An antitumor active compound, a preparation method and application thereof

Abstract

This invention relates to the field of novel compounds from the field of bald cypress, and provides an antitumor active compound, its preparation method, and its application. The antitumor active compound provided by this invention ( Taiwanin J This compound, a secondary metabolite of *Cephalotaxus fortunei*, belongs to the lignan class of compounds. Its antitumor activity enriches the diversity of lignan compounds and exhibits significant inhibitory activity against HeLa and A549 cells, showing broad application prospects in the preparation of antitumor drugs. Furthermore, it can serve as a lead compound, which is of great significance for the development of novel antitumor drugs. This invention also provides a method for preparing the antitumor active compound described above. The preparation method provided by this invention is simple to operate, low in cost, and easy to implement industrially. Moreover, this invention uses *Cephalotaxus fortunei* stem bark as raw material for extraction, achieving effective utilization of this rare plant resource, which aligns with the concepts of green environmental protection and a low-carbon economy.

Description

Technical Field

[0001] This invention relates to the field of novel compounds from bald cypress, and particularly to an antitumor active compound, its preparation method, and its application. Background Technology

[0002] Cancer is a major disease that seriously threatens human life and health, with malignant tumors receiving particular attention due to their high mortality rate. In the field of anti-tumor drug development, natural product chemistry has always played a crucial role. The diverse array of active ingredients found in nature provides an inexhaustible source for new drug discovery, and many existing anti-tumor drugs are derived from natural products or their derivatives. Therefore, searching for compounds with novel mechanisms of action from natural resources such as plants, microorganisms, and marine organisms remains an important direction for anti-cancer drug development.

[0003] bald cedar ( Taiwania flousiana Gaussen ) for Baike ( Cupressaceae ) Taiwania ( Taiwania This genus of evergreen trees is a rare and endemic species in my country. Plants in this genus are rich in chemical components, mainly including essential oils, lignans, terpenes, biflavonoids, and phenols. Studies have shown that its extracts and essential oils possess a wide range of biological activities, such as antibacterial, antioxidant, anti-inflammatory, and antitumor effects, demonstrating potential applications in medicine and agriculture. In recent years, the antitumor activity of this genus has attracted considerable attention. Research focuses on the cytotoxicity of specific chemical components on human tumor cells. For example, compounds... taiwanin A It exhibits significant inhibitory activity against tumor cell lines such as A-549, MCF-7, and HT-29, and its ED... 50 Values ​​between 0.1 and 0.2 μM indicate that it possesses potent antitumor activity even at extremely low concentrations. These findings highlight... taiwanin A Its value as a potential anticancer lead compound also provides important scientific evidence for the development of antitumor drugs based on the genus *Taxus*.

[0004] However, there are currently few known antitumor active compounds in the *Taxus* genus, and a large number of potential active compounds have not yet been discovered, so there is an urgent need to further develop new antitumor active compounds. Summary of the Invention

[0005] In view of this, the present invention provides an antitumor active compound, its preparation method, and its application. The antitumor active compound provided by the present invention is a novel lignan compound that exhibits significant inhibitory activity against cancer cells, and has broad application prospects in the preparation of antitumor drugs.

[0006] To achieve the above-mentioned objectives, the present invention provides the following technical solution: An antitumor active compound having the structure shown in Formula I: Formula I.

[0007] This invention also provides a method for preparing the antitumor active compound described above, comprising the following steps: The crude extract was obtained by extracting the bark powder from the stem of the bald cypress using an ethanol solution. The crude extract was dissolved in water and then extracted with ethyl acetate to obtain an ethyl acetate extract. The ethyl acetate extract was eluted by silica gel column chromatography, and the eluents were collected and labeled Fr.1 to Fr.10 in the order of elution. The eluent for the silica gel column chromatography was a dichloromethane-methanol system. The Fr. 8 was subjected to a first silica gel column chromatography to obtain an eluent containing an antitumor active compound with the structure shown in Formula I; The eluent was subjected to a second silica gel column chromatography to obtain the crude product; The crude product was purified by dextran gel column chromatography to obtain the antitumor active compound with the structure shown in Formula I.

[0008] Preferably, the volume concentration of the ethanol solution is 65-95%.

[0009] Preferably, the extraction includes: soaking the bald cypress bark powder in an ethanol solution followed by rotary extraction.

[0010] Preferably, the soaking time is 18-36 hours; the ratio of the bald cypress bark powder to the ethanol solution is (30g-16kg):(300mL-200L). The temperature of the rotary extraction is 40~70℃, the number of rotary extractions is 2~4 times, and the time for each rotary extraction is 10~40 minutes.

[0011] Preferably, the ethyl acetate extraction is performed 3 to 6 times; the ratio of the crude extract to the amount of ethyl acetate used in each ethyl acetate extraction is (780 to 830) g: (5 to 6) L.

[0012] Preferably, during the silica gel column chromatography elution process, the volume ratio of dichloromethane to methanol in the dichloromethane-methanol system is 100:0, 100:1, 30:1, 10:1, 5:1, and 2:1, respectively.

[0013] Preferably, the eluent for the first silica gel column chromatography is a first dichloromethane-methanol system, wherein the volume ratio of dichloromethane to methanol in the first dichloromethane-methanol system is 30:1 to 50:1; The eluent used in the second silica gel column chromatography is a second dichloromethane-methanol system, in which the volume ratio of dichloromethane to methanol is 20:1 to 40:1.

[0014] Preferably, the eluent for the dextran gel column chromatography purification is methanol.

[0015] The present invention also provides the application of the antitumor active compounds described in the above scheme or the antitumor active compounds prepared by the preparation method described in the above scheme in the preparation of antitumor drugs.

[0016] This invention provides an antitumor active compound having the structure shown in Formula I (named...). Taiwanin J The antitumor active compound provided by this invention is a secondary metabolite of *Cephalotaxus fortunei*, belonging to the lignan class of compounds. This antitumor active compound enriches the diversity of lignan compounds and exhibits significant inhibitory activity against HeLa and A549 cells, showing broad application prospects in the preparation of antitumor drugs. Furthermore, it can serve as a lead compound, which is of great significance for the development of novel antitumor drugs. The results of the examples show that at a concentration of 30 μM, the compound… Taiwanin J It showed good inhibitory activity against HeLa cells and A549 cells, with inhibition rates of 80.70% and 69.68%, respectively.

[0017] This invention also provides a method for preparing the antitumor active compound described in the above-mentioned scheme. Using *Taxus chinensis* stem bark as raw material, the method involves extraction with ethanol solution, extraction with ethyl acetate, elution by silica gel column chromatography, first silica gel column chromatography, second silica gel column chromatography, and purification by dextran gel column chromatography to obtain the antitumor active compound. The preparation method provided by this invention has the advantages of short cycle, mild conditions, few by-products, strong stereoselectivity, and low cost, making it easy to implement industrially. Furthermore, the extraction using *Taxus chinensis* stem bark as raw material achieves effective utilization of this rare plant resource, conforming to the concepts of green environmental protection and low-carbon economy. It not only efficiently obtains the target product but also provides a feasible technical path for the high-value utilization of *Taxus chinensis* resources. Attached Figure Description

[0018] Figure 1 The compound of this invention Taiwanin J HR-ESI-MS spectra; Figure 2 The compound of this invention Taiwanin J of 1 H-NMR spectrum; Figure 3 The compound of this invention Taiwanin J of 13 C-NMR; Figure 4 The compound of this inventionTaiwanin J HMBC spectrum; Figure 5 The compound of this invention Taiwanin J of 1 H- 1 H COSY spectrum. Detailed Implementation

[0019] This invention provides an antitumor active compound having the structure shown in Formula I: Formula I.

[0020] In this invention, the antitumor active compound with the structure shown in Formula I is named... Taiwanin J, It belongs to the lignan class of compounds. Taiwanin J It enriches the variety of natural lignans and shows significant inhibitory activity against HeLa and A549 cells, which has broad application prospects in the preparation of antitumor drugs and is of great significance for the development of novel antitumor lead compounds.

[0021] This invention also provides a method for preparing the antitumor active compound described above, comprising the following steps: The crude extract was obtained by extracting the bark powder from the stem of the bald cypress using an ethanol solution. The crude extract was dissolved in water and then extracted with ethyl acetate to obtain an ethyl acetate extract. The ethyl acetate extract was eluted by silica gel column chromatography, and the eluents were collected and labeled Fr.1 to Fr.10 in the order of elution. The eluent for the silica gel column chromatography was a dichloromethane-methanol system. The Fr. 8 was subjected to a first silica gel column chromatography to obtain an eluent containing an antitumor active compound with the structure shown in Formula I; The eluent was subjected to a second silica gel column chromatography to obtain the crude product; The crude product was purified by dextran gel column chromatography to obtain the antitumor active compound with the structure shown in Formula I.

[0022] Unless otherwise specified, the raw materials used in this invention are preferably commercially available products.

[0023] This invention uses an ethanol solution to extract bald cypress bark powder to obtain a crude extract. In this invention, the particle size of the bald cypress bark powder is preferably 0.02~20 mm, more preferably 0.1~10 mm, and specifically 2~5 mm. The bald cypress bark powder is obtained by pulverizing bald cypress bark, preferably naturally air-dried bald cypress bark. This invention does not specifically limit the pulverization parameters, as long as the target particle size of the bald cypress bark powder can be obtained.

[0024] In this invention, the volume concentration of the ethanol solution is preferably 65-95%, and more preferably 75%.

[0025] In this invention, the extraction preferably includes: soaking the bald cypress bark powder in an ethanol solution followed by rotary extraction; the soaking temperature is preferably room temperature, without the need for additional heating or cooling; the soaking time is preferably 18~36h, specifically 18h, 24h, 30h or 36h; the ratio of the bald cypress bark powder to the ethanol solution is preferably (30g~16kg):(300mL~200L), preferably (15~16)kg:(150~200)L, specifically 30g:300mL or 15kg:150L.

[0026] In this invention, the preferred temperature for the rotary extraction is 40-70°C, specifically 55°C; the preferred number of extractions is 2-4 times, specifically 3 times; and the preferred extraction time is 10-40 minutes, specifically 30 minutes. After soaking, the first rotary extraction is performed. Then, the extraction system is subjected to solid-liquid separation, the filtrate is collected, and ethanol is added again to the extraction residue for a second rotary extraction, and so on, until the extraction is complete. The filtrates obtained from multiple rotary extractions are combined to obtain the extract. The solid-liquid separation method is preferably filtration; the amount of ethanol solution used in each rotary extraction is preferably the same; and the rotary extraction is preferably performed in an extraction tank.

[0027] In this invention, after rotary extraction, the obtained extract is preferably concentrated under reduced pressure to obtain the crude extract. The pressure for the reduced pressure concentration is preferably -0.04 to -0.07 MPa, specifically -0.04 MPa, -0.05 MPa, -0.06 MPa, or -0.07 MPa. The temperature for the reduced pressure concentration is preferably 45 to 60°C, specifically 45°C, 50°C, 55°C, or 60°C. This invention does not specifically limit the time for the reduced pressure concentration, as long as the solvent is removed. In a specific embodiment of this invention, it is preferable to concentrate under reduced pressure until there is no alcohol odor.

[0028] After obtaining the crude extract, the present invention dissolves the crude extract in water and then extracts it with ethyl acetate to obtain an ethyl acetate extract. In the present invention, the water used for water dissolution is preferably pure water; the number of ethyl acetate extractions is preferably 3 to 6 times, specifically 4 times; the ethyl acetate extraction is preferably an equal volume extraction, that is, the volume ratio of the water used for water dissolution to the ethyl acetate used in each ethyl acetate extraction is preferably 1:1; the volume ratio of the crude extract to the ethyl acetate used in each ethyl acetate extraction is preferably (780~830) g: (5~6) L, specifically 800 g: 6 L.

[0029] In this invention, the ethyl acetate phase is collected after extraction. Preferably, the ethyl acetate in the obtained ethyl acetate phase is removed to obtain the ethyl acetate extract (i.e., containing...). Taiwanin J The preferred method for removing the ethyl acetate is vacuum concentration. The present invention does not specify the parameters for vacuum concentration, as long as the ethyl acetate can be removed.

[0030] After obtaining the ethyl acetate extract, the present invention elutes the ethyl acetate extract by silica gel column chromatography, collects each eluent, and labels each eluent sequentially as Fr.1~Fr.10 (Fr. 1, Fr. 2, Fr. 3, Fr. 4, Fr. 5, Fr. 6, Fr. 7, Fr. 8, Fr. 9 and Fr. 10) according to the elution order. In the present invention, the silica gel column chromatography elution preferably includes the following steps: dissolving the ethyl acetate extract to obtain a solution; mixing the solution with silica gel, then removing the dissolved reagent, packing the column, and performing silica gel column chromatography elution. In the present invention, the dissolved reagent is preferably a dichloromethane-methanol solution, and the volume ratio of dichloromethane to methanol in the dichloromethane-methanol solution is preferably 1:1. In the present invention, the volume ratio of the ethyl acetate extract to the dissolved reagent is preferably 300~600g:400~700mL. In this invention, the silica gel particle size is preferably 100-300 mesh, specifically 100-200 mesh or 200-300 mesh. In this invention, the mass ratio of the ethyl acetate extract to the silica gel, based on dry weight, is preferably 1:0.8-1.5, specifically 1:0.8, 1:1, 1:1.2, or 1:1.5. In this invention, the method for removing the dissolved reagent is preferably vacuum concentration. This invention does not specifically limit the method of vacuum concentration, as long as the dissolved reagent can be removed.

[0031] In this invention, the eluent for silica gel column chromatography is a dichloromethane-methanol system; the volume ratio of dichloromethane to methanol in the dichloromethane-methanol system preferably varies from 100:0 to 2:1; specifically, during the silica gel column chromatography elution process, the volume ratio of dichloromethane to methanol in the dichloromethane-methanol system is successively 100:0, 100:1, 30:1, 10:1, 5:1, and 2:1.

[0032] After obtaining Fr.8, the present invention performs a first silica gel column chromatography on Fr.8 to obtain an eluent containing an antitumor active compound with the structure shown in Formula I. In the present invention, the eluent for the first silica gel column chromatography is a first dichloromethane-methanol mixture, wherein the volume ratio of dichloromethane to methanol in the first dichloromethane-methanol mixture is preferably 30:1 to 50:1, more preferably 40:1. Before the first silica gel column chromatography, the present invention preferably further includes performing a first impurity removal and a second impurity removal sequentially on the silica gel column. In the present invention, the reagent for the first impurity removal is preferably dichloromethane; the reagent for the second impurity removal is preferably a mixture of dichloromethane and methanol with a volume ratio of 70:1. In the present invention, the first impurity removal can remove silica gel impurities; the second impurity removal can remove impurities in large quantities above the main spot.

[0033] After obtaining the eluent containing the antitumor active compound with the structure shown in Formula I, the present invention performs a second silica gel column chromatography on the eluent to obtain the crude product. In the present invention, the eluent used for the second silica gel column chromatography is preferably a second dichloromethane-methanol system, and the volume ratio of dichloromethane to methanol in the second dichloromethane-methanol system is preferably 20:1 to 40:1, more preferably 30:1.

[0034] After obtaining the crude product, the present invention purifies the crude product by dextran gel column chromatography to obtain the antitumor active compound with the structure shown in Formula I. In the present invention, the eluent for the dextran gel column chromatography purification is preferably methanol; the flow rate of the methanol is preferably 0.4~0.6 mL / min, specifically 0.5 mL / min; the chromatographic column for the dextran gel column chromatography purification is preferably a Sephadex LH-20 gel chromatography column.

[0035] This invention also provides the application of the antitumor active compounds described in the above-described schemes or the antitumor active compounds prepared by the preparation methods described in the above-described schemes in the preparation of antitumor drugs. In this invention, the tumor is preferably leukemia, liver cancer, colon cancer, breast cancer, cervical cancer, or lung cancer.

[0036] In a specific embodiment of the present invention, the cellular activity of the antitumor active compound is preferably detected by the MTS method, and the content of the antitumor active compound during the test is preferably 20~40μM, more preferably 30μM.

[0037] The technical solutions of this invention will be clearly and completely described below with reference to the embodiments thereof. Obviously, the described embodiments are only a part of the embodiments of this invention, and not all of them. Based on the embodiments of this invention, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of this invention.

[0038] Example 1 1. Extraction of antitumor active compounds Taiwanin J (1) 15.6 kg of bald cypress bark was crushed to 0.1-10 mm and soaked in a 70% ethanol solution for 24 h. The mass-to-volume ratio of the ethanol solution to the bald cypress bark powder was 15 kg: 150 L. The soaked material was subjected to constant-temperature rotary extraction at 55 °C for 3 times, each extraction lasting 30 min. The volume of ethanol solution used for each extraction was the same. After each extraction, the filtrate was collected by filtration. The filtrates were combined to obtain the extract. The extract was concentrated under reduced pressure to remove the solvent, yielding 3020 g of crude extract of secondary metabolites. The concentration under reduced pressure was 55 °C and the pressure was -0.05 to -0.06 MPa.

[0039] (2) Dissolve all crude extracts in 6 L of water, and extract the crude extracts three times with ethyl acetate, using 6 L of ethyl acetate for each extraction, with each extraction lasting 45 min. Remove the solvent from the ethyl acetate phase obtained by vacuum concentration to obtain a solution containing... Taiwanin J 800g of ethyl acetate extract; the mass-to-volume ratio of crude extract to ethyl acetate was 800g:6L.

[0040] (3) Dissolve 800g of ethyl acetate extract in 650mL of dichloromethane-methanol solution with a volume ratio of 1:1, then mix with 800g of silica gel (100~200 mesh), concentrate under reduced pressure to remove solvent, and pack into a column; perform gradient elution sequentially with dichloromethane-methanol systems with volume ratios of 100:0, 100:1, 30:1, 10:1, 5:1, and 2:1, and combine the elution fractions to obtain 10 fractions, which are named Fr. 1~Fr. 10.

[0041] (4) The Fr. 8 fraction was subjected to silica gel column chromatography. First, 300 mL of dichloromethane was used to elute to remove silica gel impurities. Then, 300 mL of dichloromethane-methanol (volume ratio of dichloromethane to methanol was 100:1) was used to elute to remove the small amount of impurities above the main spot. Finally, dichloromethane-methanol (volume ratio of dichloromethane to methanol was 40:1) was used to elute until the target compound disappeared. The eluents containing the relatively pure target compound were combined to obtain the fraction containing the target compound. Taiwanin J Eluent for crude product.

[0042] (5) Containing Taiwanin JThe eluent of the crude product was evaporated to dryness and subjected to silica gel column chromatography. First, elution was performed with 300 mL of dichloromethane-methanol (100:1 v / v) to remove small amounts of impurities above the main spot. Then, elution was performed with 200 mL of dichloromethane-methanol (30:1 v / v), at which point the target compound began to elute. Elution continued with dichloromethane-methanol (30:1 v / v) until the target compound disappeared, yielding the product. Taiwanin J The crude product.

[0043] (6) Using methanol (flow rate 0.5 mL / min) as solvent, for the reaction containing Taiwanin J The crude product was purified by dextran gel chromatography (Sephadax LH-20 gel chromatography column), dried, and 31.3 mg was obtained. Taiwanin J .

[0044] 2. Structural identification Compounds were identified using 1D / 2D NMR (one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy) and HR-ESI-MS (high-resolution electrospray ionization mass spectrometry). Taiwanin J The structure.

[0045] Taiwanin J HR-ESI-MS plot as follows Figure 1 As shown: The molecular formula of this compound is C2. 26 H 26 O 13 (m / z: 547.1444 [M+H]) + The calculated value is 547.1446, and the degree of unsaturation is 14.

[0046] Taiwanin J The results of 1D / 2D NMR detection are as follows Figures 2 - 5 As shown, Taiwanin J Compound based 1 H-NMR, 13 C-NMR spectrum ( Figures 2 - 3 The data show that the δC (ppm) values ​​are 32.0, 62.5, 73.6, 102.5, and 103.6 for five methylene groups, 43.5, 54.2, 71.3, 74.9, 78.1, 78.1, 100.1, 103.6, 108.8, 109.3, 110.6, and 127.2 for twelve methine groups, and 121.3, 132.1, 144.1, 148.3, 149.4, 151.5, 154.0, 176.2, and 194.8 for nine quaternary carbons. (The data is presented in the original text, but the translation is incomplete.) 1 H- 1 HCOSY spectrum ( Figure 5The diagram shows that, with the support of HSQC and related signals of H-1`` / H-2`` / H-3`` / H-4`` / H-5`` / H-6``, H-2` / H-3`, H-7 / H-8 / H-9 / H-8`, segments of C-1``-C-2``-C-3``-C-4``-C-5`, C-6`` are revealed. This is achieved through the HMBC diagram (…). Figure 4 The results show that H-5 is associated with C-1 and C-3, H-2 with C-4 and C-6, H-2' with C-6' and C-4', H-3' with C-1' and C-4', H-3OCH2O4' with C-3 and C-4, and H-4'OCH2O5' with C-4' and C-5'. This reveals two oxygen ring segments in 3OCH2O4 and 4'OCH2O5' and the two benzene ring segments attached to them, thus identifying the compound. Taiwanin J The carbon skeleton.

[0047] In conclusion, Taiwanin J It has the following structure: It belongs to the lignan class of compounds, and its chemical name is: 3-(benzo[d][1,3]dioxole-5-carbonyl)-4-((6-(((2S,3R,4S,5S,6R)-3,4,5-trihydroxy-6-(hydroxymethyl)tetrahydro-2H-pyran-2-yl)oxy)benzo[d][1,3]dioxol-5-yl)methyl)dihydrofuran-2(3H)-one.

[0048] compound Taiwanin J The chemical shift δ is assigned as shown in Table 1.

[0049] Table 1 Compounds Taiwanin J Chemical shift δ assignment

[0050] Example 2 compound Taiwanin J Antitumor activity test: The principle of MTS assay for cell viability detection: MTS is a novel MTT analogue, a yellow dye. Succinate dehydrogenase in the mitochondria of live cells can metabolize and reduce MTS, generating a soluble formazan compound. The formazan content can be measured using a microplate reader at 490 nm. Under normal circumstances, the amount of formazan produced is directly proportional to the number of live cells; therefore, the number of live cells can be inferred from the optical density (OD) value. This example tests IC... 50The tumor cells used for the assay were HL-60 leukemia cells, HepG2 liver cancer cells, MDA-MB-231 breast cancer cells, and SW480 colon cancer cells; the tumor cells used for the 30 μM inhibitory activity assay were HeLa cells and A549 cells, and the specific assay steps are as follows: (1) Cell seeding: Prepare a single cell suspension using culture medium (DMEM or RMPI1640) containing 10% fetal bovine serum, and seed 3,000 to 15,000 cells per well into a 96-well plate with a volume of 100 μL per well. Adherent cells should be seeded and cultured 12 to 24 hours in advance.

[0051] (2) Add the solution of the compound to be tested: Dissolve the compound in DMSO, and screen the compound at concentrations of 40 μM, 8 μM, 1.6 μM, 0.32 μM and 0.064 μM. The final volume of each well is 200 μL, and each treatment has 3 replicates.

[0052] (3) Color development: After culturing at 37 degrees Celsius for 48 hours, discard the culture medium in the wells of adherent cells, and add 20 μL of MTS solution and 100 μL of culture medium to each well; discard 100 μL of culture supernatant in the wells of suspended cells, and add 20 μL of MTS solution to each well; set up 3 blank replicates (a mixture of 20 μL of MTS solution and 100 μL of culture medium), and continue incubation for 2-4 hours to allow the reaction to proceed fully before measuring the light absorbance.

[0053] (4) Colorimetric analysis: A wavelength of 492 nm was selected, and the absorbance values ​​of each well were read using a multi-functional microplate reader (MULTISKAN FC). The results were recorded, and after data processing, a cell growth curve was plotted with concentration on the x-axis and cell viability on the y-axis. The IC50 of the compound was calculated using the Reed and Muench method. 50 value.

[0054] (5) Positive control compounds: Doxorubicin (DOX) and paclitaxel (Taxol) were used as positive control compounds in each experiment. Cell growth curves were plotted with concentration on the x-axis and cell viability on the y-axis. The IC50 of the compounds was calculated using the Reed and Muench method. 50 Values. The test results are shown in Table 2.

[0055] Table 2 Compounds Taiwanin J Tumor cell IC 50 Test Results

[0056] As can be seen from the results in Table 2, the compounds extracted in this study... Taiwanin J It has certain anti-tumor activity, especially high inhibitory activity against HL-60 cells and SW480 cells.

[0057] In addition, the other test steps were the same as the above scheme, except that the concentration of the compound was changed to 30 μM, and paclitaxel (30 μM) was used as a positive control to test the inhibition rate of the compound on HeLa cells and A549 cells. The results are shown in Table 3.

[0058] Table 3 Compounds Taiwanin J Cancer cell inhibitory activity

[0059] The experimental results in Table 3 show that, in compounds Taiwanin J In a screening test for inhibitory activity against two types of tumor cells (positive control: paclitaxel), Taiwanin J It exhibited good anti-tumor activity. Therefore, Taiwanin J It has research value as a lead compound for the development of anti-tumor drugs.

[0060] The above description is only a preferred embodiment of the present invention. It should be noted that for those skilled in the art, several improvements and modifications can be made without departing from the principle of the present invention, and these improvements and modifications should also be considered within the scope of protection of the present invention.

Claims

1. An antitumor active compound, characterized in that, It has the structure shown in Equation I: Equation I.

2. The method for preparing the antitumor active compound according to claim 1, characterized in that, Includes the following steps: The crude extract was obtained by extracting the bark powder from the stem of the bald cypress using an ethanol solution. The crude extract was dissolved in water and then extracted with ethyl acetate to obtain an ethyl acetate extract. The ethyl acetate extract was eluted by silica gel column chromatography, and the eluents were collected and labeled Fr.1 to Fr.10 in the order of elution. The eluent for the silica gel column chromatography was a dichloromethane-methanol system. The Fr. 8 was subjected to a first silica gel column chromatography to obtain an eluent containing an antitumor active compound with the structure shown in Formula I; The eluent was subjected to a second silica gel column chromatography to obtain the crude product; The crude product was purified by dextran gel column chromatography to obtain the antitumor active compound with the structure shown in Formula I.

3. The preparation method according to claim 2, characterized in that, The volume concentration of the ethanol solution is 65-95%.

4. The preparation method according to claim 2 or 3, characterized in that, The extraction process includes: soaking the bald cypress bark powder in an ethanol solution followed by rotary extraction.

5. The preparation method according to claim 4, characterized in that, The soaking time is 18-36 hours; the ratio of the amount of bald cypress bark powder to ethanol solution is (30g-16kg): (300mL-200L). The temperature of the rotary extraction is 40~70℃, the number of rotary extractions is 2~4 times, and the time for each rotary extraction is 10~40 minutes.

6. The preparation method according to claim 2, characterized in that, The ethyl acetate extraction is performed 3 to 6 times; the ratio of the crude extract to the amount of ethyl acetate used in each ethyl acetate extraction is (780 to 830) g : (5 to 6) L.

7. The preparation method according to claim 2, characterized in that, During the silica gel column chromatography elution process, the volume ratio of dichloromethane to methanol in the dichloromethane-methanol system is 100:0, 100:1, 30:1, 10:1, 5:1, and 2:1, respectively.

8. The preparation method according to claim 2, characterized in that, The eluent for the first silica gel column chromatography is a first dichloromethane-methanol system, wherein the volume ratio of dichloromethane to methanol in the first dichloromethane-methanol system is 30:1 to 50:1; The eluent used in the second silica gel column chromatography is a second dichloromethane-methanol system, in which the volume ratio of dichloromethane to methanol is 20:1 to 40:

1.

9. The preparation method according to claim 2, characterized in that, The eluent used for the purification of the dextran by gel column chromatography was methanol.

10. The use of the antitumor active compound of claim 1 or the antitumor active compound prepared by any one of claims 2 to 9 in the preparation of antitumor drugs.

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