by SRB method. lemnaliside A The cytotoxic activity of the active compound against human normal liver cells L-02, human metastatic pancreatic adenocarcinoma cells ASPC-1, human breast cancer cells MDA-MB-231, human small cell lung cancer cells NCI-H446, and drug-resistant human small cell lung cancer cells NCI-H446 / EP, where the positive drug is doxorubicin, is shown in Tables 2 and 3, respectively.

[0057] Table 2: Diterpene glycosides lemnaliside A Inhibition rate of the initial screening of anti-tumor activity %

[0058]

[0059] Table 3: Diterpene glycosides lemnaliside A Rescreening results of inhibitory effects on NCI-H446 / EP cells

[0060]

[0061] As can be seen from Tables 1 and 2, diterpene glycoside compounds lemnaliside A It has a selective inhibitory effect on the drug-resistant human small cell lung cancer cell line NCI-H446 / EP, and its IC 50 The value is 17.75 µ M, the diterpene glycoside compound lemnaliside A The invention can be used for preparing medicine for treating drug-resistant human small cell lung cancer.

[0062] The above description is merely a preferred embodiment of the present application and is not intended to limit the present application. Various modifications and variations are possible for those skilled in the art. Any modifications, equivalent substitutions, or improvements made within the spirit and principles of the present application shall be included within the scope of protection of the present application.

Claims

1. A diterpene glycoside compound, characterized in that: The chemical structural formula of the diterpene glycoside compound is: 。 2. The method for preparing the diterpene glycoside compound according to claim 1, wherein include: S01: Soft corals are chopped and soaked in methanol at room temperature to obtain an extract, which is then concentrated under reduced pressure, desalted with anhydrous methanol, and concentrated again under reduced pressure to obtain a crude extract; S02: After the crude extract was subjected to silica gel vacuum column chromatography separation, gradient elution, and thin layer chromatography color detection, the same fractions were combined and concentrated to obtain 16 primary fractions F1-F16; wherein, the silica gel vacuum column chromatography separation conditions are: the chromatography column specifications are 160mm*500mm, and the silica gel particle size is 200-300 mesh; the thin layer chromatography color detection conditions are: the developing solvent is petroleum ether and propanol in a volume ratio of 3:1, and the color developer is a sulfuric acid ethanol solution with a mass concentration of 10%; S03: After silica gel column chromatography separation, gradient elution, and thin layer chromatography color detection of F16 in the first-level fractions F1-F16, the same fractions were combined and concentrated to obtain six second-level fractions F161-F166; wherein the silica gel column chromatography separation conditions are as follows: the chromatography column specifications are 60mm*500mm; the silica gel particle size is 300-400 mesh; the thin layer chromatography color detection conditions are as follows: the developing solvent is petroleum ether and propanol in a volume ratio of 3:1, and the color developer is a sulfuric acid ethanol solution with a mass concentration of 10%; S04: After high performance liquid chromatography analysis, medium pressure preparative liquid chromatography separation and isocratic elution, F165 in the secondary components F161-F166 was obtained to obtain four tertiary components F1651-F1654; wherein, the high performance liquid chromatography analysis conditions are: the analytical column is 4.6mm*250mm, the particle size is 5μm, and the 18 The medium pressure preparative liquid chromatography separation conditions are as follows: the preparative chromatographic column is 20mm*250mm, the particle size is 5μm, and the green grass ODS C 18 column, flow rate was 10 ml / min; S05: F1654 in the tertiary fractions F1651-F1654 is subjected to HPLC analysis, HPLC separation, and isocratic elution to obtain diterpene glycoside compounds; wherein the HPLC analysis conditions are: the analytical column is 4.6mm*250mm, the particle size is 5μm, and the Lvbaicao ODS C 18 The column has a flow rate of 1 ml / min; the separation conditions of the high performance preparative liquid chromatography are as follows: the chromatographic column is 10 mm*250 mm, the particle size is 5 μm, and the ODS C 18 column, with a flow rate of 2 ml / min.

3. The method for preparing diterpene glycoside compounds according to claim 2, wherein The room-temperature methanol soaking comprises soaking the chopped soft coral in room-temperature methanol for 6 times, each soaking for 3 days; the temperature of the reduced-pressure concentration is 37° C. and the pressure is 0.1 MPa.

4. The method for preparing a diterpene glycoside compound according to claim 2, wherein In S02, gradient elution was performed using petroleum ether, petroleum ether / acetone with a volume ratio of 100 / 1, 50 / 1, 30 / 1, 20 / 1, 10 / 1, 5 / 1, 3 / 1, 2 / 1, and 1 / 1, dichloromethane, and dichloromethane / methanol with a volume ratio of 10 / 1, 5 / 1, 3 / 1, 2 / 1, 1 / 1, and 0 / 1.

5. The method for preparing diterpene glycoside compounds according to claim 2, wherein In S03, gradient elution was performed using dichloromethane / methanol with a volume ratio of 10 / 1, 5 / 1, 3 / 1, and 1 / 1 in sequence.

6. The method for preparing diterpene glycoside compounds according to claim 2, wherein In S04, isocratic elution was performed using methanol / water in a volume ratio of 80 / 20.

7. The method for preparing a diterpene glycoside compound according to claim 2, wherein: In S05, isocratic elution was performed using acetonitrile / water with a volume ratio of 35 / 65.

8. The diterpene glycoside compound according to claim 1 or the diterpene glycoside compound prepared by the preparation method according to any one of claims 2 to 7 is used for preparing a drug for treating drug-resistant human small cell lung cancer.

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