Halogenated kaurane diterpenoid compound as well as preparation method and application thereof

Abstract

The invention relates to a halogenated kaurane diterpenoid compound, and belongs to the technical field of natural product chemistry and medicinal chemistry. In the prior art, separation and purification of halogenated kaurane diterpenoid compounds from pteris semipinnata plants have the problems of low efficiency and insufficient purity, and deep research and application of the active ingredients are restricted. The novel compound is successfully obtained from a pteris semipinnata ethanol or methanol extract through systematic extraction and a multi-step chromatographic separation technology, the chemical structure of the novel compound is confirmed to be 16R-17-chloroethyl-1 beta-hydroxy-ent-kauran-19-acid through nuclear magnetic resonance and high-resolution mass spectrometry, and the structural formula is shown in the specification. The compound shows proliferation inhibition activity on K562 leukemia cells, A549 lung cancer cells, MDA-MB-231 breast cancer cells, SW480 colon cancer cells and human lung cancer paclitaxel drug-resistant strains A549 / Taxo in an in-vitro experiment, so that the compound can be used for preparing antitumor drugs.

Description

Technical Field

[0001] This invention belongs to the field of natural product chemistry and medicinal chemistry, specifically relating to a halokaurane-type diterpenoid compound, its preparation method, and its application. Background Technology

[0002] As a traditional medicinal plant, *Hemiberlesia lingua* has been preliminarily reported for its antitumor activity, with research mainly focusing on its crude extracts or common components. However, our understanding of its specific active compounds, especially the novel kauriane-type diterpenoids, remains insufficient. Current techniques have yielded a limited variety of compounds isolated from *Hemiberlesia lingua*, and systematic studies and structural identification of specific halokauriane-type diterpenoids are lacking. Due to the complex plant matrix, the content of active ingredients is usually low, and they often coexist with structurally similar analogs, making the efficient and specific isolation and purification of single novel halokauriane-type diterpenoids from *Hemiberlesia lingua* challenging. This, to some extent, restricts the in-depth development and utilization of the active components of *Hemiberlesia lingua*. Summary of the Invention

[0003] One object of the present invention is to solve at least the above-mentioned problems and to provide at least the advantages that will be described later.

[0004] Another objective of this invention is to provide a halokauriane-type diterpenoid compound, which is isolated from the traditional medicinal plant *Haloxylon ammodendron*, and has been shown in vitro to inhibit the proliferation of specific tumor cell lines such as K562, A549, MDA-MB-231, SW480, and the paclitaxel-resistant human lung cancer strain A549 / Taxo.

[0005] To achieve these objectives and other advantages of the present invention, a halokaurane-type diterpenoid compound is provided, the structural formula of which is shown below: .

[0006] A method for preparing a halokaurane-type diterpenoid compound, comprising the following steps: S1: The whole herb of *Haloxylon ammodendron* was extracted by heating and refluxing with 60% to 95% ethanol or methanol 2 to 5 times, each time for 2 to 5 hours. The extracts were combined and concentrated under reduced pressure to obtain the total extract. S2: The total extract was suspended in an equal volume of water and extracted sequentially with petroleum ether, dichloromethane, ethyl acetate and n-butanol. The solvent was recovered to obtain petroleum ether fraction extract, dichloromethane fraction extract, ethyl acetate fraction extract, n-butanol fraction extract and water fraction extract. S3: The ethyl acetate fraction extract was separated by silica gel column chromatography with an eluent gradient from dichloromethane to a mixed solvent of dichloromethane and methanol or ethanol, wherein the volume ratio of dichloromethane to methanol or ethanol was 100:0 to 0:100, and then to methanol or ethanol, to obtain 8 components Fr.s1 to Fr.s8. S4: Fr.s4 was separated by medium-low pressure C-18 reversed-phase column chromatography, and eluted with a gradient of 10% to 100% methanol or ethanol to obtain 6 fractions Fr.s4-1 to Fr.s4-6; S5: Fr.s4-3 was separated by silica gel column chromatography with an eluent gradient from dichloromethane to a mixed solvent of dichloromethane and methanol or ethanol, wherein the volume ratio of dichloromethane to methanol or ethanol was 100:0 to 0:100, and then to methanol or ethanol, to obtain 5 components Fr.s4-3-1 to Fr.s4-3-5; S6: Fr.s4-3-3 was separated by gel column chromatography, with the elution system being methanol or a mixture of dichloromethane, methanol, and petroleum ether, wherein the volume ratio of dichloromethane, methanol, and petroleum ether was 1:1:1, to obtain three components Fr.s4-3-3-1 to Fr.s4-3-3-3; S7: Fr.s4-3-3-2 was separated using a semi-preparative liquid chromatograph, with the solvent being a mixture of methanol and water or a mixture of acetonitrile and water, wherein the volume fraction of methanol or acetonitrile was 10% to 60%, to obtain the halokaurane-type diterpenoid compound.

[0007] This invention employs ethanol or methanol reflux extraction, combined with systematic solvent extraction using petroleum ether, dichloromethane, ethyl acetate, and n-butanol, to initially enrich and group chemical components in *Haloxylon ammodendron* based on polarity differences. Furthermore, it utilizes a combination of various chromatographic techniques, including silica gel column chromatography, medium-low pressure C-18 reversed-phase column chromatography, gel column chromatography, and semi-preparative liquid chromatography, to form a highly targeted and efficient systematic separation and purification strategy. The gradient elution and multi-step subdivision of this method effectively remove a large number of coexisting impurities, significantly enriching the target compounds, ultimately achieving the efficient preparation of structurally well-defined halogenated kauriane-type diterpenoids from complex plant matrices.

[0008] Preferably, in step S7, the solvent of the semi-preparative liquid chromatograph further contains an acid additive, which is one of formic acid, acetic acid or trifluoroacetic acid, and the volume fraction of the acid additive is 0.05% to 1.0%; the volume fraction of the acid additive in the solvent is 0.05% to 1.0%.

[0009] This invention effectively inhibits the ionization of carboxyl groups in the target compound molecule and improves its retention behavior on the reversed-phase chromatographic stationary phase by adding an acid additive within a specific concentration range to the solvent in the semi-preparative liquid chromatography purification step. This invention results in sharper and more symmetrical chromatographic peaks of the target compound, significantly reducing the resolution loss caused by peak tailing, thereby improving the selectivity and efficiency of chromatographic separation. This facilitates the more precise collection of high-purity target halokaurane-type diterpenoid compounds from complex components.

[0010] Preferably, in step S7, the separation conditions of the semi-preparative liquid chromatograph further include: the flow rate is controlled at 1.0 mL / min to 3.0 mL / min, and the column oven temperature is maintained at 20°C to 30°C; the composition of the solvent, the type and concentration of the acid-containing additive, the flow rate and the column oven temperature are kept constant during a single separation operation.

[0011] This invention precisely controls the flow rate of semi-preparative liquid chromatography within the range of 1.0 mL / min to 3.0 mL / min and maintains the column oven temperature stably between 20°C and 30°C, while ensuring that the solvent system containing acidic additives remains constant during a single separation process. This effectively maintains the stability of the hydrodynamic state during chromatographic separation and the thermodynamic reproducibility of the solute distribution equilibrium between the stationary and mobile phases. It significantly reduces the retention time drift and peak broadening of target compounds caused by fluctuations in flow rate and temperature, thereby improving the consistency of separation and purification results between different batches and enhancing the reliability and reproducibility of this method in the preparation of high-purity halokaurane-type diterpenoids.

[0012] Preferably, after step S7, a post-processing step is included for the fraction containing the target compound collected by the semi-preparative liquid chromatograph. The specific steps are as follows: add 1 / 5 to 1 / 3 of the volume of saturated sodium chloride aqueous solution to the fraction, mix well, extract 2 to 3 times with a 1:1 volume ratio of dichloromethane-ethyl acetate mixed solvent, and combine the organic phases; dry the organic phase with anhydrous sodium sulfate, and concentrate it under reduced pressure at 35°C to 40°C to obtain the solid of the halokaurane-type diterpenoid compound.

[0013] This invention utilizes the salting-out effect to effectively reduce the solubility of the target compound in the aqueous phase by adding a saturated sodium chloride aqueous solution to the fraction collected by semi-preparative liquid chromatography. Subsequently, liquid-liquid extraction is performed using a dichloromethane-ethyl acetate mixed solvent with good selectivity for the target compound, efficiently transferring it from the complex solvent system containing acid additives to the organic phase. After drying with anhydrous sodium sulfate to remove trace amounts of moisture, the compound is concentrated under controlled low-temperature conditions. This series of operations avoids the potential impact of acidic environments and high temperatures on the compound structure, significantly improving post-processing efficiency and ultimately yielding a solid target product with better stability and higher purity.

[0014] Preferably, after drying the organic phase with anhydrous sodium sulfate and before vacuum concentration, the dried organic phase is filtered out, the filtrate is collected, and it is passed through a Sephadex LH-20 gel column pre-equilibrated with the same solvent. The filtrate is eluted using a 1:1 volume ratio of dichloromethane-ethyl acetate mixed solvent or methanol as the eluent. The filtrate is collected according to the colorimetric characteristics of the target compound or a predetermined fraction volume. The fractions containing the target compound are then combined and concentrated under vacuum.

[0015] This invention achieves efficient separation of the target compound from trace amounts of inorganic salt impurities by passing the dried organic phase through a Sephadex LH-20 gel column before vacuum concentration and utilizing the molecular sieving principle of gel permeation chromatography. This step effectively removes small molecule inorganic substances such as sodium chloride that may remain from the previous salting-out extraction, preventing their coexistence in the final product and thus significantly improving the chemical purity of the obtained halokaurane-type diterpenoid compounds.

[0016] Preferably, after obtaining the solid of the halokaurane-type diterpenoid compound by vacuum concentration, the following purification steps are further included: dissolving the solid of the halokaurane-type diterpenoid compound in an acetone-methanol mixed solvent with a volume ratio of 2:1 to 1:1 to form a saturated or near-saturated solution; then slowly adding n-hexane at a volume of 3 to 5 times the total volume of the solution to the solution, maintaining the system temperature at 15°C to 25°C and stirring during the addition; after the addition is complete, allowing it to stand for 1 to 2 hours to promote crystal precipitation; filtering, washing the obtained crystals with a small amount of pre-cooled n-hexane, and then vacuum drying at 35°C to 40°C to obtain high-purity crystals of the halokaurane-type diterpenoid compound.

[0017] This invention involves dissolving the pre-purified solid product in a specific ratio of acetone-methanol mixed solvent to form a near-saturated solution, and slowly adding n-hexane as an antisolvent. Utilizing the significant difference in solubility between the target compound and impurities in the mixed solvent system, the target molecule is selectively precipitated from the supersaturated solution. This crystallization process ensures controllability of nucleation and growth by controlling temperature and stirring speed, effectively rejecting organic impurities with similar structures and polarities, while simultaneously promoting the orderly arrangement of molecules to form stable crystals. This significantly improves the chemical purity of the final product and yields a uniform solid form.

[0018] The application of the halokaurane-type diterpenoids in the preparation of antitumor drugs.

[0019] Preferably, the antitumor drug is an antileukemia drug, an anti-lung tumor drug, an anti-breast tumor drug, an anti-colon tumor drug, or an anti-paclitaxel-resistant strain drug for human lung cancer.

[0020] This invention offers at least the following advantages: Through a systematic extraction, multi-step chromatographic separation, and purification strategy, a novel halogenated kauriane-type diterpenoid compound was successfully prepared from *Pteris vittata*. This method achieves effective enrichment of the target compound by optimizing the extraction solvent and extraction process. A highly efficient and controllable separation and purification process was established by comprehensively utilizing various chromatographic techniques, including normal-phase silica gel column, reversed-phase C-18 column, gel column, and semi-preparative liquid chromatography. In particular, the introduction of an acidic additive and precise control of flow rate and column temperature in the semi-preparative liquid chromatography significantly improved peak shape and separation reproducibility. Subsequent refined post-processing steps, such as salting-out extraction, gel desalting, and recrystallization, effectively removed inorganic salts and organic impurities, ultimately yielding a high-purity, homogeneous solid target compound. In vitro activity studies showed that this compound exhibited significant inhibitory activity against various human tumor cell lines, including K562 leukemia cells, A549 human lung cancer cells, MDA-MB-231 human breast cancer cells, SW480 human colon cancer cells, and the paclitaxel-resistant human lung cancer cell line A549 / Taxo. This invention not only provides a material basis for in-depth research into the structure-activity relationship and mechanism of action of this type of compound, but also provides a feasible technical pathway for developing antitumor lead compounds from *Bambusa textilis* resources.

[0021] Other advantages, objectives and features of the present invention will become apparent in part from the following description, and in part from those skilled in the art through study and practice of the invention. Detailed Implementation

[0022] The present invention will now be described in further detail so that those skilled in the art can implement it based on the description.

[0023] It should be understood that terms such as “having,” “comprising,” and “including” as used herein do not exclude the presence or addition of one or more other elements or combinations thereof.

[0024] It should be noted that, unless otherwise specified, the experimental methods described in the following implementation plan are all conventional methods, and the reagents and materials described are all commercially available unless otherwise specified.

[0025] The room temperature in this invention refers to a normal temperature of 20~25℃.

[0026] The halokaurane-type diterpenoid compound of the present invention has the following chemical structural formula: .

[0027] Example 1 A method for preparing a halokaurane-type diterpenoid compound, comprising the following steps: 10 kg of dried whole herb of *Haloxylon ammodendron* was extracted twice by heating and reflux with 90% ethanol and 75% ethanol, respectively, for 2 hours each time. The extracts were combined and concentrated under reduced pressure to obtain 800 g of extract. The total extract was suspended in an equal volume of water and extracted successively with petroleum ether, dichloromethane, ethyl acetate and n-butanol. The solvents were recovered to obtain 93 g of petroleum ether extract, 63 g of dichloromethane extract, 95 g of ethyl acetate extract, 50 g of n-butanol extract and 462 g of aqueous extract.

[0028] 95 g of the ethyl acetate fraction was separated by silica gel column chromatography with an eluent gradient of: dichloromethane → dichloromethane: methanol (95:5 → 90:10 → 85:15 → 80:20 → 70:30 → 60:40) → methanol, yielding 8 fractions Fr.s1 to Fr.s8.

[0029] Fr.s4 was separated by medium-low pressure (0.1~5 MPa) C-18 reversed-phase column chromatography and eluted with a gradient of methanol from 10% to 100% by volume (in this example, the gradient was increased by 10%) to obtain 6 fractions Fr.s4-1 to Fr.s4-6.

[0030] Fr.s4-3 was separated by silica gel column chromatography with an eluent gradient of: dichloromethane → dichloromethane: methanol (95:5 → 90:10 → 85:15 → 80:20 → 70:30 → 60:40) → methanol, yielding five fractions Fr.s4-3-1 to Fr.s4-3-5.

[0031] Fr.s4-3-3 was separated by gel column chromatography with an elution system of methanol or dichloromethane:methanol:petroleum ether = 1:1:1, yielding three fractions Fr.s4-3-3-1 to Fr.s4-3-3-3.

[0032] Fr.s4-3-3-2 was separated using a semi-preparative liquid chromatograph (methanol / water = 26 / 74, tR = 95.5 min) to obtain the halokaurane-type diterpenoid compound.

[0033] The structural identification data of kauriane-type diterpenoids are as follows: 1H and 13C NMR data, Table 1; HRESIMS m / z 391.1645 [M + Na]+, calcd for 391.1647.

[0034] Table 1. NMR data (CD3OD) of compound 1 Example 2 A method for preparing a halokaurane-type diterpenoid compound, which differs from Example 1, is that the dried whole herb of *Haloxylon ammodendron* is extracted five times by heating and refluxing with 75% ethanol, each time for 4 hours; The ethyl acetate fraction was separated by silica gel column chromatography with the following eluent gradient: starting with dichloromethane, then successively using mixed solvents of dichloromethane and methanol at volume ratios of 90:10, 85:15, 80:20, 70:30, and 60:40, and finally using methanol. Fr.s4 was separated by medium-low pressure C-18 reversed-phase column chromatography, eluted with a 30%~100% methanol gradient. Fr.s4-3 was separated by silica gel column chromatography with the following eluent gradient: starting with dichloromethane, then successively using mixed solvents of dichloromethane and methanol at volume ratios of 94:6, 91:9, 88:12, and 85:15, and finally using methanol. Fr.s4-3-3 was separated by gel column chromatography with an elution system of dichloromethane:methanol:petroleum ether = 1:1:1; Fr.s4-3-3-2 was separated using a semi-preparative liquid chromatograph (methanol / water = 50 / 50) to obtain the halokaurane-type diterpenoid compound.

[0035] Example 3 A method for preparing a halokaurane-type diterpenoid compound, which differs from Example 1 in that the dried whole herb of *Haloxylon ammodendron* is extracted four times by heating and refluxing with 90% methanol, each time for 5 hours; The petroleum ether fraction was separated by silica gel column chromatography with the following eluent gradient: starting with dichloromethane, then successively using mixed solvents of dichloromethane and ethanol at volume ratios of 90:10, 85:15, 80:20, 70:30, and 60:40, and finally using ethanol. Fr.s4 was separated by medium-low pressure C-18 reversed-phase column chromatography, eluted with a gradient of 30% to 100% ethanol. Fr.s4-3 was separated by silica gel column chromatography with the following eluent gradient: starting with dichloromethane, then successively using mixed solvents of dichloromethane and ethanol at volume ratios of 94:6, 91:9, 88:12, and 85:15, and finally using ethanol. Fr.s4-3-3 was separated by gel column chromatography with an elution system of dichloromethane:methanol:petroleum ether = 1:1:1; Fr.s4-3-3-2 was separated using a semi-preparative liquid chromatograph (acetonitrile / water = 20 / 80) to obtain the halokaurane-type diterpenoid compound.

[0036] Example 4 A method for preparing a halokaurane-type diterpenoid compound differs from Example 1 in that, in step S7, the solvent of the semi-preparative liquid chromatograph further contains an acid additive, which is one of formic acid, acetic acid, or trifluoroacetic acid, and the volume fraction of the acid additive is 0.05% to 1.0%; the solvent composition of the semi-preparative liquid chromatograph is methanol-water or acetonitrile-water, wherein the volume fraction of methanol or acetonitrile is 10% to 60%, and the volume fraction of the acid additive in the solvent is 0.05% to 1.0%.

[0037] Compared with the separation conditions in Example 1, this example significantly improved the separation efficiency of semi-preparative liquid chromatography by adding 0.1% formic acid to the elution solvent, optimizing the chromatographic peak symmetry factor of the target compound from 1.8 to 1.1, increasing its resolution with nearest-neighbor impurities from 0.8 to 1.5, and increasing the purity of the target compound collected from a single injection from 85% to 95%.

[0038] Example 5 A method for preparing a halokaurane-type diterpenoid compound differs from Example 4 in that the separation conditions of the semi-preparative liquid chromatograph further include: the flow rate is controlled at 1.0 mL / min to 3.0 mL / min, and the column oven temperature is maintained at 20°C to 30°C; the composition of the solvent, the type and concentration of the acid-containing additive, the flow rate, and the column oven temperature are kept constant during a single separation operation.

[0039] This embodiment significantly improved the reproducibility of the separation process by precisely controlling the flow rate of the semi-preparative liquid chromatography to 2.0 mL / min and maintaining the column temperature at a constant 25°C. The relative standard deviation of the retention time of the target compound in three consecutive batches decreased from 8.2% in Example 4 to 1.5%, the relative standard deviation of the peak area improved from 12.5% ​​to 3.2%, and the relative standard deviation of the purity of the final product between batches also decreased significantly from 7.8% to 2.1%.

[0040] Example 6 A method for preparing a halokaurane-type diterpenoid compound differs from Example 5 in that, after step S7, a post-processing step is included for the fraction containing the target compound collected by a semi-preparative liquid chromatograph. Specifically, the method involves adding 1 / 5 to 1 / 3 of the volume of a saturated sodium chloride aqueous solution to the fraction, mixing well, and then extracting it 2 to 3 times with a 1:1 dichloromethane-ethyl acetate mixed solvent. The organic phases are then combined. After drying the organic phase with anhydrous sodium sulfate, it is concentrated under reduced pressure at 35°C to 40°C to obtain the solid halokaurane-type diterpenoid compound.

[0041] In this embodiment, by using a post-treatment step of liquid-liquid extraction with the addition of 30% saturated sodium chloride aqueous solution and a dichloromethane-ethyl acetate (1:1) mixed solvent, the content of acid-sensitive degradation products that may be generated due to the acidic environment was successfully controlled from 15% to below 3% compared to Example 5.

[0042] Example 7 A method for preparing a halokaurane-type diterpenoid compound differs from Example 6 in that, after drying the organic phase with anhydrous sodium sulfate and before vacuum concentration, the method further includes filtering out the organic phase after drying with anhydrous sodium sulfate, collecting the filtrate, and passing it through a Sephadex LH-20 gel column pre-equilibrated with the same solvent. Elution is performed using a 1:1 volume ratio of dichloromethane-ethyl acetate mixed solvent or methanol as the eluent. The eluent is collected based on the colorimetric characteristics of the target compound or a predetermined fraction volume. The fractions containing the target compound are then combined and concentrated under vacuum.

[0043] This embodiment effectively removes inorganic salt impurities that may be introduced by the previous salting-out extraction by adding a Sephadex LH-20 gel column desalting purification step, reducing the residual sodium chloride content in the product from about 2.3% in Example 6 to below 0.05%, and the inorganic ash content from 3.1% to 0.1%, while further increasing the product purity from 90% to 98%.

[0044] Example 8 A method for preparing a halokaurane-type diterpenoid compound, differing from Example 7, includes the following purification step after obtaining the solid halokaurane-type diterpenoid compound by vacuum concentration: dissolving the solid halokaurane-type diterpenoid compound in an acetone-methanol mixed solvent with a volume ratio of 2:1 to 1:1 to form a saturated or near-saturated solution; then slowly adding n-hexane at a volume of 3 to 5 times the total volume of the solution, maintaining the system temperature at 15°C to 25°C and stirring during the addition; after the addition is complete, allowing it to stand for 1 to 2 hours to promote crystal precipitation; filtering, washing the obtained crystals with a small amount of pre-cooled n-hexane, and then vacuum drying at 35°C to 40°C to obtain high-purity halokaurane-type diterpenoid compound crystals.

[0045] In this embodiment, recrystallization purification using an acetone-methanol / n-hexane system increased the chemical purity of the product from approximately 95% to over 99.5%. The relative standard deviation of the characteristic peak intensity of the powder X-ray diffraction pattern was significantly improved from 25% to 8%, indicating a substantial improvement in crystal uniformity. At the same time, the total amount of organic impurities decreased from 5% to below 0.5%, and the crystal particle size distribution became more uniform, with the D90 / D10 ratio optimized from 15.2 to 5.3.

[0046] Comparative Example 1 A method for preparing a halokaurane-type diterpenoid compound includes the following steps: 10 kg of dried whole *Haloxylon ammodendron* herb was used, with the same raw materials as in Example 1, but the extraction and separation conditions were changed as follows: The extraction solvent was changed to pure water, and the extraction was carried out twice by heating and reflux, each time for 2 hours. The combined aqueous extracts were concentrated under reduced pressure to obtain the total extract. The total extract was then directly suspended in water and extracted with ethyl acetate alone. The obtained ethyl acetate extract was separated by silica gel column chromatography, with the eluent gradient simplified to dichloromethane → methanol (no intermediate gradient change), yielding 5 first fractions.

[0047] The five first components were separated by medium-low pressure C-18 reversed-phase column chromatography and eluted isocratically with 50% methanol (instead of gradient elution). After chromatographic separation and elution, each first component yielded four second components.

[0048] The four second components were separated by silica gel column chromatography with a fixed eluent ratio of dichloromethane:methanol = 9:1. Each second component was eluted to obtain three third components.

[0049] The three third components were separated by gel column chromatography, with methanol as the only elution system (instead of the mixed solvent system in Example 1). Each third component was separated and eluted by chromatography to obtain two fourth components.

[0050] Each fourth component was separated using a semi-preparative liquid chromatograph with methanol-water = 50:50 as the solvent (no optimized ratio), and the major fractions were collected.

[0051] After the above treatment, the obtained fraction was analyzed by nuclear magnetic resonance (NMR) and high-resolution mass spectrometry (HRESIMS). No chemical structural characteristic signals (such as characteristic chemical shifts δ_H 3.64 (1H, d, J=3.1 Hz) and δ_C 70.6 (CH)) consistent with the halokaurane-type diterpenoids described in Example 1 were detected, and the mass spectrometry did not show the molecular ion peak at m / z 391.1645 [M+Na]⁺. The results indicate that this method failed to separate the target compound.

[0052] In vitro antitumor experiment The following cell lines in logarithmic growth phase were added to 96-well plates: leukemia cell line K562, non-small cell lung cancer cell line A549, human triple-negative breast cancer cell line MDA-MB-231, human colon adenocarcinoma cell line SW480, and human lung cancer paclitaxel-resistant cell line A549 / Taxo. Each well contained 3 × 10⁻⁶ cells / well. 3 ~8×10 3 Cells were cultured for 24 h, and the old culture medium was discarded. Drugs were administered to groups, with three replicates per group. The control group received fresh complete culture medium, while the experimental groups received 200 μL / well of complete culture medium containing the test sample. Monomer compounds were dissolved in DMSO. The initial screening final concentration was 50 μM for the monomer compound. Based on the initial screening results, secondary screening was performed, with final monomer compound concentrations of 50 μM, 10 μM, 2 μM, 0.4 μM, and 0.08 μM. Doxorubicin (DOX) and paclitaxel (Taxol) were used as positive controls. After drug administration, cells were incubated for 48 h. The culture medium in the wells of adherent cells was discarded, and 20 μL of MTS solution and 100 μL of culture medium were added. 100 μL of culture supernatant in the wells of suspended cells was discarded, and 20 μL of MTS solution was added. Three replicates of blank cells were set up, with 20 μL of MTS solution and 100 μL of culture medium added. The cells were incubated for another 2–4 h before measuring absorbance. The OD values ​​of each well were read and recorded at a wavelength of 492 nm using a multi-functional microplate reader. The growth inhibition rate was calculated, and the half-maximal inhibitory concentration (IC50) was determined using SPSS (13.0) software. The experimental results are shown in Table 2.

[0053] Table 2 Results of anti-tumor experiments As can be seen from the results in Table 2, the compound of Formula 1 has a significant inhibitory effect on the proliferation of K562, A549, MDA-MB-231, SW480 and the paclitaxel-resistant human lung cancer strain A549 / Taxo.

[0054] Although the embodiments of the present invention have been disclosed above, they are not limited to the applications listed in the specification and embodiments. They can be applied to various fields suitable for the present invention. For those skilled in the art, other modifications can be easily made. Therefore, without departing from the general concept defined by the claims and their equivalents, the present invention is not limited to the specific details.

Claims

1. A halokaurane-type diterpenoid compound, characterized in that, The structural formula of the compound is shown below: 。 2. The method for preparing halokaurane-type diterpenoids as described in claim 1, characterized in that, Includes the following steps: S1: The whole herb of *Haloxylon ammodendron* was extracted by heating and refluxing with 60% to 95% ethanol or methanol for 2 to 5 times, each time for 2 to 5 hours. The extracts were combined and concentrated under reduced pressure to obtain the total extract. S2: The total extract was suspended in an equal volume of water and extracted sequentially with petroleum ether, dichloromethane, ethyl acetate and n-butanol. The solvent was recovered to obtain petroleum ether fraction extract, dichloromethane fraction extract, ethyl acetate fraction extract, n-butanol fraction extract and water fraction extract. S3: The ethyl acetate fraction extract was separated by silica gel column chromatography with an eluent gradient of dichloromethane to a mixed solvent of dichloromethane and methanol or ethanol, and then to methanol or ethanol, to obtain 8 fractions Fr.s1 to Fr.s8; wherein the volume ratio of dichloromethane to methanol or ethanol was 100:0 to 0:

100. S4: Fr.s4 was separated by medium-low pressure C-18 reversed-phase column chromatography and eluted with a gradient of methanol or ethanol with a volume fraction of 10% to 100% to obtain 6 fractions Fr.s4-1 to Fr.s4-6. S5: Fr.s4-3 was separated by silica gel column chromatography with an eluent gradient from dichloromethane to a mixed solvent of dichloromethane and methanol or ethanol, and then to methanol or ethanol, to obtain five fractions Fr.s4-3-1 to Fr.s4-3-5; wherein the volume ratio of dichloromethane to methanol or ethanol was 100:0 to 0:

100. S6: Fr.s4-3-3 was separated by gel column chromatography, with the elution system being methanol or a mixture of dichloromethane, methanol, and petroleum ether, wherein the volume ratio of dichloromethane, methanol, and petroleum ether was 1:1:1, to obtain three components Fr.s4-3-3-1 to Fr.s4-3-3-3; S7: Fr.s4-3-3-2 was separated using a semi-preparative liquid chromatograph, with the solvent being a mixture of methanol and water or a mixture of acetonitrile and water, wherein the volume fraction of methanol or acetonitrile was 10% to 60%, to obtain the halokaurane-type diterpenoid compound.

3. The method for preparing halokaurane-type diterpenoids as described in claim 2, characterized in that, In step S7, the solvent of the semi-preparative liquid chromatograph also contains an acid additive, which is one of formic acid, acetic acid or trifluoroacetic acid, and the volume fraction of the acid additive in the solvent is 0.05% to 1.0%.

4. The method for preparing halokaurane-type diterpenoids as described in claim 3, characterized in that, In step S7, the separation conditions of the semi-preparative liquid chromatograph further include: the flow rate is controlled at 1.0 mL / min to 3.0 mL / min, and the column oven temperature is maintained at 20°C to 30°C; the composition of the solvent, the flow rate, and the column oven temperature of the semi-preparative liquid chromatograph remain constant during a single separation operation.

5. The method for preparing halokaurane-type diterpenoids as described in claim 4, characterized in that, Following step S7, the process further includes a post-processing step for the fraction containing the target compound collected by the semi-preparative liquid chromatograph. Specifically, the steps are as follows: 1 / 5 to 1 / 3 of the volume of saturated sodium chloride aqueous solution is added to the fraction, mixed well, and then extracted 2 to 3 times with a 1:1 volume ratio of dichloromethane-ethyl acetate mixed solvent. The organic phases are then combined. After drying the organic phase with anhydrous sodium sulfate, it is concentrated under reduced pressure at 35°C to 40°C to obtain the solid of the halokaurane-type diterpenoid compound.

6. The method for preparing halokaurane-type diterpenoids as described in claim 5, characterized in that, After drying the organic phase with anhydrous sodium sulfate and before vacuum concentration, the dried organic phase is filtered out, the filtrate is collected, and it is passed through a Sephadex LH-20 gel column pre-equilibrated with the same solvent. The eluent is a 1:1 volume ratio of dichloromethane-ethyl acetate mixture or methanol. The eluent is collected according to the colorimetric characteristics of the target compound or a predetermined fraction volume. The fractions containing the target compound are combined and then concentrated under vacuum.

7. The method for preparing halokaurane-type diterpenoids as described in claim 5, characterized in that, After obtaining the solid of the halokaurane-type diterpenoid compound by vacuum concentration, the following purification steps are further included: dissolving the solid of the halokaurane-type diterpenoid compound in an acetone-methanol mixed solvent with a volume ratio of 1:1 to 2:1 to form a saturated or near-saturated solution; then slowly adding n-hexane at a volume of 3 to 5 times the total volume of the solution to the solution, maintaining the system temperature at 15°C to 25°C and stirring during the addition; after the addition is completed, allowing it to stand for 1 to 2 hours to promote crystal precipitation; filtering, washing the obtained crystals with a small amount of pre-cooled n-hexane, and then drying them under vacuum at 35°C to 40°C to obtain high-purity crystals of the halokaurane-type diterpenoid compound.

8. The use of the halokaurane-type diterpenoid compound as described in claim 1 in the preparation of antitumor drugs.

9. The application as described in claim 8, characterized in that, Application of halogenated kauriane-type diterpenoids in the preparation of anti-leukemia drugs, anti-lung tumor drugs, anti-breast tumor drugs, anti-colon tumor drugs, or drugs against paclitaxel-resistant strains of human lung cancer.

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