CN114685430A

CN114685430A - Fenbendazole analogue, preparation method and application

Info

Publication number: CN114685430A
Application number: CN202210272948.7A
Authority: CN
Inventors: 余露山; 彭一; 曾苏
Original assignee: Jinhua Research Institute Of Zhejiang University; Zhejiang University ZJU
Current assignee: Jinhua Research Institute Of Zhejiang University; Zhejiang University ZJU
Priority date: 2022-03-18
Filing date: 2022-03-18
Publication date: 2022-07-01
Anticipated expiration: 2042-03-18
Also published as: CN114685430B

Abstract

The invention provides a fenbendazole analogue, a preparation method and application thereof. The method specifically comprises the following steps: 1- {4- [5- (phenylthio) -1H-1, 3-benzimidazol-2-yl ] piperidin-1-yl } prop-2-en-1-one; 1- [ (3R) -3- [5- (phenylthio) -1H-indazolyl-1-yl ] piperidin-1-yl ] prop-2-en-1-one; 1- [ (3R) -3- [5- (phenylthio) -2H-indazolyl-2-yl ] piperidin-1-yl ] prop-2-en-1-one; [5- (phenylthio) -1H-benzimidazol-2-yl ] acrylamide. The invention synthesizes the leflubendazole analogue by modifying the parent nucleus of the leflubendazole with different substituents. The fenbendazole and the analogue thereof can inhibit HeLa cell proliferation in vitro, improve the active oxygen concentration in the HeLa cell, further activate a downstream apoptosis pathway, and can be applied to the preparation of anti-cervical cancer drugs.

Description

Fenbendazole analogue, preparation method and application

Technical Field

The invention belongs to the field of biological medicines, and particularly relates to a fenbendazole analogue, and a preparation method and application thereof.

Background

Cancer is a major public health problem worldwide, and its incidence and mortality rate generally show a tendency to rise year by year. The high morbidity and mortality of malignant tumors make the research on tumor occurrence mechanisms, influencing factors and the like and the development of anticancer drugs a current hotspot.

In addition to the hope of developing new anti-cancer drugs in the course of cancer exploration and treatment, researchers are increasingly focusing on non-anti-tumor drugs. This opens a new window for cancer treatment, which makes it an excellent alternative strategy to classical anti-cancer drug discovery and development methods. Among these, antiparasitic drugs attract many interests, and in particular, benzimidazole antiparasitic administration has a strong potential in the development of antitumor drugs.

In experiments of various cancer cells and tumor animal models, the pleiotropic benzimidazole drugs have remarkable anticancer effect, and particularly, the pleiotropic benzimidazole drugs are used as effective microtubule damaging agents, anti-angiogenesis drugs and anti-metastasis drugs, immune checkpoint inhibitors, hypoxia inducible factors, inducers of apoptosis and M1 polarization and the like. These anti-cancer effects are attributed to a number of points of action, including induction of intrinsic apoptosis, regulation of the canonical proliferation-associated signaling pathway Wnt/β -catenin, JAK/STAT-3, JNK, MEK/ERK and hedgehog signaling pathways, regulation of cytokine and chemokine secretion, and the like.

Fenbendazole, a broad spectrum of benzimidazole antiparasitic agents, is widely used in animals to repel insects. Studies have shown that fenbendazole can interfere with tubulin formation, disrupting motor microtubules, and thereby causing G2/M phase arrest and apoptosis in cells. In addition, fenbendazole can also block glucose uptake and ATP formation, interfere with glycolipid metabolism of cells, and further participate in metabonomics of cells. In recent years, domestic and foreign researches show that fenbendazole has good research value in the aspect of tumor resistance, can effectively inhibit the proliferation and metastasis of lung cancer cells, particularly K-RAS mutant lung cancer cells, but the activity and action mechanism of fenbendazole in other cancer species are still needed to be researched and supplemented.

Based on the potential application value of fenbendazole in the anti-tumor direction, a novel compound similar to the structure is developed by carrying out structural modification on the fenbendazole, the stability, the targeting property and the toxic and side effects of the fenbendazole are improved, the treatment level can be effectively improved, a large number of tumor patients are benefited, meanwhile, a foundation is laid for the development of an anti-cancer drug from the structural modification source of fenbendazole, and a lead compound is provided for the research and development of a therapeutic drug with independent intellectual property rights.

Disclosure of Invention

The invention aims to provide fenbendazole analogues which are 1- {4- [5- (thiophenyl) -1H-1, 3-benzimidazol-2-yl ] piperidin-1-yl } propane-2-ethylenediamine-1-one with a structure shown in a formula 3, 1- [ (3R) -3- [5- (thiophenyl) -1H-indazolyl-1-yl ] piperidin-1-yl ] propane-2-ethylenediamine-1-one with a structure shown in a formula 4, 1- [ (3R) -3- [5- (thiophenyl) -2H-indazolyl-2-yl ] piperidin-1-yl ] propane-2-ethylenediamine-1-one with a structure shown in a formula 5, and a preparation method thereof, [5- (phenylthio) -1H-benzimidazol-2-yl ] acrylamides of the structure of formula 6.

Formula 3: 1- {4- [5- (phenylthio) -1H-1, 3-benzimidazol-2-yl ] piperidin-1-yl } propan-2-ethylenediamine-1-one

Formula 4: 1- [ (3R) -3- [5- (phenylthio) -1H-indazolyl-1-yl ] piperidin-1-yl ] propan-2-ediamine-1-one

Formula 5: 1- [ (3R) -3- [5- (phenylthio) -2H-indazolyl-2-yl ] piperidin-1-yl ] propan-2-ethylenediamine-1-one

Formula 6: [5- (phenylthio) -1H-benzimidazol-2-yl ] acrylamides

Another object of the present invention is to provide a preparation method of the fenbendazole analogue, which is realized by the following steps:

preparation of the compound of formula 3:

compound 3a in EtOH/THF (ethanol/tetrahydrofuran) mixed solution and Pd/C (palladium/carbon), H₂(hydrogen) to produce compounds 3b, 3b and 3c and 4-methyl-morpholine in DMF (N, N-dimethylformamide) solution, condensing agent EDCI.HCl (1-ethyl- (3-dimethylaminopropyl) carbodiimide hydrochloride) and HOBt (1-hydroxybenzotriazole) in the presence of reaction product extracted in ethyl acetate to obtain crude compounds 3d and 3e after silica gel chromatography purification. HOAc was added to a mixture of compounds 3d and 3e to react to give compound 3f, and DCM (dichloromethane) solution was used to dissolve 3f and TFA (trifluoroacetic acid) was added to give compound 3 g. 3g of compound are dissolved in 2-methyltetrahydrofuran and NaHCO is added₃The solution is reacted with a solution of acryloyl chloride in 2-methyltetrahydrofuran to form the compound of formula 3.

The reaction formula is as follows:

preparation of the compound of formula 4:

compound 4a was reacted with MsCl (methanesulfonyl chloride) (5.5g,27.5mmol) and TEA (triethanolamine) in THF to give compound 4b, which was isolated and purified. 4c, adding NaH (sodium cyanide) into the DMF solution for reaction, dropwise adding the compound 4b, reacting to obtain a mixture of the compounds 4d and 4e, and purifying the product by column chromatography. Mixing 4d, 4f and Pd₂(dba)₃A mixed dioxane (30mL) solution of (tris (dibenzylideneacetone) dipalladium), Xant-phos (4, 5-bis (diphenylphosphino) -9, 9-dimethylxanthene), DIEA (N, N-diisopropylethylamine) was reacted, and the mixture was filtered and washed with ethyl acetate. The resulting mixed solution was concentrated in vacuo and the filtrate was purified by silica gel chromatography to obtain 4g of a compound. Subsequently, 4g was reacted in HCl-MeOH (hydrochloric acid-methanol) solution and concentrated to give compound 4 h. Compound 4h as the hydrochloride salt with TEA in DCM and addedAcryloyl chloride was added to the reaction, and the reaction solution was concentrated in vacuo and then purified by preparative HPLC to give compound formula 4.

The reaction formula is as follows:

preparation of the compound of formula 5:

the compound 4e, Pd (dppf) Cl₂(1, 1' -bis-diphenylphosphinoferrocene palladium dichloride), Xant-phos, t-BuOK (potassium tert-butoxide) and the compound 4f are reacted catalytically in dioxane solution. The mixture was filtered and washed with ethyl acetate. The filtrate was concentrated in vacuo and purified by silica gel chromatography to give compound 5 a. Compound 5a was combined with HCl-EtOAc (hydrochloric acid-ethyl acetate) solution. The resulting reaction mixture was concentrated in vacuo to afford compound 5 b. THF/H to hydrochloride salt-5 b₂Adding K into O solution₃PO4 (potassium phosphate). The mixture was slowly added to the reaction as acryloyl chloride THF solution. The reaction mixture was concentrated in vacuo after the reaction was completed, and then purified by preparative HPLC to obtain compound formula 5.

The reaction formula is as follows:

preparation of a compound of formula 6:

heating and refluxing the compound 6a and 24% HBr (potassium bromide) solution at room temperature for reaction, adding NaOH solution, adjusting the pH value, extracting with ethyl acetate for three times, combining, washing the ethyl acetate with saturated sodium chloride for three times, collecting an organic phase, adding anhydrous magnesium sulfate, drying, performing suction filtration, collecting filtrate, performing vacuum concentration, purifying residues by column chromatography, collecting a target product, and performing vacuum spin drying to obtain a compound 6 b. Adding the compound 6c into a THF solution, adding DEPBT (3-diethoxyphosphoryl-1, 2, 3-benzoxazole 4(3H) -ketone), reacting at room temperature, adding the compound 6b, dissolving, adding DIPEA (N, N-diisopropylethylamine) and stirring at room temperature. After the reaction, saturated NaHCO was added₃QuenchingAnd (3) reacting, extracting with ethyl acetate with the same volume, combining, washing the ethyl acetate with saturated sodium chloride with the same volume for three times, collecting an organic phase, adding anhydrous magnesium sulfate, drying, performing suction filtration, collecting filtrate, performing vacuum concentration, purifying residues through column chromatography, collecting a target product, and performing vacuum spin drying to obtain a compound shown in formula 6.

The reaction formula is as follows:

the invention further aims to provide application of the fenbendazole analogue in preparing an anti-tumor medicament. Cell viability inhibition experiments prove that the fenbendazole analogues of the

formulae

3, 4,5 and 6 provided by the invention can inhibit the cell viability of various cancer cells, wherein the tumors are lung cancer (cell lines A549 and PC9), cervical cancer (cell lines HeLa, Caski and C-33A), colorectal cancer (cell lines HT-29 and HCT-15) and breast cancer (cell line MCF-7). Particularly, the activity inhibition effect of the compound shown in the formula 6 in HeLa cells of cervical cancer is superior to that of fenbendazole.

As a preferable medicine, the fenbendazole and the fenbendazole analogue (especially the compound formula 6) can limit the formation of HeLa cell colony of cervical cancer and inhibit the migration and invasion of HeLa cells.

As a preferred medicine, the fenbendazole and the fenbendazole analogue (especially the compound formula 6) can induce HeLa cell apoptosis.

As a preferential medicament, the fenbendazole and the fenbendazole analogue (especially the compound shown in the formula 6) can induce the HeLa G2/M phase block of the cervical cancer cells, cause the increase of the concentration of Reactive Oxygen Species (ROS) in the cells and induce the growth inhibition of the HeLa cells by destroying the oxygen metabolism balance.

Compared with the prior art, the invention has the following advantages:

the invention develops the anti-cancer effect of the fenbendazole analogue, has certain cell activity inhibition effect on various cancer cells, is a lung cancer cell, a cervical cancer cell, a breast cancer cell and a colorectal cancer cell, and can be applied to the development of anti-tumor drugs related to cell proliferation inhibition. Particularly, the fenbendazole analogue shown as the formula 6 shows a cell activity inhibiting effect superior to that of the parent drug fenbendazole in a cervical cancer cell HeLa. The parent drug fenbendazole and the compound shown in the formula 6 can inhibit the colony formation of HeLa cells, induce the apoptosis and cell cycle retardation, inhibit the migration and invasion capacity of the HeLa cells, and activate downstream apoptosis signal pathways by increasing the active oxygen concentration in the HeLa cells. The fenbendazole parent drug and the compound shown in the formula 6 can be used for preventing or treating cervical cancer caused by HeLa cell proliferation.

Drawings

FIG. 1 is a graph showing the results of inhibition of cell viability in different tumor cell lines by fenbendazole and fenbendazole analogs at three concentrations, high, medium and low.

FIG. 2 is the pharmacological effect IC of fenbendazole, compound formula 6 and the positive control drug paclitaxel in HeLa cells₅₀Concentration determination curve.

FIG. 3 is a graph showing the results of colony formation inhibition of HeLa cells by fenbendazole and compound formula 6 at three concentrations, high, medium and low.

FIG. 4 is a graph showing the results of flow apoptosis of HeLa cells after 48h treatment with 1 μ M concentration of fenbendazole, compound formula 6, positive control drug cisplatin (DDP), Paclitaxel (PTX).

FIG. 5 is a graph of DAPI staining of HeLa cell nuclei after 48h treatment with 1 μ M of the same concentration of fenbendazole, compound formula 6, the positive control drug cisplatin (DDP), Paclitaxel (PTX).

FIG. 6 is a graph of the results of HeLa cell migration after 20h treatment with the same concentration of 10nM fenbendazole, compound formula 6, the positive control drug cisplatin (DDP), Paclitaxel (PTX).

FIG. 7 is a graph of the results of HeLa cell invasion 24h after treatment with fenbendazole and compound formula 6 at three concentrations, high, medium and low.

FIG. 8 is a graph of the cell cycle results for HeLa cells after 48h treatment with fenbendazole, the fenbendazole compound formula 6, Paclitaxel (PTX) at a concentration of 1 μ M.

FIG. 9 is a graph of ROS-labeled fluorescence signals of fenbendazole, compound formula 6, at two concentrations after 24h of treatment.

Detailed Description

The invention is further explained by the accompanying drawings and examples. The following examples are illustrative of the invention and are not intended to limit the invention in any way.

Example 1 (preparation of compound 3)

Compound 3a (3.0g, 12.2mmol) was added to a mixed solution of EtOH (80.0mL)/THF (30mL) at 25 deg.C, followed by Pd/C (3.0 g). Reaction mixture is reacted in H₂Stirred at 25 ℃ for 3 h. The reaction mixture was then filtered and filtered with suction under vacuum to give 3b as a black oil (2.5g, 96.1%).

To a solution of 3b (2.5g, 11.6mmol) in DMF (30.0mL) at 25 deg.C were added 3c (3.2g, 13.9mmol) and 4-methyl-morpholine (3.8mL, 34.8 mmol). The reaction mixture was placed under nitrogen (N)₂) Stirring was carried out at 25 ℃ for 5min under protection, followed by the addition of EDCI. HCl (2.9g,15.1mmol) and HOBt (2.0g,15.1 mmol). The reaction mixture is stirred under N₂Stirring for 3h at 25 ℃ under protection. For mixtures H₂O (50mL) was diluted and extracted with ethyl acetate (100 mL). The organic layer was washed with brine (100mL) and concentrated. The crude product was purified by silica gel chromatography (EA: PE ═ 1:2) to give 3d and 3e (4.8g, 97.9%) as brown oils.

HOAc (30mL) was added to the mixture of 3d and 3e at 25 ℃. The reaction mixture was heated to 75 ℃ and at N₂Stirring for 2h under protection. The reaction mixture was concentrated in vacuo. The crude product was purified by silica gel chromatography (EA: PE ═ 1:1) to give 3f (3.8g, 82.6%) as a yellow foam.

To a stirred solution of 3f (3.8g, 9.3mmol) in DCM (30.0mL) at 25 deg.C was added TFA (30 mL). Reaction mixture is reacted with N₂Stirring at 25 ℃ for 1h under protection. The reaction mixture was concentrated in vacuo to give 3g of a brown oil (2.8g, ca. 100%). Subsequently, 3g (2.8g, 9.1mmol) were dissolved in 2-methyltetrahydrofuran (40.0ml) and cooled to 0 ℃. 40mL of NaHCO was added₃(3.8g,45.3mmol) and stirred at 0 deg.C for 5min, then acryloyl chloride (0.73mL,9.1mmol) was slowly added dropwise2-Methyltetrahydrofuran (10.0ml) solution. The solution was stirred at 0 ℃ for 1 h. For mixtures H₂O (100mL) was diluted and extracted with ethyl acetate (2X 100 mL). Combining several layers with Na₂ SO₄Dried and concentrated in vacuo. The crude product was purified by silica gel chromatography (DCM: MeOH ═ 100:1) to give compound formula 3 as a yellow foam (2.5g, 75.7%).

Compound formula 3 characterization data are as follows:¹H NMR(400MHz,DMSO-d₆)δ12.55(s,1H),12.45(s,1H),7.69-7.52(m,4H),7.29-7.17(m,12H),6.91-6.82(m,2H),6.11(t,J＝14.8Hz,2H),5.68(t,J＝10.4Hz,2H),4.74(d,J＝7.2Hz,1H),4.31-4.28(m,2H),4.08(d,J＝13.2Hz,1H),3.49-3.42(m,1H),3.16-2.81(m,5H),2.22-2.17(m,2H),1.95-1.77(m,4H),1.51(brs,2H).MS m/z[M+H]⁺(ESI):364.6。

example 2 (preparation of Compound 4)

To a solution of 4a compound (5.0g,18.3mmol) and MsCl (5.5g,27.5mmol) in THF (300mL) at 0 deg.C was added TEA (13mL,91.5 mmol). The reaction mixture was stirred at 70 ℃ for 16 h. The organic phase was dried and concentrated in vacuo and the residue was purified by column chromatography (eluting with PE in EA, 0-90% to yield 4d (5.1g, yield: 19%) 4e (3.2g, yield: 19%).

To a solution of 4c (23.0g,117.3mmol) in DMF (400mL) at 0 deg.C was added NaH (7.0g,176.0mmol) and the reaction solution was stirred at 25 deg.C for 1 h. 4b (35.0g, 125.4mmol) was added dropwise to the mixture at 0 ℃. The reaction solution was stirred at 25 ℃ for 16h, poured into ice water (1000mL), the organic phase was dried, concentrated in vacuo, and the residue was purified by column chromatography (0-90% eluted with PE in EA to give 4d (5.1g, yield: 19%) 4e (3.2g, yield: 19%).

4d (3.2g,8.41mmol), 4f (1.2g,9.25mmol) and Pd₂(dba)₃A solution of (769.4mg,0.84mmol), Xant-phos (454.1mg,0.84mmol), DIEA (2.2g,16mmol) in mixed dioxane (30mL) was stirred under Ar at 10 ℃ for 16 h. The mixture was filtered and washed with ethyl acetate (50mL)And (6) washing. The resulting mixed solution was concentrated in vacuo and the filtrate was purified by silica gel chromatography (PE: ethyl acetate ═ 5:1) to give 4g (2.5g, yield: 72.5%) as a yellow solid. Subsequently, 4g (2.5g,6.1mmol) of HCl-ethyl acetate (3.0M,30mL) solution was stirred at 25 ℃ for 18 h. The reaction mixture was concentrated to give 4h (2.7g, yield:>99%). To a solution of the hydrochloride salt form for 4h (2.7g, 8.1mmol), TEA (8.2g, 81.0mmol) was added at 0 deg.C to a solution of DCM (20mL/5mL) and acryloyl chloride (1.1g, 12.2mmol) was added. The mixture was stirred at 25 ℃ for 3 h. The reaction solution was concentrated in vacuo, and then purified by preparative HPLC to give compound formula 4(1.1g, yield: 50%) as a white solid.

Compound formula 4 characterization data are as follows:¹H NMR(400M Hz,DMSO-d₆)δ8.14(s,1H),7.96(s,1H),7.85-7.80(m,1H),7.45-7.43(m,1H),7.33-7.29(m,2H),7.22-7.17(m,3H),6.92-6.73(m,1H),6.17-6.05(m,1H),5.74-5.58(m,1H),4.80-4.57(m,1.5H),4.40-4.36(m,0.5H),4.23-4.10(m,1H),3.64-3.58(m,0.5H),3.21-3.10(m,1H),2.88-2.83(m,0.5H),2.21-2.12(m,2H),1.93-1.89(m,1H),1.66-1.59(m,1H).LCMS:m/z 364.1[M+H]。

example 3 (preparation of Compound 5)

4e (3.2g,8.41mmol), Pd (dppf) Cl₂(616.0mg,0.84mmol), Xant-phos (486.4mg,0.84mmol), t-BuOK (1.9g,16.82mmol) and 4f (2.2mmol,16.82mmol) in dioxane (30mL) were stirred under argon at 100 ℃ for 16 h. The mixture was filtered and washed with ethyl acetate (50 mL). The filtrate was concentrated in vacuo and purified by silica gel chromatography (PE: ethyl acetate ═ 5:1) to give 5a (2.9g, yield: 85%) as a yellow solid.

Compound 5a (2.9g,7.08mmol) was combined with a solution of HCl-EtOAc (3.0M,30 mL). Stirring was carried out at 10 ℃ for 3 h. The resulting reaction mixture was concentrated in vacuo to give 5b (2.7g, yield: > 99%).

To hydrochloride salt-5 b (2.79g, 8.07mmol) in THF/H₂Adding K into O (20mL/5mL) solution₃PO4(8.57g, 40.35 mmol). The mixture was stirred at 10 ℃ for 5 min. A solution of 5mL acryloyl chloride in THF (803.37mg, 8.88mmol) was added slowly. The reaction mixture was stirred at 10 ℃ for 1h, and after completion of the reaction, the reaction solution was concentrated in vacuo and then purified by preparative HPLC to give compound formula 5(1.003g, yield: 34%) as a white solid.

Compound formula 5 characterization data are as follows:¹H NMR(400M Hz,DMSO-d₆)δ8.50(s,1H),7.91(s,1H),7.67(d,J＝8.8Hz,1H),7.31(m,2H),7.20(m,4H),6.84(m,1H),6.13(m,1H),5.69(dd,J＝10.0Hz,J＝26.8Hz,1H),4.66+4.08(m,2H),4.33(m,1H),3.69+2.90(m,1H),3.33-3.18(m,1H),2.25(m,2H),1.86(m,1H),1.56(m,1H).LCMS:m/z 364.1[M+H].

example 4 (preparation of Compound 6)

Compound 6a (0.6g, 2mmol) was placed in a round bottom flask at room temperature and 25ml of 24% HBr solution was added and heated under reflux for 24 h. The reaction temperature was turned off, the temperature was cooled to room temperature, NaOH solution was added, the PH was adjusted to 12, extraction was carried out three times with 50ml ethyl acetate, after combining, ethyl acetate was washed three times with 50ml saturated sodium chloride, the organic phase was collected, dried over anhydrous magnesium sulfate, suction filtration was carried out, the filtrate was collected and concentrated in vacuo, the residue was purified by column chromatography (ethyl acetate: methanol 10:1), the desired product was collected and dried in vacuo to give 6b as a white solid (0.36g, yield: 75%).

0.72g (1 mmol) of the compound 6c was added to a solution of 5ml THF, 0.6g (2 mmol) of DEPBT was added, and after stirring at room temperature for 30min, 0.36g (1.5mmol) of the compound 6b was added, after dissolution, 0.26g (2 mmol) of DIPEA was added, and the mixture was stirred at room temperature for 16 hours. After the reaction 50ml of saturated NaHCO was added₃Quenching reaction, extracting with equal volume of ethyl acetate, mixing, washing with equal volume of saturated sodium chloride for three times, collecting organic phase, adding anhydrous magnesium sulfate, drying, vacuum filtering, collecting filtrate, vacuum concentrating, purifying residue by column chromatography (ethyl acetate: methanol 10:1), collecting target product, and vacuum drying to obtain white solid compoundFormula 6(0.36g, yield: 70%).

Compound formula 6 characterization data are as follows:¹H NMR(500MHz,DMSO-d₆)δ11.53(s,1H),7.64(d,J＝1.7Hz,1H),7.46(d,J＝8.2Hz,1H),7.31–7.26(m,2H),7.23(dd,J＝8.2,1.8Hz,1H),7.18–7.11(m,3H),4.26(t,J＝7.0Hz,2H),2.87(t,J＝7.0Hz,2H).LCMS:m/z 295[M+H]。

biological experiment 1:

cell proliferation and colony formation experiments:

the tumor cell growth inhibitory activity of the candidate compounds was determined using the CCK-8 method. The tumor cells were selected from lung cancer cell lines (A549, PC9), breast cancer cell lines (MCF-7), colorectal cancer cell lines (HT-29, HCT-15), and cervical cancer cell lines (HeLa, Caski, C-33A). Collecting human tumor cells in logarithmic growth phase, digesting with 0.25% pancreatin for a certain time, centrifuging at 1000rpm for 5min, re-suspending, counting with a blood cell counting plate, preparing cell suspension, and adjusting cell suspension to 1.0 × 10⁴～1×10⁵One per mL. Inoculating the cell suspension into 96-well culture plate at 100 μ L per well, placing at saturated humidity, 37 deg.C and 5% CO₂Culturing in an incubator for 24h until the cells are completely attached to the wall. The test compound was diluted to the desired concentration with complete medium, the complete medium of a 96-well plate inoculated with human tumor cells was aspirated, the drug-containing medium was added at 100 μ L per well, with a final DMSO concentration of less than 0.1%, and the mixture was placed in an incubator for 48 h. mu.L of 10% CCK-8 solution was added to each well of the 96-well plate, and the incubation was terminated after 2 hours of incubation. The absorbance of each well was measured at 450 nm. Setting zero adjusting hole (culture medium, CCK-8), control hole (cell, drug dissolving medium with same concentration, culture solution, CCK-8), and calculating cell growth inhibition activity condition with corresponding solvent as control. Each group is provided with 3-6 multiple holes, and the multiple holes are repeated for a plurality of times. Cell viability of control (A)_{Drug group}-A_{Zero setting hole})/(A_{Control well}-A_{Zero setting hole}) X100%. Plotting and fitting the data to a concentration response curve with variable slope, and calculating the IC from the curve₅₀The values, experimental results are shown in fig. 1 and fig. 2.

As can be seen from FIG. 1, the test compounds of formula 6 and phenMost of the bendazole has obvious inhibition effect on proliferation of cervical cancer cells HeLa, Caski and C-33A, wherein the tested compound shown in formula 6 has obvious effect on human cervical cancer tumor cells HeLa and is superior to fenbendazole control. Preferably, the compound of formula 6 has little inhibitory effect on the proliferation of human normal cervical epithelial cells, HcerEpic. In addition, the fenbendazole structurally similar compounds shown in the

formulas

3, 4,5 and 6 have secondary inhibition effects on proliferation of human colon cancer cells HCT15, HT29, lung cancer cells A549, PC-9 and human liver cancer cells MCF-7. By integrating various cells, compared with tested compounds shown in

formulas

3, 4 and 5, the tested compound shown in formula 6 has a more obvious effect on inhibiting the proliferation of HeLa cells, and provides a direction for preparing cancer treatment medicines, particularly for researching cervical cancer treatment medicines. Wherein, in HeLa cells, IC of fenbendazole and analog 6₅₀Respectively reaches 0.15 mu M and 0.083 mu M.

The tumor cells can be infinitely proliferated to form cell colonies, and the influence of the medicament on the cell proliferation can be known through colony formation experiments. HeLa cells grown in log phase at 2X 10³Cells/well were seeded in 6-well plates. 37 ℃ and 5% CO₂Removing culture medium after 24 hr administration at 37 deg.C and 5% CO₂After 14 days in the incubator, the cell colonies were fixed with 4% of a methanol polymer, stained with 0.1% of a crystal violet staining solution, and observed for cell colony formation. The results are shown in FIG. 3. The results of cell colony formation show that fenbendazole and the compound of formula 6 are able to limit tumor cell colony formation and have concentration dependence.

Biological experiment 2:

fenbendazole and compound formula 6 induce HeLa cell apoptosis and morphological apoptosis detection:

the apoptosis test of the flow cytometry detects the apoptosis effect of the preferred drug induced HeLa cells. HeLa cells grown in logarithmic phase at 1X 10⁵Each well was inoculated in 6-well plates at 37 ℃ with 5% CO₂After culturing for 24h in an incubator, adding medicine for culturing for 48 h. The supernatant and the digested cells were combined, centrifuged at 1000g for 5min, washed 2 times with PBS, the supernatant was discarded, and 150. mu.L of Annexin-V-FITC binding solution was added to gently resuspend the cells. Then 5. mu.L of Annexin-V-FITC is added and mixed gently, and then 10. mu.L of propidium iodide is added for stainingMix the solution gently. Incubating for 10-20min at room temperature in dark place, placing in ice bath, and detecting on a flow cytometer. The result of detecting HeLa cell apoptosis by flow cytometry is shown in figure 4. As shown in the results of fig. 4, the preferential drug fenbendazole and the compound shown in formula 6 can significantly induce HeLa cell apoptosis, the effect is superior to that of the positive control drug 1 cisplatin (DDP), and the apoptosis inducing effect of the compound shown in formula 6 on HeLa is equivalent to that of the positive control drug 2 Paclitaxel (PTX).

TABLE 1 Fenbendazole and Compound formula 6 Effect of inducing HeLa apoptosis

Group of	KB	DMSO	DDP	PTX	Fen		6
								Apoptosis Rate (%)	3.88	5.02	6.30	36.81	30.74	36.81

Morphological observation of apoptosis:

HeLa cells grown in log phase at 4X 10⁴Inoculating each well into a 6-well plate, culturing for 24h, adding medicine, culturing for 24h, removing liquid, washing with PBS for 3 times, fixing with pre-cooled 4% paraformaldehyde at room temperature for 10min, washing with PBS for 3 times, 2% TritonX-100, permeabilizing for 2min, washing with PBS for 3 times, dyeing with Hoechst 33258 for 20min (1mg/ml), diluting 1000 times, removing dyeing liquid after dyeing, washing with PBS for 3 times, and observing by an inverted fluorescence microscope. The results are shown in FIG. 5. As can be seen from fig. 5, the preferred compound fenbendazole and compound formula 6 showed bright blue fluorescence in HeLa cells after administration, indicating nuclear condensation and DNA fragmentation.

Biological experiment 3:

HeLa cell migration and cell invasion assay

The effect of the preferable compound fenbendazole and the compound formula 6 on the migration of HeLa cells is shown through a cell scratching experiment, and the cell scratching experiment can intuitively show the inhibition effect of the preferable medicine on the proliferation and migration of the HeLa cells after the preferable medicine acts on the HeLa cells. Firstly, a marker pen is used at the back of the 6-hole plate, and then the 6-hole plate is touched by a ruler, so that transverse lines are uniformly drawn and cross through the holes approximately every 0.5-1 cm. Each hole passes through at least 5 lines. HeLa cells grown in log phase at 5X 10 per well⁵The cells were seeded at a density and cultured overnight in a 5% CO2 incubator at 37 deg.C, the next day the tip was placed against the ruler and scratched as much as possible perpendicular to the transverse line on the back, the cells were washed 3 times with PBS, the scratched cells were removed, and serum-free media containing different drugs were added. Samples were taken at 37 ℃ and 20h in a 5% CO2 incubator and the scratch width was calculated using Image J software after photographing. Scratch width average is scratch void area/length. The cell mobility was (0h scratch width-post-incubation scratch width)/0 h scratch width × 100%, and the results are shown in fig. 6 and table 2.

TABLE 2 Fenbendazole and Compound formula 6 inhibit HeLa cell mobility

Compound (I)	DMSO	F-10nM	6-10nM	PTX-1nM	DDP-10nM
						Mobility (%)	45.99	32.78	24.91	24.62	31.56

As can be seen from fig. 6 and table 2, fenbendazole and compound formula 6 can reduce the mobility of HeLa cells after acting on HeLa cells, and as can be seen from the positive drug control, compound formula 6 is superior to fenbendazole in the effect of inhibiting HeLa cell migration.

The influence of fenbendazole and the compound of formula 6 on the invasion capacity of HeLa cells is verified through a cell invasion experiment. Inoculating HeLa cells growing in logarithmic phase into a 6-well plate, culturing for 24h in an incubator at 37 ℃, adding two positive control medicaments and complete culture media of two tested compounds in groups, adding complete culture media and blank culture media containing 0.1% DMSO in a solvent control group and a negative control group respectively, continuously culturing for 48h, and collecting the cells to prepare a cell suspension for later use. Materialgel (matrigel) was cultured in serum-free high-sugar medium according to 1: 3, taking 40ul of matrix glue, uniformly spreading the matrix glue on a polycarbonate membrane at the bottom of a Transwell chamber, and placing the matrix glue in an incubator at 37 ℃ for incubation for 1h to be solidified. The Transwell chamber was filled with 100. mu.L (40000 per well) of different cell suspensions in the upper chamber and 600. mu.L of complete medium in the lower chamber, and the 24-well plate was incubated for 24 hours, and then the Transwell chamber was removed, washed with PBS, fixed with 4% paraformaldehyde for 30min, stained with 0.1% crystal violet, washed 2 times with PBS, and the cells on the inner surface of the filter were carefully wiped off with a cotton swab, and photographed with an inverted microscope at 200X, as shown in FIG. 7. The result shows that compared with a blank control and two positive medicaments, the preferable medicament fenbendazole and the compound shown in the formula 6 can obviously inhibit the invasion and the metastasis of HeLa cells, are superior to the positive medicament cisplatin, have equivalent effect with the positive medicament taxol, and show that the compound has better activity of inhibiting the invasion and the metastasis of HeLa cells.

Biological experiment 4:

HeLa cell cycle experiments:

the cell cycle is used as a precise regulation process of signal interaction inside and outside cells and is strictly regulated and controlled, and the cell cycle comprises an interphase stage and a division stage (M stage), wherein the interphase stage is divided into three stages: a DNA synthesis early stage (G1 stage), a DNA synthesis late stage (S stage), and a DNA synthesis late stage (G2 stage). Once the cells jump out of the cycle, the cells are inevitably in a state of uncontrolled proliferation, causing inevitable damage to the body, as are tumor cells. The detection of the cell cycle change condition can define the action mechanism of the antitumor drug in regulating the cancer cell proliferation. HeLa cells in a logarithmic phase are uniformly paved in a 6-well plate, fenbendazole and a compound shown in formula 6 with different concentrations and a positive control drug Paclitaxel (PTX) are added after the cells are attached to the wall, the cells are collected after 24 hours of action, and 1mL of precooled 70% EtOH4 ℃ is added for fixation for 2 hours. Centrifuging at 1000g for 3-5min, precipitating, and collecting cells. The cells were washed with 1mL PBS, 500. mu.L of ethidium bromide (PI) was added and incubated for 30min at 37 ℃ in the dark, and cell cycle distribution was detected on a flow cytometer, and the results are shown in FIG. 8. The results show that the preferred drug fenbendazole and compound formula 6 are capable of inducing G2/M phase arrest in HeLa cells. Fenbendazole has more obvious effect of retarding the HeLa cell cycle and is close to a positive control drug paclitaxel.

Biological experiment 5:

ROS active oxygen detection in HeLa cells:

intracellular Reactive Oxygen Species (ROS) were detected by 2, 7-dichlorofluorescein diacetate (DCFH-DA). Firstly, HeLa is addedCell (1X 10)⁵Cells/well) were seeded overnight in 12-well plates to allow cells to adhere and treated with different doses (0, 0.1, and 0.5 μ M) of fenbendazole and compound formula 6 for 48 h. Then, cells in containing 10 u M DCFH-DA serum-free medium at 37 degrees C protected from light were incubated for 30 min. Next, excess DCFH-DA was removed and the cells were washed 3 times with PBS. Cells were immediately observed by fluorescence microscopy. The fluorescence intensity was analyzed using Image J. The results are shown in FIG. 9. The results show that after the fenbendazole and the compound shown as the formula 6 are used for treating cells, the generation of active oxygen in HeLa cells can be promoted, and the normal oxygen metabolism balance of the HeLa cells can be destroyed.

Claims

1. A fenbendazole analog, wherein said fenbendazole analog is:

1- {4- [5- (phenylthio) -1H-1, 3-benzimidazol-2-yl ] piperidin-1-yl } prop-2-en-1-one;

1- [ (3R) -3- [5- (phenylthio) -1H-indazolyl-1-yl ] piperidin-1-yl ] prop-2-en-1-one;

1- [ (3R) -3- [5- (phenylthio) -2H-indazolyl-2-yl ] piperidin-1-yl ] prop-2-en-1-one;

[5- (phenylthio) -1H-benzimidazol-2-yl ] acrylamide.

2. A process for the preparation of fenbendazole analogues according to claim 1, characterized by the following steps:

(1) preparation of the compound of formula 3:

compound 3a in EtOH/THF mixed solution and Pd/C, H₂Reacting in the presence of a solvent to form compounds 3b, compound 3c and 4-methyl-morpholine in DMF solution in the presence of EDCI, HCl and HOBt, extracting the reaction product in ethyl acetate to obtain crude compounds 3d and 3e after purification by silica gel chromatography, adding HOAc to the mixture of compounds 3d and 3e to obtain compound 3f, dissolving 3f in DCM solution and adding TFA to obtain compound 3g, dissolving 3g of NaHCO in 2-methyltetrahydrofuran, adding NaHCO, dissolving in 2-methyltetrahydrofuran, and adding₃Reacting the solution with a 2-methyltetrahydrofuran solution of acryloyl chloride to generate a compound of formula 3; the reaction formula is as follows:

(2) preparation of the compound of formula 4:

compound 4a was reacted with MsCl and TEA in THF to give a mixture of compounds 4b and 4c, which was isolated and purified. 4c, adding NaH into DMF solution for reaction, dropwise adding a compound 4b for reaction to obtain a mixture of compounds 4d and 4e, purifying the product by column chromatography, and mixing 4d, 4f and Pd₂(dba)₃A mixed dioxane solution of xanth-phos, DIEA was reacted, and the mixture was filtered and washed with ethyl acetate. The resulting mixed solution was concentrated in vacuo to the filtrate and purified by silica gel chromatography to give 4g of compound, then 4g was reacted in HCl-MeOH solution and concentrated to give 4h of compound, 4h of hydrochloride form of compound with TEA in DCM and acryloyl chloride was added to react and the reaction solution was concentrated in vacuo and then purified by preparative HPLC to give compound formula 4; the reaction formula is as follows:

(3) preparation of the compound of formula 5:

the compound 4e, Pd (dppf) Cl₂Xant-phos, t-BuOK and the like 4f in dioxane. The mixture was filtered and washed with ethyl acetate, the filtrate was concentrated in vacuo and purified by silica gel chromatography to give compound 5a, compound 5a was combined with HCl-EtOAc solution and the resulting reaction mixture was concentrated in vacuo to give compound 5b as the hydrochloride salt-5 b in THF/H₂Adding K into O solution₃PO4, this mixture was added slowly to the reaction as a solution of acryloyl chloride in THF. Concentrating the reaction mixture in vacuum after the reaction is finished, and then purifying by preparative HPLC to obtain a compound of formula 5; the reaction formula is as follows:

(4) preparation of a compound of formula 6:

heating and refluxing the compound 6a and 24% HBr solution at room temperature for reaction, adding NaOH solution, adjusting pH value, extracting with ethyl acetate for three times, mixing, washing ethyl acetate with saturated sodium chloride for three times, collecting organic phase, adding anhydrous magnesium sulfate for drying, performing suction filtration, collecting filtrate, performing vacuum concentration, purifying residue by column chromatography, collecting target product, performing vacuum spin-drying to obtain compound 6b, adding compound 6c into THF solution, adding DEPBT, reacting at room temperature, adding compound 6b, dissolving, adding DIEPA, stirring at room temperature, adding saturated NaHCO, reacting, and adding₃Quenching reaction, extracting with ethyl acetate of the same volume, combining, washing the ethyl acetate with saturated sodium chloride of the same volume for three times, collecting an organic phase, adding anhydrous magnesium sulfate for drying, performing suction filtration, collecting filtrate for vacuum concentration, purifying residues through column chromatography, collecting a target product, and performing vacuum spin drying to obtain a compound shown in formula 6; the reaction formula is as follows:

3. the use of fenbendazole analogs and fenbendazole in the preparation of antineoplastic drugs according to claim 1, wherein the neoplasm is cervical cancer, colorectal cancer, breast cancer.

4. The use of claim 3, wherein said anti-tumor is by inhibiting the proliferation of Hela cells from cervical cancer, thereby limiting Hela colony formation.

5. The use of claim 3, wherein the anti-tumor agent is an agent that induces G2/M phase arrest in Hela cells.

6. The use of claim 3, wherein said anti-tumor agent is an agent that induces apoptosis of Hela cells.

7. The use according to claim 3, wherein said anti-tumor is the inhibition of HeLa cell migration and invasion.

8. The use of claim 3, wherein the anti-tumor agent is an agent that increases the level of reactive oxygen species in Hela cells.