Benzimidazole derivatives containing an aryl urea structure, methods of manufacture and uses thereof

Abstract

The application discloses a kind of benzimidazole derivatives containing aryl urea structure, manufacturing method and its role in anticancer aspect.The indazole ring of HIF-1 alpha classic inhibitor YC-1 is changed into benzimidazole ring, then according to the principle of pharmacophore splicing, aryl urea structure is introduced into the structure of benzimidazole to obtain a new benzimidazole derivative containing aryl urea structure with higher biological activity, which widens the scope of existing anticancer compounds, and can be used as a lead compound for further optimization.The application has good anticancer effect and meets the needs of the medical field.

Description

TECHNICAL FIELD

[0001] The present application relates to a class of benzimidazole compounds containing aryl urea structure, manufacturing method and use thereof, in particular to a class of benzimidazole derivatives containing aryl urea structure with anticancer effect. BACKGROUND

[0002] Hypoxia is a significant feature of tumor microenvironment, which leads to high expression of HIF-1a and its downstream target gene VEGF, and is highly related to tumor growth, metastasis, drug resistance and immune escape (You L et al. Mol Med Rep, 2021, 41(3): 1622-1643). Notably, VEGFR-2 inhibitors such as sorafenib have achieved excellent therapeutic effect in clinical treatment, but its inhibition of tumor neovascularization further aggravates tumor hypoxia, making its long-term therapeutic effect decline (Zhao Q et al. Pancreas, 2007, 34(2): 242-7.). There is no report on the design and synthesis of small molecule inhibitors targeting HIF-1a and VEGFR-2 as new anti-tumor drugs. YC-1 is a classic HIF-1a inhibitor, and its modification strategy is mainly to replace the indazole nucleus of YC-1 with other structures. Replacing indazole with benzimidazole can maintain good HIF-1a inhibitory activity and anti-tumor activity, and greatly weaken the side effect of YC-1 on inhibiting platelet aggregation (Zhang Z et al. Mol Med Rep, 2018, 18(4): 3547-3554). Studies have shown that YC-1 combined with VEGFR-2 inhibitor sorafenib has better efficacy (Chen YF et al. Bioorg Med Chem Lett, 2015, 25(18): 3873-7). The aryl urea structure is a classic pharmacophore of VEGFR-2 inhibitors, so benzimidazole derivatives containing aryl urea are expected to become better dual-target anti-tumor drugs. SUMMARY

[0003] The technical problem to be solved by the present application is to disclose a new compound with benzimidazole structure to overcome the weak anti-tumor activity of the prior art;

[0004] Another technical problem to be solved by the present application is to disclose a preparation method of the compound;

[0005] Still another technical problem to be solved by the present application is to disclose the application of the compound in the medicine for treating cancer, to meet the needs of the medical field.

[0006] The technical idea of the present application is as follows:

[0007] The inventors changed the indazole ring of the classic HIF-1α inhibitor YC-1 to a benzimidazole ring, and then according to the pharmacophore combination principle, the pharmacophore of sorafenib, aryl urea structure, was introduced into the structure of benzimidazole to obtain a series of new antitumor small molecule compounds targeting HIF-1α and VEGFR-2.

[0008] The benzimidazole derivative containing aryl urea structure of the present application is a compound having one of the following general structural formulas:

[0009]

[0010] Wherein: R1, R2, R3, R4, R 5， R6, R7, R8, R9, R 10 R1, R2, R3, R4, R 5， R6, R7, R8, R9, R 10 Represent hydrogen, oxygen, halogen, alkyl, alkoxy, trifluoromethyl, trifluoromethoxy.

[0011] R1, R2, R3, R4, R 5， R6, R7, R8, R9, R 10 Including C1-C5 halogen, alkoxy, trifluoromethyl, C6-C 10 Halogen, alkyl, alkoxy, trifluoromethyl.

[0012] The preferred compounds of the present application include but are not limited to:

[0013] 1-(3-bromophenyl)-3-(1-(4-bromobenzyl)-1H-benzo[d]imidazol-2-yl) urea (code YCB01)

[0014] 1-(3-bromophenyl)-3-(1-(4-bromobenzyl)-1H-benzo[d]imidazol-2-yl) urea (code YCB01)

[0015] 1-(3-bromophenyl)-3-(1-(4-bromobenzyl)-1H-benzo[d]imidazol-2-yl) urea (code YCB01)

[0016] 1-(1-(4-bromobenzyl)-1H-benzo[d]imidazol-2-yl)-3-(3-(trifluoromethyl) phenyl) urea (code YCB04)

[0017] 1-(1-(4-bromobenzyl)-1H-benzo[d]imidazol-2-yl)-3-(3-(trifluoromethyl) phenyl) urea (code YCB04)

[0018] 1-(1-(4-methylbenzyl)-1H-benzo[d]imidazol-2-yl)-3-(3-(trifluoromethyl)phenyl)urea (Code YCB06)

[0019] 1-(1-(3-fluorobenzyl)-1H-benzo[d]imidazol-2-yl)-3-(3-(trifluoromethyl)phenyl)urea (Code YCB07)

[0020] 1-(1-(3-bromobenzyl)-1H-benzo[d]imidazol-2-yl)-3-(3-(trifluoromethyl)phenyl)urea (Code YCB08)

[0021] The present application also includes the salts of the above-mentioned compounds, and the salts include hydrochloride, sulfate, hydrobromide or methanesulfonate.

[0022] The preparation method of the benzimidazole derivative having aryl urea structure of the present application is characterized in that the compound of general formula (1) comprises the following steps:

[0023] (1) reacting the compound having general formula a with o-phenylenediamine in the presence of a solvent and an acid binding agent to obtain the compound having general formula b.

[0024] (2) the reaction temperature is 0-50°C, and the reaction time is 0-2 hours; the solvent is one or a mixture of N,N-dimethylformamide (DMF), acetone, methanol or ethanol; and the acid binding agent is one or a mixture of triethylamine, N,N-diisopropyl ethylamine, potassium carbonate, sodium carbonate or sodium hydroxide.

[0025] (3) reacting the compound having general formula b with cyanogen bromide in the presence of a solvent to obtain the compound having general formula c.

[0026] (4) the reaction temperature is 0-40°C, and the reaction time is 0-6 hours; the solvent is one or a mixture of ethanol or methanol.

[0027] (5) reacting the compound having general formula d with triphosgene in the presence of a solvent and an acid binding agent to obtain the compound having general formula e.

[0028] (6) the reaction temperature is -10°C-30°C, and the reaction time is 0-2 hours; the solvent is one or a mixture of dichloromethane, tetrahydrofuran or acetonitrile; and the acid binding agent is one or a mixture of triethylamine, N,N-diisopropyl ethylamine, potassium carbonate, sodium carbonate or sodium hydroxide.

[0029] (7) reacting the compound having general formula c with the compound having general formula e in the presence of a solvent to obtain the compound of general formula (1).

[0030] (8) the reaction temperature is 0-60℃, the reaction time is 1-12 hours; the solvent is one or mixture of dichloromethane, tetrahydrofuran or acetonitrile

[0031]

[0032] The in-vitro anti-tumor experiment of the benzimidazole derivative with aryl urea structure of the present application is carried out:

[0033] The MTT method and cell colony formation experiment show that the compound with general formula (1) has strong anti-proliferation effect, and the IC 50 value is 4.78-82.75 μM. The VEGFR-2 kinase inhibition experiment shows that the IC 50 value of YCB01-YCB08 is 1.86-73.56 nM. The HRE double luciferase reporter gene experiment shows that the inhibition rate of YCB01-YCB08 on HIF-1α transcription activity is 2.89%-83.38%.

[0034] From the above disclosed technical solutions, it can be seen that the compound of the present application has excellent anti-cancer effect, and the preparation method is easy, which is convenient for industrialized production and can meet the needs of the medical field. The compound of the present application has the advantages of clear target and high curative effect. BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 A is the anti-proliferation effect of the compound of Example 4 (code YCB04) with different concentrations on HCT-116 cells under normal culture conditions; Figure 1 B is the anti-proliferation effect of the compound of Example 4 (code YCB04) with different concentrations on HCT-116 cells under hypoxic conditions. DETAILED DESCRIPTION

[0036] Example 1: Preparation of 1-(3-bromophenyl)-3-(1-(4-bromobenzyl)-1H-benzo[d]imidazol-2-yl)urea (code YCB01).

[0037] Take 1.26 g of o-phenylenediamine into a 100 mL round-bottom flask, dissolve with 5 mL of DMF, then add 1.5 mL of DIPEA, and place in a 50°C water bath for heating and stirring. Dissolve 1 g of 1-bromo-4-(bromomethyl)-benzene in 2 mL of DMF, and slowly drop into the reaction system. The reaction is monitored by TLC (eluent: dichloromethane). After the reaction is completed, the reaction solution is extracted with water and ethyl acetate, the ethyl acetate layer is retained, anhydrous sodium sulfate is added for dehydration, then anhydrous sodium sulfate is removed by reduced pressure filtration, and the obtained crude product is purified by silica gel column chromatography, with dichloromethane as the eluent, to obtain 0.83 g of intermediate 3a, with a yield of 86%. Then the obtained product is dissolved in 20 mL of anhydrous ethanol solution, 0.55 g of cyanogen bromide is added, and the reaction is carried out at room temperature for 6 h, with the reaction progress monitored by TLC (eluent volume ratio: methanol:dichloromethane = 1:20). After the reaction is completed, 10% sodium hydroxide solution is added to adjust the pH to 9, anhydrous ethanol is removed by distillation under reduced pressure, 30 mL of water is added, and then white solid intermediate 4a is obtained by reduced pressure filtration, with a yield of 89%.

[0038] Take 0.05 g of triphosgene and add it to a round-bottom flask, dissolve with 20 mL of dichloromethane, and stir in an ice bath environment. Dissolve 0.04 g of 3-bromoaniline in 2 mL of dichloromethane, add 0.5 mL of DIPEA, mix well, then slowly drop it into the triphosgene, and react for 0.5 h to obtain intermediate 6a, 0.032 g, with a yield of 71%. Add 0.12 g of intermediate 4a to the reaction solution of 6a, and react at room temperature for 1 h, with the reaction progress monitored by TLC (dichloromethane). After the reaction is completed, add an appropriate amount of silica gel powder, remove the solvent by evaporation under reduced pressure, purify by column chromatography with dichloromethane as the eluent. After passing through the column, remove the eluent by evaporation under reduced pressure to obtain a colorless oily liquid. Dissolve the oily liquid in 1 mL of dichloromethane, then slowly drop it into 20 mL of petroleum ether, and wait for the solid to precipitate. Then filter by reduced pressure to obtain white solid 1-(3-bromophenyl)-3-(1-(4-bromobenzyl)-1H-benzo[d]imidazol-2-yl)urea, 0.14 g, with a yield of 69%.

[0039] The relevant data are as follows: white solid, yield 69%, Mp: 168-170°C. 1H NMR (500 MHz, DMSO-d6) δ 12.22 (s, 1H), 9.31 (s, 1H), 8.16 (s, 1H), 7.54 (d, J = 8.3 Hz, 3H), 7.43 (d, J = 7.5 Hz, 1H), 7.34 (d, J = 8.3 Hz, 2H), 7.25 (d, J = 7.2 Hz, 1H), 7.18 (t, J = 8.0 Hz, 1H), 7.15 - 7.02 (m, 3H), 5.31 (s, 2H).13C NMR (126 MHz, DMSO-d6) δ 161.56, 152.89, 143.49, 136.48, 132.04, 130.80, 130.12, 129.89, 129.51, 123.80, 122.68, 122.50, 122.04, 121.18, 120.63, 117.14, 111.77, 109.54, 43.98. HRMS (ESI, m / z): [M+H]+calcd for C 21 H 17 Br2N4O, 498.9769; found, 498.9771.

[0040] Example 2: Preparation of 1-(3-bromophenyl)-3-(1-(4-methylbenzyl)-1H- benzo[d]imidazol-2-yl)urea (Code YCB02).

[0041] Using 1-bromomethyl-4-methylbenzene instead of 1-bromo 4-(bromomethyl)- benzene, 1-(3-bromophenyl)-3-(1-(4-methylbenzyl)-1H-benzo[d]imidazol-2- yl)urea was prepared according to the procedure of Example 1. 0.13 g, yield 66%.

[0042] The relevant data are as follows: Mp: 175-176 °C. 1H NMR (500 MHz, DMSO-d6) δ 12.24 (s, 1H), 9.37 (s, 1H), 8.19 (s, 1H), 7.55 (d, J = 8.2 Hz, 1H), 7.42 (d, J = 7.2 Hz, 1H), 7.27 (d, J = 7.8 Hz, 2H), 7.23 (d, J = 7.3 Hz, 1H), 7.19 (t, J = 8.0 Hz, 1H), 7.14 (d, J = 7.9 Hz, 2H), 7.12 - 7.04 (m, 3H), 5.29 (s, 2H), 2.24 (s, 3H).13C NMR (126 MHz, DMSO-d6) δ 161.66, 152.95, 143.56, 137.20, 134.01, 130.79, 129.86, 129.65, 127.87, 123.76, 122.53, 122.41, 122.04, 120.64, 117.14, 111.69, 109.66, 44.36, 21.15. HRMS (ESI, m / z): [M+H]+calcd for C 22 H 20 BrN4O, 435.0820; found, 435.0812.

[0043] Example 3: Preparation of 1-(3-bromophenyl)-3-(1-(3-fluorobenzyl)-1H- benzo[d]imidazol-2-yl)urea (Code YCB03).

[0044] Using 1-bromomethyl-3-fluorobenzene instead of 1-bromo 4-(bromomethyl)-benzene, 1-(3-bromophenyl)-3-(1-(3-fluorobenzyl)-1H-benzo[d]imidazol-2-yl)urea was prepared according to the procedure of Example 1. 0.14 g, 75% yield.

[0045] The relevant data are as follows: Mp: 165-166 °C. 1H NMR (500 MHz, DMSO-d6) δ 12.23 (s, 1H), 9.32 (s, 1H), 8.17 (s, 1H), 7.55 (d, J = 8.2 Hz, 1H), 7.48 - 7.34 (m, 2H), 7.27 (dd, J = 14.5, 8.7 Hz, 2H), 7.19 (dd, J = 14.3, 7.4 Hz, 2H), 7.16 - 7.01 (m, 4H), 5.35 (s, 2H).13C NMR (126 MHz, DMSO-d6) δ 162.66 (d, J = 243.73 Hz), 161.56, 152.92, 143.49, 139.91 (d, J = 7.3 Hz), 131.22 (d, J = 8.3 Hz), 130.80, 129.89, 129.55, 123.84 (d, J = 5.0 Hz), 122.70, 122.53, 122.04, 120.65, 117.15, 114.86 (d, J = 4.73 Hz), 114.85 (d, J = 37.88 Hz), 111.77, 109.53, 44.10. HRMS (ESI, m / z): [M+H]+calcd for C 21 H 17 BrFN4O, 439.0570; found, 439.0564.

[0046] Example 4: Preparation of 1-(1-(4-chlorobenzyl)-1H-benzo[d]imidazol-2-yl)-3-(3- (trifluoromethyl)phenyl)urea (Code YCB04).

[0047] Using 1-bromomethyl-4-chlorobenzene instead of 1-bromo-4-(bromomethyl)-benzene and 3-(trifluoromethyl)-aniline instead of 3-bromoaniline, 1-(1-(4-chlorobenzyl)-1H- benzo[d]imidazol-2-yl)-3-(3-(trifluoromethyl)phenyl)urea was prepared according to the procedure of Example 1. 0.12 g, 71% yield.

[0048] The relevant data are as follows: Mp: 161-162 °C. 1H NMR (500 MHz, DMSO-d6) δ 12.28 (s, 1H), 9.51 (s, 1H), 8.42 (s, 1H), 7.74 (d, J = 8.2 Hz, 1H), 7.51 - 7.36 (m, 6H), 7.25 (dd, J = 20.5, 7.5 Hz, 2H), 7.12 (p, J = 7.2 Hz, 2H), 5.34 (s, 2H).13C NMR (126 MHz, DMSO-d6) δ 161.70, 152.95, 142.60, 136.05, 132.67, 129.91 (d, J = 1.99 Hz), 129.81, 129.60, 129.52, 129.12, 124.92 (d, J = 271.82 Hz), 122.69, 122.52, 121.89, 117.54 (d, J = 3.52 Hz), 114.34, 111.83, 109.56, 43.97. HRMS (ESI, m / z): [M+H]+calcd for C 22 H 17 ClF3N4O, 445.1043; found, 445.1039.

[0049] Example 5: Preparation of 1-(1-(4-bromobenzyl)-1H-benzo[d]imidazol-2-yl)-3-(3- (trifluoromethyl)phenyl)urea (Code YCB05).

[0050] Using 3-(trifluoromethyl)-benzenamine instead of 3-bromoaniline, 1-(1-(4- bromobenzyl)-1H-benzo[d]imidazol-2-yl)-3-(3-(trifluoromethyl)phenyl)urea was prepared according to the procedure of Example 1. 0.121 g, 75% yield.

[0051] The relevant data are as follows: Mp: 160-161 °C. 1H NMR (500 MHz, DMSO-d6) δ 12.28 (s, 1H), 9.51 (s, 1H), 8.41 (s, 1H), 7.74 (d, J = 8.2 Hz, 1H), 7.55 (d, J = 8.2 Hz, 2H), 7.45 (dd, J = 11.2, 7.8 Hz, 2H), 7.36 (d, J = 8.2 Hz, 2H), 7.25 (dd, J = 16.8, 7.5 Hz, 2H), 7.17 - 7.05 (m, 2H), 5.33 (s, 2H).13C NMR (126 MHz, DMSO-d6) δ 161.70, 152.95, 142.59, 136.47, 132.04, 130.13, 129.91, 129.56 (d, J = 10.3 Hz), 124.92 (d, J = 272.62 Hz), 122.70, 122.52, 121.89, 121.18, 117.54 (d, J = 4.34 Hz), 114.32, 111.83, 109.56, 44.03. HRMS (ESI, m / z): [M+H]+calcd for C 22 H 17 BrF3N4O, 489.0538; found, 489.0540.

[0052] Example 6: Preparation of 1-(1-(4-methylbenzyl)-1H-benzo[d]imidazol-2-yl)-3-(3- (trifluoromethyl)phenyl)urea (Code YCB06).

[0053] Using 1-bromomethyl-4-methylbenzene instead of 1-bromo 4-(bromomethyl)-benzene and 3-(trifluoromethyl)-aniline instead of 3-bromoaniline, 1-(1-(4-methylbenzyl)-1H- benzo[d]imidazol-2-yl)-3-(3-(trifluoromethyl)phenyl)urea 0.126 g was prepared according to the procedure of Example 1 in 71% yield.

[0054] The relevant data are as follows: Mp: 180-181 °C. 1H NMR (500 MHz, DMSO-d6) δ 12.26 (s, 1H), 9.50 (s, 1H), 8.43 (s, 1H), 7.75 (d, J = 8.3 Hz, 1H), 7.45 (dd, J = 14.7, 7.3 Hz, 2H), 7.28 (d, J = 7.9 Hz, 2H), 7.23 (t, J = 5.7 Hz, 2H), 7.14 (d, J = 7.9 Hz, 2H), 7.10 (dd, J = 13.9, 6.8 Hz, 2H), 5.30 (s, 2H), 2.25 (s, 3H).13C NMR (126 MHz, DMSO-d6) δ 161.78, 152.98, 142.65, 137.21, 133.99, 129.86 (d, J = 4.46 Hz), 129.64, 127.87, 124.93 (q, J = 272.58 Hz), 122.55, 122.42, 121.88, 117.49 (d, J = 4.73 Hz), 114.34, 111.74, 109.67, 44.40, 21.13. HRMS (ESI, m / z): [M+H]+calcd for C 23 H 20 F3N4O, 425.1589; found, 425.1582.

[0055] Example 7: Preparation of 1-(1-(3-fluorobenzyl)-1H-benzo[d]imidazol-2-yl)-3-(3- (trifluoromethyl)phenyl)urea (Code YCB07).

[0056] Using 1-(bromomethyl)-3-fluorophenyl instead of 1-bromo-4-(bromomethyl)-benzene and 3-(trifluoromethyl)-aniline instead of 3-bromoaniline, 1-(1-(3-fluorobenzyl)-1H- benzo[d]imidazol-2-yl)-3-(3-(trifluoromethyl)phenyl)urea was prepared according to the procedure of Example 1. 0.13 g, 72% yield.

[0057] The relevant data are as follows: Mp: 151-152 °C. 1H NMR (500 MHz, DMSO-d6) δ 12.29 (s, 1H), 9.52 (s, 1H), 8.42 (s, 1H), 7.75 (d, J = 8.2 Hz, 1H), 7.46 (t, J = 8.1 Hz, 2H), 7.39 (dd, J = 14.4, 7.7 Hz, 1H), 7.28 (dd, J = 15.5, 8.7 Hz, 2H), 7.22 (t, J = 8.2 Hz, 2H), 7.17 - 7.06 (m, 3H), 5.36 (s, 2H).13C NMR (126 MHz, DMSO-d6) δ 162.67 (d, J = 245.41 Hz), 152.96, 142.58, 139.92, 139.86, 131.21 (d, J = 8.3 Hz), 129.90, 129.56, 124.92 (d, J = 273.51 Hz), 123.88 (d, J = 1.95 Hz), 122.72, 122.55, 121.90, 117.55 (d, J = 3.57 Hz), 115.00, 114.85 (d, J = 4.84 Hz), 114.69, 114.36, 111.83, 109.55, 49.06. HRMS (ESI, m / z): [M+H]+calcd for C 22 H 17 F4N4O, 429.1339; found, 429.1350.

[0058] Example 8: Preparation of 1-(1-(3-bromobenzyl)-1H-benzo[d]imidazol-2-yl)-3-(3- (trifluoromethyl)phenyl)urea (Code YCB08).

[0059] Using 1-bromo-3-(bromomethyl)-benzene instead of 1-bromo 4-(bromomethyl)-benzene and 3-(trifluoromethyl)-aniline instead of 3-bromoaniline, 1-(1-(3-bromobenzyl)-1H- benzo[d]imidazol-2-yl)-3-(3-(trifluoromethyl)phenyl)urea 1.17 g was prepared according to the procedure of Example 1 in 73% yield.

[0060] The relevant data are as follows: Mp: 168-169 °C. 1H NMR (500 MHz, DMSO-d6) δ 12.29 (s, 1H), 9.52 (s, 1H), 8.42 (s, 1H), 7.74 (d, J = 8.2 Hz, 1H), 7.63 (s, 1H), 7.49 (d, J = 8.1 Hz, 1H), 7.45 (d, J = 8.2 Hz, 2H), 7.37 (d, J = 7.8 Hz, 1H), 7.31 (t, J = 7.8 Hz, 2H), 7.23 (d, J = 7.6 Hz, 1H), 7.18 - 7.06 (m, 2H), 5.35 (s, 2H).13C NMR (126 MHz, DMSO-d6) δ 161.68, 152.95, 142.57, 139.83, 131.39, 130.98, 130.54, 129.88 (d, J = 6.53 Hz), 129.60, 129.53, 124.92 (d, J = 270.86 Hz), 122.76, 122.59, 122.31, 121.91, 117.57 (d, J = 3.76 Hz), 114.36, 111.86, 109.54, 43.99. HRMS (ESI, m / z): [M+H]+calcd for C 22 H 17 BrF3N4O, 489.0538; found, 489.0530.

[0061] Example 9: Anti-tumor proliferation activity of compounds in Examples 1-8 was determined by MTT colorimetric method

[0062] The specific method is as follows, HCT-116 cells, GES-1 cells, MCF-7 cells. MCF-7 cells use culture medium containing 10% fetal bovine serum, and need to add 1% double antibody additionally; HCT-116 cells use culture medium containing 15% FBS, and need to add 1% double antibody additionally; GES-1 cells use culture medium containing 10% FBS, and need to add 1% double antibody additionally. When culturing, the temperature of the incubator is set to 37°C, and the CO2 concentration is set to 5%.

[0063] When the cells are recovered, they need to be quickly transferred from -80°C to 37°C warm water. In order to accelerate the dissolution of the frozen solution, it can be gently shaken in water. After the frozen solution is completely dissolved, it is transferred to a 10-fold volume of serum-containing medium, mixed in a centrifuge tube and centrifuged for 5 min, and then the supernatant is discarded after the end, and serum-containing medium is added, transferred to a sterile culture bottle, and then placed in a carbon dioxide incubator for culture.

[0064] Newly revived cells will contain some dead cells and metabolic waste, so the medium needs to be changed after being placed in the incubator for a period of time. Discard the culture medium in the bottle, wash away the dead cells and cellular metabolic waste with PBS, and add fresh culture medium to continue culturing.

[0065] Cell passage: Once the adherent cells in the culture flask have covered the cell culture surface, the cells need to be digested and transferred out to allow the remaining cells sufficient space to continue growing. After discarding the culture medium, wash away any residual medium with PBS, add a certain amount of trypsin, and spread it evenly on the cell surface. Observe the cell morphology changes under a microscope during digestion. Once the cells detach from the cell wall, discard the trypsin and add fresh culture medium to stop the digestion effect of the residual trypsin. Then, mix the cells by pipetting with culture medium, leaving one-quarter of the cells to continue culturing, and using the remaining cells for related experiments.

[0066] The test compound and positive control drugs benzimidazole and sorafenib were dissolved in DMSO to prepare a stock solution with a concentration of 33333 μmol / L. Confluent adherent cells were digested and suspended into a cell suspension. 200 μL of PBS was added to each well around the outer edge of a 96-well plate, and 4 × 10⁴ cells were evenly seeded in the inner wells. 3 Cells were incubated in a cell culture incubator for 24 hours. Eight drug concentrations were set up in a gradient of decreasing proportions, with the highest concentration being 128 μmol / L. The stock solution was diluted with culture medium to obtain drug-containing mediums of these concentrations. Each concentration was used in triplicate, with 200 μL of drug-containing medium added to each replicate. In addition, six blank control groups were set up with equal volumes of drug-free medium after seeding cells, and six negative control groups were set up with equal volumes of medium containing 3% DMSO but no cells. After labeling the 96-well plates with added drugs, they were incubated in an incubator for 48 hours. Then, 20 μL of 5% MMT solution was added to each well (except for the outer ring of the 96-well plate), and incubation was continued for another 4 hours. Then, the 96-well plate was removed, the culture medium in the wells was aspirated, and 150 μL of DMSO was added. DMSO can dissolve the formazan crystals formed by the live cells and MTT at the bottom of the well. To ensure more complete dissolution, the plate was placed on a shaker in the dark and shaken for 10 minutes. The absorbance (OD value) at a wavelength of 490 nm was detected using a multi-functional microplate reader.

[0067] Inhibition rate = (OD value of negative control group - OD value of experimental group) / (OD value of negative control group - OD value of blank group) × 100%

[0068] The inhibition rate of drug growth at different concentrations on each cell type was calculated using the above formula, and the IC50 was calculated based on the relationship between concentration and inhibition rate. 50 Values. Primarily calculated using GraphPad Prism 8.0. All experiments were performed independently three times, and the results are shown in Table 1.

[0069] Table 1. In vitro antiproliferative activity of benzimidazole derivatives containing aryl urea structure against tumor cells

[0070]

[0071]

[0072] Example 10: Cell colony formation assay to determine the antiproliferative effect of the compound of Example 4

[0073] The detailed procedure is as follows, and the results are shown in the attached figures Figure 1 .

[0074] After the cells adhered and covered the surface of the flask, the dead cells were washed away with PBS, and trypsin was added to digest the cells into single cell suspension. The cells were then evenly distributed into 6-well plates at a density of 1000 cells per well. The plates were incubated in a CO2incubator for 24 hours. After the suspension cells adhered to the surface, fresh culture medium containing different concentrations of the compound of Example 4 (0, 2.5, 5 and 10 μmol / L) was added, and a group containing 3% DMSO was used as a control group. The culture medium was replaced every three days. After 9 days of drug treatment, the culture medium was discarded, and 4% paraformaldehyde was added to fix the cells for 15 minutes. Then 0.1% crystal violet was added to stain the cells for 15 minutes. After the crystal violet was discarded, the cells were washed with 3 mL PBS for 3 times, and the excess water was evaporated in a dry oven. Finally, the staining results were photographed.

[0075] Example 11: VEGFR-2 kinase inhibition assay

[0076] Thaw 5x Kinase assay buffer, ATP and 50x PTK substrate on ice. Add 6 μL 5x Kinase assay buffer + 1 μL ATP (500 μM) + 1 μL 50x PTK substrate + 17 μL autoclaved water per well in a non-transparent 96-well plate. Mix well. Set up 8 dilution concentrations for each drug, with the highest concentration being 500 nmol / L. Add 5 μL of each drug at different concentrations to each well in the experimental group, and add 5 μL of DMSO at the same concentration to each well in the blank control group and the positive control group. Finally, add 20 μL of diluted VEGFR-2 kinase to each well in the experimental group and the positive control group, and add 20 μL of 1x Kinase assay buffer to each well in the blank control group. Then incubate at 30°C for 45 min. After 45 min, add 50 μL of Kinase-Glo Max reagent to each well. Cover the plate with aluminum foil and incubate at room temperature for 15 min, and then measure the luminescence intensity using a microplate reader.

[0077] Example 12: HRE Dual-Luciferase Reporter Assay

[0078] Seeding: Seed the cells in the logarithmic growth phase in a 96-well plate after trypsinization, with a seeding density of 8 x 104cell / mL per well. Add 100 μL per well. Incubate in a normoxic incubator (37°C, 5% CO2) for 24 h.

[0079] Transfection: When the cells are incubated to a density of 70%-90%, dilute the LipofectamineTM 3000 reagent using Opti-MEMTM medium. Dilute pGL4.42 and pGL4.47 using Opti-MEMTM medium, and prepare a DNA premix at a ratio of 10:1, then add P3000TM reagent and mix well. Add the prepared diluted DNA to the diluted LipofectamineTM 3000 reagent in each tube, incubate for 15 min to prepare a DNA-lipid complex, and then add it to the cells for further incubation for 2 days.

[0080] Drug addition: Dilute the drug stock solution in gradient to prepare a series of drug solutions at different concentrations. Add 100 μL of the drug at the target concentration to each well, and add 100 μL of culture medium to each well in the blank control group. After drug addition, incubate in a hypoxic incubator (37°C, 5% CO2, 1% O2) for 24 h.

[0081] Test: suck off the culture medium, add 100 μL PBS to wash, and suck off the PBS. Add 20 μL PLB lysis solution to each well for lysis, and shake for 10 min at room temperature. Add 100 μL LAR II reagent to each well, and transfer to the white plate for luminescence test after blowing for 3 times. After reading, add 100 μL Stop & Glo® Reagent to each well of the white plate, blow for 3 times, and measure the luminescence value of Renilla luciferase. Data processing: normalize the measured data by F / R, calculate the relative luminescence value, and substitute into the formula to calculate the inhibition rate of the test compound on HIF-1α transcriptional activity.

[0082]

[0083] Table 2 Inhibition rate of VEGFR-2 and HIF-1α transcriptional activity of the benzimidazole derivatives containing aryl urea structure

[0084]

Claims

1. A benzimidazole derivative containing an arylurea structure, characterized in that, The derivative is a compound having the following general formula: Where R1, R2, R3, R4, R5, R6, R7, R8, R9, and R10 can be the same or different, representing hydrogen, halogen, trifluoromethyl, and trifluoromethoxy, and the compound is not:

2. A benzimidazole derivative containing an arylurea structure, characterized in that, The derivatives are selected from the following compounds: 1-(3-bromophenyl)-3-(1-(4-bromobenzyl)-1H-benzo[d]imidazol-2-yl)urea 1-(3-bromophenyl)-3-(1-(4-methylbenzyl)-1H-benzo[d]imidazol-2-yl)urea 1-(3-Bromophenyl)-3-(1-(3-fluorobenzyl)-1H-benzo[d]imidazol-2-yl)urea 1-(1-(4-chlorobenzyl)-1H-benzo[d]imidazol-2-yl)-3-(3-(trifluoromethyl)phenyl)urea 1-(1-(4-bromobenzyl)-1H-benzo[d]imidazol-2-yl)-3-(3-(trifluoromethyl)phenyl)urea 1-(1-(4-methylbenzyl)-1H-benzo[d]imidazol-2-yl)-3-(3-(trifluoromethyl)phenyl)urea 1-(1-(3-fluorobenzyl)-1H-benzo[d]imidazol-2-yl)-3-(3-(trifluoromethyl)phenyl)urea 1-(1-(3-bromobenzyl)-1H-benzo[d]imidazol-2-yl)-3-(3-(trifluoromethyl)phenyl)urea.

3. A salt of the derivative according to any one of claims 1 or 2.

4. The salt according to claim 3, characterized in that, The salt is selected from hydrochloride, sulfate, hydrobromide or methanesulfonate.

5. The method for preparing the derivative according to claim 1, characterized in that, Includes the following steps: (1) A compound having general formula a is reacted with o-phenylenediamine in the presence of a solvent and an acid-binding agent to obtain a compound having general formula b; (2) The reaction temperature is 0-50℃ and the reaction time is 0-2 hours; the solvent is one or a mixture of N,N-dimethylformamide (DMF), acetone, methanol or ethanol; the acid-binding agent is one or a mixture of triethylamine, N,N-diisopropylethylamine, potassium carbonate, sodium carbonate or sodium hydroxide. (3) A compound having general formula b reacts with cyanide bromide in the presence of a solvent to give a compound having general formula c; (4) The reaction temperature is 0-40℃ and the reaction time is 0-6 hours; the solvent is ethanol or methanol. (5) Compounds with general formula d react with triphosgene in the presence of solvent and acid-binding agent to give compounds with general formula e; (6) The reaction temperature is -10℃ to 30℃ and the reaction time is 0 to 2 hours; the solvent is one or a mixture of dichloromethane, tetrahydrofuran or acetonitrile, and the acid-binding agent is one or a mixture of triethylamine, N,N-diisopropylethylamine, potassium carbonate, sodium carbonate or sodium hydroxide. (7) A compound having general formula c reacts with a compound having general formula e in the presence of a catalyst and a solvent to give a compound having general formula (1). (8) The reaction temperature is 0℃~60℃, and the reaction time is 0~1 hour; the solvent is one or a mixture of dichloromethane, tetrahydrofuran or acetonitrile.

6. A composition containing a therapeutically effective amount of the derivative according to claim 1 or 2 and a pharmaceutically acceptable carrier.

7. The use of the derivative of claim 1 or 2 in the preparation of a medicament for treating cancer, characterized in that, The cancer cells described are HCT-116 cells, GES-1 cells, or MCF-7 cells.

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