Terpenoid as well as preparation method and application thereof

Abstract

The invention provides terpenoids as well as a preparation method and application thereof. The structural formula of the terpenoids is shown in the specification, wherein R1, R2, R3, R4, R5 and R6 are respectively and independently selected from hydrogen, C1-6 alkyl groups and C1-6 alkoxy groups. The terpenoid shows a remarkable anti-inflammatory effect in lipopolysaccharide-induced inflammatory reaction, has a remarkable effect in the aspect of neuroinflammation regulation and control, and shows a good clinical application prospect in treatment of inflammatory diseases. In addition, the terpenoid further shows an inhibiting effect on proliferation of various tumor cells.

Description

Technical Field

[0001] This invention relates to the field of pharmaceutical technology, and in particular to a terpenoid compound, its preparation method, and its application. Background Technology

[0002] Inflammation is a defensive response of the body to injury, infection, or other harmful stimuli. However, excessive or persistent inflammatory responses can lead to tissue damage and various diseases. In the nervous system, neuroinflammation is closely related to a variety of neurodegenerative diseases, such as Alzheimer's disease and Parkinson's disease. Overactivation of microglia and astrocytes releases large amounts of pro-inflammatory factors (such as TNF-α, IL-6, and IL-1β), exacerbating neuronal damage and dysfunction. Currently, although nonsteroidal anti-inflammatory drugs (NSAIDs) and glucocorticoids can be used for anti-inflammatory treatment, they suffer from significant side effects and limited efficacy, making it difficult to meet the clinical needs for neuroinflammation treatment.

[0003] Therefore, developing novel anti-inflammatory drugs with fewer side effects and significant therapeutic effects is of great importance. Summary of the Invention

[0004] The present invention aims to at least partially solve one of the technical problems existing in the prior art. Therefore, in a first aspect, the present invention provides a terpene compound, or an isomer thereof, or a pharmaceutically acceptable salt thereof, the structural formula of which is shown below: , ; Among them, R1, R2, R3, R4, R5, and R6 are independently selected from hydrogen, C1-6 alkyl, and C1-6 alkoxy, respectively.

[0005] Preferably, R1, R2, R3, R4, R5, and R6 are each independently selected from hydrogen, C1-3 alkyl, and C1-3 alkoxy.

[0006] Preferably, the terpenoid compound is selected from the following structures: .

[0007] In a second aspect, the present invention provides a method for preparing the above-mentioned terpene compounds, comprising the following steps: Step 1): Chop the short-finger soft coral, extract it with a ketone organic solvent, concentrate it under reduced pressure to obtain a crude ketone organic solvent extract, extract it with an ether organic solvent, concentrate it under reduced pressure to obtain an ether organic solvent extract. Step 2): The ether organic solvent extract was separated and purified by silica gel column chromatography, gel column chromatography, and high performance liquid chromatography to obtain the terpenoid compounds.

[0008] Preferably, the silica gel column chromatography uses a mixed solvent of petroleum ether and ethyl acetate for elution, and the gel column chromatography uses dichloromethane for elution.

[0009] Preferably, the ketone organic solvent is acetone, and the ether organic solvent is diethyl ether.

[0010] Preferably, in step 2), the ether-based organic solvent extract is separated and purified by silica gel column chromatography, gel column chromatography, and high-performance liquid chromatography, including the following steps: S1. Separate the ether organic solvent extract by silica gel column chromatography, and perform gradient elution in the petroleum ether (PE) / ethyl acetate (EA) system (the EA ratio is gradually increased from 0% to 100%). Collect the entire 5:1 elution fraction as component C and the entire 3:1 elution fraction as component E. S2. Substance C was subjected to Sephadex LH-20 gel column chromatography, eluted and then subjected to TLC color development to obtain substance CF; Substance E was subjected to Sephadex LH-20 gel column chromatography, eluted and then subjected to TLC color development to obtain substance EF. S3. Subcomponent CF was subjected to silica gel column chromatography and eluted with a gradient of petroleum ether / ethyl acetate at a volume ratio of 20:1-5:1. The eluted fraction was collected to obtain subcomponent CFD. S4 and subcomponent CFD were separated and purified by high performance liquid chromatography to obtain compound 1; subcomponent EF was separated and purified by high performance liquid chromatography to obtain compounds 2 and 3.

[0011] Preferably, the high-performance liquid chromatography uses an Agilent Eclipse XDB-C18 semi-preparative column.

[0012] Preferably, the subcomponent CFD is eluted isocratically; the mobile phase is methanol:water = 80:20 (v / v); the flow rate is 3 mL / min.

[0013] Preferably, the subcomponent EF is eluted isocratically; the mobile phase is methanol:water = 65:35 (v / v); the flow rate is 3 mL / min.

[0014] Preferably, the detection wavelength is 210 nm.

[0015] In a third aspect, the present invention provides a pharmaceutical preparation comprising the above-described terpenoid compound or its isomers, and a pharmaceutically acceptable salt.

[0016] Preferably, in the pharmaceutical preparation, the concentration of the terpene compound or its isomer, or a pharmaceutically acceptable salt, is 5-50 μM.

[0017] In a fourth aspect, the present invention provides the use of the above-mentioned terpenoid compound or its isomer, or a pharmaceutically acceptable salt, in the preparation of an anti-inflammatory drug.

[0018] Preferably, the anti-inflammatory drug is an anti-neuroinflammatory drug.

[0019] In a fifth aspect, the present invention provides the use of the above-mentioned terpenoid compound or its isomer, or a pharmaceutically acceptable salt, in the preparation of an antitumor drug.

[0020] Preferably, the tumor is a solid tumor or a hematoma.

[0021] Preferably, the tumor is leukemia, liver cancer, or lung cancer.

[0022] The beneficial effects of this invention are as follows: 1. This invention provides a terpenoid compound that exhibits significant anti-inflammatory effects against lipopolysaccharide-induced inflammation. This compound can be used to prepare novel drugs for treating inflammatory diseases, demonstrating significant neuroinflammatory regulatory effects and showing good clinical application potential in the treatment of inflammatory diseases. The aforementioned terpenoid compound also exhibits inhibitory effects on the growth of various tumor cells, providing lead compounds for the research of novel antitumor drugs.

[0023] 2. This invention provides the application of the above-mentioned terpenoid compounds in the preparation of antitumor drugs or anti-inflammatory drugs. Detailed Implementation

[0024] The present invention will be further described below with reference to specific embodiments. However, the following embodiments are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified, specific conditions in the following embodiments are performed under conventional conditions or conditions recommended by the manufacturer. Unless otherwise specified, the methods used are conventional methods known in the art, and the consumables and reagents used are commercially available. Unless otherwise stated, the technical and scientific terms used herein have the same meaning as those familiar with the art. Furthermore, any methods or materials similar to or equivalent to those described herein may also be applied to the present invention.

[0025] The reagents and equipment used in this application are shown in Table 1 below: Table 1

[0026] Example 1: Preparation of terpenoid compounds 1-3 I. Preparation of Extracts and Powders Short-finned soft corals collected in 2023 from the waters off Ximao Island in the South China Sea. SinulariaThe sp. was thawed and chopped, rinsed with acetone, and extracted with acetone by sonication four times (300 W power, 40 kHz frequency, 30 min each time). The extracts were combined and concentrated under reduced pressure to obtain a crude extract.

[0027] II. Isolation and Purification of Compounds (1) The crude extract was suspended in water at a mass ratio of 1:30 to water. An equal volume of anhydrous ether was added and the extract was extracted four times. The ether extracts were combined and concentrated under reduced pressure to obtain the ether extract.

[0028] (2) Pass the ether extract through silica gel (200-300 mesh) and elute with a gradient of petroleum ether / ethyl acetate (EA ratio gradually increases from 0% to 100%). Collect the entire 5:1 elution fraction as component C and the entire 3:1 elution fraction as component E. (3) Component C was subjected to Sephadex LH-20 gel column chromatography, eluted with dichloromethane, and then developed by TLC (eluted with dichloromethane, collected and combined, and developed on TLC using a petroleum ether / ethyl acetate solvent system with a volume ratio of 5:1). f The fraction with a value of 0.4-0.6 and UV absorption at a wavelength of 254 nm was used to obtain subfraction CF; fraction E was subjected to Sephadex LH-20 gel column chromatography, eluted with dichloromethane and developed by TLC (eluted with dichloromethane, collected and combined on TLC with a petroleum ether / ethyl acetate solvent system of 3:1 v / v). f The subcomponent EF is obtained by considering components with a value of 0.4~0.5 and exhibiting ultraviolet absorption at a wavelength of 254nm under ultraviolet light. (4) Column chromatography: The subfraction CF was subjected to silica gel (200-300 mesh) column chromatography, using petroleum ether / ethyl acetate (20:1) Gradient elution was performed at a ratio of 5:1, and the eluted fraction was collected to obtain subcomponent CFD. (5) Subcomponent CFD was separated and purified by high performance liquid chromatography (HPLC) to obtain compound 1, and subcomponent EF was separated and purified by HPLC to obtain compounds 2 and 3. The chromatographic column was an Agilent Eclipse XDB-C18 semi-preparative column, and the detection wavelength was 210 nm. The mobile phase used for separating dihydrocembrene C (compound 1) was methanol:water = 80:20, with isocratic elution at a flow rate of 3 mL / min. The retention time of compound 1 was approximately 14.3 min. Separation (1) E ,3 E 7 R 8 S ,11 EThe mobile phase used for 1-(2-methoxy-propan-2-yl)-4,8,12trimethyloxabicyclo[12.1.0]-pentadeca-1,3,11-triene (compound 2) and oculatolide (compound 3) was methanol:water = 65:35, with isocratic elution at a flow rate of 3 mL / min, and retention times of 14.3 min (compound 2) and 23.5 min (compound 3).

[0029] III. Structural Identification of Compounds Compound 1 1 H and 13 The C10 NMR data are shown in Table 2. The compound was identified as dihydrocembrene C1 (compound 1), and its structural formula is shown below: .

[0030] Table 2. Compound 1 1 H and 13 12000 C NMR data (CDCl3)

[0031] Compound 2 1 H and 13 The C NMR data are shown in Table 3, and the identification is (1) E ,3 E 7 R 8 S ,11 E )-1-(2-methoxy-propan-2-yl)-4,8,12-trimethyloxabicyclo[12.1.0]-pentadeca-1,3,11-triene. The structural formula is shown below: .

[0032] Table 3. Compound 2 1 H and 13 12C NMR data (CDCl3)

[0033] Compound 3 1 H and 13 The C10 NMR data are shown in Table 4, and the sample was identified as oculatolide. The structural formula is shown below: .

[0034] Table 4. Compound 3 1 H and 1312C NMR data (CDCl3)

[0035] Example 2: Study on the anti-inflammatory activity of the terpenoid compounds provided in this application I. Model Construction Small gelatinous BV BV cells are widely distributed in the central nervous system and are the main cells responsible for the immune function of the nervous system. Under physiological conditions, they play a surveillance role and help stabilize the internal environment. Microglia are sensitive to external stimuli; even minor pathological changes in the central nervous system can activate them, leading to the activation of BV cells. On the one hand, cells perform phagocytosis, engulfing cell debris and secreting growth factors to promote nerve repair. On the other hand, they can also secrete inflammatory factors, glutamate, NO, etc., exacerbating the inflammatory response and causing secondary damage. BV was stimulated with 100 ng / mL lipopolysaccharide (LPS). Two cells release a large number of inflammatory factors, including NO. The NO content in the supernatant was detected using the Griess reagent method to preliminarily determine BV. 2. Cellular inflammatory response level. In this experiment, the anti-inflammatory activity of the terpenoid compounds (compound 1, compound 2, and compound 3) provided in this application was tested using an LPS-induced pro-inflammatory differentiation model of BV-2 cells and dexamethasone as a positive control.

[0036] II. Experimental Methods BV-2 cells were digested with 0.25% trypsin and then suspended in DMEM high-glucose culture medium containing 10% fetal bovine serum. The culture was then incubated at a rate of 2 × 10⁶ cells / day. 5Cells were seeded at a density of 100 µL / well in 96-well plates and incubated at 37 °C with 5% CO2 for 24 h. The medium was then replaced with fresh DMEM high-glucose medium containing 10% fetal bovine serum. Different concentrations (2 µM, 20 µM) of the test compound sample (10 µL / well, DMSO) were added to each well in the drug treatment group, while an equal volume of blank solvent (10 µL / well, DMSO) was added to the normal control group and the LPS model group. After 2 h of incubation, LPS solution was added to the drug treatment group and the LPS model group to achieve a final concentration of 100 ng / mL in the culture system. An equal volume of DMEM high-glucose medium containing 10% fetal bovine serum was added to the normal control group. After another 24 h of incubation, 50 µL of supernatant was mixed with 50 µL of Griess reagent from each well and reacted at room temperature in the dark for 15 min. The absorbance was measured at 540 nm to evaluate the amount of NO generated. After sampling the supernatant, the remaining cells were used for cell viability assay. Cell-free culture medium was used as a blank control. Then, 20 µL of MTT solution was added to each well of the 96-well plate, and the plates were incubated at room temperature in the dark for 1 h. After incubation, the supernatant was discarded, and 150 µL of DMSO (to dissolve the generated formazan crystals) was added to each well. The absorbance was measured at 570 nm to assess cell viability. NO production was calculated using the following formula: NO generation (%) = (OD 药物组 OD 正常对照组 ) / (OD 模型组 OD 正常对照组 ) × 100% In the formula, OD 药物组 The absorbance value of the drug group was measured at a wavelength of 540 nm, OD 模型组 The absorbance values ​​of the model group measured at a wavelength of 540 nm are OD values. 正常对照组 The absorbance values ​​for the normal control group were measured at a wavelength of 540 nm.

[0037] Cell viability is calculated using the following formula: Cell viability (%) = (OD) 药物组 -OD 空白组 ) / (OD 正常对照组 -OD 空白组 ) × 100% In the formula, OD 药物组 The absorbance value of the drug group was measured at a wavelength of 570 nm, OD 正常对照组 The absorbance value was measured at a wavelength of 570 nm for the normal control group.

[0038] III. Experimental Results Three groups were measured, and the average value was taken. The results are shown in Table 5.

[0039] Table 5. Results of LPS-induced anti-inflammatory activity assay in the BV-2 cell model.

[0040] The experimental results showed that compounds 1 and 2 at a concentration of 20 µM did not exhibit significant toxicity to BV-2 cell viability (cell survival ≥80%), and demonstrated extremely significant, dose-dependent anti-inflammatory activity against LPS-induced BV-2 cell inflammation. This indicates that dihydrocembrene C (compound 1) and (1) E ,3 E 7 R 8 S ,11 E Compound 2 (12.1.0)-1-(2-methoxy-propan-2-yl)-4,8,12trimethyloxabicyclo[12.1.0]-pentadeca-1,3,11-triene) effectively inhibited LPS-induced inflammatory responses in BV-2 cells, demonstrating its great potential as a target molecule or lead compound. Low concentrations of compound 3 also effectively inhibited LPS-induced inflammatory responses in BV-2 cells.

[0041] Example 3: Antitumor cell proliferation activity of the terpenoid compounds provided in this application I. Experimental Methods Cell lines MV-4-11, Hep3B, HepG2, and A549 in logarithmic growth phase were used. Cells were digested with 0.25% trypsin and counted at a ratio of 3 × 10⁻⁶ cells / cells. 3 Cells were seeded per well in 96-well plates, with 100 µL of DMEM medium added to each well. The plates were incubated overnight at 37 °C with 5% CO2. Subsequently, different concentration gradients (1.5625, 3.125, 6.25, 12.5, 25, and 50 µM) of the test compounds (compound 1, compound 2, and compound 3) were added, with doxorubicin used as a positive control. After 72 h of further incubation, 20 µL of MTT solution was added to each well, and the plates were incubated for 4 h. After discarding the supernatant, 150 µL of LDMSO (to dissolve the generated formazan crystals) was added to each well, and the absorbance was measured at 570 nm using a microplate reader. A blank control group without the test compounds was also included. Cell viability was calculated based on the absorbance values, and the IC50 was calculated using GraphPad Prism 10 software. 50 value.

[0042] Cell viability (%) = (OD)药物组 OD 空白对照组 ) / (OD 对照组 OD 空白对照组 ) × 100% II. Experimental Results The results are shown in Table 6.

[0043] Table 6 Results of anti-tumor cell proliferation activity (IC50) 50 (Unit: μM)

[0044] Note: DOX: Doxorubicin, "-": IC50 of the compound. 50 >50 μM The results showed that compounds 1 and 3 exhibited inhibitory effects on the growth of various tumor cells and possessed broad-spectrum anti-tumor cell proliferation capabilities.

[0045] In summary, compounds 1-3 have the potential to be developed into anti-inflammatory and / or anti-tumor drugs, and deserve further in-depth research to develop them into anti-inflammatory and / or anti-tumor drugs or lead compounds.

[0046] Although embodiments of the present invention have been shown and described above, it is understood that the above embodiments are exemplary and should not be construed as limiting the present invention. Those skilled in the art can make changes, modifications, substitutions and variations to the above embodiments within the scope of the present invention, all of which should be included within the protection scope of the present invention.

Claims

1. A terpenoid compound, or an isomer thereof, or a pharmaceutically acceptable salt thereof, characterized in that, The structural formula of the terpenoid compound is shown below: 、 ； Among them, R1, R2, R3, R4, R5, and R6 are independently selected from hydrogen, C1-6 alkyl, and C1-6 alkoxy, respectively.

2. The terpenoid compound according to claim 1, or its isomers or pharmaceutically acceptable salts, characterized in that, R1, R2, R3, R4, R5, and R6 are each independently selected from hydrogen, C1-3 alkyl, and C1-3 alkoxy, respectively.

3. The terpenoid compound according to claim 1, or its isomers or pharmaceutically acceptable salts, characterized in that, The terpenoid compounds are selected from the following structures: 。 4. A method for preparing a terpene compound according to any one of claims 1-3, characterized in that, Includes the following steps: Step 1): Chop the short-finger soft coral, extract it with a ketone organic solvent, concentrate it under reduced pressure to obtain a crude ketone organic solvent extract, extract it with an ether organic solvent, concentrate it under reduced pressure to obtain an ether organic solvent extract. Step 2): The ether organic solvent extract was separated and purified by silica gel column chromatography, gel column chromatography, and high performance liquid chromatography to obtain the terpenoid compounds.

5. The method for preparing terpenoid compounds according to claim 4, characterized in that, The silica gel column chromatography uses a mixed solvent of petroleum ether and ethyl acetate for elution, while the gel column chromatography uses dichloromethane for elution.

6. A pharmaceutical preparation comprising the terpenoid compound or its isomer as described in any one of claims 1-3, or a pharmaceutically acceptable salt thereof, wherein the concentration of the terpenoid compound or its isomer, or the pharmaceutically acceptable salt thereof, in the pharmaceutical preparation is 5-50 μM.

7. The use of a terpenoid compound or its isomer, or a pharmaceutically acceptable salt, as described in any one of claims 1-3 in the preparation of an anti-inflammatory drug.

8. The application according to claim 7, characterized in that, The anti-inflammatory drug is an anti-neuroinflammatory drug.

9. The use of a terpenoid compound or its isomer, or a pharmaceutically acceptable salt, as described in any one of claims 1-3 in the preparation of an antitumor drug.

10. The application according to claim 9, characterized in that, The tumor is a solid tumor or a hematologic tumor.

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