Process for the preparation of a clerodane diterpene from the roots of sphaeranthus indicus and its antitumor applications

Abstract

The present application relates to the structure of a clero dene diterpene compound in fierce taste foot bone crisp, preparation method and its application in antitumor, the compound has the structure as shown in figure 1.The compound of the present application has tumor proliferation and metastasis inhibitory activity and angiogenesis inhibitory activity, and can be used for the development and application of antitumor drugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically relating to the preparation method of clerodane diterpenoid compounds from the spicy bone fibrous material and their antitumor and antiangiogenic applications. Background Technology

[0002] Cancer is one of the leading causes of death worldwide, and its incidence is rising year by year. According to the International Agency for Research on Cancer (IARC), cancer has become the second leading cause of death after cardiovascular disease. Despite continuous innovation and development in modern medical treatments, cancer recurrence and metastasis remain major challenges in clinical treatment. At the same time, the resistance of tumor cells to conventional drugs is becoming increasingly prominent, necessitating the development of new anti-tumor drugs to improve patients' survival rates and quality of life.

[0003] Tumor growth and metastasis depend on angiogenesis. Tumor cells stimulate the formation of new blood vessels in surrounding normal tissues by secreting various factors (such as vascular endothelial growth factor VEGF), thereby ensuring their own oxygen and nutrient supply. Angiogenesis not only supports tumor growth and proliferation but also provides channels for tumor cell spread. Therefore, inhibiting angiogenesis can effectively suppress the occurrence, development, and metastasis of tumors, and is an important strategy in anti-tumor therapy.

[0004] Natural products, due to their structural diversity and biological activity, have become an important source of anti-tumor drugs. In traditional medicine, many medicinal plants have been proven to possess various biological activities, including anti-tumor activity. Diterpenoids, as an important class of natural products, are widely found in both plants and animals and possess a variety of significant biological activities, such as antioxidant, anti-tumor, and anti-angiogenic effects. Compared with conventional drugs, their complex structures and multi-target characteristics give them greater potential in cancer treatment.

[0005] To screen and identify natural products with antitumor and anti-angiogenic activities, and to develop novel, highly effective, and low-toxicity tumor therapeutics, we established screening models using human acute myeloid leukemia cell line K562, human hepatocellular carcinoma cell line HepG2, human lung cancer cell line A549, and human cervical cancer cell line HeLa. During the cell exponential growth phase, different concentrations of the test drug were added, and after a certain treatment time, the optical density was measured using the MTT assay to assess the cytotoxicity of the compounds. This process aimed to screen compounds with strong cytotoxic activity and identify the most sensitive cell lines. Based on the results of the cytotoxicity experiments, we constructed a zebrafish tumor xenograft model. Fluorescently labeled tumor cells were microinjected into the yolk sac of juvenile zebrafish, and the proliferation and metastasis of tumor cells were observed. The growth and metastasis of tumor cells were assessed by calculating fluorescence intensity and cell number, further evaluating the in vivo antitumor effects of the in vitro screened active compounds. Simultaneously, we established a transgenic zebrafish model and co-incubated the test drug with the zebrafish to observe the in vivo angiogenesis of the juvenile fish and explore the effects of the compounds on angiogenesis.

[0006] Through the above bioactivity screening, we have identified lead compounds that may have anticancer and antiangiogenic activity, laying the foundation for the further development of novel, highly effective, and low-toxicity tumor therapeutics. Summary of the Invention

[0007] The purpose of this invention is to provide the compound casearlucin F in the brittle bone of the strong-tasting foot, its preparation method and application.

[0008] The compound casearlucin F provided by this invention belongs to the closan type diterpenoid compound, and its structure is as follows: Figure 1 As shown.

[0009] The present invention also provides a method for preparing the compound casearlucin F, the method comprising the following steps: (1) The dried leaves of Casearia graveolens were extracted three times with methanol; (2) The three crude extracts obtained in step (1) are combined and concentrated by vacuum distillation; (3) The extract obtained in step (2) is added to distilled water to disperse it into a suspension, and then extracted using an organic solvent; (4) The extract obtained in step (3) is separated by silica gel column chromatography, using a mixture of multiple organic solvents as the elution system; (5) The fractions obtained in step (4) are separated by MPLC (medium-pressure liquid chromatography, with ODS as the chromatographic packing material) using a gradient elution with methanol / water or acetonitrile / water mixed solvent as the mobile phase. (6) The fraction obtained in step (5) above is separated by HPLC-RI (high performance liquid chromatography-differential detection) chromatography, eluted with methanol / water as the mobile phase, or eluted with acetonitrile / water as the mobile phase gradient, to obtain the compound casearlucin F.

[0010] The compound preparation method provided by the present invention is an extract of the leaves of Casearia graveolens, a plant belonging to the genus Casearia in the family Flacotyleaceae.

[0011] The preparation method of the compound provided by the present invention, wherein the extraction method in step (1) is heating reflux extraction or ultrasonic extraction, and the solvent used is at least one of dichloromethane, chloroform, ethyl acetate, methanol, and ethanol, and the weight-volume ratio of medicinal material to solvent is 1:5 to 1:15.

[0012] The preparation method of the compound provided by the present invention, the extraction method in step (3), uses any one of petroleum ether, dichloromethane, chloroform and ethyl acetate as the organic solvent, and the volume ratio of the aqueous solution to the organic solvent is 1:1 to 1:2.

[0013] In the preparation method of the compound provided by the present invention, in step (4), the elution solvent is a mixture of petroleum ether / acetone or petroleum ether / ethyl acetate, with a ratio of 100:2 to 100:30.

[0014] In the preparation method of the compound provided by the present invention, in step (5), the ratio of the methanol / water mixed solvent is 6:4 to 9:1, preferably 7:3 to 8:2, or the ratio of the acetonitrile / water mixed solvent is 6:4 to 9:1, preferably 7:3 to 8:2.

[0015] The preparation method of the compound provided by the present invention, wherein the volume ratio of the mobile phase methanol and water mixed solvent or acetonitrile and water mixed solvent in step (6) is 6:4 to 9:1, preferably 7:3 to 9:1.

[0016] The closan-type diterpenoid compounds provided by this invention possess antitumor and anti-angiogenic activities. They can be used to prepare antitumor drugs. Attached Figure Description

[0017] Figure 1 The structural formula of the compound of this invention; Figure 2 1H NMR spectra of the compounds of this invention; Figure 3 13C NMR spectra of the compounds of this invention; Figure 4 DEPT135 spectrum of the compounds of this invention; Figure 5In vivo antitumor effects of the compound in a zebrafish tumor xenograft model; Figure 6 Anti-angiogenic activity of the compound in a transgenic zebrafish model. Detailed Implementation

[0018] The following embodiments will further illustrate the present invention, but are not intended to limit the invention. Example

[0019] (1) 14.0 kg of dried leaves of the strong-flavored foot bone were extracted three times by heating and refluxing with methanol for 2 h, 1.5 h and 1.5 h respectively to obtain crude extract; (2) The methanol extract obtained in step (1) was concentrated by vacuum distillation to obtain 2.4 kg of extract; (3) The extract obtained in step (2) was added to distilled water at a ratio of 1:1 to form a suspension, and then extracted with petroleum ether and ethyl acetate solvents in sequence to obtain 300 g of petroleum ether extract and 280 g of ethyl acetate extract. (4) The ethyl acetate extract in step (3) was separated by silica gel column chromatography with petroleum ether-acetone as the elution system and the eluent ratios were 100:2, 100:4, 100:8, 100:12, 100:17, 100:25, and 100:38, respectively. (5) The component F3 (6.0 g) obtained in step (4) above was separated by medium-pressure liquid chromatography (MPLC) and eluted with 83% and 92% methanol / water in sequence. (6) The subcomponent F3-5 obtained in step (5) above was separated by semi-preparative high performance liquid chromatography (HPLC) using 93% methanol / water as the mobile phase to obtain the compound casearlucin F.

[0020] The structure of casearlucin F was identified based on its physicochemical properties and spectroscopic data (spectral data of casearlucin F can be found in...). Figures 1-3 ).

[0021] The structural identification data for compound casearlucin F are as follows: Colorless oil. The 1H-NMR spectrum of this compound shows a total of seven methyl hydrogen signals: δH 1.65 (3H, s, H3-16), 0.88 (3H, d, J = 7.4 Hz, H3-17), 0.82 (3H, s, H3-20), 0.96 (3H, t, J = 7.6 Hz, H3-24), 1.17 (3H, d, J = 6.8 Hz, H3-25), 1.92 (3H, s, H3-27), 2.07 (3H, s, H3-29); in addition, there are two oxygen-bound methine hydrogen signals in the mid-to-low field: δH 6.66 (1H, t, J = 1.6 Hz, H-18), 6.35 (1H, s, J = 7.4 Hz, H3-17), 0.82 (3H, s, H3-20), 0.96 (3H, t, J = 7.6 Hz, H3-24), 1.17 (3H, d, J = 6.8 Hz, H3-25), 1.92 (3H, s, H3-27), 2.07 (3H, s, H3-29); H-19); there are 5 olefinic hydrogen signals in the low field: δH 5.89 (1H, d, J = 3.0, H-3), 5.36 (1H, brs, H-12), 6.25 (1H, dd, J = 10.6, 17.4 Hz, H-14), 5.09 (1H, d, J = 17.4 Hz, H-15a), 4.91 (1H, d, J = 10.8 Hz, H-15b).

[0022] The 13C-NMR spectrum showed a total of 29 carbon signals for this compound, including 3 carbonyl carbon signals: δC 176.0, 170.3, 169.7; 6 alkene carbon signals: δC 120.4, 147.0, 129.3, 135.6, 141.3, 111.8; and 3 oxygen-bonded carbon signals: δC 66.3, 94.5, 98.8. In addition to these 12 carbon signals, 17 other carbon signals were observed. Combined with the DEPT 135 spectrum, these were further classified into 7 methyl carbon signals, 5 methylene carbon signals, 3 methine carbon signals, and 2 quaternary carbon signals. Based on these hydrocarbon data, it is speculated that this compound may be a closan-type diterpene. The 1H NMR (400 MHz, CDCl3) and 13C NMR (100 MHz, CDCl3) data are shown in Table 1.

[0023] Example

[0024] (1) 10.0 kg of dried leaves of the strong-flavored foot bone crisp were extracted three times with ethanol (3 × 30 L), and the crude extract was obtained by vacuum recovery. (2) The ethanol extract obtained in step (1) was made into a suspension by adding water and extracted with ethyl acetate to obtain the ethyl acetate extract; (3) Step (2) was separated by silica gel column chromatography, and eluted sequentially with petroleum ether:acetone 100:4, 100:6, 100:8, 100:11, 100:16, 100:23, 100:30. (3) The petroleum ether: ethyl acetate fractions of 100:2 to 100:23 obtained in step (2) above were separated by medium-pressure liquid chromatography (MPLC) with a gradient elution of methanol / water 7:3 to 9:1 as the mobile phase; (4) The methanol / water (8:2) fraction obtained in step (3) above was separated by HPLC-RI and eluted with methanol / water 70:30 to 90:10 as the mobile phase to obtain casearlucin F.

[0025] The method for identifying the structure of the compound casearlucin F is described in Example 1. Example

[0026] (1) 10.0 kg of dried leaves of the strong-flavored foot bone crisp were extracted with acetone three times (3 × 30 L), and the crude extract was obtained by vacuum recovery of the extract. (2) The acetone extract obtained in step (1) was mixed with water to form a suspension, and then extracted with dichloromethane to obtain a dichloromethane extract; (3) Step (2) was separated by silica gel column chromatography, and eluted sequentially with petroleum ether:acetone 100:4, 100:6, 100:8, 100:11, 100:16, 100:23, 100:30. (3) The petroleum ether:acetone fractions obtained in step (2) above were separated by medium-pressure liquid chromatography (MPLC) with a gradient elution of methanol / water 7:3 to 9:1 as the mobile phase; (4) The methanol / water (8:2) fraction obtained in step (3) above was separated by HPLC-RI and eluted with acetonitrile / water 70:30 to 90:10 as the mobile phase to obtain the compound casearlucin F.

[0027] The method for identifying the structure of the compound casearlucin F is described in Example 1. Example

[0028] Cytotoxic activity assay of compound casearlucin F.

[0029] (1) Experimental principle The MTT assay primarily assesses cell growth based on the activity of intracellular dehydrogenases. When cells are in their growth phase and metabolism is normal, viable cells can reduce MTT (a yellow, water-soluble dye) to form a purple formazan precipitate. Intracellular dehydrogenases, such as mitochondrial reductases, catalyze the reduction of MTT. After drug treatment, cell death or apoptosis occurs, leading to a decrease in dehydrogenase activity, affecting the degree of MTT reduction and thus reducing the production of formazan. Therefore, the formazan precipitate is dissolved, and the optical density (OD) value is measured using a microplate reader; the OD value is directly proportional to cell viability. Finally, by comparing the OD values ​​of different treatment groups, the cytotoxicity of the compounds is evaluated, helping to screen for potential antitumor active compounds.

[0030] (2) Experimental methods ① Culture of tumor cells Cells (human acute myeloid leukemia cell line K562, human liver cancer cell line HepG2, human lung cancer cell line A549, and human cervical cancer cell line HeLa) were cultured at 37°C in a 5% CO2 incubator until the cells basically covered the bottom of the culture flask. They were then passaged or subjected to experimental treatments. The culture medium used was DMEM high glucose medium containing 10% fetal bovine serum (FBS) and 1% penicillin:streptomycin = 1:1.

[0031] ② Preparation methods of compounds The dried compound was dissolved in DMSO to prepare a 30 mM stock solution, which was stored at -20°C. Before administration, it was properly diluted with fresh culture medium, successively to 100 μM, 30 μM, 10 μM, or other concentration gradients as needed.

[0032] ③ Cytotoxic activity test of the test compound Tumor cells in the exponential growth phase were collected and adjusted to a density of 1×10⁴ cells / mL. 100 μL of cell suspension was seeded into each well of a 96-well plate and incubated at 37°C in a 5% incubator for 24 h. Different concentrations of the target drug were then added for further incubation for 48 h. Afterward, 20 μL of MTT solution (5 mg / mL) was added to each well. After 3-4 h, the supernatant was discarded, and 150 μL of DMSO was added. The mixture was shaken to dissolve the bottom precipitate, and the absorbance (OD value) at 490 nm was measured using a microplate reader. The inhibition rate of each compound on tumor cell proliferation was calculated based on the OD value.

[0033] ④ Calculation method of IC50 Based on the inhibition rate of different concentrations of the compound, the IC50 value of the compound inhibiting tumor cell proliferation was calculated using nonlinear regression fitting, and each experiment was repeated three times.

[0034] (3) Experimental results: The IC50 of the cytotoxic activity of compound casearlucin F is shown in Table 2.

[0035] Example

[0036] Casearlucin F in Zebrafish inhibits the proliferation and metastasis of tumor cells in zebrafish.

[0037] (1) Experimental principle Zebrafish, as an important experimental model organism, are widely used in biomedical research due to their small size, transparent embryos, large sample size, high genetic homology with humans, and short experimental cycle. In this model, tumor cells were labeled using CM-Dil live cell staining agent. CM-Dil can effectively bind to lipid molecules in the cell membrane structure to form a stable red fluorescent label, with a positive labeling rate exceeding 98%. The labeled tumor cells were injected into the yolk sac of zebrafish, and the proliferation and migration of the transplanted cells could be observed in the transparent juvenile fish. Real-time imaging and fluorescence intensity analysis were used to analyze the growth and metastasis of the transplanted tumor cells in the zebrafish, thus providing important data support for the efficacy evaluation of anti-tumor drugs.

[0038] (2) Experimental methods Collect A549 cells in the logarithmic growth phase, add CM-DiI staining solution (final concentration 2 μM), incubate at 37 ℃ for 5 min, then incubate at 4 ℃ for 15 min to complete cell staining. After incubation, resuspend the cells in an appropriate amount of cell culture medium and adjust the cell density to 1×107 cells / mL for microinjection.

[0039] Two normal zebrafish embryos with a dpf (day pass rate) were randomly selected and microinjected sequentially after tricaine anesthesia. Approximately 5 nL of cell suspension was injected per embryo. Four hours later, different concentrations of drug solutions were added for treatment. Etoposide was used as a positive control. The embryos were incubated in a 28.5℃ constant temperature and light incubator for 48 hours. Finally, the development of tumors in each group of embryos was observed under a laser confocal microscope. Fluorescence intensity and metastatic lesions were counted, and embryonic death or malformation was carefully observed.

[0040] (3) Experimental results: casearlucin F inhibited the proliferation and metastasis of tumor cells in zebrafish, such as Figure 5As shown in the figure, the blank control group exhibited the highest fluorescence intensity. Besides the red fluorescence emitted by HepG2 cells at the injected yolk sac, fluorescence was also observed in the zebrafish tail, indicating that HepG2 cells migrated in vivo. In the xenograft zebrafish model, after treatment with casearlucin F for 48 h, the relative intensity of red fluorescence gradually decreased with increasing concentration, and the number of metastatic fluorescent foci also decreased. Quantitative analysis using ImageJ software revealed that different concentrations of casearlucin F inhibited the proliferation of HepG2 cells by 25.3% (5 μM), 76.6% (10 μM), and 87.2% (20 μM). Furthermore, the migration inhibition rate of HepG2 cells also increased with increasing dose, from 18.2% (5 μM), 69.7% (10 μM), and 84.8% (20 μM). When the concentration of casearlucin F reached 20 μM, its inhibitory effect on HepG2 cell proliferation was comparable to that of the positive control etoposide, with no statistically significant difference between the two; however, its inhibitory effect on HepG2 cell migration was superior to that of etoposide. These results indicate that casearlucin F can inhibit the proliferation and migration of HepG2 cells in zebrafish. Example

[0041] Casearlucin F in Zebrafish inhibits angiogenesis in zebrafish.

[0042] (1) Experimental principle This study used Tg(fli1:EGFP) transgenic zebrafish as a model. The vascular endothelial cells of this strain express enhanced green fluorescent protein (EGFP) controlled by the fli1 promoter, resulting in a distinct green hue in their blood vessels under a fluorescence microscope. This characteristic allows for direct observation of the growth and development of blood vessels in live zebrafish juveniles. In this experiment, fluorescence microscopy was used to observe the vascular development of transgenic zebrafish under different drug concentrations. By analyzing changes in fluorescence intensity and vascular structure, the effects of the test compounds on angiogenesis and development were evaluated.

[0043] (2) Experimental methods Healthy Tg(fli1:EGFP) transgenic zebrafish embryos (6 hpf) were randomly selected and exposed to different concentrations of casearlucin F. They were incubated for 48 h in a 28.5℃ constant temperature and light incubator. The embryos were anesthetized with 0.02% tricaine, and the development of intersegmental vessels (ISVs) was imaged using a confocal microscope. ISV length was measured and quantified using ImageJ software.

[0044] (3) Experimental results: The results of casearlucin F inhibiting angiogenesis in transgenic zebrafish are as follows: Figure 6 As shown in the figure. Quantitative analysis using ImageJ software revealed that the embryos in the blank control group developed normally with intact blood vessels; however, the zebrafish ISVs and DLAVs treated with casearlucin F showed varying degrees of fragmentation. The average length of the ISVs in the control group was 3547.08±80.33 μm. With increasing compound concentration, the anti-angiogenic effect became increasingly significant, with the average ISV length gradually decreasing to 3450.46±29.09 μm (5 μM), 3028.65±17.01 μm (10 μM), and 2597.96±116.38 μm (20 μM). At a compound concentration of 20 μM, the inhibition rate of ISV length was 26.76%. These results indicate that casearlucin F can inhibit tumor growth and metastasis by blocking angiogenesis in zebrafish.

Claims

1. A clerodane diterpenoid compound in the spicy and brittle bone extract, characterized by: It has the following structure .

2. A method for preparing the compound according to claim 1, characterized in that: The method includes the following steps: (1) The strong-tasting bone brittle (Casearia graveolens) was extracted with solvent and the crude extract was recovered. (2) The crude extract obtained in step (1) is dissolved in water and extracted with an organic solvent that is immiscible with water. The solvent is recovered under reduced pressure to obtain the extract. (3) The extract obtained in step (2) was separated by silica gel column chromatography and eluted with a gradient of petroleum ether / acetone or petroleum ether / ethyl acetate. (4) The fraction obtained in step (3) above is separated by medium-pressure liquid chromatography (MPLC, with ODS as the chromatographic packing material) using a gradient elution with methanol / water or acetonitrile / water mixed solvent as the mobile phase. (5) The fraction obtained in step (4) above is separated by HPLC-RI (high performance liquid chromatography-differential detection) chromatography, and gradient elution is performed with methanol / water as the mobile phase or acetonitrile / water as the mobile phase to obtain compound 1.

3. The method for preparing the compound according to claim 2, characterized in that: The aforementioned "Lièwèi Jǔgǔchéng" is an extract from the stems and leaves of Casearia graveolens, a plant belonging to the genus Casearia in the family Flacotiaceae.

4. The method for preparing the compound according to claim 2, characterized in that: The extraction method described in step (1) is heating and reflux extraction or ultrasonic extraction 1 to 3 times. The solvent used is at least one of petroleum ether, cyclohexane, dichloromethane, chloroform, ethyl acetate, acetone, methanol, and ethanol. The weight-volume ratio of medicinal material to solvent is 1:5 to 1:

15.

5. The method for preparing the compound according to claim 2, characterized in that: The extraction method described in step (2) uses an aqueous solution and an organic solvent in a volume ratio of 1:1 to 1:2, and the extraction solvent used is one of petroleum ether, dichloromethane, chloroform, or ethyl acetate.

6. The method for preparing the compound according to claim 2, characterized in that: The ratio of the elution solvent, petroleum ether / acetone or petroleum ether / ethyl acetate, in step (3) is 100:2 to 100:

30.

7. The method for preparing the compound according to claim 2, characterized in that: The ratio of methanol / water or acetonitrile / water mixed solvent in step (4) is 3:2 to 9:

1.

8. The method for preparing the compound according to claim 2, characterized in that: In step (5), the mobile phase is a methanol / water or acetonitrile / water mixed solvent. The ratio of the mixed solvent in the mobile phase is 3:2 to 9:1 to obtain compound 1; the ratio of the mixed solvent in the mobile phase is 3:2 to 9:1 to obtain compound 2; the ratio of the mixed solvent in the mobile phase is 3:2 to 9:1 to obtain compound 3; and the ratio of the mixed solvent in the mobile phase is 3:2 to 9:1 to obtain compound 4.

9. A pharmaceutical formulation comprising the compound or pharmaceutically acceptable salt of claim 1 and pharmaceutically acceptable excipients, diluents and carriers.

10. The use of the novel compound of claim 1 in the preparation of a drug for treating malignant tumors.

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