CN104910894B - Benzimidazole hERG potassium ion channel small-molecular fluorescent probe and preparation method and applications thereof - Google Patents
Benzimidazole hERG potassium ion channel small-molecular fluorescent probe and preparation method and applications thereof Download PDFInfo
- Publication number
- CN104910894B CN104910894B CN201510224724.9A CN201510224724A CN104910894B CN 104910894 B CN104910894 B CN 104910894B CN 201510224724 A CN201510224724 A CN 201510224724A CN 104910894 B CN104910894 B CN 104910894B
- Authority
- CN
- China
- Prior art keywords
- preparation
- fluorescent probe
- probe
- luorobenzyl
- herg potassium
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Active
Links
Landscapes
- Plural Heterocyclic Compounds (AREA)
- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention relates to a benzimidazole hERG potassium ion channel small-molecular fluorescent probe and a preparation method and applications thereof; the fluorescent probe has the structural general formula represented by the formula (I), wherein in the formula, R1 is hydroxyl, halogen, alkyl or alkoxy monosubstituent or polysubstituent, R2 is fluorophore, n is 1-6, particularly, R1 is p-halogen, and R2 is coumarin, naphthalene diimide, NBD, Cy5 or fluorescein isothiocyanate fluorophore. The invention discloses the applications of the probe in marking of hERG potassium ion channels and high-expression tumor cells or tissues, high-throughput screening of hERG potassium ion channel inhibitors, evaluation of new drug cardiotoxicity, use as a probe for identification of the hERG potassium ion channels and studies on hERG potassium ion channel physiological, pathological and related diseases.
Description
Technical field
The invention belongs to technical field of pharmaceuticals, and in particular to a kind of small molecule of benzimidazole hERG potassium-channels is glimmering
Light probe and preparation method and application.
Background technology
Mankind's fruit bat related gene (human ether-a-go-go related gene, hERG) is from human hippocampal base
Isolation identification draws there is homology with fruit bat EAG genes because in.HERG gene codes heart quickly activates delayed rectification (Ikr)
The α subunits of potassium-channel, this passage is primarily present in heart tissue and nervous system, in heart action potential process of repolarization
In play a significant role.Recently it is found that hERG high expression, the propagation and apoptosis to tumor cell in many tumor cells
With adjustment effect.When the mutation of hERG genes or blocked hERG passages, the phase between heritability or acquired long QT can be caused comprehensive
Simulator sickness (LQTS), and then cause torsade de pointeses, ventricular fibrillation even to die suddenly.
For the cardiac toxicity that some clinical medicines or new drug drug candidate are shown, many studies have shown that being due to right
The hERG potassium-channels of myocardial cell produce retardance so as to cause LQTS.HERG passages are used as III class antiarrhythmic drug
Action target spot, the predominantly heart medication of inhibitory action, such as anti-arrhythmic (dofetilide), anti-anginal drug are produced to it
(bepridil) and cardiac tonic (perhexiline), in addition, some other non-cardiac medicine can also block the passage, such as anti-spirit is lost
Normal medicine (chlorpromazine), antihistaminic (terfenadine), digestive tract power reinforcing medicine (cisapride) etc..Because medicine and hERG potassium ions lead to
The higher affinity in road and produce Long-QT syndrome, at present, cardiac toxicity should be all carried out in new drug preclinical study
Safety evaluatio, and hERG potassium-channels are as the important target of cardiotoxicity of medicine screening.
Research shows that the expression of hERG genes is significantly raised in many tumor cell lines, such as colon cancer cell (HT-
29), neuroblastoma cell (SH-SY5Y), stomach cancer cell (SGC7901), human breast cancer cell (MCF-7) etc..HERG potassium
Ion channel is closely related with tumor cell proliferation, apoptosis, differentiation and aggressive, and in the normal cell in corresponding source low table
Up to or do not express.In addition, there is the medicine that specific inhibition is acted on to suppress the propagation of tumor cell to hERG passages.Thus
HERG potassium-channels would be possible as the drug targets of oncotherapy, and can be used as swollen neoplastic biomarker
Thing.
In recent years, the high-throughput screening method for hERG potassium-channels has radioligand-binding study, voltage film
Piece tongs technology and the detection method based on fluorescent probe etc..Radioligand-binding study, can cause radiation pollution.Voltage film
Piece tongs technology is high due to its technical requirements, need to put into larger man power and material, is not suitable for the screening of quantitatively compound.Fluorescence
Probe-detection methods sensitivity is high, simple and efficient, special installation is not required to, in being widely used in various bioanalysiss.It is presently used for
The related fluorescent probe of potassium-channel is mainly visited using fluorescent protein labeling technology, immunofluorescence technique, voltage-sensitive fluorescence
Pin etc., and the application of high selectivity, highly sensitive small-molecule fluorescent probe in potassium-channel field is less.Small molecule fluorescent
The features such as probe has quick, sensitive, high flux and is easy to automatization, has been widely used for the important life such as protein, nucleic acid
Development tool in the biology of thing molecule and pharmacology's detection, to fields such as disease mechanisms discussion, clinical diagnosises and drug screenings
There is important meaning.Small-molecule probe is typically made up of two parts：Pharmacophore portion and fluorophor moiety.Pharmacophoric group with
Target biological molecules are combined into high-affinity, and fluorophor launches fluorescence by exciting with labelled protein.Therefore, Ren Menkai
Effect of the beginning trial with small-molecule fluorescent probe come detection compound to hERG ion channels.
The method that the fluorescent probe high flux screening for hERG potassium-channels and labelling are set up in research, will directly reflect
Inhibitory activity of the compound to hERG potassium-channels, has important meaning to the safety evaluatio of medicament research and development and Preclinical Drug
Justice.In recent years, it has been found that the high expression hERG potassium-channels of some tumor cells, this is that study tumor cell and its tissue are carried
New enlightenment is supplied.
The content of the invention
In order to solve above-mentioned problems of the prior art, the purpose of the present invention be a kind of benzimidazole hERG potassium from
Small-molecule fluorescent probe of subchannel and preparation method and application.
For achieving the above object, the present invention adopts following technical proposals：
A kind of small-molecule fluorescent probe of benzimidazole hERG potassium-channels, the general structure such as formula of the fluorescent probe
(I) shown in：
In formula, R1For the monosubstituted or multi-substituent of hydroxyl, halogen, alkyl or alkoxyl；R2For fluorogen；N=1-6.
Preferably, the R1For para-position halogen；R2For Coumarinses, benzene-naphthalene diimide class, NBD classes, Cy5 classes and different sulfur cyanogen
Sour fluoresceins fluorogen；N=4.
Preferably, the small-molecule fluorescent probe is selected from the compound with following structural formula：
The benzimidazole small-molecule fluorescent probe is in hERG potassium-channels and its tumor cell or group of high expression
Knit the application in labelling.
The benzimidazole small-molecule fluorescent probe can directly reflect that suppression of the compound to hERG potassium-channels is lived
Property.
The benzimidazole small-molecule fluorescent probe hERG potassium channel inhibitors high flux screening and
Application in the evaluation of new drug cardiac toxicity.
The benzimidazole small-molecule fluorescent probe is in the probe as identification hERG potassium-channels and in hERG
Application in the research of potassium-channel physiology, pathology and relevant disease.
A kind of preparation method of benzimidazole small-molecule fluorescent probe, comprises the following steps：
(1) preparation of recognition group：2-Chlorobenzimidazole and is reacted fluorobenzyl bromide under alkalescence condition (pH=10～14)
3～5h generate 1- to luorobenzyl -2-Chlorobenzimidazole, then with 4- amino -1- ethoxycarbonyls phenylpiperidines microwave (35～50W,
150～180 DEG C) under conditions of react 0.5～1min, generate 4- ((1- is to luorobenzyl -1H-2- benzimidazolyls) amino) -1-
Piperidine ethyl formate, then be that 30%～60% hydrobromic acid generates 1- to luorobenzyl-N- in 80～100 DEG C of backflows with mass fraction
(4- piperidyls) -1H-2- aminobenzimidazole bromates, then with N- (4- brombutyls) phthalimide in alkalescence condition
8～9h is reacted under (pH=9～13), N- (4- ((4- ((1- is to luorobenzyl -1H-2- benzimidazolyls) amino) 1- piperidines) are generated
Butyl) phthalimide, then be 70%～80% hydration 4～5h of hydrazine reaction with mass fraction, obtain N- (1- (4- ammonia fourths
Base) piperidyl) -1- is to luorobenzyl -1H-2- aminobenzimidazoles；
(2) preparation of probe molecule：Various fluorogens are prepared, by introducing carboxyl on fluorogen, then with N- (1-
(4- ammonia butyl) piperidyl) -1- to luorobenzyl -1H-2- aminobenzimidazoles in EDCI/HOBT/Et3Contract in the presence of N
Reaction is closed, 12～14h of room temperature reaction is obtained probe molecule；Or halogen is introduced on fluorogen, with N- (1- (4- ammonia butyl)
Piperidyl) to luorobenzyl -1H-2- aminobenzimidazoles, potassium carbonate makees acid binding agent, acetonitrile as solvents, in 70～80 DEG C of backflows to -1-
, there is substitution reaction in 6～7h of reaction, prepare probe molecule.
Preferably, in the step (1), 2-Chlorobenzimidazole, to fluorobenzyl bromide and 4- amino -1- ethoxycarbonyl phenylpiperidines
Addition mol ratio be 0.5～1.2：1～1.8：1～2.
Preferably, in the step (1), 4- amino -1- ethoxycarbonyl phenylpiperidines, hydrobromic acid, N- (4- brombutyls) neighbour's benzene
The mol ratio that dicarboximide and hydrazine hydrate are added is 1～1.8：50～70：1.2～2：60～80.
Preferably, in the step (1), return time is 30～40h.
Preferably, in the step (2) N- (1- (4- ammonia butyl) piperidyl) -1- to luorobenzyl -1H-2- amino benzo miaows
The mol ratio that azoles, carboxylic fluorogen, EDCI, HOBT and triethylamine are added is 0.5～1.2：0.5～1.2：1～2：1～
2：1.5～3.
Preferably, in the step (2), N- (1- (4- ammonia butyl) piperidyl) -1- is to luorobenzyl -1H-2- amino benzos
Imidazoles, halogen-containing fluorogen, the mol ratio of potassium carbonate are 0.5～1.2：1～2：1.5～3.
The present invention Advantageous Effects be：
The benzimidazole small-molecule fluorescent probe of the present invention has high selectivity and highly sensitive to hERG potassium-channels
Degree, can be used for the identification of hERG potassium-channels, and the research of hERG potassium-channel physiology, pathology and relevant disease；
Can be used for the screening of hERG potassium channel inhibitors, inhibitory activity of the direct reaction compound to hERG potassium-channels is used
In the research that new drug cardiac toxicity is evaluated.
Description of the drawings
Fig. 1 is the HEK293 cells of L1 and hERG transfections and the imaging results for adding inhibitor；
Fig. 2 is L1 and neuroblastoma cell and the imaging results of addition inhibitor.
Specific embodiment
With reference to specific embodiments and the drawings, the present invention is further illustrated.
Embodiment 1：The preparation of Coumarinses fluorescence probes：
Concrete synthetic route is as follows：
(1) preparation of intermediate 1：
To add in 2-Chlorobenzimidazole (5g, 32.7mmol) 30ml acetonitriles and potassium hydroxide (2.62g, 46.6mmol, plus
Heat stirs 30min, solution clarification to 80 DEG C；After being cooled to room temperature, add to fluorobenzyl bromide (9.29g, 49.1mmol), be warming up to
80 DEG C of backflow 5h, the white dirty solution of reactant liquor；Reactant liquor is extracted into three times (100mL × 3), distillation washing with dichloromethane
Wash, merge organic layer, anhydrous magnesium sulfate is dried, sucking filtration, concentration obtains white solid, and solid acetone/petroleum ether recrystallization is cold
Hide, sucking filtration, be dried, obtain white solid, yield is 91.1%.1H-NMR(300MHz,DMSO):δ=7.64 (d, J=6.6Hz,
2H), 7.32-7.24 (m, 4H), 7.19 (t, J=4.9Hz, 2H), 5.53 (s, 2H)；ESI-MS:([M+H]+):261.6。
(2) preparation of intermediate 2：
By intermediate 1 (0.5g, 1.9mmol) and colorless oil 4- amino -1- ethoxycarbonyl phenylpiperidines (0.495g,
2.8mmol) add into microwave tube, stir, 180 DEG C of reaction 1min of microwave are cooled to room temperature, in red brown solid, use
Sample is mixed after methanol 15mL and dichloromethane 5mL dissolvings, column purification is crossed, white solid is obtained, yield is 34.0%.1H-NMR
(300MHz,DMSO):δ=7.21 (m, 3H), 7.14 (t, J=6.6Hz, 2H), 7.06 (d, J=5.8Hz, 1H), 6.93 (t, J
=5.6Hz, 1H), 6.84 (t, J=5.6Hz, 1H), 6.64 (d, J=5.6Hz, 1Hz), 5.26 (s, 2H), 4.02 (m, 5H),
2.96 (s, 2H), 1.99 (d, J=7.4Hz, 2H), 1.42 (m, 2H), 1.19 (t, J=5.3Hz, 3H)；ESI-MS:([M+H
]+):397.5。
(3) preparation of intermediate 3：
Add 30ml mass fractions to be 40% hydrobromic acid in intermediate 1 (1.2g, 3.02mmol), be warming up to 100 DEG C and return
Stream 15h, it is in yellowish-brown settled solution, reactant liquor is concentrated in vacuo, with re-crystallizing in ethyl acetate, sucking filtration, dry white solid,
Yield is 95%.1H-NMR(300MHz,DMSO):δ=7.17 (m, 5H), 7.05 (d, J=13.8Hz, 1H), 6.91 (td, J=
7.5Hz, 1.2Hz, 1H), 6.81 (td, J=7.5Hz, 0.9Hz, 1H), 6.61 (d, J=18.9Hz, 1H), 3.81 (brs, 1H),
2.97 (d, J=12.3Hz, 2H), 2.55 (t, J=9.9Hz, 2H), 1.92 (d, 9.6Hz, 2H), 1.38 (qd, J=11.7Hz,
3.6Hz, 2H)；ESI-MS:([M+H]+):325.3。
(4) preparation of intermediate 4：
Potassium phthalimide (2.0g, 10.7mmol) and Isosorbide-5-Nitrae-dibromobutane (2.8g, 12.9mmol) are dissolved in
25mL is dried N, and in N- dimethyl acid imides, pH is 10, and 26h is stirred at room temperature.Three times (100mL × 3) are extracted with dichloromethane, is steamed
Distilled water is washed, and merges organic layer, and anhydrous magnesium sulfate is dried, and concentration, dichloromethane/distilled water recrystallization, sucking filtration is whitely dry
Solid, yield is 92.4%.
(5) preparation of intermediate 5：
By intermediate 3 (1.20g, 2.96mmol) and intermediate 4 (1.67g, 5.92mmol) be dissolved in 30ml be dried acetonitrile and
10ml is dried N, in the imido mixed liquor of N- dimethyl, adds Anhydrous potassium carbonate (0.82g, 5.92mmol), in 80 DEG C of backflows
Reaction 10h.Question response liquid is cooled to room temperature, and with dichloromethane three times (100mL × 3) are extracted, and distilled water wash merges organic
Layer, is dried with anhydrous magnesium sulfate, is filtered, is concentrated, and crosses column purification, obtains white solid, and yield is 63%.1H-NMR(300MHz,
CDCl3):δ=7.84 (dd, J=6.0Hz, 3.0Hz, 2H), 7.71 (dd, J=5.4Hz, 3.0Hz, 2H), 7.51 (d, J=
7.8Hz,1H),7.16-7.09(m,3H),7.07-6.99(m,4H),5.06(s,2H),3.98-3.90(m,2H),3.70(t,J
=6.9Hz, 2H), 2.86 (d, J=10.2Hz, 2H), 2.46 (t, J=7.5Hz, 2H), 2.27 (t, J=10.8Hz, 2H),
2.12-2.03 (m, 3H), 1.72 (q, J=6.6Hz, 2H), 1.63-1.50 (m, 2H)；ESI-MS:([M+H]+):526.5。
(6) preparation of intermediate 6：
Intermediate 5 (0.50g, 0.95mmol) is dissolved in 30mL dehydrated alcohol, the hydration that mass fraction is 80% is added
Hydrazine 5mL, back flow reaction 5h.Reactant liquor is concentrated into into about 5mL, dichloromethane 30mL is added, White Flocculus hydrazides is separated out, is taken out
Filter, collects filtrate, concentration, with dichloromethane/petroleum ether recrystallization, sucking filtration, dry white solid.Yield 90.0%.1H-
NMR (600MHz, DMSO)：δ=7.23-7.18 (m, 3H), 7.14 (t, J=12.0Hz, 2H), 7.05 (d, J=12.0Hz,
1H), 6.92 (t, J=12.0Hz, 1H), 6.82 (t, J=12.0Hz, 1H), 6.57 (d, J=12.0Hz, 1H), 5.26 (s,
2H), 3.73 (brs, 1H), 2.84 (d, J=18.0Hz, 2H), 2.55 (s, 2H), 2.25 (t, J=12Hz, 2H), 1.96 (m,
4H),1.57-1.35(m,7H)；ESI-MS:([M+H]+):396.5。
(7) preparation of intermediate 7 and 8：
4- (diethylin) salicylide (0.50g, 2.59mmol) is dissolved in 20mL dehydrated alcohol, malonic acid two is added
Ethyl ester (0.42g, 2.59mmol), obtains mixed liquor 1, and morpholine (22.56mg, 0.26mmol) is dissolved in 10 μ L glacial acetic acids
In 4mL dehydrated alcohol, mixed liquor 2 is obtained, mixed liquor 1 and mixed liquor 2 are mixed, back flow reaction 8h.Ice bath is cooled down, and it is heavy to separate out
Form sediment, sucking filtration obtains intermediate 7, direct plunges into next step.20mL2M NaOH, room temperature reaction 18h are added in product Intermediate 7,
Reactant liquor is adjusted to acid pH=3, separate out a large amount of precipitations with 2M HCl, sucking filtration, with dichloromethane/petroleum ether recrystallization, sucking filtration,
It is dried, obtains orange/yellow solid, yield is 94.2%.
1H-NMR(300MHz,CDCl3):δ=12.33 (s, 1H), 8.66 (s, 1H), 7.45 (d, J=9.0Hz, 1H),
6.71 (dd, J=9.0Hz, 2.4Hz, 1H), 6.53 (d, J=2.4Hz, 1H), 3.49 (q, J=7.2Hz, 4H), 1.26 (t, J=
7.2Hz,6H)；ESI-MS:([M+H]+):262.3.
(8) preparation of compound L 1：
By intermediate 8 (66mg, 0.25mmol) and 1- ethyls-(3- dimethylaminopropyls) phosphinylidyne diimmonium salt hydrochlorate
(EDCI) (47mg, 0.30mmol), I-hydroxybenzotriazole (HOBT) (47mg, 0.30mmol) are dissolved in 20mL dry methylene chlorides
In, then ice bath stirring 30min adds intermediate 6 (100mg, 0.25mmol) and triethylamine (40 μ L, 0.50mmol) above-mentioned
In solution, continue to stir 30min, move to room temperature reaction 14h.Reactant liquor dichloromethane is extracted twice (100mL × 2), is steamed
Distilled water is washed, and merges organic layer, and anhydrous magnesium sulfate is dried, and column purification is crossed in sucking filtration, concentration, obtains fluorescent yellow-green solid, yield
40.0%.1H-NMR(300MHz,CD3OD):δ=8.65 (s, 1H), 7.57 (d, J=12Hz, 1H), 7.34 (m, 1H), 7.16
(dd, J=18Hz, 12Hz, 2H), 7.07 (m, 4H), 6.99 (m, 1H), 6.84 (dd, J=12Hz, 6Hz, 1H), 6.60 (d, J=
6Hz, 1H), 5.29 (s, 2H), 3.95 (brs, 1H), 3.55 (q, J=12Hz, 4H), 3.49 (t, J=12Hz, 2H), 2.86 (m,
2H), 2,75 (t, J=18Hz, 2H), 2.23 (d, J=18Hz, 2H), 1.74 (m, 7H), 1.25 (t, J=12Hz, 6H)；
163.62,163.36,162.80,161.17,157.65,153.20,152.61,148.12,141.93,134.41,131.28,
131.25,131.18,128.41,128.32,121.58,119.88,116.32,116.15,115.94,110.15,110.01,
108.41,107.40,96.58,57.55,52.08,48.93,45.08,45.04,38.88,31.12,27.40,23.12,
12.43.ESI-MS:([M+H]+)calcd for C37H43FN5O3:639.3453,found:639.3452。
Embodiment 2：The preparation of benzene-naphthalene diimide fluorogen class probe
Concrete synthetic route is as follows：
In the synthetic route of the present embodiment, the preparation of intermediate 1-3 with the intermediate 1-3 in embodiment 1 preparation method.
(1) preparation of intermediate 4：
4mL glacial acetic acid, 20mL pyridines, back flow reaction are added in 4- amino -1,8- benzene-naphthalene diimides (2g, 9.3mmol)
1h.It is subsequently adding 30mL acetic anhydride, back flow reaction 5h.Question response liquid is down to room temperature, and in pouring 200mL frozen water into, sucking filtration, filter cake is used
Water washing, obtains crude product brown solid, and yield is 71.3%.
(2) preparation of intermediate 5：
Intermediate 4 (1.7g, 6.66mmol) and 4- amino-n-butyl alcohol (0.71g, 7.96mmol) are dissolved in into dehydrated alcohol
In, back flow reaction 5h.Reactant liquor is concentrated, with re-crystallizing in ethyl acetate, sucking filtration, brown solid is obtained, yield is 73.7%.1H-
NMR(300MHz,CDCl3):δ 8.63 (dd, J=7.2,0.9Hz, 1H), 8.59 (d, J=8.4Hz, 1H), 8.37 (brs, 1H),
8.19 (d, J=8.1Hz, 1H), 7.83-7.75 (m, 2H), 4.24 (t, J=7.2Hz, 2H), 3.76 (t, J=6.3Hz, 2H),
1.89-1.79(m,2H),1.74-1.57(m,2H).ESI-MS:([M+H]+):327.5。
(3) preparation of intermediate 6：
Intermediate 5 (0.60g, 1.84mmol) is dissolved in and is dried in ethyl acetate, then 0.5mL phosphorus tribromides are dissolved in into 5mL
It is dried in ethyl acetate and obtains mixture 1, mixture 1 is added dropwise in the solution of intermediate 5 under ice bath, after completion of dropping,
It is transferred to 80 DEG C of oil bath, back flow reaction 6h.Question response liquid is cooled to room temperature, in being poured into trash ice, separates out brown solid, takes out
Filter, filter cake washes with water, dry brown solid.Yield 95.2%.1H-NMR(300MHz,CDCl3):δ 8.63 (d, J=
7.2Hz, 1H), 8.60 (d, J=8.1Hz, 1H), 8.39 (brs, 1H), 8.20 (d, J=8.4Hz, 1H), 7.81-7.75 (m,
2H), 4.24 (t, J=6.9Hz, 2H), 3.49 (t, J=6.3Hz, 2H), 2.38 (s, 3H), 2.04-1.85 (m, 4H)；ESI-
MS:([M+H]+):389.4。
(4) preparation of compound L 1：
By intermediate 3 (90mg, 0.22mmol) and intermediate 6 (110mg, 0.28mmol) be dissolved in 18mL be dried acetonitrile and
2mL is dried N, in the imido mixed solution of N- dimethyl, adds Anhydrous potassium carbonate (70mg, 0.50mmol), returns in 80 DEG C
Stream reaction 6h.Question response liquid is cooled to room temperature, filters off potassium carbonate, concentrates the filtrate to about 2mL, crosses column purification, obtains faint yellow
Solid, yield is 50.5%.1H-NMR(300MHz,DMSO):δ 10.45 (s, 1H), 9.26 (brs, 1H), 8.74 (d, J=
12Hz, 1H), 8.55 (d, J=12Hz, 1H), 8.50 (d, J=12Hz, 1H), 8.33 (d, J=12Hz, 1H), 7.91 (t, J=
12Hz, 1H), 7.23 (s, 3H), 7.16 (t, J=12Hz, 3H), 6.96 (d, J=36Hz, 2H), 5.34 (s, 2H), 4.11 (t, J
=12Hz, 2H), 4.00 (brs, 1H), 3.56 (d, J=12Hz, 2H), 3.10 (brs, 4H), 2.29 (s, 3H), 2.23 (d, J=
18Hz,2H),2.03(brs,1H),1.74(m,5H)；13C-NMR(75MHz,CD3OD):δ 171.26,164.37,163.88,
140.06,133.22,131.80,131.48,131.02,128.78,128.73,128.34,128.25,126.43,125.08,
122.42,121.94,120.75,120.65,118.45,115.31,115.09,114.21,108.54,44.31,38.78,
24.86,22.51,21.43.ESI-MS:([M+H]+)calcd for C37H37FN5O3:633.2945,found:633.3081。
Embodiment 3：Optically active measure
Table 1：The optical signature of probe molecule
Note：All of above optical property is measured in the phosphate buffer of pH=7.4.
Embodiment 4：The measure of biological activity
With astemizole (Astemizole) as positive drug, using radioligand comptetetion Binding experiment, fluorescence is determined
The binding activity of probe molecule and hERG potassium-channels, as a result as shown in table 2.The activity of probe molecule L1, L3, L5 of synthesis
Low compared with positive drug astemizole, the activity of probe L4 is suitable with astemizole activity.
The affinity of the probe molecule of table 2 and hERG potassium-channels
Note：IC50For the concentration that compound is combined 50% with hERG potassium-channels, KiRepresentation compound and hERG potassium ions
The binding constant of passage.
Embodiment 5：Application of the probe molecule in hERG potassium-channel cell imagings
With probe molecule L1 as object of study, its application in cell imaging is investigated, select the HEK293 of hERG transfections
Cell, neuroblastoma cell (SH-SY5Y) are positive cell, under the same terms, add inhibitor astemizole as the moon
Property control.
Concretely comprise the following steps：The HEK293 cells of hERG transfections are with containing 10% hyclone and 400 μ g/mL G418
DMEM culture medium；1640 culture medium of the neuroblastoma cell (SH-SY5Y) containing 10% hyclone, in 5%CO2It is empty
Cultivated in gas and 37 DEG C of environment, before imaging, cell is inoculated in copolymerization Jiao's capsule, 12-24h is cultivated, by culture medium
Sop up, washed once with the culture medium without serum, add probe L1 (culture medium without serum is prepared, and concentration is 1 μM)；Phase
With under the conditions of, another capsule add probe L1 and inhibitor astemizole (serum-containing media is not prepared, and L1 concentration is 1 μM, Ah
Department's imidazole concentration is 50 μM) make negative control, it is incubated, culture medium is then suctioned out, washed once, use Zeiss Axio
Observer A1 are imaged.
As depicted in figs. 1 and 2, probe molecule being capable of labelling hERG potassium-channels and its high nerve expressed for imaging results
Blastoma cell (SH-SY5Y), the probe has wide in the research of hERG potassium-channel physiology, pathology and relevant disease
Application prospect.
Although the above-mentioned accompanying drawing that combines is described to the specific embodiment of the present invention, not to invention protection domain
Restriction, one of ordinary skill in the art should be understood that on the basis of technical scheme those skilled in the art are not required to
The various modifications or deformation made by paying creative work are still within the scope of the present invention.
Claims (8)
1. a kind of small-molecule fluorescent probe of benzimidazole hERG potassium-channels, the general structure such as formula of the fluorescent probe
(I) shown in：
In formula, R1For the monosubstituted or multi-substituent of hydroxyl, halogen, alkyl or alkoxyl；R2For benzene-naphthalene diimide class, NBD classes,
Cy5 classes and Fluorescein isothiocyanate class fluorogen；N=1-6.
2. small-molecule fluorescent probe according to claim 1, it is characterised in that：The R1For para-position halogen；N=4.
3. small-molecule fluorescent probe according to claim 2, it is characterised in that：The small-molecule fluorescent probe is selected to be had
The compound of following structural formula：
4. the preparation method of the arbitrary small-molecule fluorescent probe of claim 1-3, comprises the following steps：
(1) preparation of recognition group：2-Chlorobenzimidazole in the basic conditions, and is reacted fluorobenzyl bromide, generate 1- to luorobenzyl-
2-Chlorobenzimidazole, then react under conditions of 150～180 DEG C of microwave with 4- amino -1- ethoxycarbonyls phenylpiperidines, generate 4-
((1- is to luorobenzyl -1H-2- benzimidazolyls) amino) -1- piperidine ethyl formates, then be 30%～60% hydrogen with mass fraction
Bromic acid backflow generates 1- to luorobenzyl-N- (4- piperidyls) -1H-2- aminobenzimidazole bromates, then with N- (4- bromine fourths
Base) phthalimide reacts in the basic conditions, generates N- (4- ((4- ((1- is to luorobenzyl -1H-2- benzimidazolyls)
Amino) 1- piperidines) butyl) phthalimide, then be that 70%～80% hydration hydrazine reaction obtains N- (1- with mass fraction
(4- ammonia butyl) piperidyl) -1- is to luorobenzyl -1H-2- aminobenzimidazoles；
(2) preparation of probe molecule：Various fluorogens are prepared, carboxyl is introduced on fluorogen, then with N- (1- (4- ammonia butyl)
Piperidyl) -1- by condensation reaction, is obtained probe molecule to luorobenzyl -1H-2- aminobenzimidazoles；Or on fluorogen
Halogen is introduced, substitution reaction, system occur to luorobenzyl -1H-2- aminobenzimidazoles with N- (1- (4- ammonia butyl) piperidyl) -1-
Standby probe molecule.
5. preparation method according to claim 4, it is characterised in that：In the step (1), 2-Chlorobenzimidazole, to fluorine
The mol ratio of the addition of benzyl bromine and 4- amino -1- ethoxycarbonyl phenylpiperidines is 0.5～1.2：1～1.8：1～2.
6. preparation method according to claim 4, it is characterised in that：In the step (1), 4- amino -1- ethoxycarbonyls
The mol ratio that phenylpiperidines, hydrobromic acid, N- (4- brombutyls) phthalimides and hydrazine hydrate are added is 1～1.8：50～
70：1.2～2：60～80.
7. preparation method according to claim 4, it is characterised in that：In the step (1), return time is 30～40h.
8. high pass of the arbitrary benzimidazole small-molecule fluorescent probe of claim 1-3 in hERG potassium channel inhibitors
Amount screening and the application in new drug cardiac toxicity is evaluated.
Priority Applications (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510224724.9A CN104910894B (en) | 2015-05-05 | 2015-05-05 | Benzimidazole hERG potassium ion channel small-molecular fluorescent probe and preparation method and applications thereof |
Applications Claiming Priority (1)
| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN201510224724.9A CN104910894B (en) | 2015-05-05 | 2015-05-05 | Benzimidazole hERG potassium ion channel small-molecular fluorescent probe and preparation method and applications thereof |
Publications (2)
| Publication Number | Publication Date |
|---|---|
| CN104910894A CN104910894A (en) | 2015-09-16 |
| CN104910894B true CN104910894B (en) | 2017-05-03 |
Family
ID=54080349
Family Applications (1)
| Application Number | Title | Priority Date | Filing Date |
|---|---|---|---|
| CN201510224724.9A Active CN104910894B (en) | 2015-05-05 | 2015-05-05 | Benzimidazole hERG potassium ion channel small-molecular fluorescent probe and preparation method and applications thereof |
Country Status (1)
| Country | Link |
|---|---|
| CN (1) | CN104910894B (en) |
Families Citing this family (4)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| CN107011444B (en) * | 2016-01-28 | 2021-11-16 | 中国科学院脑科学与智能技术卓越创新中心 | Method for screening hERG potassium ion channel agonist and detecting toxicity |
| CN113444047B (en) * | 2016-07-20 | 2023-06-16 | 纳莹(上海)生物科技有限公司 | Fluorescent probe and preparation method and application thereof |
| CN106701061B (en) * | 2016-11-15 | 2019-01-29 | 山东大学 | A kind of GPR120 small-molecule fluorescent probe and its application |
| CN109293673A (en) * | 2018-10-10 | 2019-02-01 | 中国药科大学 | A kind of prolyl hydroxylase small-molecule fluorescent probe and preparation method thereof |
Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4588722A (en) * | 1984-01-09 | 1986-05-13 | Janssen Pharmaceutica N.V. | N-(4-piperidinyl) bicyclic condensed 2-imidazolamine derivatives |
| CN103012381A (en) * | 2013-01-10 | 2013-04-03 | 山东大学 | Benzofuran compound, preparation method thereof and application of benzofuran compound in preparation of antiarrhythmic drugs |
| CN104449670A (en) * | 2014-11-11 | 2015-03-25 | 山东大学 | Micro-molecule fluorescent probe of phenyl furan hERG potassium channel and application thereof |
Family Cites Families (1)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2015007615A1 (en) * | 2013-07-18 | 2015-01-22 | Fondazione Istituto Italiano Di Tecnologia | Phenyl carbamates and their use as inhibitors of the fatty acid amide hydrolase (faah) enzyme and modulators of the d3 dopamine receptor (d3dr) |
-
2015
- 2015-05-05 CN CN201510224724.9A patent/CN104910894B/en active Active
Patent Citations (3)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| US4588722A (en) * | 1984-01-09 | 1986-05-13 | Janssen Pharmaceutica N.V. | N-(4-piperidinyl) bicyclic condensed 2-imidazolamine derivatives |
| CN103012381A (en) * | 2013-01-10 | 2013-04-03 | 山东大学 | Benzofuran compound, preparation method thereof and application of benzofuran compound in preparation of antiarrhythmic drugs |
| CN104449670A (en) * | 2014-11-11 | 2015-03-25 | 山东大学 | Micro-molecule fluorescent probe of phenyl furan hERG potassium channel and application thereof |
Non-Patent Citations (2)
| Title |
|---|
| "A Short Synthesis of Astemizole";C. Parrodi et al.，;《SYNTHETIC COMMUNICATIONS》;20060821;第26卷(第17期);第3323-3329页 * |
| "Kv11.1 (hERG)-induced cardiotoxicity: a molecular insight from a binding kinetics study of prototypical Kv11.1 (hERG) inhibitors";Z. Yu et al.，;《British Journal of Pharmacology》;20141215;第172卷(第3期);第940-955页 * |
Also Published As
| Publication number | Publication date |
|---|---|
| CN104910894A (en) | 2015-09-16 |
Similar Documents
| Publication | Publication Date | Title |
|---|---|---|
| CN107540608B (en) | 4-substituted naphthalimide compound and application thereof | |
| CN104910894B (en) | Benzimidazole hERG potassium ion channel small-molecular fluorescent probe and preparation method and applications thereof | |
| CN107174584B (en) | Application of piperazine structure-containing compound in preparation of LSD1 inhibitor | |
| CN110526881A (en) | A kind of naphthylamine compounds and its acceptable salt of biology, preparation method and application | |
| JP2021522317A (en) | Histone Acetyltransferase (HAT) Inhibitors and Their Applications | |
| CN104449670A (en) | Micro-molecule fluorescent probe of phenyl furan hERG potassium channel and application thereof | |
| CN109776379A (en) | It is a kind of to can be used for responding the near infrared fluorescent probe and preparation method thereof that in living cells and pH changes in chronic wounds development process | |
| CN111423379B (en) | Substituted 3-indazole Mcl-1 protein inhibitor, preparation method and application | |
| CN105062465B (en) | The α of a kind of environment sensitive type1Adrenergic receptor near-infrared fluorescent ligand and its application | |
| JP6021148B2 (en) | Compound transported by LST-1 and / or LST-2 | |
| CN103664876B (en) | Quinoline derivatives and uses thereof | |
| CN105924403A (en) | Cyclomalonamide compound with antitumor activity, and preparation method and application thereof | |
| CN102277154A (en) | Phenyl piperazine small-molecule fluorescent probe of alpha1-adrenergic receptor and application thereof | |
| CN115677545A (en) | anti-HBV sulfanilamide benzamide derivative and preparation method and application thereof | |
| CN106543194A (en) | Narciclasine derivative and its preparation and the application in antineoplastic is prepared | |
| WO2020246616A1 (en) | Fluorescent probe for detection of enpp activity | |
| JP7410567B2 (en) | New non-fluorescent rhodamines | |
| CN115043833B (en) | Bcl-2 fluorescent probe and preparation method and application thereof | |
| JP7126547B2 (en) | 2-oxo-1-pyrrolidinyl imidazothiadiazole derivatives | |
| CN111606888B (en) | Pyrrole derivative and preparation method and application thereof | |
| WO2016197959A1 (en) | Compound for detecting dihydroorotate dehydrogenase | |
| CN107207480A (en) | Coumarin based compounds and correlation technique | |
| CN105778896B (en) | Phenyl（4 piperidyls）The small-molecule fluorescent probe of ketone class hERG potassium-channels | |
| CN110343088A (en) | A kind of derivative and its preparation method and application based on PARP inhibitor Niraparib | |
| CN112300106B (en) | Protein kinase inhibitor, preparation method and application |
Legal Events
| Date | Code | Title | Description |
|---|---|---|---|
| C06 | Publication | ||
| PB01 | Publication | ||
| C10 | Entry into substantive examination | ||
| SE01 | Entry into force of request for substantive examination | ||
| GR01 | Patent grant | ||
| GR01 | Patent grant |