CN106928207A - Double target spot inhibitor of the α of tumour tubulin HIF 1 and preparation method thereof - Google Patents

Abstract

The invention discloses a tumor tubulin HIF‑1α dual-target inhibitor and a preparation method thereof. The synthetic compound blocks tumor blood vessels while inhibiting the glycolytic ability of tumor cells. The blood vessel blocking agent uses tubulin colchicum Alkali-binding site as the target, glycolysis inhibition with hypoxia-inducible factor 1α as the target, design and synthesis of flavonoid natural compounds as the mother core, modification of the corresponding group synthesis series of tumor blood blockers 3', 4', 5 '-Trimethoxy-7-hydroxyflavone derivatives or 2',3',4'-trimethoxy-7-hydroxyflavone derivatives or 4',5',6'-trimethoxy-7-hydroxyflavone derivatives Derivatives are screened by anti-tumor activity in vitro and inhibition level of target protein, and compounds that simultaneously act on tubulin and inhibit hypoxia-inducible factor 1α are screened out. Select candidate drugs that simultaneously act on tumor blood vessels and inhibit glycolysis; provide potential new candidate drugs for the treatment of tumors.

Description

Tumor tubulin HIF-1α dual-target inhibitor and preparation method thereof

technical field

The invention belongs to the technical field of malignant tumors, in particular to a dual-target inhibitor of tumor tubulin and HIF-1α and a preparation method thereof.

Background technique

Tumor is a common and frequently occurring disease, among which malignant tumors are currently the most serious hazard to human health. Blood vessels provide sufficient oxygen and nutrients for tumor growth and survival, and eliminate metabolic waste products. Therefore, tumor vascular targeting therapy has become an effective tumor treatment strategy. Research Progress of Tumor Vascular Inhibitors In the early 1970s, Folkman first proposed that tumor angiogenesis plays an important role in the growth and spread of tumors. Further studies such as Bergers have also shown that: tumor cells need the support of new blood vessels and functional blood vessels in the process of proliferation and metastasis; moreover, a large number of studies in recent years have confirmed that: the growth and survival of tumors need to be provided with sufficient oxygen and blood vessels. Without the support of blood vessels and new blood vessels, the growth of tumors will not exceed 2mm. Therefore, tumor blood vessel targeting therapy has emerged as the times require and has become an effective tumor treatment strategy. Different from traditional anti-tumor drugs with poor selectivity, high toxicity and easy drug resistance, tumor blood vessel targeted therapy has many advantages such as strong selectivity, low toxicity, and broad anti-tumor spectrum. It can directly act on tumor vascular endothelial cells, inhibit All types of tumors depend on the vasculature for growth. In addition, targeted blood vessel therapy has little impact on the normal physiological functions of the body, and compared with normal tissue blood vessels, the structure of tumor blood vessels is incomplete, and endothelial cells are in a state of proliferation, making them more vulnerable to attack by blood vessel-targeted drugs. According to different mechanisms of action, tumor vascular targeting agent (VTA) can be divided into two types: angiogenesis inhibitor (AI) and vascular disrupting agent (VDA). (1) Inhibiting tumor angiogenesis means inhibiting the proliferation and migration of endothelial cells and inhibiting tumor angiogenesis by interfering with the signal transduction between tumor cells, endothelial cells and stromal cells and the function of vascular endothelial cells. These include two classes of drugs, one is a monoclonal antibody and the other is a small molecule tyrosine kinase inhibitor. Bevacizumab, sorafenib, and sunitinib are currently in clinical use. Angiogenesis inhibitors are suitable for early-stage tumors with small volume, and mainly work on the periphery of the tumor. In theory, angiogenesis inhibitors are suitable for conditions with a high tendency to angiogenesis, such as advanced tumors, which require long-term medication. However, angiogenesis inhibitors have no therapeutic effect on the formed tumor blood vessels, and cannot completely eradicate all tumor cells. Therefore, current neovascularization inhibitors usually require combination therapy. (2) Block the existing tumor blood vessels, rapidly cut off the tumor blood supply by selectively destroying the endothelial cells and pericytes of the formed tumor vascular network, and induce ischemic necrosis of tumor cells. This concept was proposed by Denekamp in 1982. The corresponding drugs are called vascular blockers. Among the VDA drug candidates currently in the clinical research stage, the main targets of action are tubulin, which is shown to inhibit the aggregation of tubulin to form microtubules. For example, CA4P and its analogues OXi4503, AVE8062, colchicine analogue ZD6126, BNC-105, CKD-516 (having a common structure of benzenetrimethoxyl group), etc. have been approved as rare disease drugs in the United States in May 2006 , for the treatment of ovarian cancer and thyroid cancer, it has also been granted the title of rare disease drug in Europe for anaplastic thyroid cancer. At present, blood vessel blocking agents have become a research hotspot.

In clinical medicine, the effect of using vascular blocking agents alone is not good, and the tumor can still survive after vascular blocking, and it is believed that the reason may be the Warburg effect. The Warburg effect is the observation that most cancer cells generate energy primarily through high-rate glycolysis with or without oxygen, whereas in most normal cells energy is generated primarily through aerobic respiration in the mitochondria. Malignant fast-growing tumor cells often have a glycolytic rate up to 200 times that of the tissue of their normal origin, which occurs even when oxygen is adequate. The energy of tumor cells mainly comes from glycolysis. Hypoxia is a common feature of solid tumors and plays a key role in tumor progression. Otto Warburg scholars found that many tumor cells produced too much lactic acid even under normoxia, which was called pseudo-hypoxia. The rapid proliferation of tumors requires a continuous supply of oxygen and nutrients. Hypoxia occurs when the rate of tissue growth far outstrips the ability of surrounding blood vessels to supply these nutrients. Hypoxia is closely related to tumor angiogenesis, invasion and metastasis, resistance to radiotherapy and chemotherapy, and poor prognosis. The pyruvate produced by the glycolytic pathway can directly enter the tricarboxylic acid cycle pathway or be converted into lactate by lactate dehydrogenase (lactatedehydrogenase, LDH). In the process of lactic acid production from glucose through the glycolytic pathway, key metabolic enzymes such as HK, PFK, PK, and LDH are related to tumors, and are regulated by transcription factors such as hypoxia inducible factor-1α (hypoxia inducible factor-1α) in the oncogenic factor signal transduction pathway. 1α, HIF-1α) and so on. (1) Hypoxia-inducible factor inhibitor Hypoxia-inducible factor 1 (HIF-1) is the most critical nuclear transcriptional regulator in the regulation of hypoxia effects. HIF-1 is selectively and continuously highly expressed in solid tumor tissues, and the downstream key regulatory genes are closely related to the occurrence and development of tumors, such as promoting angiogenesis, cell survival, inhibiting tumor cell apoptosis, metabolic remodeling, and regulation of pH homeostasis. Glycolysis is an important means for tumor cells to obtain energy during hypoxia. HIF-1α binds to the DNA binding site on the target gene, induces the expression of glycolytic enzyme genes, increases glycolysis, and promotes anaerobic metabolism, so it is beneficial to the survival of tumor cells under hypoxia. Drugs affecting the synthesis and degradation of HIF-1α The flavonoid chrysin increases ubiquitination and degrades HIF-1α by increasing its prolyl hydroxylation. Furthermore, chrysin interacted with HIF-1α and HSP90. Chrysin was also found to inhibit the expression of HIF-1α through the Akt signaling pathway. Chrysin can inhibit the expression level of vascular endothelial growth factor by inhibiting HIF-1α. Mirzoeva et al. found that in the human prostate cancer cell line PC3-M, apigenin down-regulates the mRNA and protein expression levels of HIF-1α, and reduces the stability of its protein structure, thereby preventing the activation of HIF-1 downstream target gene VEGF, down-regulating VEGF mRNA and protein expression levels inhibit angiogenesis in prostate cancer. Liu et al. also found that although acacetin had no significant effect on the expression level of HIF-1α mRNA, it inhibited the activation of VEGF by down-regulating its protein expression level, thereby preventing the formation of neovascularization in ovarian cancer, and this effect on VEGF The inhibitory effect can be abolished by overexpressing HIF-1α.

Contents of the invention

The purpose of the present invention is to provide a tumor tubulin HIF-1α dual-target inhibitor and its preparation method, aiming at solving the problem of poor effect of using blood vessel blocking agents alone in clinical medicine, and the tumor blood vessels still remain after blockade. survivable issues.

The present invention is achieved in this way, a tumor tubulin HIF-1α dual-target inhibitor, the tumor tubulin HIF-1α dual-target inhibitor has the following general formula:

n=1,2,3; R1=R2=R3=OCH3 or R2=R3=R4=OCH3 or R3=R4=R5=OCH3.

Another object of the present invention is to provide a preparation method for the preparation method of the tumor tubulin HIF-1α dual-target inhibitor, the preparation method of the tumor tubulin HIF-1α dual-target inhibitor comprises the following step:

Step 1, add 3', 4', 5'-trimethoxy-7-hydroxyflavone or 2', 3', 4'-trimethoxy-7-hydroxyflavone or 4' sequentially in a 250mL round bottom flask, 5',6'-trimethoxy-7-hydroxyflavone, anhydrous potassium carbonate and acetone, heated and stirred to reflux, then added dropwise 1,2-dibromoethane or 1,3-dibromopropane or 1,4 -Dibromobutane, heated and condensed to reflux at 60°C, the solution became clear and then cloudy; the reaction process was detected by thin-layer chromatography, purified by column chromatography, and the eluent was: methanol: dichloromethane = 1:50;

Step 2: Add benzimidazole derivatives to a 250 mL three-necked flask, dissolve them in 100 mL of acetone, add potassium carbonate, stir for 10 min, add the purified flavonoid derivatives obtained in Step 1, tetrabutylammonium bromide, and heat to 60 Stir the reaction at ℃, extract with ethyl acetate, wash the organic layer with saturated brine, and then dry with anhydrous sodium sulfate. After the solvent is evaporated to dryness under reduced pressure, it is separated with a silica gel column to obtain the target compound. The eluent is: methanol: dichloromethane =1:50.

Further, in step one: add 3', 4', 5'-trimethoxy-7-hydroxyflavone or 2', 3', 4'-trimethoxy-7-hydroxyl Flavone or 4', 5', 6'-trimethoxy-7-hydroxyflavone 3.31g, 0.01mol, anhydrous potassium carbonate 5.52g, 0.04mol and acetone 100ml; 1,2-dibromoethane or 1,3 - 0.04 mol of dibromopropane or 1,4-dibromobutane.

Further, in the second step: 0.015 mol of benzimidazole derivatives, 5.52 g of anhydrous potassium carbonate, 0.04 mol, 0.01 mol of the purified flavone derivatives obtained in step 1, tetrabutyl bromide Ammonium 0.03g, 0.0001mol and acetone 100ml.

The tumor tubulin HIF-1α dual-target inhibitor and its preparation method provided by the present invention are designed to synthesize compounds that block tumor blood vessels while inhibiting the glycolytic ability of tumor cells. The blood vessel blocking agent uses tubulin colchicum Alkali-binding site as the target, glycolysis inhibition with hypoxia-inducible factor 1 as the target, design and synthesis of flavonoid natural compounds as the mother core, modify the corresponding groups of tumor blood blockers to synthesize a series of compounds, and use the in vitro anti-tumor activity , the inhibitory level of the target protein was screened, and the compounds acting on tubulin and inhibiting hypoxia-inducible factor 1α were screened out. The compound drug candidates that simultaneously act on tumor blood vessels and inhibit glycolysis are selected to provide potential new candidate drugs for the treatment of tumors. Among the tumor blood vessel blocking agents, most of them target the colchicine binding site of tubulin. This kind of compounds are analyzed. A common structural group found in the structure is benzenetrimethoxy. Therefore, the designed compound will retain the benzenetrimethoxy group. From the aspect of reducing the glycolysis of tumor cells, the most important thing is HIF-1α. There are many types of HIF-1α inhibitors. We found that the inhibition of HIF-1α synthesis Flavonoids and benzimidazoles were found in flavonoids, and the earliest discovered vascular blocker was flavone acetate, which took flavone as the core structure, and introduced trimethoxy in the A ring or B ring of flavone, and introduced benzene on the other side. The synthesis of imidazole or benzimidazole derivatives is expected to block blood vessels and also inhibit glycolysis by inhibiting HIF-1α.

In the invention, the same compound has an effective group for inhibiting tumor tubulin and an effective group for inhibiting HIF-1α, and the compound can block tumor blood vessels and inhibit tumor glycolysis.

Description of drawings

Fig. 1 is a flow chart of the preparation method of the tumor tubulin HIF-1α dual-target inhibitor provided by the embodiment of the present invention.

Fig. 2 is a schematic diagram of the experimental results provided by the embodiment of the present invention.

detailed description

In order to make the object, technical solution and advantages of the present invention more clear, the present invention will be further described in detail below in conjunction with the examples. It should be understood that the specific embodiments described here are only used to explain the present invention, not to limit the present invention.

The application principle of the present invention will be described in detail below in conjunction with the accompanying drawings.

The tumor tubulin HIF-1α dual-target inhibitor provided by the embodiment of the present invention

As shown in Figure 1, the preparation method of the tumor tubulin HIF-1α dual-target inhibitor provided by the embodiment of the present invention includes the following steps:

S101: Add 3', 4', 5'-trimethoxy-7-hydroxyflavone or 2', 3', 4'-trimethoxy-7-hydroxyflavone or 4', 5 ', 6'-trimethoxy-7-hydroxyflavone (3.31g, 0.01mol), anhydrous potassium carbonate (5.52g, 0.04mol) and acetone (100ml), heated and stirred to reflux, then added dropwise 1,2- Dibromoethane or 1,3-dibromopropane or 1,4-dibromobutane (0.04mol) was heated and condensed to reflux at 60°C, the solution became clear and then cloudy. TCL (thin-layer chromatography) detects the reaction process, and column chromatography purifies, and the eluent is: methanol: dichloromethane=1:50;

S102: add benzimidazole derivative (0.015mol) in 250mL there-necked flask, make it dissolve in 100mL acetone, add potassium carbonate (5.52g, 0.04mol), after stirring for 10min, add the flavone derivative ( 0.01mol), tetrabutylammonium bromide (TBAB) (0.03g, 0.0001mol), be heated to 60 ℃ and stir reaction, (TCL detects reaction process), extract with ethyl acetate, organic layer is washed with saturated saline, then with After drying with anhydrous sodium sulfate, the solvent was evaporated to dryness under reduced pressure, and the target compound was obtained by separation with a silica gel column. The eluent was methanol:dichloromethane=1:50.

The chemical reaction equation of the preparation method of the tumor tubulin HIF-1α dual-target inhibitor provided in the embodiment of the present invention is as follows:

The application principle of the present invention will be further described below in combination with specific embodiments.

Example 1: 7-(2-bromoethoxy)-2-(3,4,5-trimethylphenyl)-4H-benzopyran-4-one

In a 250mL round bottom flask, add 3', 4', 5'-trimethoxy-7-hydroxyflavone (3.31g, 0.01mol), anhydrous potassium carbonate (5.52g, 0.04mol) and acetone (30ml) successively, Heat and stir to reflux, then add 1,2-dibromoethane (0.04mol) dropwise, heat and condense to reflux at 60°C, the solution becomes clear and then turbid. TCL (thin-layer chromatography) detects the reaction process, and purifies by column chromatography, the eluent is: methanol:dichloromethane=1:50. Yellow powder, the yield is 80%. ¹ H NMR (400MHz, cdcl ₃ ) δ7.72(d, J=8.5Hz, 1H), 7.14(s, 2H), 6.78(d, J=2.1Hz, 1H), 6.76(d, J=2.2Hz , 1H), 6.75–6.73(m, 2H), 4.40(t, J=6.2Hz, 2H), 3.92(d, J=10.7Hz, 9H), 3.68(t, J=6.2Hz, 2H).

Example 2: 7-(2-(1H-benzo[d]imidazol-1-yl)ethoxy)-2-(3,4,5-trimethoxyphenyl)-4H-benzopyran -4-one

Add benzimidazole (0.0015mol) in 100mL three-necked flask, make it dissolve in 30mL acetone, add potassium carbonate (0.552g, 0.004mol), after stirring for 10min, add the pale yellow solid 7-(2- Bromoethoxy)-2-(3,4,5-trimethylphenyl)-4H-benzopyran-4-one (0.001mol), tetrabutylammonium bromide (TBAB) (0.03g, 0.0001mol), heated to 60°C and stirred for reaction, (TCL detection reaction progress), extracted with ethyl acetate, washed the organic layer with saturated brine, then dried with anhydrous sodium sulfate, evaporated the solvent to dryness under reduced pressure and separated it with a silica gel column. The target compound was obtained, and the eluent was: methanol:dichloromethane=1:50. Yellow powder, yield 51%. ¹ H NMR (400MHz, cdcl ₃ ) δ8.03(s, 1H), 7.82(d, J=7.0Hz, 1H), 7.68(d, J=8.6Hz, 1H), 7.47(d, J=7.3Hz , 1H), 7.32(dq, J=7.2, 6.0Hz, 2H), 7.11(s, 2H), 6.74–6.70(m, 2H), 6.70(d, J=2.1Hz, 1H), 6.65(d, J=2.0Hz, 1H), 4.64(t, J=5.2Hz, 2H), 4.41(t, J=5.1Hz, 2H), 3.91(d, J=7.6Hz, 9H).

Example 3: 7-(2-(2-methyl-1H-benzo[d]imidazol-1-yl)ethoxy)-2-(3,4,5-trimethoxyphenyl)-4H -Benzopyran-4-one

Add 2-methylbenzimidazole (0.0015mol) into a 100mL three-necked flask, dissolve it in 30mL of acetone, add potassium carbonate (0.552g, 0.004mol), stir for 10min, then add the light yellow solid 7 obtained in Example 1 -(2-Bromoethoxy)-2-(3,4,5-trimethylphenyl)-4H-benzopyran-4-one (0.001mol), tetrabutylammonium bromide (TBAB) (0.03g, 0.0001mol), heated to 60°C and stirred for reaction, (TCL detection reaction process), extracted with ethyl acetate, washed the organic layer with saturated brine, then dried with anhydrous sodium sulfate, evaporated the solvent to dryness under reduced pressure and used The target compound was obtained by separation on a silica gel column, and the eluent was methanol:dichloromethane=1:50. Yellow powder, yield 48%. ¹ H NMR (400MHz, cdcl ₃ ) δ7.67(d, J=8.6Hz, 1H), 7.10(s, 2H), 6.73(s, 1H), 6.68(d, J=2.0Hz, 1H), 6.66 (d, J=2.1Hz, 1H), 6.60(d, J=2.0Hz, 1H), 4.59(t, J=5.3Hz, 3H), 4.39(t, J=5.2Hz, 3H), 3.90(d , J=7.4Hz, 9H), 2.72(s, 3H).

Example 4: 7-(2-(2-chloro-1H-benzo[d]imidazol-1-yl)ethoxy)-2-(3,4,5-trimethoxyphenyl)-4H- Benzopyran-4-one

Add 2-chlorobenzimidazole (0.0015mol) to a 100mL three-necked flask, dissolve it in 30mL acetone, add potassium carbonate (0.552g, 0.004mol), stir for 10min, then add the light yellow solid 7- (2-Bromoethoxy)-2-(3,4,5-trimethylphenyl)-4H-benzopyran-4-one (0.001mol), tetrabutylammonium bromide (TBAB) ( 0.03g, 0.0001mol), heated to 60°C and stirred for reaction, (TCL detection of reaction progress), extracted with ethyl acetate, washed the organic layer with saturated brine, then dried with anhydrous sodium sulfate, evaporated the solvent to dryness under reduced pressure and washed it with silica gel The target compound was obtained by column separation, and the eluent was: methanol: dichloromethane = 1:50. Yellow powder, yield 49%. ¹ H NMR (400MHz, cdcl ₃ ) δ7.70 (d, J=7.1Hz, 1H), 7.66 (d, J=8.5Hz, 1H), 7.44 (d, J=7.4Hz, 1H), 7.38–7.26 (m, 2H), 7.11(s, 2H), 6.73(s, 1H), 6.67(dd, J=8.5, 2.1Hz, 1H), 6.63(d, J=2.0Hz, 1H), 4.66(t, J=5.4Hz, 2H), 4.41 (dd, J=10.6, 5.1Hz, 2H), 3.91 (d, J=7.9Hz, 9H).

Example 5: 7-(2-(5-nitro-1H-benzo[d]imidazol-1-yl)ethoxy)-2-(3,4,5-trimethoxyphenyl)-4H -Benzopyran-4-one

Add 6-nitrobenzimidazole (0.0015mol) to a 100mL three-necked flask, dissolve it in 30mL acetone, add potassium carbonate (0.552g, 0.004mol), stir for 10min, then add the light yellow solid 7 obtained in Example 1 -(2-Bromoethoxy)-2-(3,4,5-trimethylphenyl)-4H-benzopyran-4-one (0.001mol), tetrabutylammonium bromide (TBAB) (0.03g, 0.0001mol), heated to 60°C and stirred for reaction, (TCL detection reaction process), extracted with ethyl acetate, washed the organic layer with saturated brine, then dried with anhydrous sodium sulfate, evaporated the solvent to dryness under reduced pressure and used The target compound was obtained by separation on a silica gel column, and the eluent was methanol:dichloromethane=1:50. Yellow powder, yield 57%. ¹ H NMR (400MHz, cdcl ₃ ) δ8.74(s, 1H), 8.54(d, J=2.0Hz, 1H), 8.30(d, J=11.2Hz, 1H), 8.27–8.20(m, 1H) , 7.88(d, J=9.0Hz, 1H), 7.69(d, J=8.4Hz, 1H), 7.57(d, J=9.0Hz, 1H), 7.11(d, J=2.1Hz, 2H), 6.72 (d, J=12.5Hz, 4H), 4.72 (dt, J=9.9, 4.7Hz, 2H), 4.45 (dd, J=9.8, 5.0Hz, 2H), 3.94–3.88 (m, 9H).

Example 6: 7-(2-(5,6-Dimethyl-1H-benzo[d]imidazol-1-yl)ethoxy)-2-(3,4,5-trimethoxyphenyl )-4H-benzopyran-4-one

Add 5,6-dimethylbenzimidazole (0.0015mol) into a 100mL three-necked flask, dissolve it in 30mL of acetone, add potassium carbonate (0.552g, 0.004mol), stir for 10min, then add the dilute solution obtained in Example 1 Yellow solid 7-(2-bromoethoxy)-2-(3,4,5-trimethylphenyl)-4H-chromen-4-one (0.001mol), tetrabutylammonium bromide (TBAB) (0.03g, 0.0001mol), heated to 60 ℃ and stirred for reaction, (TCL detection reaction process), extracted with ethyl acetate, washed the organic layer with saturated brine, then dried with anhydrous sodium sulfate, and the solvent was evaporated under reduced pressure After drying, the target compound was obtained by separation with a silica gel column. The eluent was: methanol: dichloromethane = 1:50. Yellow powder, yield 43%. ¹ H NMR (400MHz, cdcl ₃ ) δ7.91(s, 1H), 7.68(d, J=8.5Hz, 1H), 7.57(s, 1H), 7.21(s, 1H), 7.11(s, 2H) , 6.73(s, 1H), 6.70(dd, J=8.6, 2.0Hz, 1H), 6.65(d, J=2.0Hz, 1H), 4.58(t, J=5.1Hz, 2H), 4.38(t, J=5.2Hz, 2H), 3.91(d, J=7.8Hz, 9H), 2.38(d, J=13.9Hz, 6H).

Embodiment 7: the in vitro antitumor activity test of the compound of the present invention

The anti-tumor cell proliferation activity test is carried out on the compound of the present invention, and the test method adopts the conventional MTT method.

The cell lines are human gastric cancer cell MGC-803, human liver cancer cell HepG2 and human breast cancer cell MCF-7. The culture medium is DMEM+15%NBS+double antibody.

The preparation method of the drug solution: after dissolving in DMSO (Merck), add PBS (-) to make a 1mmol/mL solution or a uniform suspension, then dilute with DMSO in PBS (-), the final concentration is 384μmol/L , 192 μmol/L, 96 μmol/L, 48 μmol/L, 24 μmol/L, 12 μmol/L, 6 μmol/L, 3 μmol/L.

The listed antineoplastic drugs 5-fluorouracil and chrysin were prepared as reference solution under the same conditions.

experiment procedure

Add 100 μL of cell suspension with a concentration of 3×10 ⁴ cells/mL to each well of a 96-well plate, that is, 3000 cells/well, and place in a 37° C., 5% CO ₂ incubator. After 24 hours, add the sample solution and reference solution respectively, 10 μL/well, and react at 37°C for 72 hours. Add 5mg/mL of MTT (3-(4,5-dimethylthiazole-2-

Base)-2,5-diphenyltetrazolium bromide) solution 20μL, add solution DMSO after 4 hours of action, 100μL/well, put it in the incubator, and measure 570nm with MK-2 automatic microplate reader the next day OD value. Calculate the half inhibitory concentration IC50.

The test results are shown in Table 1, wherein the samples refer to newly synthesized flavonoid benzimidazole derivatives C1-C24, and Examples 1-6 are only the specific synthesis methods and results of some of the compounds.

The half inhibitory concentration IC50 (unit: μ mol/L) of the compound of table 1 to tumor cell

Anti-proliferative activity of compound against the cancer cell lines

SD=standard deviation,

N.D = not detected

The above experimental results show that the compound of the present invention has good antitumor activity, and 3', 4', 5'-trimethoxy-7-hydroxyflavone benzimidazole derivatives have anti-tumor effect on human gastric cancer cell MGC-803, human liver cancer cell HepG2 , human breast cancer cell MCF-7 all showed a certain degree of inhibitory activity, and compound C14 had a significant effect on human gastric cancer cell MGC-803, human liver cancer cell HepG2, and human breast cancer cell MCF-7, so C14 No. compound for further in vivo anti-tumor activity test. Because the effect of the newly synthesized compound on human gastric cancer cell MGC-803 is better than others, mouse gastric cancer cell MFC was used to build the tumor model in the anti-tumor activity test in vivo.

Embodiment 8: In vivo anti-tumor activity test of No. C14 compound of the present invention

Experimental animal preparation: experimental animals 38 healthy male mice aged 3 to 4 weeks were randomly divided into 5 groups: administration group (7 in each of the three doses of high, middle and low), positive control group (pentafluorouracil) 7, negative control group 10 rats were fed with autoclaved water and feed, the relative humidity of the breeding room was 55%±10%, the temperature was (22±2)°C, the light was 12h, and light and dark alternated.

The DMEM medium and fetal bovine serum required in the cell culture process were purchased from Gibco, USA. Experimental instruments for microsurgery were purchased from Shanghai Medical Instrument Corporation and used after autoclaving.

After the cryopreserved mouse-derived gastric cancer cells MFC were revived, they were cultured and subcultured in DMEM medium containing 10% fetal bovine serum in a 37°C, 5% CO ₂ incubator until the logarithmic growth phase, and the pancreas After enzymatic digestion, the cells were resuspended into 1×10 ⁷ /mL cell suspension with normal saline, and the cell suspension was injected subcutaneously in the middle of the left side of the mouse to form a mound containing 0.2 mL of cell suspension (2×10 ⁶ ), a total of 38 mice were inoculated, and the experiment started when the tumor volume reached about 125 mm ³ (defined as day 0).

Observation and collection of materials: All mouse models were given free access to water, and the body weight, mental state, food intake, activity and nutrition of the mice were observed every two days. Observe the subcutaneous tumor formation of the mice every two days after modeling, measure the long diameter (a) and wide diameter (b) of the subcutaneous tumor, calculate the volume (V=a×b ² ), and use the tumor volume and growth time as coordinates to draw Growth curve. At the same time, the tumor formation time, tumor shape, and skin condition on the surface of the tumor were recorded. After 2 weeks, all the mice were sacrificed and the tumors were excised to observe the tumor size.

The experimental results are shown in Figure 2. The experimental results show that the C14 compound has a good inhibitory effect on the growth of tumors in mice, and it is concentration-dependent. The mechanism of action of the compound can be further studied and discussed.

Figure 2 is a growth curve drawn with tumor volume and growth time as coordinates.

The above descriptions are only preferred embodiments of the present invention, and are not intended to limit the present invention. Any modifications, equivalent replacements and improvements made within the spirit and principles of the present invention should be included in the protection of the present invention. within range.

Claims (4)

1. double target spot inhibitor of a kind of tumour tubulin HIF-1 α, it is characterised in that the tumour tubulin HIF-1 α are double Target spot inhibitor：

N=1,2,3；R1=R2=R3=OCH3 or R2=R3=R4=OCH3 or R3=R4=R5=OCH3.

2. the preparation method of the preparation method of the double target spot inhibitor of a kind of tumour tubulin HIF-1 α as claimed in claim 1, Characterized in that, the preparation method of the double target spot inhibitor of the tumour tubulin HIF-1 α is comprised the following steps：

Step one, sequentially adds 3', 4', 5'- trimethoxy -7- flavonols or 2', 3', 4'- tri- in 250mL round-bottomed flasks Methoxyl group -7- flavonols or 4', 5', 6'- trimethoxy -7- flavonols, Anhydrous potassium carbonate and acetone, heating stirring are returned Stream, then 1,2- Bromofumes or 1,3- dibromopropane or Isosorbide-5-Nitrae-dibromobutane are added dropwise over, 60 DEG C of heating condensing refluxes, solution becomes Clarification becomes cloudy again；Thin-layered chromatography detects reaction process, column chromatography purifying, and eluant, eluent is：Methyl alcohol：Dichloromethane=1:50；

Step 2, benzimidizole derivatives are added in 250mL there-necked flasks, it is dissolved in 100mL acetone, add potassium carbonate, After stirring 10min, add the chromocor derivative of step one gained purifying, TBAB to be heated to 60 DEG C of stirring reactions, use Ethyl acetate is extracted, and organic layer is washed with saturated common salt, and then with anhydrous sodium sulfate drying, solvent under reduced pressure uses silicagel column after being evaporated Separate and obtain target compound, eluant, eluent is：Methyl alcohol：Dichloromethane=1:50.

3. the preparation side of the preparation method of the double target spot inhibitor of the as claimed in claim 2 tumour tubulin HIF-1 α Method, it is characterised in that in the step one：3', 4', 5'- trimethoxy -7- hydroxyls are sequentially added in 250mL round-bottomed flasks Flavones or 2', 3', 4'- trimethoxy -7- flavonols or 4', 5', 6'- trimethoxy -7- flavonol 3.31g, 0.01mol, Anhydrous potassium carbonate 5.52g, 0.04mol and acetone 100ml；1,2- Bromofume or 1,3- dibromopropane or Isosorbide-5-Nitrae-two NBB 0.04mol.

4. the preparation side of the preparation method of the double target spot inhibitor of the as claimed in claim 2 tumour tubulin HIF-1 α Method, it is characterised in that in the step 2：Benzimidizole derivatives 0.015mol, nothing are sequentially added in 250mL round-bottomed flasks Aqueous carbonate potassium 5.52g, 0.04mol, chromocor derivative 0.01mol, the TBAB 0.03g of step one gained purifying, 0.0001mol and acetone 100ml.