CN104876878B - - 2 alkyl substituted urea benzimidazoles compound of 5- aryl phenols and its application - Google Patents
- 2 alkyl substituted urea benzimidazoles compound of 5- aryl phenols and its application Download PDFInfo
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- CN104876878B CN104876878B CN201410069823.XA CN201410069823A CN104876878B CN 104876878 B CN104876878 B CN 104876878B CN 201410069823 A CN201410069823 A CN 201410069823A CN 104876878 B CN104876878 B CN 104876878B
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- benzimidazolyl
- radicals
- methylurea
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- alkyl substituted
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- 0 C*C(C)(NC1=NC2=N*(Oc3cccc(F)c3)=CCC=C2N1)O Chemical compound C*C(C)(NC1=NC2=N*(Oc3cccc(F)c3)=CCC=C2N1)O 0.000 description 1
- XUUPZAFNFBMPQR-UHFFFAOYSA-N CNC(Nc1nc(cc(cc2)Oc3cc(C=C)ccc3)c2[nH]1)=O Chemical compound CNC(Nc1nc(cc(cc2)Oc3cc(C=C)ccc3)c2[nH]1)=O XUUPZAFNFBMPQR-UHFFFAOYSA-N 0.000 description 1
- WYMYIGVUVYNDNS-UHFFFAOYSA-N COC(Nc1nc2cc(Oc3ccccc3)ccc2[nH]1)=O Chemical compound COC(Nc1nc2cc(Oc3ccccc3)ccc2[nH]1)=O WYMYIGVUVYNDNS-UHFFFAOYSA-N 0.000 description 1
Classifications
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D235/00—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings
- C07D235/02—Heterocyclic compounds containing 1,3-diazole or hydrogenated 1,3-diazole rings, condensed with other rings condensed with carbocyclic rings or ring systems
- C07D235/04—Benzimidazoles; Hydrogenated benzimidazoles
- C07D235/24—Benzimidazoles; Hydrogenated benzimidazoles with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached in position 2
- C07D235/30—Nitrogen atoms not forming part of a nitro radical
Abstract
The invention discloses a kind of 5 aryl phenol, 2 alkyl substituted urea benzimidazoles compound and its application, the compound to have formula（Ⅰ）Structure.5 aryl phenol, 2 alkyl substituted urea benzimidazoles compound of the present invention can significantly inhibit the propagation of kinds of tumor cells in vitro, and the retardance for forming the micro-pipe of tumour cell with phase cell cycle G2/M shows dose dependent；New medicament selection is provided for clinical treatment tumour at the same time.
Description
Technical field
The present invention relates to chemical medicine, more particularly to a kind of -2 alkyl substituted urea benzimidazole of 5- aryl phenols
Compound and its application.
Background technology
Tumour, especially malignant tumour, are the major diseases of serious threat human health and life security.According to statistics, dislike
Property tumour that global 8,200,000 people were caused in 2012 is dead；And it was predicted that in following 20 years, the annual cases of cancer in the whole world
22,000,000 will be risen to by 14,000,000 in 2012（Globocan2012,IARC）.In China, tumour is equally most important cause
One of dead cause of disease.Thus, it is found that and it is the important problem of Present Global to find new effective antitumour medicine.
Microtubule inhibitors are important antitumor drugs, and which includes microtubule stabilizer and microtubule destabilizer.Micro-pipe
Inhibitor medicaments are mainly by plant origin, such as vincaleukoblastinum analog and paclitaxel analogs.These medicines all have complicated
Chemical constitution, poor solubility, and resistance problems.New small molecule microtubule inhibitors are developed, are expected to solve existing medicine
Deficiency.
ErnestLacey et al. has found（BiochemicalPharmacology,34,7,1073-1077;
BiochemicalPharmacology,34,19,3603-3605）Benzimidazoles compound, which has, preferably suppresses tubulin
Activity and suppress associated tumor cells growth.
The content of the invention
Based on this, it is necessary to provide a kind of antitumor activity and good -2 alkyl substituted urea benzo miaow of 5- aryl phenols of solubility
Azole compounds.
A kind of -2 alkyl substituted urea benzimidazoles compound of 5- aryl phenols, has formula（Ⅰ）Structure：
Wherein, Ar is naphthyl or phenyl, and the phenyl is can appoint by 0,1,2 or 3 optionally from fluorine, chlorine, bromine, iodine, methyl,
Ethyl, isopropyl, hydroxyl, methoxyl group, ethyoxyl, trifluoromethyl, cyano group, alkynyl, phenyl ring, benzyloxy substituents substitution；
R is methyl or ethyl.
In wherein some embodiments, when the Ar is phenyl, the 2nd, 6 of substituent not substituted-phenyl
In wherein some embodiments, the Ar is phenyl, and the phenyl is appoints by 0,1,2 or 3 optionally from fluorine, chlorine,
Bromine, iodine, methyl, ethyl, isopropyl, hydroxyl, methoxyl group, ethyoxyl, trifluoromethyl, cyano group, alkynyl, phenyl ring, benzyloxy substitution
Base substitutes;
The R is methyl.
In wherein some embodiments, the phenyl is substituted to appoint selected from chlorine, isopropyl, hydroxyl substituent.
In wherein some embodiments, the compound is selected from：
1-（5- phenoxy group -1H- benzimidazolyl-2 radicals）- 3- methylurea；
1-（5- phenoxy group -1H- benzimidazolyl-2 radicals）- 3- ethyl carbamides；
1-（5-（2- chlorophenoxies）- 1H- benzimidazolyl-2 radicals）- 3- methylurea；
1-（5-（3- chlorophenoxies）- 1H- benzimidazolyl-2 radicals）- 3- methylurea；
1-（5-（4- chlorophenoxies）- 1H- benzimidazolyl-2 radicals）- 3- methylurea；
1-（5-（2- methoxyphenoxies）- 1H- benzimidazolyl-2 radicals）- 3- methylurea；
1-（5-（3- methoxyphenoxies）- 1H- benzimidazolyl-2 radicals）- 3- methylurea；
1-（5-（4- methoxyphenoxies）- 1H- benzimidazolyl-2 radicals）- 3- methylurea；
1-（5-（3,4- dimethoxy phenoxy groups）- 1H- benzimidazolyl-2 radicals）- 3- methylurea；
1-（5-（3,5- dimethoxy phenoxy groups）- 1H- benzimidazolyl-2 radicals）- 3- methylurea；
1-（5-（3,4,5- trimethoxy phenoxy groups）- 1H- benzimidazolyl-2 radicals）- 3- methylurea；
1-（5-（3- fluorophenoxies）- 1H- benzimidazolyl-2 radicals）- 3- methylurea；
1-（5-（5- fluorophenoxies）- 1H- benzimidazolyl-2 radicals）- 3- methylurea；
1-（5-（3- bromobenzene epoxides）- 1H- benzimidazolyl-2 radicals）- 3- methylurea；
1-（5-（3- methylphenoxies）- 1H- benzimidazolyl-2 radicals）- 3- methylurea；
1-（5-（3- 4-trifluoromethylphenopendants）- 1H- benzimidazolyl-2 radicals）- 3- methylurea；
1-（5-（3- ethyl phenoxy groups）- 1H- benzimidazolyl-2 radicals）- 3- methylurea；
1-（5-（3- cumene epoxides）- 1H- benzimidazolyl-2 radicals）- 3- methylurea；
1-（5-（3- cyano-benzene oxygens）- 1H- benzimidazolyl-2 radicals）- 3- methylurea；
1-（5-（3- phenyl biphenylyloxies）- 1H- benzimidazolyl-2 radicals）- 3- methylurea；
1-（5-（4- phenyl biphenylyloxies）- 1H- benzimidazolyl-2 radicals）- 3- methylurea；
1-（5-（3- acetylenylbenzene epoxides）- 1H- benzimidazolyl-2 radicals）- 3- methylurea；
1-（5-（Naphthalene 1- epoxides）- 1H- benzimidazolyl-2 radicals）- 3- methylurea；
1-（5-（4- hydroxyphenoxies）- 1H- benzimidazolyl-2 radicals）- 3- methylurea；
1-（5-（4- ethoxy phenoxies）- 1H- benzimidazolyl-2 radicals）- 3- methylurea；
1-（5-（4- benzyloxy phenoxyls）- 1H- benzimidazolyl-2 radicals）- 3- methylurea.
Another object of the present invention is to provide a kind of answering for -2 alkyl substituted urea benzimidazoles compound of 5- aryl phenols
With.
A kind of -2 alkyl substituted urea benzimidazoles compound of 5- aryl phenols or its pharmaceutically acceptable salt are being prepared in advance
Application in anti-and tumor.
In wherein some embodiments, the tumour include liver cancer, leukaemia, lung cancer, cervical carcinoma, colon cancer, stomach cancer,
The cancer of the esophagus, oophoroma, breast cancer, melanoma, sarcoma or cancer of pancreas.
Another object of the present invention is to provide the composition of a kind of Prevention and tumor.
The composition of a kind of Prevention and tumor, its active ingredient are the 5- virtues described in claim 1-4
- 2 alkyl substituted urea benzimidazoles compound of base phenol or its pharmaceutically acceptable salt.
Another object of the present invention is to provide a kind of answering for -2 alkyl substituted urea benzimidazoles compound of 5- aryl phenols
With.
A kind of -2 alkyl substituted urea benzimidazoles compound of 5- aryl phenols or its pharmaceutically acceptable salt are preparing suppression
The inhibitor of tubulin polymerization processed, mitotic inhibitor, chemotherapy, radiotherapeutic sensitizer, inducing cell apoptosis derivant, retardance
Application in the retarding agent of cell cycle.
The present invention has the advantages that compared with the prior art：
（1）- 2 alkyl substituted urea benzimidazoles compound of 5- aryl phenols of the present invention can significantly inhibit in vitro
The propagation of kinds of tumor cells, the retardance for forming the micro-pipe of tumour cell with phase cell cycle G2/M show dose-dependant
Property.
（2）- 2 alkyl substituted urea benzimidazoles compound of 5- aryl phenols of the present invention, carries for clinical treatment tumour
New medicament selection has been supplied, new selection is further provided for more effective, safer medicine, has helped to solve day
The serious drug resistance etc. that becomes clinical treatment problem.
（3）- 2 alkyl substituted urea benzimidazoles compound of 5- aryl phenols of the present invention, can be as follows
Medicine.
（a）, be used to prepare pharmaceutical composition, described pharmaceutical composition is used to suppress growth of tumour cell or treatment tumour；
（b）, be used to prepare suppress tubulin polymerization inhibitor；
（c）, be used to prepare mitotic inhibitor；
（d）, be used to prepare chemotherapy, radiotherapeutic sensitizer；
（e）, be used to prepare inducing cell apoptosis derivant；
（f）, be used to prepare the retarding agent of arresting cell cycle.
（4）- 2 alkyl substituted urea benzimidazoles compound of 5- aryl phenols of the present invention, raw material sources are wide, cost
It is low.
Brief description of the drawings
Fig. 1 is for compound described in the embodiment 19 of various concentrations in dosing 8 as a child afterwards to tumour cell NCI460 micro-pipes
The experimental result schematic diagram that structure influences；
Fig. 2 is for compound described in the embodiment 19 of various concentrations in dosing 24 as a child afterwards to tumour cell NCI460 cells
The experimental result schematic diagram of cycle influences；
Fig. 3 is for compound described in the embodiment 1 of various concentrations in dosing 8 as a child afterwards to HepG2 cellular microtubules structure shadows
Loud experimental result schematic diagram；
Fig. 4 is for compound described in the embodiment 1 of various concentrations in dosing 24 as a child afterwards to HepG2 Cell cycle influences
Experimental result schematic diagram.
Embodiment
The present invention provides a kind of preparation method of -2 alkyl substituted urea benzimidazoles compound of 5- aryl phenols,
Include the following steps, such as（Ⅱ）It is shown：
（1）React using aryl phenol as raw material and the fluoro- 2- nitroanilines of 5- under the action of Anhydrous potassium carbonate generate 5- aryl-
2- nitrobenzene amine compounds；
（2）Step（1）Products therefrom 5- aryl -2- nitrobenzene amine compounds conventional 10% palladium carbon of nitro reducing condition,
Hydrogen（Or iron powder, zinc powder reduction）Reduction, bis- amido benzene class compound of generation 5- aryl -1,2-；
（3）To step（2）Bis- acid methyl -2- of 1.3- are added in gained reduzate 5- aryl -2- nitrobenzene amine compounds
Thio isourea, ring closure reaction generation 5- aryl benzimidazoles compounds；
（4）Step（3）Products therefrom 5- aryl benzimidazoles compound reacts under microwave condition with alkylamine again, raw
Into -2 alkyl substituted urea benzimidazoles compound of 5- aryl phenols.
Following embodiments further instruction will specifically be passed through
Embodiment 1
1-（5- phenoxy group -1H- benzimidazolyl-2 radicals）The synthesis of -3- methylurea
The synthesis of step 12- nitro -5- phenoxybenzamines
The phenol of 0.9mL (10mmol) is dissolved in 10mLN, in dinethylformamide, it is anhydrous to add 1.4g (10mmol)
Potassium carbonate, 100 DEG C of stirring 1h.Then add the fluoro- 2- nitroanilines of 0.8g (5mmol) 5-, 100 DEG C reaction about 6h, TLC detection with
Track reacts, it is found that the reaction was complete for raw material.It is poured into after being cooled to room temperature in frozen water, separates out precipitation, filtered with Buchner funnel
To 1.1g crude product 2- nitro -5- phenoxybenzamines, yield 98.5%, crude product is directly used in next without further purification
Step reaction.
The synthesis of step 24- phenoxy group -1,2- phenylenediamines
1.1g (4.9mmol) 2- nitro -5- phenoxybenzamines that previous step synthesizes are dissolved in 10mL ethanol, are added
0.03g (0.24mmol) palladium carbon, argon gas are replaced 3 times, and hydrogen is replaced 3 times, normal-temperature reaction about 3h, are spin-dried for ethanol, add 20mL water,
Repeatedly extracted with ethyl acetate, merge organic phase, it is dry, solvent is spin-dried for, column chromatography obtains 0.74g product 4- phenoxy groups -1,2-
Phenylenediamine, yield 75.4%.
The synthesis of step 3 methyl -5- benzyloxy -1H- benzimidazole-2-carbamates
0.74g (3.7mmol) 4- phenoxy group -1,2- phenylenediamines that previous step synthesizes are dissolved in 10mL ethanol, are added
The bis- thio isourea of carboxymethyl -2- methyl -2- of 1.2g (5.6mmol) 1,3-, is heated to 95 DEG C of back flow reactions and stays overnight, and reaction produces heavy
Form sediment.It is cooled to room temperature, is filtered to obtain 0.68g product methyl -5- benzyloxies -1H- benzimidazolyl-2 radicals-amino first with Buchner funnel
Acid esters, yield 65.0%.1HNMR（400MHz, DMSO-d6）δ7.40(d,J=8.8Hz,1H),7.36-7.31(m,2H),
7.08-7.04(m,2H),6.94-6.92(d,J=8.0Hz,2H),6.81-6.79(dd,J=8.8,2.0Hz,1H),3.74(s,
3H)。
Step 41-（5- phenoxy group -1H- benzimidazolyl-2 radicals）The synthesis of -3- methylurea
Methyl -5- benzyloxy -1H- the benzimidazole-2-carbamates for weighing 0.6g (2.1mmol) add 5mL second
In nitrile, 33% methylamine alcohol solutions of 0.6mL (4.2mmol), 140 DEG C of reaction 2h of microwave, are poured into frozen water, separate out precipitation, decompression filters
Obtain 0.37g products, yield 62.5%.1HNMR（400MHz, DMSO-d6）δ11.51(s,1H),9.96(s,1H),7.36-
7.31(m,2H),7.16(s,1H),7.06-7.02(m,2H),6.91(d,J=8.0Hz,2H),6.75(dd,J=8.4,1.6Hz,
1H),2.74(d,J=4.4Hz,3H).HRMS (ESI), [M+H]+：283.1192.
Embodiment 2
1-（5- phenoxy group -1H- benzimidazolyl-2 radicals）The synthesis of -3- ethyl carbamides
Methyl -5- benzyloxy -1H- the benzimidazole-2-carbamates for weighing 0.6g (2.1mmol) add 5mL second
In nitrile, 70% ethylamine solutions of 0.54mL (4.2mmol), 140 DEG C of reaction 2h of microwave, are poured into frozen water, separate out precipitation, decompression is taken out
Filter obtains 0.32g products 1-（5- phenoxy group -1H- benzimidazolyl-2 radicals）- 3- ethyl carbamides, yield 51.0%.1HNMR（400MHz,
DMSO-d6）δ11.53(s,1H),9.85(s,1H),7.36-7.31(m,3H),7.20(s,1H),7.06-7.03(m,2H),
6.91(d,J=8.4Hz,2H),6.75(d,J=8.4Hz,1H),3.22-3.18(m,2H),1.10(t,J=7.2Hz,3H)。HRMS
(ESI), [M+H]+：297.1347.
Embodiment 3
1-（5-（2- chlorophenoxies）- 1H- benzimidazolyl-2 radicals）The synthesis of -3- methylurea
Using 2- chlorophenols as starting material, the same 1- of step（5- phenoxy group -1H- benzimidazolyl-2 radicals）The synthesis of -3- methylurea,
Total recovery is 8.5%.The method of wherein nitro reduction uses iron powder reducing, and concrete operations are as follows：By 1.4g（24.6mmol）Iron powder
Add in 2mL water, instill concentrated hydrochloric acid 0.5mL, 100 DEG C of reflux 1h.Add the 2- nitros -5- being dissolved in 6mL ethanol（2- chlorobenzene oxygen
Base）Aniline 1.3g (4.9mmol), reaction is overnight.Filter while hot, for filtrate with saturated solution of sodium bicarbonate tune pH to neutrality, there have to be heavy
Precipitation goes out, and decompression filters, and filtrate is spin-dried for solvent, and column chromatography obtains 1.0g products 1-（5-（2- chlorophenoxies）- 1H- benzimidazoles-
2）- 3- methylurea, yield 87.2%.1HNMR（400MHz, DMSO-d6）δ11.52(s,1H),9.97(s,1H),7.56(dd,
J=8.0,2.0Hz,1H),7.36(d,J=8.0Hz,1H),7.30-7.26(m,1H),7.13-7.10(m,2H),6.97(s,
1H),6.90(d,J=8.0Hz,2H),3.72(dd,J=8.4,2.0Hz,1H),2.74(d,J=4.4Hz,3H).HRMS (ESI),
[M+H]+：317.0802.
Embodiment 4
1-（5-（3- chlorophenoxies）- 1H- benzimidazolyl-2 radicals）The synthesis of -3- methylurea
Using 3- chlorophenols as starting material, the same 1- of step（5- phenoxy group -1H- benzimidazolyl-2 radicals）The synthesis of -3- methylurea,
Total recovery is 30.0%.The method of wherein nitro reduction uses iron powder reducing, and concrete operations are as follows：By 1.2g（20.8mmol）Iron
Powder is added in 2mL water, instills concentrated hydrochloric acid 0.4mL, 100 DEG C of reflux 1h.Add the 2- nitros -5- being dissolved in 6mL ethanol（3- chlorobenzenes
Epoxide）Aniline 1.1g（4.1mmol）, reaction is overnight.Filter while hot, filtrate has with saturated solution of sodium bicarbonate tune pH to neutrality
Precipitation, decompression filter, and filtrate is spin-dried for solvent, and column chromatography obtains 0.65g products 1-（5-（3- chlorophenoxies）- 1H- benzo miaows
Azoles -2）- 3- methylurea, yield 68.1%.1HNMR（400MHz, DMSO-d6）δ7.38-7.32(m,2H),7.25(s,1H),
7.10-7.06(m,2H),6.91-6.87(m,2H),6.77(dd,J=8.4,2.0Hz,1H),2.74(d,J=4.4Hz,3H)。
HRMS (ESI), [M+H]+：317.0803.
Embodiment 5
1-（5-（4- chlorophenoxies）- 1H- benzimidazolyl-2 radicals）The synthesis of -3- methylurea
Using 4- chlorophenols as starting material, the same 1- of step（5- phenoxy group -1H- benzimidazolyl-2 radicals）The synthesis of -3- methylurea,
Total recovery is 29.5%.The method of wherein nitro reduction uses iron powder reducing, and concrete operations are as follows：By 1.2g（21.2mmol）Iron
Powder is added in 2mL water, instills concentrated hydrochloric acid 0.5mL, 100 DEG C of reflux 1h.Add the 2- nitros -5- being dissolved in 6mL ethanol（4- chlorobenzenes
Epoxide）Aniline 1.1g（4.1mmol）, reaction is overnight.Filter while hot, filtrate has with saturated solution of sodium bicarbonate tune pH to neutrality
Precipitation, decompression filter, and filtrate is spin-dried for solvent, and column chromatography obtains 0.6g products 1-（5-（4- chlorophenoxies）- 1H- benzimidazoles-
2）- 3- methylurea, yield 62.7%.1HNMR（400MHz, DMSO-d6）δ11.53(s,1H),9.97(s,1H),7.36(m,
3H),7.13(s,1H),7.03(s,1H),6.93(d,J=8.8Hz,2H),6.76(dd,J=8.4,2.0Hz,1H),2.74(d,J
=4.4Hz,3H).HRMS (ESI), [M+H]+：317.0801.
Embodiment 6
（1-（5-（2- methoxyphenoxies）- 1H- benzimidazolyl-2 radicals）The synthesis of -3- methylurea
Using 2- metoxyphenols as starting material, the same 1- of step（5- phenoxy group -1H- benzimidazolyl-2 radicals）- 3- methylurea
Synthesis, total recovery 14.4%.1HNMR（400MHz, DMSO-d6）δ11.39(s,1H),9.90(s,1H),7.28(d,J=
8.4Hz,1H),7.20-7.09(m,3H),6.93-6.87(m,3H),6.62(dd,J=8.4,2.4Hz,1H),3.76(s,3H),
2.73(d,J=3.2Hz,3H).HRMS (ESI), [M+H]+：313.1296.
Embodiment 7
1-（5-（3- methoxyphenoxies）- 1H- benzimidazolyl-2 radicals）The synthesis of -3- methylurea
Using 3- metoxyphenols as starting material, the same 1- of step（5- phenoxy group -1H- benzimidazolyl-2 radicals）- 3- methylurea
Synthesis, total recovery 17.9%.1HNMR（400MHz, DMSO-d6）δ11.51(s,1H),9.98(s,1H),7.35(d,J=
8.4Hz,1H),7.21(t,J=4.0Hz,1H),7.15(s,1H),7.03(s,1H),6.76(dd,J=8.8,2.0Hz,1H),
6.28(dd,J=8.4,2.0Hz,1H),6.49-6.44(m,2H),3.71(s,3H),2.74(d,J=4.8Hz,3H)。HRMS
(ESI), [M+H]+：313.1299.
Embodiment 8
1-（5-（4- methoxyphenoxies）- 1H- benzimidazolyl-2 radicals）The synthesis of -3- methylurea
Using 4- metoxyphenols as starting material, the same 1- of step（5- phenoxy group -1H- benzimidazolyl-2 radicals）- 3- methylurea
Synthesis, total recovery 20.5%.1HNMR（400MHz, DMSO-d6）δ11.43(s,1H),9.91(s,1H),7.30(d,J=
8.8Hz,1H),7.17(s,1H),6.91(s,5H),6.70(d,J=3.6Hz,1H),3.72(s,3H),2.74(d,J=4.4Hz,
3H).HRMS (ESI), [M+H]+：313.1297.
Embodiment 9
1-（5-（3,4- dimethoxy phenoxy groups）- 1H- benzimidazolyl-2 radicals）The synthesis of -3- methylurea
With 3,4- syringol for starting material, the same 1- of step（5- phenoxy group -1H- benzimidazolyl-2 radicals）- 3- methyl
The synthesis of urea, total recovery 21.7%.1HNMR（400MHz, DMSO-d6）δ11.42(s,1H),9.91(s,1H),7.31(d,J=
8.4Hz,1H),7.18(s,1H),6.95(s,1H),6.89(d,J=8.4Hz,2H),6.73-6.68(m,2H),6.40(dd,J=
8.8,2.8Hz,1H),3.70(d,J=4.0Hz,6H),2.73(d,J=4.4Hz,3H).HRMS (ESI), [M+H]+：
343.1403。
Embodiment 10
1-（5-（3,5- dimethoxy phenoxy groups）- 1H- benzimidazolyl-2 radicals）The synthesis of -3- methylurea
With 3,5- syringol for starting material, the same 1- of step（5- phenoxy group -1H- benzimidazolyl-2 radicals）- 3- methyl
The synthesis of urea, total recovery 15.8%.1HNMR（400MHz, DMSO-d6）δ11.52(s,1H),9.96(s,1H),7.34(d,J=
8.0Hz,1H),7.15(s,1H),7.04(s,1H),6.75(d,J=8.0Hz,1H),6.20(s,1H),6.05(s,1H),3.68
(s,6H),2.74(d,J=3.2Hz,3H).HRMS (ESI), [M+H]+：343.1402.
Embodiment 11
1-（5-（3,4,5- trimethoxy phenoxy groups）- 1H- benzimidazolyl-2 radicals）The synthesis of -3- methylurea
With 3,4,5- trimethoxy phenol for starting material, the same 1- of step（5- phenoxy group -1H- benzimidazolyl-2 radicals）- 3- first
The synthesis of base urea, total recovery 31.5%.1HNMR（400MHz, DMSO-d6）δ11.47(s,1H),9.94(s,1H),7.33(d,
J=7.6Hz,1H),7.16(s,1H),7.00(s,1H),6.75(d,J=8.4Hz,1H),6.27(s,2H),3.67(s,6H),
3.61(s,3H),2.73(d,J=4.0Hz,3H).HRMS (ESI), [M+H]+：373.1509.
Embodiment 12
1-（5-（3- fluorophenoxies）- 1H- benzimidazolyl-2 radicals）The synthesis of -3- methylurea
Using 3- fluorophenols as starting material, the same 1- of step（5- phenoxy group -1H- benzimidazolyl-2 radicals）The synthesis of -3- methylurea,
Total recovery is 17.1%.The method of wherein nitro reduction uses zinc powder reduction, and concrete operations are as follows：By 0.8g（3.2mmol）2- nitre
Base -5-（3- fluorophenoxies）Aniline is dissolved in 10mL ethanol, adds 1.2g（16.0mmol）Zinc powder, argon gas protection, is slowly dropped into
Saturated ammonium chloride solution 8mL, normal-temperature reaction 6h,
Decompression filters, and filtrate is spin-dried for solvent, and column chromatography obtains product 1-（5-（3- fluorophenoxies）- 1H- benzimidazolyl-2 radicals）-3-
Methylurea, yield 93.2%.1HNMR（400MHz, DMSO-d6）δ11.57(s,1H),9.98(s,1H),7.38-7.31(m,
2H),7.12(s,1H),7.07(s,1H),6.87(t,J=8.0Hz,1H),6.78(d,J=8.4Hz,1H),6.73(d,J=
8.0Hz,2H),2.74(d,J=3.2Hz,3H)。MS(ESI):m/z300.1[M+H]+。
Embodiment 13
1-（5-（5- fluorophenoxies）- 1H- benzimidazolyl-2 radicals）The synthesis of -3- methylurea
Using 5- fluorophenols as starting material, the same 1- of step（5- phenoxy group -1H- benzimidazolyl-2 radicals）The synthesis of -3- methylurea,
Total recovery is 20.5%.The method of wherein nitro reduction uses zinc powder reduction, and concrete operations are as follows：By 1.0g（4.0mmol）2- nitre
Base -5-（4- fluorophenoxies）Aniline is dissolved in 10mL ethanol, adds 1.3g（20.0mmol）Zinc powder, argon gas protection, is slowly dropped into
Saturated ammonium chloride solution 7mL, normal-temperature reaction 4h, decompression filter, and filtrate is spin-dried for solvent, and column chromatography obtains product 1-（5-（5- fluorobenzene oxygen
Base）- 1H- benzimidazolyl-2 radicals）- 3- methylurea, yield 95.2%.1HNMR（400MHz, DMSO-d6）δ11.48(s,1H),
9.95(s,1H),7.34(d,J=8.4Hz,1H),7.16(m,3H),6.97(m,3H),6.74(d,J=8.4Hz,1H),2.74
(d,J=2.8Hz,3H)。MS(ESI):m/z300.1[M+H]+。
Embodiment 14
1-（5-（3- bromobenzene epoxides）- 1H- benzimidazolyl-2 radicals）The synthesis of -3- methylurea
Using 3- bromophenols as starting material, the same 1- of step（5- phenoxy group -1H- benzimidazolyl-2 radicals）The synthesis of -3- methylurea,
Total recovery is 38.7%.The method of wherein nitro reduction uses zinc powder reduction, and concrete operations are as follows：By 1.3g（4mmol）2- nitre
Base -5-（3- bromobenzene epoxides）Aniline is dissolved in 10mL methanol, adds 1.3g（20mmol）Zinc powder, argon gas protection, is slowly dropped into full
With ammonium chloride solution 8mL, normal-temperature reaction 2h, decompression filters, and filtrate is spin-dried for solvent, and column chromatography obtains product 1-（5-（3- bromobenzene oxygen
Base）- 1H- benzimidazolyl-2 radicals）- 3- methylurea, yield 71.7%.1HNMR（400MHz, DMSO-d6）δ7.65(s,1H),7.35
(d,J=8.4Hz,1H),7.28(t,J=8.0Hz,1H),7.21(d,J=8.0Hz,1H),7.04(s,1H),6.93(dd,J=
6.4,1.6Hz,1H),6.73(dd,J=6.4,2.0Hz,1H),2.75(d,J=4.4Hz,3H)。MS(ESI):m/z360.0[M+
H]+。
Embodiment 15
1-（5-（3- methylphenoxies）- 1H- benzimidazolyl-2 radicals）The synthesis of -3- methylurea
Using 3- methylphenols as starting material, the same 1- of step（5- phenoxy group -1H- benzimidazolyl-2 radicals）The conjunction of -3- methylurea
Into total recovery 24.4%.1HNMR（400MHz, DMSO-d6）δ11.50(s,1H),9.94(s,1H),7.34(d,J=8.4Hz,
1H),7.22-7.16(m,2H),6.70(s,1H),6.86(d,J=7.6Hz,1H),6.75-6.70(m,3H),2.74(d,J=
4.4Hz,3H),2.25(s,1H).HRMS (ESI), [M+H]+：
Embodiment 16
1-（5-（3- 4-trifluoromethylphenopendants）- 1H- benzimidazolyl-2 radicals）The synthesis of -3- methylurea
Using 3- trifloro methyl phenols as starting material, the same 1- of step（5- phenoxy group -1H- benzimidazolyl-2 radicals）- 3- methylurea
Synthesis, total recovery 25.7%.1HNMR（400MHz, DMSO-d6）δ11.60(s,1H),9.99(s,1H),7.56(t,J=
8.0Hz,1H),7.39(d,J=7.6Hz,1H),7.22-7.10(m,4H),6.81(d,J=8.4Hz,1H),2.74(d,J=
3.2Hz,3H).HRMS (ESI), [M+H]+：351.1067.
Embodiment 17
1-（5-（3- ethyl phenoxy groups）- 1H- benzimidazolyl-2 radicals）The synthesis of -3- methylurea
Using 3- ethyl -phenols as starting material, the same 1- of step（5- phenoxy group -1H- benzimidazolyl-2 radicals）The conjunction of -3- methylurea
Into total recovery 7.8%.1HNMR（400MHz, DMSO-d6）δ11.49(s,1H),9.94s,1H),7.34(d,J=8.4Hz,
1H),7.22(t,J=8.0Hz,1H),7.15(s,1H),7.00(s,1H),6.89(d,J=7.2Hz,1H),6.78(s,1H),
6.74(d,J=8.4Hz,1H),6.70(d,J=8.4Hz,1H),2.74(d,J=2.4Hz,3H),2.55(q,J=7.2Hz,2H),
1.14(t,J=7.2Hz,3H)。MS(ESI):m/z310.1[M+H]+
Embodiment 18
1-（5-（3- cumene epoxides）- 1H- benzimidazolyl-2 radicals）The synthesis of -3- methylurea
Using 3- isopropyl-phenols as starting material, the same 1- of step（5- phenoxy group -1H- benzimidazolyl-2 radicals）- 3- methylurea
Synthesis, total recovery 18.5%.1HNMR（400MHz, DMSO-d6）δ11.50(s,1H),9.94(s,1H),7.34(d,J=
8.0Hz,1H),7.24-7.20(m,1H),7.16(s,1H),7.00(s,1H),6.93(d,J=7.6Hz,1H),6.74(d,J=
8.4Hz,2H),6.67(d,J=8.0Hz,1H),2.87-2.80(m,1H),2.74(d,J=4.0Hz,3H),1.16(d,J=
6.8Hz,6H).HRMS (ESI), [M+H]+：325.1660.
Embodiment 19
1-（5-（3- cyano-benzene oxygens）- 1H- benzimidazolyl-2 radicals）The synthesis of -3- methylurea
Using 3- cyanophenols as starting material, the same 1- of step（5- phenoxy group -1H- benzimidazolyl-2 radicals）The conjunction of -3- methylurea
Into total recovery 24.8%.The method of wherein nitro reduction uses zinc powder reduction, and concrete operations are as follows：By 1.0g（4.1mmol）
2- nitros -5-（3- cyano-benzene oxygens）Aniline is dissolved in 10mL ethanol, adds 1.4g（20.5mmol）Zinc powder, argon gas protection, delays
Slow to instill saturated ammonium chloride solution 6mL, normal-temperature reaction 4h, decompression filters, and filtrate is spin-dried for solvent, and column chromatography obtains product 1-（5-（3-
Cyano-benzene oxygen）- 1H- benzimidazolyl-2 radicals）- 3- methylurea, yield 90.9%.1HNMR（400MHz, DMSO-d6）δ11.60
(s,1H),9.99(s,1H),7.55-7.51(m,2H),7.39-7.35(m,2H),7.24(d,J=6.8Hz,1H),7.18-
7.03(m,2H),6.79(d,J=8.0Hz,1H),2.74(d,J=3.6Hz,3H).HRMS (ESI), [M+H]+：308.1144.
Embodiment 20
1-（5-（3- phenyl biphenylyloxies）- 1H- benzimidazolyl-2 radicals）The synthesis of -3- methylurea
The synthesis of step 13- phenyl xenols
By 0.54mL（5mmol）M -bromoacetophenone and 0.61g（5mmol）Phenyl boric acid is dissolved in 5mL1, in 4- dioxane, adds
0.5mL water, adds 2.1g (15mmol) Anhydrous potassium carbonates and 0.2g (0.15mmol) tetra-triphenylphosphine palladium, and argon gas is protected, 70 DEG C
React 8h.Dilute hydrochloric acid tune pH is added to neutrality, is repeatedly extracted with ethyl acetate, merges organic phase, drying, is spin-dried for solvent, column layer
Analyse to obtain product 3- phenyl xenol 0.32g, yield 75.2%.
Step 21-（5-（3- phenyl biphenylyloxies）- 1H- benzimidazolyl-2 radicals）The synthesis of -3- methylurea
Using 3- phenyl xenol as substrate, the same 1- of step（5- phenoxy group -1H- benzimidazolyl-2 radicals）The synthesis of -3- methylurea,
Total recovery is 31.1%.1HNMR（400MHz, DMSO-d6）δ7.73(s,1H),7.44(d,J=7.2Hz,2H),7.41-7.32
(m,6H),7.18(t,J=6.0Hz,1H),7.06(d,J=2.4Hz,1H),6.90(dd,J=8.0,2.0Hz,1H),6.76(dd,
J=8.4,2.4Hz,1H),2.74(d,J=4.4Hz,3H).HRMS (ESI), [M+H]+：359.1500.
Embodiment 21
31-（5-（4- phenyl biphenylyloxies）- 1H- benzimidazolyl-2 radicals）The synthesis of -3- methylurea
Using 4- phenyl xenol as starting material, the same 1- of step（5- phenoxy group -1H- benzimidazolyl-2 radicals）- 3- methylurea
Synthesis, total recovery 6.7%.1HNMR（400MHz, DMSO-d6）δ11.53(s,1H),9.96(s,1H),7.63-7.60(m,
4H),7.43(t,J=7.6Hz,2H),7.37(d,J=8.0Hz,1H),7.32(t,J=7.2Hz,1H),7.15(s,1H),7.07
(s,1H),6.99(d,J=8.8Hz,2H),6.80(dd,J=8.4,2.0Hz,1H),2.74(d,J=4.8Hz,3H)。MS(ESI):
m/z359.2[M+H]+
Embodiment 22
1-（5-（3- acetylenylbenzene epoxides）- 1H- benzimidazolyl-2 radicals）The synthesis of -3- methylurea
Step 12- nitros -5-（The bromo- phenoxy groups of 3-）The synthesis of aniline
Using 3- bromophenols as starting material, step is the same as the synthesis of 2- nitro -5-- phenoxybenzamines, yield 67.3%.
Step 22- nitros -5-（3- trimethylsilyl acetylenes base-phenoxy group）The synthesis of aniline
0.47g (1.5mmol) 2- nitros -5- that previous step is synthesized（The bromo- phenoxy groups of 3-）Aniline and 0.23g
(2.25mmol) trimethylsilyl acetylene is dissolved in 5mL acetonitriles, addition 0.052g (0.075mmol) two triphenyl phosphorus palladium chloride,
0.014g (0.075mmol) cuprous iodides and 0.5mL (3mmol) n,N-diisopropylethylamine, argon gas protection, 70 DEG C of reaction 8h.
It is cooled to room temperature, decompression filters, and filtrate is spin-dried for, and adds 20mL water, is repeatedly extracted with ethyl acetate, merges organic phase, dry, rotation
Dry solvent, column chromatography obtain 0.4g products, yield 81.8%.
Step 32- nitros -5-（3- acetenyls-phenoxy group）The synthesis of aniline
2- nitros-the 5- that previous step is synthesized（3- trimethylsilyl acetylenes base-phenoxy group）Aniline is dissolved in 10mL methanol, is added
Enter 0.34g (2.46mmol) Anhydrous potassium carbonate, react at room temperature 2h.Solvent is spin-dried for, 20mL water is added, is repeatedly extracted with ethyl acetate
Take, merge organic phase, it is dry, solvent is spin-dried for, column chromatography obtains product 0.28g, yield 89.6%.
Step 44-（3- acetenyls-phenoxy group）The synthesis of -1,2- phenylenediamines
2- nitros-the 5- that previous step is synthesized（3- acetenyls-phenoxy group）Aniline is dissolved in 10mL methanol, adds 0.36g
(5.5mmol) zinc powder, argon gas protection, is slowly dropped into 1.7mL saturated ammonium chloride solutions, reacts at room temperature 2h.Decompression filters, filtrate rotation
It is dry, dissolved with 20mL ethyl acetate, filter off insoluble matter, filtrate is spin-dried for, and column chromatography obtains product 0.23g, yield 93.3%.
Step 51-（5-（3- acetylenylbenzene epoxides）- 1H- benzimidazolyl-2 radicals）The synthesis of -3- methylurea
With 4-（3- acetenyls-phenoxy group）- 1,2- phenylenediamine are substrate, cyclization, the same 1- of amination steps（5- phenoxy groups -1H-
Benzimidazolyl-2 radicals）The synthesis of -3- methylurea, two step total recoverys are 9.8%.1HNMR（400MHz, DMSO-d6）δ11.55(s,
1H),9.98(s,1H),7.38-7.32(m,2H),7.15(d,J=7.6Hz,2H),7.05(s,1H),6.98(d,J=8.4Hz,
1H),6.91(s,1H),6.77(d,J=7.2Hz,1H),4.18(s,1H),2.73(d,J=4.4Hz,3H)。MS(ESI):m/
z306.1[M+H]+
Embodiment 23
1-（5-（Naphthalene 1- epoxides）- 1H- benzimidazolyl-2 radicals）The synthesis of -3- methylurea
Using alpha-Naphthol as starting material, the same 1- of step（5- phenoxy group -1H- benzimidazolyl-2 radicals）The synthesis of -3- methylurea, always
Yield is 28.3%.1HNMR（400MHz, DMSO-d6）δ11.50(s,1H),9.95(s,1H),8.21(d,J=7.6Hz,1H),
7.96(d,J=7.2Hz,1H),7.63(d,J=8.4Hz,1H),7.53-7.59(m,2H),7.42-7.37(m,2H),7.16(s,
1H),7.04(s,1H),6.84(dd,J=8.4,2.0Hz,1H),6.79(d,J=7.6Hz,1H),2.73(d,J=4.4Hz,3H)。
HRMS (ESI), [M+H]+：333.1346.
Embodiment 24
1-（5-（4- hydroxyphenoxies）- 1H- benzimidazolyl-2 radicals）The synthesis of -3- methylurea
Using hydroquinone as starting material, the same 1- of step（5- phenoxy group -1H- benzimidazolyl-2 radicals）The synthesis of -3- methylurea,
Total recovery is 3.6%.1HNMR（400MHz, DMSO-d6）δ11.38(s,1H),9.90(s,1H),9.19(s,1H),7.28(d,J
=8.4Hz,1H),7.19(s,1H),6.88(s,1H),6.81(d,J=8.8Hz,2H),6.73(d,J=8.8Hz,2H),6.67
(dd,J=8.8,2.2Hz,2H),2.73(d,J=4.4Hz,3H)。MS(ESI):m/z298.1[M+H]+
Embodiment 25
1-（5-（4- ethoxy phenoxies）- 1H- benzimidazolyl-2 radicals）The synthesis of -3- methylurea
Using 4- thanatols as starting material, the same 1- of step（5- phenoxy group -1H- benzimidazolyl-2 radicals）- 3- methylurea
Synthesis, total recovery 30.6%.1HNMR（400MHz, DMSO-d6）δ11.42(s,1H),9.92(s,1H),7.30(d,J=
8.8Hz,1H),7.18(s,1H),6.90(s,5H),6.70(dd,J=8.4,2.0Hz,1H),3.98(d,J=6.8Hz,2H),
2.73(d,J=4.4Hz,3H),1.31(t,J=6.8Hz,3H)。MS(ESI):m/z326.1[M+H]+
Embodiment 26
1-（5-（4- benzyloxy phenoxyls）- 1H- benzimidazolyl-2 radicals）The synthesis of -3- methylurea；
Step 12- nitros -5-（4- hydroxyphenoxies）The synthesis of aniline
5.77g (52.5mmol) hydroquinone is dissolved in 30mLN, in dinethylformamide, adds 7.24g
(52.5mmol) Anhydrous potassium carbonate, 100 DEG C of reaction 30min, adds the fluoro- 2- nitroanilines of 2.8g (17.5mmol) 5-, reacted
Night.It is cooled to room temperature, is poured into frozen water, separate out solid, decompression filters, dry 2.1g products, yield 48.8%.
Step 25-（4- benzyloxyphenoxies）The synthesis of -2- nitroanilines
0.1g (0.41mmol) 2- nitros -5- for taking previous step to synthesize（4- hydroxyphenoxies）Aniline is dissolved in 5mLN, N- bis-
In methylformamide, 0.12g (0.82mmol) Anhydrous potassium carbonates and 0.11g (0.62mmol) bromobenzyl are added, room temperature reaction is overnight.
30mL water is added, is repeatedly extracted with ethyl acetate, merges organic phase, it is dry, it is spin-dried for solvent and obtains 0.31g products, yield is
91.9%。
Step 31-（5-（4- benzyloxy phenoxyls）- 1H- benzimidazolyl-2 radicals）The synthesis of -3- methylurea
With 5-（4- benzyloxyphenoxies）- 2- nitroanilines are substrate, nitro reduction, cyclization, amination steps 1-（5-（2-
Chlorophenoxy）- 1H- benzimidazolyl-2 radicals）- 3- methylurea, three step total recoverys are 26.6%.1HNMR（400MHz, DMSO-d6）δ
11.42(s,1H),9.91(s,1H),7.44-7.30(m,6H),7.18(br,1H),7.00(d,J=5.2Hz,2H),6.91(d,
J=5.2Hz,2H),6.71(d,J=8.8Hz,1H),5.06(s,2H),2.74(d,J=4.4Hz,3H)。MS(ESI):m/z388.2
[M+H]+
Embodiment 27
The compounds of this invention Proliferation of Tumor Cells In Vitro inhibitory activity is present embodiments provided to be tested
Experimental method
1）Respectively with being counted after different tumour cell Colo205 and NCI-H460 digestion, according to 1000-2000/hole,
For kind into 96 orifice plates, each concentration sets 6 multiple holes.
Passage is no less than not more than 15 times twice after cell recovery.Cell state is observed before kind plate, it is good using cell state
Good, the long cells that liquid completely, had recently just been changed to about 6 seventy percent.
2）In cell incubator culture 24 it is small when after, by 100 μ L compound working solutions add, continue culture 72 it is small when.
Compound is made into the mother liquor of 10mol/l with DMSO.10 μ L mother liquors are added in 90 μ LDMSO, are shaken using vortex
Swing device mixing, successively step by step dilute 5 times, be configured to DMSO storing liquids, be positioned over 4 DEG C it is stand-by.Using it is preceding again mix and according to
1:500 are added to storing liquid in complete medium as working solution, are mixed using turbula shaker.Working solution needs current existing
Match somebody with somebody, cause to separate out to prevent from placing for a long time.
3）Culture medium is sucked, 100 μ L is added and contains MTT（0.5mg/ml）Culture medium, place in incubator 4 it is small when.Culture
Base removes clean, 100 μ LDMSO of addition, concussion mixing.
The preparation of MTT：1.25gMTT is added in 250mlPBS, is mixed, 4 DEG C are kept in dark place.
4）Use the light absorption value of porous plate microplate reader detection 570nm or 490nm.
5）The OD values of detection are with prism software analysis and calculate the IC of each compound50Value.
Target compound determination of activity result is as shown in the table
Meanwhile embodiment Isosorbide-5-Nitrae, the IC50 of the compound on tumor cell K562 described in 18,19 are less than 50nM；It is thin to tumour
The IC50 of born of the same parents A431 is less than 100nM；10 μM are less than to the IC50 of tumour cell HepG2；2 are less than to the IC50 of tumour cell Hela
μM；100nM is less than to the IC50 of tumour cell MDA-MB-435S.Specific data are as follows：
There is the chemical combination that can be seen that from activity data above in general formula of the present invention very strong external suppression to swell
The activity of oncocyte.And electrical position of substituent and substituent on phenyl ring etc. is little to the activity influence of compound, wherein
The non-ortho substituted compound of phenyl ring has higher antitumor activity.
Embodiment 28
The present embodiment is the compound 1- described in the embodiment of the present invention 19（5-（3- cyano-benzene oxygens）- 1H- benzo miaows
Azoles -2）Experiment of -3- the methylurea to tumour cell NCI460 micro-tubular structures and Cell cycle influences：
1st, cell cycle analysis
（1）Drug-treated cell：The cell inoculation of suitable concentration is taken into 6 orifice plates, cell confluency degree is waited after 24h 60%
Left and right adds compound 1- described in various concentrations embodiment 19（5-（3- cyano-benzene oxygens）- 1H- benzimidazolyl-2 radicals）- 3- methyl
Urea medicine, continues to cultivate 24-48h.
（2）Collect cell：In predetermined time point collecting cell.1.5EP pipes are transferred to after cell is digested using pancreatin
In, room temperature 1500rpm is centrifuged 10 minutes.Washed more than twice with PBS, 1500rpm is centrifuged 10 minutes.
The PBS of 300 μ L4 DEG C precoolings is first added, the 700 μ L of absolute ethyl alcohol that -20 DEG C of precoolings are added after mixing are resuspended, use rifle
Head mix, be put into -20 DEG C of refrigerators fix 24 it is small when more than.
3000rpm centrifuges 10min.Washed 2 times using PBS, 3000rpm centrifugations 10min.Weight is carried out with the PBS of about 200ul
It is outstanding（Need to ensure cell concentration in 10-6 or so）
The RNase to 50 μ g/ml of final concentration of 50mg/ml is added, cell is placed in 37 DEG C of digestion 30min.
（3）Staining analysis：Add PI dyestuffs to 10 μ g/ml of final concentration, the room temperature lucifuge prepared and dye 30min.In 1h
Carry out FCM analysis.Flow cytometer：FACSCalibur, BD.
Carry out data analysis.
2nd, immunofluorescence
（1）Drug-treated cell：The cell inoculation of suitable concentration waits cell confluency degree left 30% into 6 orifice plates, after 24h
The right medicine for adding various concentrations, continues to cultivate 6-24h.
（2）Cell is fixed：4% 300 μ L of paraformaldehyde fixer are added into every hole, place 30 minutes at room temperature with solid
Determine cell.
（3）Cell-permeant：The penetrating 300 μ L of liquid of 0.2%TritonX-100 are added per hole, are placed 10 minutes at room temperature so that thin
After birth is penetrating.
（4）Antigen blockade：3% normal goat serum（NormalGoatSerum, NGS）20 μ L are as confining liquid, during closing
When room temperature 1 is small or 4 DEG C are stayed overnight, and are influenced with removing intracellular non-specific binding.
（5）Primary antibody is incubated：The primary antibody of immunofluorescence（a-tubulin）Use confining liquid（3%NGS）Dilution, dilution ratio
Determined according to antibody titer and intracellular antigen abundance（Ordinary antibody is to endogenous antigen generally with 1:50 dilutions）, it is incubated at room temperature
1.5 it is small when.
（6）Secondary antibody is incubated：Different selection goat antirabbits or goat anti-mouse antibody of the secondary antibody according to primary antibody, and according to reality
Border requires to select coupled FITC（Green fluorescence）Or TRITC（Red fluorescence）；Use confining liquid（3%NGS）Dilution, dilution ratio
For 1:100~1:200.When incubation 1 is small at room temperature.
（7）Contaminate core：1 × PBS is according to 1:4000 dilution proportion DAPI stostes, suck in 24 orifice bores
After washingbuffer, the DAPI dyeing liquors of 200 μ L are rapidly joined in every hole, were absorbed at once after 5-6 seconds after all adding
Dyeing liquor simultaneously adds rapidly 500 μ L1 × PBS in every hole, is cleaned twice according to step 3, pays attention to lucifuge.
（8）Mounting：80% glycerine of 2 μ L is added dropwise on glass slide in darkroom, coverslip is tipped upside down on glycerine, is slightly dried in the air
One preserves after the meeting；Fluorescence microscope or laser confocal microscope detection.
It will be seen from figure 1 that in the case of the compound being not added with described in embodiment 19, tumour cell NCI460 micro-tubular structures
Uniform sequential elongate, threadlike structure is showed outside nucleus（Shown in Figure 1A）, when the compound concentration described in embodiment 19 from
25nM（Shown in Figure 1B）Increase to 75nM（Shown in Fig. 1 C）, 100nM（Shown in Fig. 1 D）When NCI460 micro-tubular structures occur it is different
The normal elongated filament being distributed in order disappears（Shown in Figure 1B, C, D）, while do not occur in cellular morphology.The result is demonstrate,proved
Bright the compounds of this invention can influence the performance of the tubulin normal function of tumour cell in a manner of dose-dependent.
Figure it is seen that in the case of the compound described in various dose embodiment 19 is added, tumour cell NCI460
Gradually decreased in G0/G1 phases and the cell of S phases, and the cell in the G2/M phases gradually increases（Shown in Fig. 2A, B, C, D）.Should
As a result prove that tumour cell can be arrested in the G2/M phases by the compounds of this invention in a manner of dose-dependent, so that it is thin to influence tumour
The operating of born of the same parents' normal cycle ultimately results in cell death.
Embodiment 29
The present embodiment is the compound 1- described in the embodiment of the present invention 1（5- phenoxy group -1H- benzimidazolyl-2 radicals）- 3- first
Base urea is to the micro-tubular structure of HepG2 cells and the experiment of the influence in cycle, and specific experimental procedure is with reference to embodiment 28.
From figure 3, it can be seen that in the case of the compound being not added with described in embodiment 1, HepG2 cellular microtubules structures are in cell
Uniform sequential elongate, threadlike structure is showed outside core（Shown in Fig. 3 A）, when the compound concentration described in embodiment 1 is from 0.04uM
（Shown in Fig. 3 B）Increase to 0.2uM（Shown in Fig. 3 C）, 1uM（Shown in Fig. 3 D）When HepG2 cellular microtubules structures occur it is abnormal
The elongated filament being distributed in order disappears（Shown in Fig. 3 B, C, D）, while do not occur in cellular morphology.The result proves
The compounds of this invention can influence the performance of the tubulin normal function of HepG2 cells in a manner of dose-dependent.
From fig. 4, it can be seen that in the case of the compound described in various dose embodiment 1 is added, HepG2 cells are in
G0/G1 phases and the cell of S phases gradually decrease, and the cell in the G2/M phases gradually increases（Shown in Fig. 4 A, B, C, D）.The result
Prove that tumour cell can be arrested in the G2/M phases by the compounds of this invention in a manner of dose-dependent, so as to influence tumour cell just
The operating in normal cycle ultimately results in cell death.
In conclusion -2 alkyl substituted urea benzimidazoles compound of 5- aryl phenols of the present invention can be shown in vitro
The propagation for suppressing kinds of tumor cells is write, the retardance for forming the micro-pipe of tumour cell with phase cell cycle G2/M shows dosage
Dependence；Therefore compound of the present invention can be used for preparing pharmaceutical composition, described pharmaceutical composition is thin for suppressing tumour
Intracellular growth or treatment tumour, new medicament selection are provided for clinical treatment tumour, further to be more effective, safer
Medicine provide new selection, help to solve the problems, such as the clinical treatments such as the drug resistance that is on the rise.
Embodiment described above only expresses the several embodiments of the present invention, its description is more specific and detailed, but simultaneously
Therefore the limitation to the scope of the claims of the present invention cannot be interpreted as.It should be pointed out that for those of ordinary skill in the art
For, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the guarantor of the present invention
Protect scope.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.
Claims (7)
1. a kind of -2 alkyl substituted urea benzimidazoles compound of 5- aryl phenols, it is characterised in that there is formula (I) structure：
Wherein, Ar is phenyl, and the phenyl is to appoint by 0,1,2 or 3 optionally from fluorine, bromine, methyl, ethyl, isopropyl, fluoroform
Base, cyano group, the substitution of alkynyl substituted base；Or the 3rd of the phenyl and the 5th all by methoxy substitution；Or the phenyl
The 4th be substituted by phenyl；
R is methyl.
2. -2 alkyl substituted urea benzimidazoles compound of 5- aryl phenols according to claim 1, it is characterised in that described
When Ar is phenyl, the 2nd, 6 of substituent not substituted-phenyl.
3. -2 alkyl substituted urea benzimidazoles compound of 5- aryl phenols according to claim 1, it is characterised in that described
Compound is selected from：
1- (5- phenoxy group -1H- benzimidazolyl-2 radicals) -3- methylurea；
1- (5- (3,5- dimethoxys phenoxy group) -1H- benzimidazolyl-2 radicals) -3- methylurea；
1- (5- (3- fluorophenoxies) -1H- benzimidazolyl-2 radicals) -3- methylurea；
1- (5- (5- fluorophenoxies) -1H- benzimidazolyl-2 radicals) -3- methylurea；
1- (5- (3- bromobenzenes epoxide) -1H- benzimidazolyl-2 radicals) -3- methylurea；
1- (5- (3- methylphenoxies) -1H- benzimidazolyl-2 radicals) -3- methylurea；
1- (5- (3- 4-trifluoromethylphenopendants) -1H- benzimidazolyl-2 radicals) -3- methylurea；
1- (5- (3- ethyls phenoxy group) -1H- benzimidazolyl-2 radicals) -3- methylurea；
1- (5- (3- cumenes epoxide) -1H- benzimidazolyl-2 radicals) -3- methylurea；
1- (5- (3- cyano-benzene oxygens) -1H- benzimidazolyl-2 radicals) -3- methylurea；
1- (5- (4- phenyl biphenylyloxy) -1H- benzimidazolyl-2 radicals) -3- methylurea；
1- (5- (3- acetylenylbenzenes epoxide) -1H- benzimidazolyl-2 radicals) -3- methylurea.
4. -2 alkyl substituted urea benzimidazoles compound of 5- aryl phenols or its pharmacy described in claims 1 to 3 any one
Upper acceptable salt is preparing the application in preventing and treating tumour medicine.
5. application according to claim 4, it is characterised in that the tumour includes liver cancer, leukaemia, lung cancer, uterine neck
Cancer, colon cancer, stomach cancer, the cancer of the esophagus, oophoroma, breast cancer, melanoma, sarcoma or cancer of pancreas.
6. the composition of a kind of Prevention and tumor, it is characterised in that its active ingredient is claim 1-3 institutes
- 2 alkyl substituted urea benzimidazoles compound of 5- aryl phenols stated or its pharmaceutically acceptable salt.
7. -2 alkyl substituted urea benzimidazoles compound of 5- aryl phenols or its pharmacy described in claims 1 to 3 any one
Upper acceptable salt is preparing inhibitor, mitotic inhibitor, chemotherapeutic sensitizer, the radio therapy sensitization of suppression tubulin polymerization
Agent, inducing cell apoptosis derivant, arresting cell cycle retarding agent in application.
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| CN1481368A (en) * | 2000-12-15 | 2004-03-10 | ��̩��˹ҩ��ɷ�����˾ | Gyrase imhibitors and uses thereof |
| CN101538263A (en) * | 2002-06-13 | 2009-09-23 | 沃泰克斯药物股份有限公司 | 2-ureido-6-heteroaryl-3H-benzoimidazole-4-carboxylic acid derivatives and related compounds as gyrase and/or topoisomerase IV inhibitors for the treatment of bacterial infections |
| CN101827827A (en) * | 2007-10-19 | 2010-09-08 | 赛诺菲-安万特 | 6-aryl/heteroalkyloxy benzothiazole and benzimidazole derivatives, method for preparing same, application thereof as drugs, pharmaceutical compositions and novel use in particular as C-MET inhibitors |
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| CN1481368A (en) * | 2000-12-15 | 2004-03-10 | ��̩��˹ҩ��ɷ�����˾ | Gyrase imhibitors and uses thereof |
| CN101538263A (en) * | 2002-06-13 | 2009-09-23 | 沃泰克斯药物股份有限公司 | 2-ureido-6-heteroaryl-3H-benzoimidazole-4-carboxylic acid derivatives and related compounds as gyrase and/or topoisomerase IV inhibitors for the treatment of bacterial infections |
| CN101827827A (en) * | 2007-10-19 | 2010-09-08 | 赛诺菲-安万特 | 6-aryl/heteroalkyloxy benzothiazole and benzimidazole derivatives, method for preparing same, application thereof as drugs, pharmaceutical compositions and novel use in particular as C-MET inhibitors |
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