CN109867644B - Benzoquinone compound, preparation method thereof and application thereof in preparation of antitumor drugs - Google Patents

Abstract

The invention provides a benzoquinone compound, which has the following structural formula:

JNU-144. The invention also provides application of the benzoquinone compound in preparation of antitumor drugs and a preparation method of the benzoquinone compound. The technical proposal provided by the invention successfully extracts natural compounds with anti-tumor activity, namely compounds JNU-144, from the root of arnebia root; the invention also provides the application of the compounds JNU-144 in preparing antitumor drugs, and proves the feasibility and effectiveness of the medical clinical application; particularly, the compounds JNU-144 can provide new treatment medicines and treatment approaches for patients with liver cancer and other tumor metastases, and the compounds JNU-144 have the advantages of moderate effective dose, obvious curative effect, small toxic and side effects and the like, so the compounds have wide application prospects.

Description

Benzoquinone compound, preparation method thereof and application thereof in preparation of antitumor drugs

Technical Field

The invention relates to the technical field of pharmaceutical chemistry, in particular to a benzoquinone compound, a preparation method of the benzoquinone compound, and application of the benzoquinone compound in preparation of antitumor drugs.

Background

Tumor refers to a new organism formed by local tissue cell proliferation under the action of various tumorigenic factors, because the new organism mostly presents space-occupying block-shaped protrusions, which is also called neoplasm. As is well known, cancer is one of the major diseases endangering human life and health, according to the latest WHO statistics, about 820 million people die of cancer in the world in 2012, wherein 307 new cancer patients in china and about 220 million people die of cancer patients account for 21.9% and 26.8% of the total amount of the world respectively. In recent years, the introduction of preoperative or preoperative chemotherapy of tumors is more and more emphasized by scholars at home and abroad, and with the increasing perfection of tumor therapeutics and the continuous generation of new chemotherapeutics, people gradually recognize the important position of chemotherapy in cancer treatment, and adjuvant chemotherapy and synchronous chemoradiotherapy become indispensable important treatment means for cancer treatment. Although chemotherapy can reduce cancer mortality, the existing antitumor drugs often have great killing property on normal cells while killing tumor cells, so that toxic and side effects are great, and therefore, tumors are still one of diseases seriously threatening the life and quality of life of people. Therefore, in view of such serious practical problems, for a long time, government departments, research institutions, and pharmaceutical enterprises in various countries have been highly concerned about the research and development of therapeutic methods for malignant tumors and antitumor drugs, and a huge amount of money is invested. Among them, natural drugs have important value in this field and can form proprietary intellectual property rights.

Sinkiang Arnebia euchroma (Royle) Johnst is a perennial herb of Boraginaceae, and the traditional Chinese medicine considers that Sinkiang Arnebia euchroma has the effects of clearing heat and cooling blood, promoting blood circulation and removing toxin, promoting eruption and eliminating spots, and is clinically used for treating blood heat and toxicity, burn, eruption purple black, measles imperviousness, fever macula, eczema, bloody stranguria, bloody dysentery, pyocutaneous disease, erysipelas and the like. Modern pharmacological research shows that arnebia euchroma has the effects of sterilization, anti-inflammation, HIV resistance, anti-tumor, aging delay, immunoregulation and the like. Alkannin purified from arnebia euchroma (Royle) Johnst can completely inhibit the growth of ascites sarcoma 180 cells at 50mg/kg, and can prolong the life of tumor-bearing mice by 92.5% at 10 mg/kg. Naphthoquinone and mixed source terpene compounds can affect growth, proliferation, invasion and migration of malignant tumors such as leukemia, human melanoma, cervical cancer, colon cancer, human choriocarcinoma, breast cancer, etc. In addition, alkannin can enhance the radiotherapy sensitivity of mouse liver cancer H22 and Lewis lung cancer and reverse the drug resistance of glioblastoma; mechanistic studies have shown that shikonin induces non-small cell lung cancer death by inhibiting autophagy.

Disclosure of Invention

The invention aims to provide a brand-new natural compound which is a benzoquinone compound and shows excellent antitumor activity.

Specifically, the invention provides a benzoquinone compound, which has the following structural formula:

and the structure of the compound is determined to be 2-methoxy-Arnebinone B through detection, so the compound is named as compound JNU-144.

The second aspect of the invention provides an application of the benzoquinone compound in preparation of an anti-tumor drug.

Preferably, in said use, said tumor is selected from the group consisting of: liver cancer, lung cancer, esophageal cancer, gastric cancer, colorectal cancer, pancreatic cancer, leukemia, brain tumor and skin cancer.

The application of the benzoquinone compound in preparing the antitumor drugs can comprise the following steps: the compound is used for preparing products (such as medicines, pharmaceutical preparations and pharmaceutical compositions) for treating tumors, products (such as medicines, pharmaceutical preparations and pharmaceutical compositions) for inducing apoptosis of tumor cells, products (such as medicines, pharmaceutical preparations and pharmaceutical compositions) for inhibiting proliferation of tumor cells, products (such as medicines, pharmaceutical preparations and pharmaceutical compositions) for inhibiting invasion and migration of tumor cells and products (such as medicines, pharmaceutical preparations and pharmaceutical compositions) for inhibiting mTOR activity.

The third aspect of the present invention provides a method for preparing benzoquinone compounds, comprising the following steps:

s1: cold soaking and percolating the Sinkiang alkannin root for multiple times by using 95% ethanol at room temperature, then recovering the solvent under reduced pressure, and concentrating to obtain ethanol extract;

s2: suspending the ethanol extract in water, and extracting with chloroform, ethyl acetate and n-butanol respectively to obtain chloroform extract AEC, ethyl acetate extract AEE and n-butanol extract AEB;

s3: dissolving the chloroform extract AEC with chloroform and methanol, mixing with column chromatography silica gel, loading onto column, and sequentially adding cyclohexane in a volume ratio of 50: 1 cyclohexane-ethyl acetate, volume ratio 25: 1 cyclohexane-ethyl acetate, volume ratio 10: 1 cyclohexane-ethyl acetate, volume ratio 5: 1 cyclohexane-ethyl acetate, in a volume ratio of 1: 1 cyclohexane-ethyl acetate gradient elution; performing TLC analysis, and merging the same fractions to obtain 5 components AEC 1-AEC 5;

s4: dissolving AEC3 with chloroform, and performing Sephadex LH-20(40 × 830mm) column chromatography, wherein the eluent is chloroform-methanol; performing TLC analysis, and combining the same fractions to obtain 4 components AEC 3-1-AEC 3-4;

s5: preparing a liquid chromatographic column by taking the component AEC3-2 and passing through Waters XTerra RP-18, wherein the mobile phase is a mixture of 82: 18 methanol-water, pH 2.5, ultraviolet detection wavelength 205nm, flow rate 8mL/min, and the benzoquinone compound is obtained at a retention time of 37.42 min.

Preferably, in the preparation method, the column chromatography silica gel is 100-200 mesh column chromatography silica gel.

Preferably, in the above preparation method, the eluent is a mixture of water and an organic solvent at a volume ratio of 7: 3 chloroform-methanol.

In conclusion, the benzoquinone compounds, i.e., compounds JNU-144, of the present invention have the following technical advantages:

the compounds JNU-144 are natural small molecular compounds, and have low toxicity to normal cells and small side effect; the compounds JNU-144 have strong antitumor activity, and can inhibit proliferation and invasion and migration of liver cancer cells and induce apoptosis of the cancer cells within a short action time.

In the process of treating cancers, such as liver cancer, the problem of drug tolerance is always a problem, but the benzoquinone compound JNU-144 provided by the invention can show good inhibition effect on cell proliferation in a 5-FU drug-resistant cell line of SMMC-7721, so that the drugs prepared from the compounds JNU-144 can be used for patients who are resistant to chemotherapy; and compounds JNU-144 can also be used in combination with other drugs. Experiments prove that the inhibition rate of the compounds JNU-144 on liver cancer cells is increased along with the increase of the concentration of the compounds JNU-144 under the same treatment time; at the same concentration, the inhibition rate of compounds JNU-144 on hepatoma cells increased with the treatment time of compounds JNU-144. In particular, experiments prove that the compounds JNU-144 can effectively inhibit the proliferation of liver cancer cells, and the inhibition effect is increased along with the increase of the concentration of the compounds JNU-144. In addition, a series of experiments carried out by the inventor prove that the compounds JNU-144 can inhibit the activation of mTOR, induce liver cancer cells to undergo apoptosis, inhibit the invasion and migration of the liver cancer cells and inhibit the growth of tumors.

In conclusion, the technical scheme provided by the invention successfully extracts natural compounds with anti-tumor activity, namely compounds JNU-144 (benzoquinone compounds), from the root of arnebia euchroma; in addition, the invention also provides the application of the compounds JNU-144 in the preparation of antitumor drugs, and proves the feasibility and effectiveness of the medical clinical application of the compounds; particularly, the compounds JNU-144 can provide new treatment medicines and treatment approaches for patients with liver cancer and other tumor metastases, and the compounds JNU-144 have the advantages of moderate effective dose, obvious curative effect, small toxic and side effects and the like, so the compounds have wide application prospects.

Drawings

FIG. 1 is the NMR of compounds JNU-144 ¹ H NMR chart;

FIG. 2 shows the NMR of compounds JNU-144 ¹³ C NMR chart;

FIG. 3 is a graph showing the inhibitory effect of compounds JNU-144 at various concentrations on SMMC-7721 and HepG2 cells; wherein, FIG. 3A shows the inhibitory effect of various concentrations of compounds JNU-144 on HepG2 cells at 12 hours of treatment; FIG. 3B shows the inhibitory effect of various concentrations of compound JNU-144 on SMMC-7721 cells at 12 hours; FIG. 3C shows the inhibitory effect of the same concentration of compounds JNU-144 on HepG2 cells at different times; FIG. 3D shows the inhibitory effect of compound JNU-144 at the same concentration on SMMC-7721 cells at different times;

FIG. 4 is a graph showing the inhibitory effect of compounds JNU-144 at various concentrations on SMMC-7721 and HepG2 cell clone formation; wherein the left panel shows trypan blue staining results and the right panel shows statistics of the number of colonies formed by SMMC-7721 and HepG2 cells treated with different concentrations of compounds JNU-144 as a percentage of the number of colonies formed by the DMSO-treated control group;

the left panel in FIG. 5 shows the expression levels of mTOR and p-mTOR in SMMC-7721 cells treated with compounds JNU-144 at different concentrations; the right panel in FIG. 5 shows the expression levels of mTOR and p-mTOR in SMMC-7721 cells treated with compound JNU-144 at different times; wherein, p-mTOR represents phosphorylated mTOR, and Actin protein is used as an internal reference protein;

FIG. 6 is a micrograph of SMMC-7721 cells undergoing apoptosis in response to treatment with compounds JNU-144; wherein FIG. 6A shows the morphological changes of SMMC-7721 cells treated with compound JNU-144 and FIGS. 6B/6C show the protective effect of z-VAD-fmk on the clonality inhibition of SMMC-7721 cells treated with compound JNU-144;

FIG. 7 is a graph showing the inhibitory effect of compound JNU-144 treatment on EMT and invasive migration ability of SMMC-7721 cells; wherein, FIG. 7A shows the change of migration ability of SMMC-7721 cells under the treatment of compound JNU-144 in the scratch test result compared with the control group; FIG. 7B is a statistical graph showing the change in the invasion capacity of SMMC-7721 cells treated with compound JNU-144 as compared to a control group in the transwell test, wherein the upper half is the crystal violet staining result and the lower half is the change ratio of the number of stained cells counted in the upper half;

FIG. 8 is a graph showing the results of experiments in which compounds JNU-144 inhibited liver cancer in mice; wherein, fig. 8A shows the appearance of the tumor of the nude mouse with liver cancer transplantation tumor, and fig. 8B shows the tumor weight of the nude mouse with liver cancer transplantation tumor.

Detailed Description

The present invention will be further described with reference to specific embodiments, but the present invention is not limited to the following embodiments. The experimental methods in the following examples are all conventional methods unless otherwise specified; materials, reagents and the like used in the following examples are commercially available from public unless otherwise specified.

The benzoquinone compound has the following structural formula:

the invention provides application of the benzoquinone compound (i.e. the compounds JNU-144) in preparation of antitumor drugs. And, the tumor is preferably selected from: liver cancer, lung cancer, esophageal cancer, gastric cancer, colorectal cancer, pancreatic cancer, leukemia, brain tumor and skin cancer.

In a preferred embodiment of the present invention, the tumor is liver cancer, and the tumor cells are liver cancer cell lines SMMC-7721 and HepG 2. Experiments conducted in the detailed description show that: on a molecular level, the treatment of the compounds JNU-144 can effectively inhibit the phosphorylation of mTOR so as to inhibit the proliferation of liver cancer cells, can also inhibit the invasion and migration of EMT and liver cancer cells, and can induce tumor cells to undergo apoptosis, thereby achieving the purpose of treating cancers.

In a preferred embodiment of the present invention, the benzoquinone like compounds (i.e., compounds JNU-144) can be prepared by the following method:

carrying out cold soaking and percolation extraction on 7.5kg of Sinkiang alkannis root by 95 percent ethanol for multiple times at room temperature, then recovering the solvent under reduced pressure, and concentrating to obtain 1150g of ethanol extract; suspending the ethanol extract in water, and extracting with chloroform, ethyl acetate and n-butanol respectively to obtain 178.21g of chloroform extract AEC, 35.3g of ethyl acetate extract AEE and 230.3g of n-butanol extract AEB. And then dissolving the chloroform extract AEC with chloroform and methanol, stirring the sample with (100-200 mesh) column chromatography silica gel, dissolving the silica gel with cyclohexane, packing the column with the silica gel, balancing the column with cyclohexane until the silica gel surface does not descend, loading the sample by a dry method, and sequentially adding cyclohexane in a volume ratio of 50: 1 cyclohexane-ethyl acetate, volume ratio 25: 1 cyclohexane-ethyl acetate, volume ratio 10: 1 cyclohexane-ethyl acetate, volume ratio 5: 1 cyclohexane-ethyl acetate, in a volume ratio of 1: 1 cyclohexane-ethyl acetate gradient elution; TLC analysis was used, and the same fractions were combined to give 5 fractions AEC 1-AEC 5. Thereafter, the fraction AEC3(21.34g, and each eluted with cyclohexane-ethyl acetate 25: 1) was taken, dissolved with chloroform, and subjected to Sephadex LH-20(40 × 830mm) column chromatography using chloroform-methanol (7: 3) as an eluent; TLC analysis is adopted, and 4 components AEC3-1 to AEC3-4 are obtained after the same fractions are combined; a liquid chromatography column (19X 250mm) was prepared by passing the component AEC3-2 through a Waters XTerra RP-18, wherein the mobile phase was 82: 18 methanol-water, pH 2.5, ultraviolet detection wavelength 205nm, flow rate 8mL/min, and retention time 37.42min to obtain the benzoquinone like compounds (i.e., compounds JNU-144).

Of course, the compounds JNU-144 of the present invention can also be prepared by other extraction processes or chemical synthesis methods.

In the following examples, the non-small cell lung cancer cell lines SMMC-7721 and HepG2 were ATCC products, fetal bovine serum was Hyclone, DMEM medium was Gibco, and MTT was Solarbio.

EXAMPLE 1 characterization of Compounds JNU-144

The novel benzoquinone compound provided by the invention is red amorphous powder, and ESI-MS (ESI-MS) (reactive) gives an excimer ion peak M/z 339[ M + Na ]] ⁺ Suggesting a molecular weight of 316; bonding of ¹ H and ¹³ c NMR confirmed its molecular formula as C ₁₈ H ₂₀ O ₅ The unsaturation was calculated to be 9. In that ¹ H NMR(400MHz,CDCl ₃ ) There are 20 hydrogen signals in the hydrogen signal, ¹³ c NMR and DEPT spectra showed 10 carbons attached directly to 20 hydrogens and the compound was found to have 3 methyl groups [ delta ] present _H 1.50(3H,s,H-16),12.8(C-16)；δ _H 4.02(3H,s,2-OCH ₃ ),61.4(2-OCH ₃ )；δ _H 3.98(3H,s,3-OCH ₃ ),61.4(3-OCH ₃ )]4 methylene [ delta ] _H 1.95(1H,dd,J＝12.8,1.2Hz,H-9ax),2.11(1H,m,H-9eq),27.0(C-9)；δ _H 2.02(1H,d,J＝10.4Hz,H-10ax),2.30(1H,dd,J＝12.0,3.2Hz,H-10eq),24.2(C-10)；δ _H 2.74(1H,d,J＝13.2Hz,H-13ax),3.36(1H,d,J＝13.2Hz,H-13eq),26.4(C-13)；δ _H 4.75(2H,br s,H-15),77.6(C-15)]3 methine radicals [ delta ] _H 6.35(1H,t,J＝4.8Hz,H-6),79.5(C-6)；δ _H 5.10(1H,s,H-7),124.1(C-7)；δ _H 5.07(1H,br s,H-11),122.3(C-11)]And 8 quaternary carbons [ delta ] _C 185.2(C-1),145.2(C-2),144.7(C-3),184.1(C-4),141.3(C-5),136.3(C-8),139.5(C-12),144.2(C-14)]。

The above information shows that the compound is a heterpene. Of the compound ¹ H and ¹³ c NMR and of shikonin ¹ H and ¹³ c NMR is very similar, and the greatest difference between the structure of this compound and shikonin is found by analysis: carbon in position 2 being substituted by methoxy [ delta ] _H 4.02(3H,s,2-OCH ₃ ),61.4(2-OCH ₃ )]Is substituted simultaneously by ¹ H- ¹ The H COSY, HMQC and HMBC spectra confirmed that the structure of this compound was identified as 2-methoxy-Arnebinone B, from which the inventors named it as compound JNU-144.

Specifically, the nuclear magnetic data for compounds JNU-144 are as follows (see FIGS. 1 and 2): ¹ H NMR(400MHz,CDCl ₃ )δ _H 6.35(1H,t,J＝4.8Hz,H-6),5.10(1H,s,H-7),1.95(1H,dd,J＝12.8,1.2Hz,H-9ax),2.11(1H,m,H-9eq),2.02(1H,d,J＝10.4Hz,H-10ax),2.30(1H,dd,J＝12.0,3.2Hz,H-10eq),5.07(1H,br s,H-11),2.74(1H,d,J＝13.2Hz,H-13ax),3.36(1H,d,J＝13.2Hz,H-13eq),4.75(2H,br s,H-15),1.50(3H,s,H-16),4.02(3H,s,2-OCH ₃ ),3.98(3H,s,3-OCH ₃ )； ¹³ C NMR(100MHz,CDCl ₃ ):δ _C 185.2(C-1),145.2(C-2),144.7(C-3),184.1(C-4),141.3(C-5),79.5(C-6),124.1(C-7),136.3(C-8),27.0(C-9),24.2(C-10),122.3(C-11),139.5(C-12),26.4(C-13),144.2(C-14),77.6(C-15),23.4(C-16),61.4(2-OCH ₃ ),61.4(3-OCH ₃ )。

example 2 inhibition of viability and proliferation of hepatoma cells by Compounds JNU-144

SMMC-7721 and HepG2 cells were divided into nine groups of three parallel wells, each seeded with 1X 10 cells ⁴ The cells were cultured in DMEM medium containing 10% fetal bovine serum in 5% CO ₂ The culture is carried out in an incubator at 37 ℃; the nine groups of cells were treated after cell inoculation as follows: group 1 DMSO was added; group 2 was added JNU-144 at a final concentration of 10. mu.g/mL; group 3 was added JNU-144 to a final concentration of 20. mu.g/mL; group 4 was added JNU-144 at a final concentration of 40. mu.g/mL. The above 4 groups of cells were treated for 12 hours after adding the drugs according to the above method, then 10 μ L of 5mg/mL MTT aqueous solution was added, the culture was continued for 4 hours, then the medium containing MTT was aspirated, 150 μ L of DMSO was added, shaking was performed on a decolorization shaker for 10 minutes under dark room temperature conditions, and then the absorbance of the solution in each well at 590nm wavelength was measured with a microplate reader, with the results shown in fig. 3A and 3B.

The above experimental results show that: the inhibition of the activity of the compounds JNU-144 on the liver cancer cells is strengthened along with the increase of the concentration of the compounds JNU-144.

SMMC-7721 and HepG2 cells were divided into nine groups of three parallel wells, each seeded with 1X 10 cells ⁴ The cells were cultured in DMEM medium containing 10% fetal bovine serum in 5% CO ₂ The culture was carried out in an incubator at 37 ℃ and the nine groups of cells were treated as follows after cell inoculation: group 1 was not treated; adding DMSO into groups 2-5, and sequentially treating for 4/8/12/16 hours; JNU-144 with the final concentration of 20 mug/mL are added into the groups 6-9, and the treatment time is 4/8/12/16 hours in sequence; DMSO was added in group 5; JNU-144 were added to groups 6-9 to a final concentration of 20. mu.g/mL. The nine groups of cells were treated as described above, 10. mu.L of aqueous MTT solution at 5mg/mL concentration was added, the culture was continued for 4 hours, the medium containing MTT was aspirated, 150. mu.L of DMSO was added, shaking was performed on a decolorizing shaker for 10 minutes under dark room temperature, and the absorbance of the solution in each well at 590nm wavelength was measured with a microplate reader, with the results shown in FIGS. 3C and 3D.

The above experimental results show that: inhibition of hepatoma cell viability by compounds JNU-144 was enhanced with prolonged treatment time by compounds JNU-144.

EXAMPLE 3 inhibitory Effect of Compounds JNU-144 on the clonogenic formation of hepatoma cells

SMMC-7721 and HepG2 cells were divided into 4 groups of three parallel wells, cultured in DMEM medium containing 10% fetal bovine serum in 5% CO ₂ The cells of the 4 groups were treated in an incubator at 37 ℃ as follows: group 1 DMSO was added; group 2 was added JNU-144 at a final concentration of 10. mu.g/mL; group 3 was added JNU-144 at a final concentration of 20. mu.g/mL; group 4 was added JNU-144 at a final concentration of 40. mu.g/mL. The above 4 groups of cells were treated by adding the above drugs for 12 hours, the cells were digested, counted, and the number of cells per well was 1X 10 ³ The individual cells were seeded at density in 12-well plates, 1mL fresh DMEM complete medium was added per well, shaken well and then incubated at 5% CO ₂ The culture chamber of (1) was cultured at 37 ℃ for 10 days, the medium was removed, after methanol was fixed for half an hour, and then stained with 0.4% trypan blue, and the number of colonies formed in each well was counted, and the results are shown in FIG. 4, in which the left graph is an image of colonies formed in each well and the right graph is a statistical graph based on the counting results after staining.

As can be seen, the experimental results show that the inhibition of compounds JNU-144 on the proliferation of hepatoma cells is enhanced with the increase of the concentration of compounds JNU-144.

EXAMPLE 4 Compounds JNU-144 inhibit the activation of mTOR in hepatoma cells

SMMC-7721 cells were divided into 8 groups and seeded at 5X 10 cells per well ⁵ The individual cells were cultured in DMEM medium containing 10% fetal bovine serum in 5% CO ₂ The culture was carried out in an incubator at 37 ℃ and the 8 groups of cells were treated as follows after cell inoculation: group 1 DMSO was added; group 2 was added JNU-144 at a final concentration of 10. mu.g/mL; group 3 was added JNU-144 to a final concentration of 20. mu.g/mL; group 4 was added JNU-144 to a final concentration of 40. mu.g/mL; the 4 groups of cells are treated for 12 hours after being added with medicine according to the method; DMSO was added in group 5; JNU-144 were added to groups 6-8 to a final concentration of 20. mu.g/mL for 4/8/12 hours. After each group of cells was treated as described above, the medium was removed and 1X appliedThe cells were washed twice with PBS, then lysed with RIPA lysate containing PMSF at a final concentration of 1mM (200. mu.L of RIPA lysate per well), centrifuged at 12000rpm and 4 ℃ for 5 minutes, and the pellet was discarded to give a supernatant containing total cytoplasmic protein in each group of cells. And detecting the expression levels of mTOR and p-mTOR in the total protein of each group of cells by using a western blot method. The primary antibody for detecting the expression level of mTOR is Cell Signaling Technology product with the product code of 2972, the primary antibody for detecting the expression level of p-mTOR is Cell Signaling Technology product with the product code of 2974, the internal reference is GAPDH, the primary antibody for detecting the expression level of GAPDH is protein within tech product with the product code of 60004-1-lg. The experimental results are shown in fig. 5, and it can be seen through analysis that with the increase of the concentration of compounds JNU-144 and the prolongation of the treatment time, compounds JNU-144 can effectively inhibit the phosphorylation of mTOR, i.e., effectively inhibit the activation of mTOR.

Example 5 Compounds JNU-144 induce apoptosis in hepatoma cells

SMMC-7721 cells were divided into 2 groups and seeded at 1X 10 cells per well ⁵ The cells were cultured in DMEM medium containing 10% fetal bovine serum in 5% CO ₂ The cells were inoculated and then the 2 groups of cells were treated as follows: group 1 DMSO was added; group 2 was added JNU-144 at a final concentration of 20. mu.g/mL; the difference in cell morphology observed microscopically after 12 hours of drug treatment resulted in the appearance of typical apoptotic cell characteristics of JNU-144 treated cells as shown in FIG. 6A, for example: cell shrinkage, cell membrane blebbing, apoptotic body formation, and the like. Dividing SMMC-7721 cells into 4 groups each having three parallel wells, culturing in DMEM medium containing 10% fetal calf serum under 5% CO ₂ The cells of the 4 groups were treated in an incubator at 37 ℃ as follows: group 1 DMSO was added; group 2 was added JNU-144 to a final concentration of 20. mu.g/mL; group 3 was supplemented with z-VAD-fmk at a final concentration of 20. mu.M; group 4 was added JNU-144 at a final concentration of 20. mu.g/mL and z-VAD-fmk at a final concentration of 20. mu.M. The above 4 groups of cells were treated by adding the above drugs for 12 hours, the cells were digested, counted, and the number of cells per well was 1X 10 ³ Individual cells were seeded at density in 12-well plates, and 1mL of fresh DMEM complete medium was added to each well and shakenAfter shaking evenly, the mixture is in 5 percent CO ₂ The cells were cultured at 37 ℃ for 10 days, then the medium was removed, the cells were fixed with methanol for half an hour, and then stained with 0.4% trypan blue, and the number of colonies formed in each well was counted, and the results are shown in fig. 6B and 6C, in which fig. 6B is an image of colonies formed in each well and fig. 6C is a statistical chart based on the count results after staining.

The experimental result of the embodiment shows that the inhibition of compounds JNU-144 on the proliferation of the liver cancer cells can be blocked by caspase inhibitor z-VAD-fmk, so that compounds JNU-144 can induce the liver cancer cells to generate apoptosis.

Example 6 ability of Compounds JNU-144 to inhibit migration of hepatoma cells

SMMC-7721 cells were divided into 2 groups and seeded at 2X 10 cells per well ⁵ The cells were cultured in DMEM medium containing 10% fetal bovine serum in 5% CO ₂ The culture is carried out in an incubator at 37 ℃, and DMSO is added into the group 1 when the cell confluency is 80-90%; group 2 was added JNU-144 to a final concentration of 20. mu.g/mL; after 8 hours of treatment, the cells were scratched with a tip in the middle of the cell, which was set to 0h, and then 24h and 48h were taken under a microscope to photograph the cell migration at the scratch. The results of the experiment are shown in FIG. 7A, which shows that the migration ability of the liver cancer cells treated with compounds JNU-144 was inhibited.

Example 7 ability of Compounds JNU-144 to inhibit invasion of hepatoma cells

SMMC-7721 cells were divided into 2 groups of 3 parallel wells, and cultured in DMEM medium containing 10% fetal bovine serum in 5% CO ₂ The culture is carried out in an incubator at 37 ℃, and DMSO is added into the group 1 when the cell confluency is 80-90%; group 2 was added JNU-144 at a final concentration of 20. mu.g/mL; after 8 hours of treatment, the cells were digested, counted, and diluted with serum-free medium at 1X 10 per 100. mu.L ⁵ The density of individual cells was seeded into a transwell chamber coated with 100ng matrigel, the chamber was placed in a 24-well plate, and 500. mu.L of medium containing 10% FBS was added to the 24-well plate. After 24 hours, the cells in the upper layer of the chamber were gently scraped off with a cotton swab, and the cells in the lower layer were fixed with methanol for half an hour, stained with 0.1% crystal violet, and then observed under a microscope, each chamber was followed byThe machine takes 6 fields and takes pictures and counts the number of cells. The experimental results are shown in FIG. 7B, wherein the upper half is the image of each group of cells after invading the lower chamber layer, and the lower half is the statistical image based on the counting results after staining. As can be seen by analysis, the experimental result shows that the invasion capacity of the hepatoma cells treated by the compounds JNU-144 is inhibited.

EXAMPLE 8 inhibition of liver tumor growth in nude mice by Compounds JNU-144

14 SPF-grade BALB/c-nu/nu female mice (product of the center of Guangdong provincial medical laboratory animals) 6 weeks old were injected subcutaneously 5X 10 to the left ventral side of each mouse ⁶ Obtaining the liver cancer nude mouse by using SMMC-7721 cells. The size of the tumor to be detected is 100mm ³ On the order of size, 14 nude mice with liver cancer were randomly divided into two groups, one group was a control group (Vehicle), the other group was JNU-144 treatment groups, and each group consisted of 7 nude mice. JNU-144 was dissolved in olive oil and administered at a dose of 15mg JNU-144 per kg body weight in a volume of 100. mu.L, and the control group (Vehicle) was administered an equal volume of Vehicle per peritoneal cavity once every two days for a total of 5 administrations. The tumor volume and the weight of the nude mice were measured on days 0, 2, 4, 6 and 10 with the first administration time as day 0, the nude mice were sacrificed after the tumor volume and the weight of the nude mice were measured on day 10, the tumor tissue was taken down to take a picture, and then the tumor tissue was taken to detect the Vimentin condition in the tumor tissue by western blot and immunohistochemistry, and the results are shown in fig. 8, in which fig. 8A is the result of taking a picture of the tumor tissue and fig. 8B is a statistical chart based on the result of the tumor tissue weight.

The experimental results of this example show that compounds JNU-144 are effective in inhibiting the growth of tumor tissue in nude mice.

The embodiments of the present invention have been described in detail, but the embodiments are merely examples, and the present invention is not limited to the embodiments described above. Any equivalent modifications and substitutions to those skilled in the art are also within the scope of the present invention. Accordingly, equivalent changes and modifications made without departing from the spirit and scope of the present invention should be covered by the present invention.

Claims (5)

1. A benzoquinone compound is characterized by having the following structural formula:

2. the use of the benzoquinone compound of claim 1 in the preparation of an anti-tumor medicament; the tumor is liver cancer.

3. The method for preparing benzoquinone compounds according to claim 1, comprising the steps of:

s1: cold soaking and percolating the Sinkiang alkannin root for multiple times by using 95% ethanol at room temperature, then recovering the solvent under reduced pressure, and concentrating to obtain ethanol extract;

s2: suspending the ethanol extract in water, and extracting with chloroform, ethyl acetate and n-butanol respectively to obtain chloroform extract AEC, ethyl acetate extract AEE and n-butanol extract AEB;

s3: dissolving the chloroform extract AEC with chloroform and methanol, mixing with column chromatography silica gel, loading onto column, and sequentially adding cyclohexane in a volume ratio of 50: 1 cyclohexane-ethyl acetate, volume ratio 25: 1 cyclohexane-ethyl acetate, volume ratio 10: 1 cyclohexane-ethyl acetate, volume ratio 5: 1 cyclohexane-ethyl acetate, in a volume ratio of 1: 1 cyclohexane-ethyl acetate gradient elution; performing TLC analysis, and combining the same fractions to obtain 5 components AEC 1-AEC 5;

s4: dissolving AEC3 with chloroform, and performing SephadexLH-20 column chromatography with specification of 40 × 830mm, wherein the used eluent is chloroform-methanol; performing TLC analysis, and combining the same fractions to obtain 4 components AEC 3-1-AEC 3-4;

s5: preparing a liquid chromatographic column by taking the component AEC3-2 and passing through Waters XTerra RP-18, wherein the mobile phase is a mixture of 82: 18 methanol-water, pH 2.5, ultraviolet detection wavelength 205nm, flow rate 8mL/min, and the benzoquinone compound is obtained at a retention time of 37.42 min.

4. The preparation method according to claim 3, wherein the column chromatography silica gel is 100-200 mesh column chromatography silica gel.

5. A method of preparation according to claim 3, wherein the eluent is a mixture of water in a volume ratio of 7: 3 chloroform-methanol.

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