CN106946864A - Suppress the α of HIF 1 antitumor drug candidate and preparation method - Google Patents

Abstract

The invention discloses an anti-tumor drug candidate for inhibiting HIF-1α and a preparation method. It is proposed to design and synthesize a compound capable of inhibiting the glycolysis ability of tumor cells. Glycolysis inhibition takes hypoxia-inducible factor 1α as the target, and the designed and synthesized compound is Flavonoid natural compounds were used as the mother nucleus, and benzimidazole derivatives were modified to synthesize a series of chrysin derivatives. The in vitro anti-tumor activity and the inhibition level of target proteins were screened to screen out compounds that act on hypoxia-inducible factor 1α. Select candidate drugs that inhibit glycolysis; provide potential new candidate drugs for the treatment of tumors.

Description

Anti-tumor drug candidate for inhibiting HIF-1α and preparation method

technical field

The invention belongs to the technical field of malignant tumors, and in particular relates to a candidate antitumor drug for inhibiting HIF-1α and a preparation method.

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.

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 (lactate dehydrogenase, LDH). In the process of glucose producing lactic acid through glycolysis pathway, key metabolic enzymes such as HK, PFK, PK and LDH are related to tumors, and are affected by transcription factors such as hypoxia inducible factor-1α (hypoxia inducible factor) 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 preparation method of anti-tumor candidate drug inhibiting HIF-1α, aiming to solve the problem that in clinical medicine, the effect of using blood vessel blocking agents alone is not good, and tumor blood vessels can still survive after blocking.

The present invention is achieved in this way, a candidate drug for inhibiting HIF-1α anti-tumor, said drug candidate for inhibiting HIF-1α anti-tumor has the following general formula:

Another object of the present invention is to provide a preparation method of the anti-tumor drug candidate for inhibiting HIF-1α, which comprises the following steps:

Step 1: Add chrysin, anhydrous potassium carbonate and acetone in sequence in a 250mL round bottom flask, heat and stir to reflux, then add 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 progress was checked and purified by column chromatography, and the eluent was: methanol: dichloromethane = 1:50;

Step 2, add benzimidazole derivatives in a 250mL three-necked flask, dissolve it in 100mL acetone, add potassium carbonate, stir for 10min, add the purified chrysin derivatives obtained in step 1, tetrabutylammonium bromide, and heat to Stir the reaction at 60°C, 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: dichloro Methane = 1:50.

Further, in step one: add 2.54g of chrysin, 0.01 (modified) mol, 5.52g of anhydrous potassium carbonate, 0.04mol and 100ml of acetone in a 250mL round bottom flask successively; 1,2-dibromoethane or 0.04mol of 1,3-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 purified chrysin derivatives obtained in step 1, tetrabutyl bromide Ammonium chloride 0.03g, 0.0001mol and acetone 100ml.

The anti-tumor candidate drug for inhibiting HIF-1α provided by the present invention and its preparation method are intended to design and synthesize compounds that can inhibit the glycolysis ability of tumor cells. Glycolysis inhibition takes hypoxia-inducible factor 1α as the target, and the design and synthesis is based on natural flavonoids. The compound is the mother nucleus, and a series of chrysin derivatives are synthesized by modifying benzimidazole derivatives. The anti-tumor activity in vitro and the inhibition level of the target protein are screened, and the compounds that inhibit hypoxia-inducible factor 1α are screened out. Candidate drugs that inhibit glycolysis are selected to provide potential new candidate drugs for the treatment of tumors. In terms of reducing the glycolysis of tumor cells, the most important one is HIF-1α. There are many types of HIF-1α inhibitors. Among the inhibitors that inhibit the synthesis of HIF-1α, flavonoids and benzimidazoles were found, and they were the first to be discovered. The vascular blocking agent in the study is flavone acetate, so it is proposed to use flavone as the core structure, and introduce benzimidazole or benzimidazole derivatives on the other side. The synthesis is expected to inhibit glycolysis by inhibiting HIF-1α.

Description of drawings

Fig. 1 is a flow chart of the preparation method of the HIF-1α-inhibiting anti-tumor candidate drug 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 anti-tumor drug candidates for inhibiting HIF-1α provided by the embodiments of the present invention have the following general formula:

As shown in Figure 1, the preparation method of the anti-tumor drug candidate for inhibiting HIF-1α provided by the embodiment of the present invention comprises the following steps:

S101: Add chrysin (2.54g, 0.01mol), anhydrous potassium carbonate (5.52g, 0.04mol) and acetone (100ml) in sequence in a 250mL round bottom flask, heat and stir to reflux, and then add 1,2-di Bromoethane or 1,3-dibromopropane or 1,4-dibromobutane (0.04mol), heated at 60°C and condensed to reflux, the solution became clear and then cloudy. TCL (thin-layer chromatography) detects the reaction process, and purifies by column chromatography, the eluent is: methanol:dichloromethane=1:50.

S102: Add a benzimidazole derivative (0.015mol) to a 250mL three-neck flask, dissolve it in 100mL acetone, add potassium carbonate (5.52g, 0.04mol), stir for 10min, then add the purified chrysin derivative obtained in step 1 (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 brine, then Dry with anhydrous sodium sulfate, evaporate the solvent to dryness under reduced pressure, and then separate with a silica gel column to obtain the target compound. The eluent is methanol:dichloromethane=1:50.

The reaction equation of the preparation method of the HIF-1α anti-tumor candidate drug provided by 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)-5-hydroxy-2-phenyl-4H-benzopyrone-4-one

Add chrysin (2.54g, 0.01mol), anhydrous potassium carbonate (5.52g, 0.004mol) and acetone (100ml) successively in a 250mL round bottom flask, heat and stir to reflux, then add 1,2-dibromoethyl Alkanes (0.04mol), heated at 60°C to condense to reflux, the solution became clear and then cloudy. TCL (thin-layer chromatography) detects the reaction process, and purifies by column chromatography, the eluent is: methanol:dichloromethane=1:50.

Example 2: 7-(3-bromopropoxy)-5-hydroxy-2-phenyl-4H-benzopyrone-4-one

Add chrysin (2.54g, 0.01mol), anhydrous potassium carbonate (5.52g, 0.004mol) and acetone (100ml) successively in a 250mL round bottom flask, heat and stir to reflux, then add 1,3-dibromopropane dropwise (0.04mol), heated and condensed at 60°C to reflux, the solution became clear and then cloudy. TCL (thin-layer chromatography) detects the reaction process, and purifies by column chromatography, the eluent is: methanol: dichloromethane = 1:50.

Example 3: 7-(4-bromobutoxy)-5-hydroxy-2-phenyl-4H-benzopyrone-4-one

Add chrysin (2.54g, 0.01mol), anhydrous potassium carbonate (5.52g, 0.004mol) and acetone (100ml) successively in a 250mL round bottom flask, heat and stir to reflux, then add 1,4-dibromobutyl dropwise Alkanes (0.04mol), heated at 60°C to condense to reflux, the solution became clear and then cloudy. TCL (thin-layer chromatography) detects the reaction process, and purifies by column chromatography, the eluent is: methanol:dichloromethane=1:50.

Example 4: 5-hydroxy-7-(2-(2-chloro-1H-benzo[d]imidazol-1-yl)ethoxy)-2-phenyl-4H-chromen-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)-5-hydroxy-2-phenyl-4H-benzopyrone-4-one (0.36g, 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 separated it with a silica gel column. The target compound was obtained, and the eluent was: methanol:dichloromethane=1:50. Pale yellow powder, the yield is 56%. ¹ H NMR (400MHz, cdcl ₃ ) δ12.71(s, 1H), 7.84(dd, J=8.1, 1.6Hz, 2H), 7.69(d, J=7.6Hz, 1H), 7.55–7.47(m, 3H), 7.44(d, J=7.3Hz, 1H), 7.37–7.26(m, 2H), 6.64(s, 1H), 6.39(d, J=2.2Hz, 1H), 6.28(d, J=2.3 Hz, 1H), 4.64(t, J=5.3Hz, 2H), 4.37(t, J=5.4Hz, 2H).

Example 5: 5-Hydroxy-7-(2-(5-nitro-1H-benzo[d]imidazol-1-yl)ethoxy)-2-phenyl-4H-chromen-4-one

Add 6-nitrobenzimidazole (0.0015mol) to a 100mL three-necked flask, dissolve it in 30mL acetone, add potassium carbonate (0.0552g, 0.004mol), stir for 10min, then add the light yellow solid 7 obtained in Example 1 -(2-Bromoethoxy)-5-hydroxy-2-phenyl-4H-benzopyrone-4-one (0.36g, 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, and separated the solvent with a silica gel column after evaporating to dryness under reduced pressure. The target compound was obtained, and the eluent was: methanol: dichloromethane = 1:50. Pale yellow powder, the yield is 52%. ¹ H NMR (400MHz, cdcl ₃ ) δ12.75 (d, J=3.9Hz, 1H), 8.74 (s, 1H), 8.52 (s, 1H), 8.33–8.18 (m, 2H), 7.86 (dd, J=13.7, 8.3Hz, 2H), 7.58(d, J=9.0Hz, 1H), 7.52(t, J=8.1Hz, 2H), 6.65(s, 1H), 6.43(dd, J=17.4, 2.2 Hz, 1H), 6.30 (s, 1H), 4.74–4.64 (m, 2H), 4.41 (dd, J=10.4, 5.3Hz, 2H).

Example 6: 5-Hydroxy-7-(2-(5-nitro-1H-benzo[d]imidazol-1-yl)propoxy)-2-phenyl-4H-chromen-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 2 -(3-Bromopropoxy)-5-hydroxy-2-phenyl-4H-benzopyrone-4-one (0.37g, 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, and separated the solvent with a silica gel column after evaporating to dryness under reduced pressure. The target compound was obtained, and the eluent was: methanol: dichloromethane = 1:50. Pale yellow powder, the yield is 47%. ¹ H NMR (400MHz, cdcl ₃ ) δ12.75(d, J=3.8Hz, 1H), 8.73(d, J=2.1Hz, 1H), 8.41(d, J=2.1Hz, 1H), 8.22(dd , J=13.0, 5.8Hz, 1H), 8.10(d, J=18.5Hz, 1H), 7.89–7.83(m, 2H), 7.52(dd, J=15.8, 8.4Hz, 2H), 6.67(d, J=1.6Hz, 1H), 6.47(d, J=2.2Hz, 1H), 6.41(d, J=2.2Hz, 1H), 6.36–6.32(m, 1H), 4.57–4.48(m, 2H), 4.00 (dd, J=11.8, 6.0 Hz, 2H), 2.42 (dd, J=11.1, 5.3 Hz, 2H).

Example 7: 5-hydroxy-7-(2-(2-methyl-1H-benzo[d]imidazol-1-yl)propoxy)-2-phenyl-4H-chromen-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 2 -(3-Bromopropoxy)-5-hydroxy-2-phenyl-4H-benzopyrone-4-one (0.37g, 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, and separated the solvent with a silica gel column after evaporating to dryness under reduced pressure. The target compound was obtained, and the eluent was: methanol: dichloromethane = 1:50. Pale yellow powder, the yield is 49%. ¹ H NMR (400MHz, cdcl ₃ ) δ12.73(s, 1H), 7.94–7.81(m, 2H), 7.68(d, J=6.7Hz, 1H), 7.57–7.45(m, 3H), 7.32– 7.27(m, 1H), 7.22–7.18(m, 2H), 6.67(s, 1H), 6.43(d, J=2.2Hz, 1H), 6.36(d, J=2.2Hz, 1H), 4.37(t , J=6.6Hz, 2H), 3.96(t, J=5.5Hz, 2H), 2.58(s, 3H), 2.38–2.28(m, 2H).

Example 8: 5-Hydroxy-7-(2-(1H-benzo[d]imidazol-1-yl)butoxy)-2-phenyl-4H-chromen-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 light yellow solid 7-(4- Bromobutoxy)-5-hydroxyl-2-phenyl-4H-benzopyrone-4-one (0.39g, 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 separated it with a silica gel column to obtain the target compound , the eluent is: methanol: dichloromethane = 1:50. Pale yellow powder with a yield of 51%. ¹ H NMR (400MHz, cdcl ₃ ) δ12.71(s, 1H), 7.92(s, 1H), 7.89-7.83(m, 2H), 7.81(d, J=7.2Hz, 1H), 7.55-7.47( m, 3H), 7.41(d, J=7.1Hz, 1H), 7.34–7.25(m, 3H), 6.65(s, 1H), 6.44(d, J=2.2Hz, 1H), 6.33(d, J =2.2Hz, 1H), 4.28(t, J=7.0Hz, 2H), 4.03(t, J=6.0Hz, 2H), 2.11(dt, J=14.7, 7.4Hz, 2H), 1.85(dd, J = 15.2, 5.8Hz, 2H).

Example 9: 5-Hydroxy-7-(2-(2-methyl-1H-benzo[d]imidazol-1-yl)butoxy)-2-phenyl-4H-chromen-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 3 -(4-bromobutoxy)-5-hydroxy-2-phenyl-4H-benzopyrone-4-one (0.39g, 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, and separated the solvent with a silica gel column after evaporating to dryness under reduced pressure. The target compound was obtained, and the eluent was: methanol: dichloromethane = 1:50. Pale yellow powder, the yield is 48%. ¹ H NMR (400MHz, cdcl ₃ ) δ12.72(s, 1H), 7.89–7.84(m, 2H), 7.72–7.65(m, 1H), 7.52(t, J=7.2Hz, 3H), 7.33– 7.27(m, 1H), 7.24–7.18(m, 2H), 6.65(s, 1H), 6.44(d, J=2.2Hz, 1H), 6.33(d, J=2.1Hz, 1H), 4.21(t , J=7.1Hz, 2H), 4.03(t, J=6.0Hz, 2H), 2.63(s, 3H), 2.02(dd, J=15.0, 7.7Hz, 2H), 1.88(dd, J=14.8, 5.7Hz, 2H).

Embodiment 10: 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 20 μL of 5 mg/mL MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) solution to each well, and add the solution DMSO after acting for 4 hours , 100 μL/well, put it in the incubator, and measure the 570nm OD value with MK-2 automatic microplate reader the next day. Calculate the half inhibitory concentration IC50.

The test results are shown in Table 1, where the sample refers to the newly synthesized chrysin benzimidazole derivatives 1-21, and Examples 1-9 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 the chrysin benzimidazole derivatives have a certain degree of antitumor activity on human gastric cancer cell MGC-803, human liver cancer cell HepG2, and human breast cancer cell MCF-7. Inhibitory activity, among which compound 16 has a significant effect on human gastric cancer cell MGC-803, human liver cancer cell HepG2, and human breast cancer cell MCF-7, so compound No. 16 will be used for further in vivo anti-tumor activity tests. Because the effect of the newly synthesized compound on human gastric cancer cell line MGC-803 is better than others, mouse-derived gastric cancer cell line MFC was used to establish a normal mouse tumor model in the in vivo anti-tumor activity test. Embodiment 11: In vivo anti-tumor activity test of No. 16 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 compound 16 has a good inhibitory effect on the growth of mouse tumors in a concentration-dependent manner. The mechanism of action of the compounds 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. an antitumor drug candidate for inhibiting HIF-1α, characterized in that, the antitumor drug candidate for inhibiting HIF-1α is: R ₁ =CH ₃ , R ₂ =R ₃ =H R ₁ =OCH ₃ , R ₂ =R ₃ =H R ₁ =Cl, R ₂ =R ₃ =H R ₁ =C ₆ H ₅ , R ₂ =R ₃ =H R ₁ =CF ₃ , R ₂ =R ₃ =H R ₂ =CH ₃ , R ₁ =R ₃ =H R ₂ =OCH ₃ , R ₁ =R ₃ =H R ₃ =NO ₂ , R ₁ =R ₂ =H R ₂ =R ₃ =CH ₃ , R ₁ =H. 2. a kind of preparation method that suppresses HIF-1α antitumor candidate drug as claimed in claim 1, is characterized in that, the preparation method that described suppresses HIF-1α antitumor candidate drug comprises the following steps: Step 1: Add chrysin, anhydrous potassium carbonate and acetone in sequence in a 250mL round bottom flask, heat and stir to reflux, then add 1,2-dibromoethane or 1,3-dibromopropane or 1,4- Dibromobutane, heated and condensed at 60°C to reflux, the solution became clear and then cloudy; the reaction progress was checked and purified by column chromatography, and the eluent was methanol:dichloromethane=1:50; Step 2, add benzimidazole derivatives in a 250mL three-necked flask, dissolve it in 100mL acetone, add potassium carbonate, stir for 10min, add the purified chrysin derivatives obtained in step 1, tetrabutylammonium bromide, and heat to Stir the reaction at 60°C, extract with ethyl acetate, wash the organic layer with saturated brine, then dry with anhydrous sodium sulfate, evaporate the solvent to dryness under reduced pressure and separate it with a silica gel column to obtain the target compound. The eluent is: methanol: dichloro Methane = 1:50. 3. the preparation method of described suppressing HIF-1α antineoplastic candidate drug as claimed in claim 2, is characterized in that, in described step 1: in 250mL round bottom flask, add chrysin 2.54g successively, 0.01mol, without Potassium carbonate water 5.52g, 0.04mol and acetone 100ml; 1,2-dibromoethane or 1,3-dibromopropane or 1,4-dibromobutane 0.04mol. 4. the preparation method of described inhibition HIF-1α antineoplastic candidate drug as claimed in claim 2, is characterized in that, in described step 2: in 250mL round bottom flask, add benzimidazole derivative 0.015mol successively, without Potassium carbonate water 5.52g, 0.04mol, 0.01mol of the purified chrysin derivative obtained in Step 1, 0.03g, 0.0001mol of tetrabutylammonium bromide and 100ml of acetone.