CN113072526B - Anthraquinone/coumarin dimer compound and preparation method and application thereof - Google Patents

Abstract

The present invention relates to an anthraquinone/coumarin dimer type new skeleton compound and its preparation method and application. The structural formula of the dimer compound is:

It is a novel skeleton compound formed by the coupling of anthraquinone and coumarin through a methylene carbon bridge. The compound can effectively and selectively inhibit the activity of protein tyrosine phosphatase 1B. The present invention relates to the compound, its preparation method, and its application in the preparation of PTP1B inhibitor, medicine for treating type 2 diabetes and antitumor medicine.

Description

An anthraquinone/coumarin dimer compound and its preparation method and application

technical field

The invention belongs to the field of pharmaceutical compounds, and in particular relates to a new skeleton compound formed by coupling of anthraquinone and coumarin with carbon-carbon bonds derived from Euphorbia sp., a preparation method of the compound, and the use of the compound in the preparation of PTP1B enzyme inhibitor , Type 2 diabetes treatment drugs and/or anti-tumor drugs.

Background technique

Protein kinases and phosphatases are respectively responsible for the post-translational phosphorylation and dephosphorylation of proteins, and coordinately regulate cell signaling pathways to maintain normal life activities. Therefore, both are important targets for drug development. Protein tyrosine phosphatase 1B (PTP1B) is a subtype of PTP family distributed in cells. It was first isolated from human placenta and widely distributed in liver, kidney and placenta. PTP1B is closely related to insulin resistance and obesity, plays a negative regulatory role in the insulin and leptin signaling pathways, and reduces the body's glucose and lipid metabolism by promoting insulin receptor dephosphorylation, down-regulating insulin and leptin levels. By inhibiting the activity of PTP1B, the sensitivity of peripheral tissues to insulin can be improved, so PTP1B is an important target for the treatment of type 2 diabetes and obesity. In recent years, studies have reported that PTP1B is highly expressed in a variety of tumor cells, and inhibitors designed for PTP1B can significantly inhibit the proliferation of breast cancer, lung cancer and other cells, and are new targets for antitumor drugs.

由α3和α6螺旋构成，具有保守性低、主要由亲脂性氨基酸组成的特点，结合于变构口袋的抑制剂显示出更佳的理化性质和显著的选择性。但PTP1B变构抑制剂报道非常少，作用于变构位点的先导化合物极度缺乏。天然产物中的化学成分具有独特的分子骨架和空间构型，表现出多样的生物学活性，是创新药物和新颖结构先导化合物的重要来源。However, two characteristics of PTP1B greatly limit the development of its inhibitors. One is that the catalytic active site of PTPs is highly conserved, which makes the inhibitor have low selectivity between PTP1B and other PTPs. For example, T-cell PTP (TCPTP) has nearly 80% identical amino acid sequences in the catalytic region of the two. TCPTP plays an important role in hematopoiesis and immune system, and cross-inhibition of TCPTP can cause obvious side effects. The other is that the phosphate group of its substrate is negatively charged, and the active site of PTP1B is positively charged. The inhibitor structure designed for the active site has groups that can dissociate negative charges, such as carboxyl group, sulfonic acid group, etc., Such inhibitors that mimic substrate synthesis have strong polarity, are difficult to penetrate cell membranes, and have poor bioavailability, which seriously affects their druggability. An allosteric site of PTP1B was reported in 2004, which is approximately approx.

It is composed of α3 and α6 helices, and has the characteristics of low conservation and mainly composed of lipophilic amino acids. The inhibitor bound to the allosteric pocket shows better physicochemical properties and significant selectivity. However, there are very few reports of allosteric inhibitors of PTP1B, and the lead compounds acting on the allosteric site are extremely lacking. The chemical components in natural products have unique molecular skeletons and spatial configurations, exhibit diverse biological activities, and are an important source of innovative drugs and novel structural lead compounds.

Knoxia valerianoides is a perennial herb of the Rubiaceae family, distributed in the southern provinces of China, mainly in Guangxi, and in other regions such as Guangdong, Yunnan, Fujian, Guizhou and Hainan. The root tuber of the plant is used as medicine, and has the effects of diarrhoea and drinking, attacking toxins, reducing swelling and dispersing knots; treating mainly pleural effusion, unfavorable stools, carbuncle, swollen sore, scrofula and phlegm core embolism. The inventor's research on Euphorbia for many years has shown that the plant is rich in anthraquinone and triterpenoids, as well as a small amount of lignans, coumarin and phenolic acids, among which anthraquinones have antiviral, anti-inflammatory and The protective activity of hepatocytes, the research results provide material basis and scientific basis for the application of Euphorbia chinensis.

In the continuous research on Euphorbia, the inventor discovered a new backbone compound, Euphorbia quinone A, which is formed by coupling anthraquinone and coumarin through a methylene bridge. The core structures of anthraquinone and coumarin derived from natural sources are benzocyclodienone and benzoα-pyrone, respectively, and most of their derivatives contain simple substituents such as hydroxyl, methoxy, and isopentenyl, or are combined with Sugars combine to form glycosides. Many anthraquinone dimers and coumarin dimers have been reported in the literature, mainly including anthraquinone-anthraquinone, anthraquinone-koushanone, coumarin-coumarin and coumarin-chalcone dimer. and other types, the connection methods include phenyl-phenyl coupling between parent nuclei and oxygen atoms between parent nuclei. The structure of the compound formed by anthraquinone-coumarin through methylene coupling has not yet been seen in the present invention. Preparation method and application report. The compound involved in the present invention has efficient and highly selective PTP1B inhibitory activity, is an allosteric inhibitor of PTP1B with novel structure, can inhibit the proliferation of various liver cancer cells, and can be used for research and development with high specificity and good bioavailability Novel hypoglycemic drugs, type 2 diabetes treatment drugs and/or antitumor drugs with new mechanisms and targets.

SUMMARY OF THE INVENTION

In the present invention, a new skeleton compound in which anthraquinone and coumarin are coupled by carbon-carbon bonds is isolated and obtained by carrying out systematic chemical composition and biological activity research on Euphorbia sp.

One of the objects of the present invention is to provide a dimer compound of anthraquinone coupled coumarin, named as spurge quinone A, and its structure is as follows:

The structural feature of the compound is that it contains anthraquinone and coumarin structural fragments, wherein anthraquinone is a benzocyclodienone structure, coumarin is a benzo α-pyrone structure or a benzohexamethylene lactone ring structure, anthracene is a benzocyclodienone structure The 2-methyl group of quinone is coupled with C-3 of coumarin through carbon-carbon bond to form a novel dimer compound.

The second object of the present invention is to provide a preparation method of the compound, comprising the following steps:

1) The roots of Euphorbia are pulverized and extracted with ethanol, and the extract is evaporated to dryness under reduced pressure to obtain an extract;

2) The extract is dissolved in water, the aqueous solution is extracted with ethyl acetate, the ethyl acetate is evaporated to dryness under reduced pressure to obtain the ethyl acetate extraction part, and the ethyl acetate extraction part is successively subjected to silica gel column chromatography, MCI column chromatography, and gel column chromatography. Separation to obtain a site containing Euphorbia quinone A;

3) Recrystallize the part containing quinone A of spurge with chloroform-methanol mixed solution, or purify with high performance liquid chromatography to obtain quinone A of spurge.

Preferably, in step 1), the ethanol concentration is 95%, and the extraction temperature is normal temperature.

Preferably, in step 2), in silica gel column chromatography, firstly use 100-200 mesh silica gel and petroleum ether/acetone as mobile phases for separation, and then use 200-300 mesh silica gel and chloroform/methanol as mobile phases for separation. In MCI column chromatography, the stationary phase is MCI gelCHP20p and the mobile phase is methanol/water. In gel column chromatography, the gel is Sephadex LH-20, and the mobile phase is chloroform/methanol.

Preferably, in step 3), the ratio of chloroform/methanol in the recrystallization method is 3:1.

Preferably, in step 3), in the high performance liquid chromatography, the chromatographic column is ODS filler, and the mobile phase is 80% methanol/water.

More preferably, in step 2), the volume ratio of petroleum ether/acetone used in the silica gel column chromatography is successively 10:0, 10:1, 10:2, 2:1, 1:1, 0:1, The chloroform/methanol volume ratios used in the collection of petroleum ether/acetone 2:1 elution part and silica gel column chromatography were followed by 100:1, 10:1, 2:1, 1:1, 0:1; the chloroform/methanol 1 : 1 The methanol concentration used in the separation of the eluted part by MCI column chromatography was 30%, 50%, 70%, 90%, 100%; the 70% methanol/water eluted part was collected by gel column chromatography. The chloroform/methanol volume ratio was 1:1.

The third object of the present invention is to provide the application of such compounds in the preparation of PTP1B inhibitors, type 2 diabetes treatment drugs and/or antitumor drugs.

The compound of the present invention is an allosteric inhibitor of PTP1B with novel structure, and has high-efficiency and high-selective PTP1B inhibitory activity. The _IC50 values for PTP1B and TCPTP inhibition were 2.42 μM and 73.33 μM, respectively, with greater than 30-fold selectivity. The results of enzyme inhibition kinetic experiments, molecular docking and effects on the glucose uptake capacity of insulin-resistant skeletal muscle C2C12 cells showed that the compound is a mixed-type inhibitor of PTP1B and can bind to the allosteric site of PTP1B to form a stable molecule- A protein complex that increases glucose consumption in insulin-resistant skeletal muscle cells. Under the circumstance that there are few reports of PTP1B allosteric inhibitors, the compounds disclosed in the present invention provide a novel structural template, which provides a reference for further synthesis and structural modification to develop PTP1B inhibitors with high specificity and good bioavailability , has a good application prospect in the development of new hypoglycemic drugs, type 2 diabetes treatment drugs and/or anti-tumor drugs targeting PTP1B.

Description of drawings

Figure 1: Enzyme Inhibition Kinetic Experiment Results

Figure 2: Molecular docking results

Figure 3: Changes in glucose uptake in insulin-resistant skeletal muscle C2C12 cells

Detailed ways

The compounds of the present invention, preparation steps and activity screening experiments are further described below through specific examples. The following examples are used to illustrate the technical content of the present invention, not to limit the content of the present invention. All changes made based on the present invention Or equivalent replacement, all should belong to the protection scope of the present invention.

Example 1 Preparation of Euphorbia quinone A

Red spurge 20Kg, crushed and then extracted with 95% ethanol by ultrasonic at room temperature, the extract was combined with the solvent under reduced pressure and evaporated to dryness to obtain 3.9Kg of extract. After the extract was dissolved in water, it was extracted three times with ethyl acetate, and the solvent was evaporated to obtain 400 g of ethyl acetate extract. The ethyl acetate fraction was separated by silica gel column chromatography and eluted successively with petroleum ether/acetone in volume ratios of 10:0, 10:1, 10:2, 2:1, 1:1, and 0:1. The eluted fraction (30 g) of petroleum ether/acetone 2:1 was separated again by silica gel column chromatography, and washed with chloroform/methanol with a volume ratio of 100:1, 10:1, 2:1, 1:1 and 0:1 in turn. take off. The chloroform/methanol 1:1 elution fraction (4.7 g) was separated by MCI column chromatography, eluting sequentially with 30%, 50%, 70%, 90%, 100% methanol/water. The 70% methanol/water eluted part was evaporated to dryness under reduced pressure, dissolved in a small amount of chloroform/methanol, and purified by Sephadex LH-20 gel column chromatography, chloroform/methanol (V/V) 1:1 was the elution solvent, and thin The eluates were analyzed by layer chromatography, combined and evaporated to dryness to obtain an orange solid, a part of which was recrystallized with an appropriate amount of chloroform/methanol (V/V) 1:1 to obtain Euphorbia quinone A (3.1 mg). The remaining part was purified by HPLC, the flow rate was 2.5 mL/min, the detection wavelength was 280 nm, and the mobile phase was 80% methanol/water to obtain Euphorbia quinone A (15.3 mg). The structure was identified by the following spectroscopic information and physicochemical properties.

Valeriaquinone A: orange-yellow amorphous powder; UV(MeOH)max(logε)204(6.2), 239(4.4), 281(4.3), 336(3.6), 407(3.3) nm; IR max 3743(OH), 3304(OH), 3216(OH), 2924, 2854, 1663(C=O), 1590, 1512, 1462, 1396, 1365, 1331, 1301, 1280, 1163, 1120, 1092, 1030,994,953,829,790,712cm ^-1 ; ¹ H NMR (DMSO-d ₆ , 500MHz) δ 7.37 (1H, s, H-4), 8.19 (1H, dd, J=7.0, 1.75, H-5), 7.95 ( 1H,dd,J=7.0,7.0,1.95,H-6),7.93(1H,dd,J=7.0,7.0,1.75,H-7),8.24(1H,dd,J=7.0,1.95,H- 8), 3.77 (2H, d, J=3.77, H-11), 7.18 (2H, t, J=3.77, H-4'), 6.68 (1H, s, H-5'), 13.18 (1H, s, 1-OH), 11.47 (1H, s, 3-OH), 3.70 (3H, s, 6'-CH ₃ ), 9.36 (1H, brs, 7'-OH), 9.41 (1H, brs, 8 '-OH); ¹³ C NMR (DMSO-d ₆ , 125MHz) δ 163.3 (C-1), 117.7 (C-2), 163.9 (C-3), 108.2 (C-4), 133.5 (C- 4a), 127.3(C-5), 135.2(C-6), 135.1(C-7), 126.9(C-8), 133.3(C-8a), 186.8(C-9), 109.8(C-9a ), 182.4(C-10), 133.5(C-10a), 23.4(C-11), 161.8(C-2′), 121.9(C-3′), 139.0(C-4′), 100.3(C -5'), 145.8(C-6'), 138.7(C-7'), 133.1(C-8'), 138.4(C-9'), 111.0(C-10'), 156.3(4'- OCH3);(-)-HRESIMS m/z459.0710(calcd for C25H15O9,459.0722).

Example 2 Evaluation of PTP1B and TCPTP inhibitory activity

Compounds were formulated in DMSO at gradient concentrations (50, 25, 12.5, 6.2, 3.1, 1.6 μM final concentrations, respectively). The pre-prepared enzyme solution (buffer containing 25mM Hepes pH 7.5, 150mM NaCl, 5mM DTT, 2mM EDTA, containing 0.1μg PTP1B or 0.15μg TCPTP) was added to the 96-well plate, 90 μL per well, and 1 μL compound solution was added to each well, Using DMSO as a blank control, three parallel duplicate wells were set for each concentration, and the 96-well plate was placed in a constant temperature shaker and shaken at 37°C for 15 min. After 15 min, 10 μL of p-NPP solution with a final concentration of 4 mM was added, and the solution was shaken at 37° C. for 30 min in a constant temperature shaker. Take out after 30 min, add 20 μL of 3M NaOH solution in turn to stop the reaction, and finally measure the OD value at 405 nm of the microplate reader, and calculate the inhibition rate and IC ₅₀ value of the compound on PTP1B. Ursolic acid was used as the positive control drug.

Calculation formula: inhibition rate=(OD _blank -OD _sample )/OD _blank ×100%

Statistical analysis was performed on the above data using GraphPad Prism 8.0 software, and three independent experiments were repeated, and the data were expressed as mean ± standard deviation.

Table 1 Inhibitory effects of euphorbia quinone A on PTP1B and TCPTP

The results in Table 1 show that spurge quinone A has a significant inhibitory effect on PTP1B, and is stronger than the control drug ursolic acid, but has a weak effect on TCPTP, and shows selective inhibitory activity on PTP1B.

Example 3 PTP1B enzyme inhibition kinetics experiment

The pre-prepared PTP1B solution was added to the 96-well plate, 90 μL per well, and 1 μL of the test compound solution was added to the final concentration of 0, 1, 2.5, 5, and 10 μM, respectively. As a blank control, the 96-well plate was placed in a constant temperature shaker and shaken at 37°C for 15 min. Then add p-NPP solution (final concentrations are 2, 4, 8mM), immediately measure the change of OD value at 405nm within 15min with a microplate reader, and calculate the initial rate of enzymatic hydrolysis. The compound concentration and the initial rate of enzymatic hydrolysis were plotted by Dixon plot, and the slope of the straight line at each concentration was obtained by Lineweaver-Burk plot, and the inhibition constant Ki value was obtained by plotting the compound concentration and the slope. The type of inhibition of PTP1B by compounds was analyzed.

Figure 1 shows the results of the kinetic experiment. In Figure 1(A), the curves of each group converge to the xy-axis region, indicating that Euphorbia quinone A is a mixed-type inhibitor of PTP1B, which can interact with other sites outside the catalytically active region of PTP1B. point combination.

Example 4 Molecular docking

Using the C-DOCKER module of the molecular docking software BIOVIA Discovery Studio 3.0, the molecular docking simulation was carried out on the active site and allosteric site of the crystal structure of Euphorbia quinone A and PTP1B. The crystal structure of PTP1B comes from the Protein Crystal Library, numbered 2HB1 (crystal formed by the combination of the substrate with the active site of PTP1B), 1T49 (crystal formed by the combination of the compound with the allosteric site of PTP1B). First, the 3D structure of Euphorbia quinone A was calculated using Discovery Studio 3.0 software, and the optimal conformation was obtained. The structure of PTP1B protein was optimized by CHARM force field after dehydration and hydrogenation. Then, the binding site was defined based on the receptor site. After the receptor was removed, the optimal conformation of the compound was calculated by docking with the crystal structure of PTP1B. The software was scored to analyze the action site and binding mode of the highest scoring conformation.

Figure 2 shows the results of molecular docking. Euphorbia quinone A can successfully dock with the active site of 2HB1 and the allosteric site of 1T49. Figure 2(a, b, c) are the overall molecule-protein binding map (2a) after docking with the active site, the docking map with the active site (2b) and the two-dimensional map (2c) of the mode of action, respectively. Euphorbia quinone A only forms weak π-alkyl interactions with Ala217 in the catalytic active site P-loop (residues 215-222), but has no effect on the key amino acid residues Cys215 and Arg221. It can interact with other sites near the active site, including Tyr46 in pTyr-loop (residues 47-49) that recognize and bind to auxiliary substrates to form π-π stacking and hydrogen bonds, and it can interact with Lys-loop (119-121 Residues) Lys120 forms hydrogen bonds and interacts with the _NH2 of Gln262 in the second binding site Q-loop (residues 261-262). It has been reported that inhibitors that bind to pTyr-loop, Lys-loop and Q-loop show better selectivity for PTP1B. Figure 2(d, e, f) are the overall molecule-protein binding map after docking with the allosteric site (2d), the docking map with the active site (2e), and the two-dimensional map of the mode of action (2f), respectively. The results show that spurge quinone A can bind well to the allosteric site, the anthraquinone fragment is inserted into the hydrophobic pocket formed by Phe280 and Phe196 in the α3 and α6 helices, and the three rings in its structure can form a strong π- π stacking, and the lactone ring of coumarin also forms π-π stacking with the non-conserved, key amino acid residue Phe280 in the allosteric site (this position is cysteine Cys280 in TCPTP). It presents a conformation that the compound wraps around Phe280, forms a stable molecule-protein complex, and further stabilizes the complex in an inactive conformation through the hydrogen bond with Asn193, the hydrogen bond with Gln276 and the hydrophobic interaction with Ala189, Make PTP1B unable to convert to a catalytically active conformation and inactivate. The overall molecular docking results show that the ability of quinone A to compete with the substrate for the active site is not strong, but it has a better binding effect with the allosteric site, which may be the main reason for its selective inhibitory effect. A structurally novel allosteric inhibitor that can be developed as a potent and selective PTP1B inhibitor.

Example 5 Evaluation of tumor cytotoxic activity

The cytotoxic activity of Euphorbia quinone A on three human hepatoma HepG2, SMMC-7721 and QGY-7703 cells was tested by MTT method. After the tumor cells were recovered, they were passaged with DMEM medium containing 10% fetal bovine serum and 1% double antibody, and the cells in the logarithmic growth phase were used for the experiment. The final concentrations of the compounds were 50, 25, 12.5, 6.25, and 3.13 μM in sequence. Cells in good growth state were taken and seeded at 2×10 ⁴ cells/100 μl; after culturing for 12 h, the supernatant was discarded, and each concentration of drugs was added to the cells in a volume of 100 μl per well, and three replicate wells were set for each concentration. After culturing for 24 h, discard the supernatant, add 100 μl of MTT solution prepared in serum-free medium to each well, and continue to culture for 4 h, discard the supernatant, add 150 μl of DMSO to each well, shake on a microplate shaker for 10 min, and use The OD value at 570nm was measured by a microplate reader and the cell viability was calculated. Cell survival rate=OD value of experimental group/OD value of control group×100%. The experiment was repeated three times.

Table 2 Cytotoxic activity of Euphorbia quinone A

The test results show that spurge quinone A has significant antitumor activity, and its inhibitory effect on HepG2 is higher than that of the positive control drug doxorubicin.

Example 6 Effects of compounds on glucose consumption in insulin-resistant skeletal muscle C2C12 cells

Cell Differentiation: C2C12 mouse myoblasts were passaged at 80% density. After culturing a sufficient number of cells, digest the cells, centrifuge, resuspend them, seed them at 2×10 ⁴ cells/100 μl, and culture them in a cell incubator for 12 h. After the cells adhere to the wall, discard the supernatant and add 100 μl PBS to each well. After rinsing three times, 200 μl of fresh differentiation medium was added to each well, and the culture was continued for 24 h. After that, fresh differentiation medium was replaced every 24h, and co-differentiation was cultured for 96h until single spindle cells were differentiated into regularly arranged multinucleated fusion myocytes.

Construction of a muscle cell model of insulin resistance: the modeling method used in the reference, the differentiated muscle cell culture medium was aspirated, and 100 μl of PBS was added to each well to rinse the cells 3 times, and 200 μl of high-glucose DMEM medium was added to each well of normal group cells. 200 μl of the prepared modeling insulin solution was added to each well of the cells of the model group and the drug-added group, and the cells were placed in a cell incubator at 37°C and 5% CO ₂ for 48 hours. After 48 hours, if the model group compared with the blank group, the cellular glucose uptake was significantly reduced, which proved that the modeling was successful.

Administration: The compounds were diluted with high-glucose DMEM medium, and the final concentrations were 50 μM, 33.3 μM, and 22.2 μM in turn. After aspirating the culture medium from each well, 200 μl of high-glucose DMEM medium was added to each well of cells in the normal group and model group. In the drug-added group, 200 μl of each concentration of drugs was added to each well, and three duplicate wells were set for each concentration, and the independent experiments were repeated three times.

Glucose content detection: Place the cells in an incubator to continue culturing. After 48 hours, prepare the glucose content detection solution according to the operation method in the kit. In a new 96-well plate, add 180 μl of the glucose content detection solution to each well, and add 20 μl of the cell supernatant from each well respectively. Incubate in a shaker for 15 min. After 15 minutes, the OD value at 505 nm of the 96-well plate was measured on a microplate reader, and the glucose content in each well was calculated according to the formula in the kit instructions. 20 μl of pre-prepared MTT solution was added to the supernatant of the 96-well plate containing cells, and after culturing in a cell incubator for 4 h, the OD value at 490 nm was measured with a microplate reader. In order to remove the influence of cell number on the detection results, the glucose content/MTT value was used as the final glucose content.

Figure 3 shows the changes of glucose consumption in insulin-resistant skeletal muscle C2C12 cells after treatment with different concentrations of spurge quinone A. Compared with the normal group, the glucose consumption of the cells in the model group was significantly reduced, while the glucose consumption of the cells in the administration group was significantly increased, and the glucose consumption at medium and high concentrations was even close to the normal group. It can be seen that the compound can significantly reverse the glucose consumption capacity of insulin resistant cells, and is expected to be developed into a new type of hypoglycemic drug or a drug for the treatment of type 2 diabetes.

Claims (5)

1. an anthraquinone/coumarin dimer compound, its structural formula is:

2. the preparation method of a kind of anthraquinone/coumarin dimer compound as claimed in claim 1, is characterized in that, concrete steps are: (1) Extraction: pulverize Euphorbia rubellae, extract with ethanol as a solvent, filter to obtain an extract, and concentrate to obtain an extract; the ethanol concentration is 95%, and the extraction temperature is normal temperature; (2) separation: the extract is dissolved in water, the aqueous solution is extracted with ethyl acetate, the ethyl acetate is evaporated to dryness under reduced pressure to obtain the ethyl acetate extraction part, and the ethyl acetate extraction part is successively used silica gel column chromatography, MCI column chromatography, gel Column chromatography separation to obtain the part containing the target compound; in silica gel column chromatography, first use 100-200 mesh silica gel, petroleum ether/acetone as mobile phase for separation, and then use 200-300 mesh silica gel, chloroform/methanol as mobile phase Separation; in MCI column chromatography, the stationary phase is MCI gel CHP20p, and the mobile phase is methanol/water; in gel column chromatography, the gel is Sephadex LH-20, and the mobile phase is chloroform/methanol; (3) Purification: the part containing the target compound is recrystallized with chloroform-methanol mixed solution, or purified by high performance liquid chromatography to obtain the target compound; the ratio of chloroform/methanol in the recrystallization method is 3:1; high performance liquid chromatography The chromatographic column is ODS packing, and the mobile phase is 80% methanol/water. 3. The application of the anthraquinone/coumarin dimer compound as claimed in claim 1 in the preparation of PTP1B enzyme inhibitor. 4. The application of the anthraquinone/coumarin dimer compound as claimed in claim 1 in the preparation of a medicine for the treatment of type 2 diabetes. 5. The application of the anthraquinone/coumarin dimer compound according to claim 1 in the preparation of anti-cancer drugs.

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