CN103694268A - Kinase activity probe and method for treating related diseases - Google Patents
Kinase activity probe and method for treating related diseases Download PDFInfo
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- CN103694268A CN103694268A CN201310689503.XA CN201310689503A CN103694268A CN 103694268 A CN103694268 A CN 103694268A CN 201310689503 A CN201310689503 A CN 201310689503A CN 103694268 A CN103694268 A CN 103694268A
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Images
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Abstract
The invention provides a kinase activity probe and a method for treating related diseases. The activity of bruton's tyrosine kinase (Btk) can be well inhibited by the kinase activity probe, and the kinase activity probe and cysteine residues at an ATP (adenosine triphosphate) binding bag in a kinase structure domain of the Btk form covalent binding. By adopting the kinase activity probe, the Btk can be marked in vitro, cells or tissues expressing the Btk can be marked in vitro, and the efficacy of a Btk inhibitor in an animal can be evaluated.
Description
Technical field
The present invention relates to the method for kinase activity probe and treatment relative disease.
Background technology
Protein kinase is one of gene family maximum in eukaryote, is just widely studied.There is at present more than the 500 kind of kinases of surpassing to be found, accounted for 2% of Human genome.The electronegative phosphate group that it shifts ATP γ position by catalysis, to its substrate protein, is modified its biological activity, thereby is regulating most signal transduction in eukaryotic cell.Kinases is being controlled a lot of cellular processes, as metabolism, transcribe, cell cycle, cytoskeleton reset and activity, apoptosis and the differentiation etc. of cell, thereby as cancer, chronic inflammatory diseases close relation, become in the past few decades the popular drug target that people competitively chase with human diseases.Most of kinases target spots are relevant to cancer, and the imbalance of kinase function is also with relevant such as the disease of immunity, nerve, metabolism, infection aspect, thereby people have lighted the interest of this serial disease of development small molecules kinase inhibitor for treating.
Bruton's tyrosine kinase (Bruton ' s tyrosine kinase, Btk) be a class nonreceptor tyrosine kinase that belongs to Tec family, in all hematopoietic cells except T cell, natural killer cell and plasmocyte, there is expression.Btk has very important effect to the growth of B cell, differentiation and signal transduction.The expression of Btk is considered to the key condition of B cell proliferation and survival.The genovariation of Btk in human body can cause the disappearance of recycle system bone-marrow-derived lymphocyte, because can not produce the immunoglobulin (Ig) of all categories, therefore can not increase people's humoral immune reaction.This basic immune deficiency is called as the third blood cell deficiency disease (XLA) that X is relevant.
The molecule mechanism that Btk is activated in cell is not also illustrated at present completely.It is generally acknowledged, posttranslational modification and subcellular being positioned at have wherein been brought into play vital role.In steady state, Btk is mainly in kytoplasm, once B-cell receptor participates in signal enabling, Btk will be transferred to cytolemma and be activated by upstream Src family kinase Blk, Lyn and Fyn, its Tyr551 that is positioned at kinase domain can be by upstream kinases Lyn phosphorylation, and an autophosphorylation process that comprises the Tyr223 on SH3 structural domain can occur Btk subsequently.Subsequently, Btk is by linking the close cooperation of albumen (BLNK), phosphorylated substrate albumen Phospholipase C γ 2(PLC γ 2 with its adaptin B cell), thus transfer, the activation of transcription factor NF-KB and the map kinase signal path of calcium ion caused.
The activation of B cell antigen receptor (BCR) signalling channel is for the activation of B cell malignancies and autoimmune disease and maintain it and vital role.In the mutating experiment of people and mouse, confirmed that Btk is very crucial for BCR signalling channel.Therefore, for the selectivity of B cell, to suppress be an attractive especially kinases target spot to Btk.Separately confirm on evidence that BCR signal path playing an important role aspect the growth of non-Hodgkin lymphoma (NHL) tumour cell.Recently, Btk is determined to be the signal kinases of the disperse large B cell lymphoid tumor survival of subtype.
B lymph Tyrosylprotein kinase (B lymphoid tyrosine kinase, Blk) is the nonreceptor tyrosine kinase relevant with bone-marrow-derived lymphocyte growth, differentiation and signal.B-cell receptor signalling channel (BCR) needs closely cooperating between several Tyrosylprotein kinases, phosphoesterase and corresponding coreceptor.Antigen is combined on B cell antigen receptor (BCR) can triggering signal, finally causes B cell activation.Blk is in the signal transmission by surface immumoglobulin and help pro-B to play the part of key player in the transition of pre-B, and plays respective action in the retarded growth of B-cell receptor and apoptosis.The activation of the NF-κ B that Blk connection mediates for pre-B cell receptor (pre-BCR) with Fyn and Lyn plays keying action.
EGF-R ELISA (Epidermal Growth Factor Receptor, EGFR) is the acceptor of epidermal growth factor (EGF) cell proliferation and signal transduction.EGFR belongs to a kind of of ErbB receptor family, is also known as HER1, ErbB1, suddenlys change or crosses to express and generally can cause tumour.After EGFR dimerization, can activate it and be positioned at intracellular kinase pathway, comprise that Tyr992, Tyr1045, Tyr1068, Tyr1148 and Tyr1173 etc. activate site.This autophosphorylation can guide the phosphorylation in downstream, comprises MPAK, Akt and JNK path, brings out cell proliferation.
Research shows to exist high expression level or the unconventionality expression of EGFR in many noumenal tumours.EGFR is relevant with propagation, vasculogenesis, tumor invasion, transfer and the apoptotic inhibition of tumour cell.Its mechanism has: the high expression level of EGFR causes the enhancing of downstream signal conduction; The increase of mutant egf R acceptor or ligand expression causes the continuous activation of EGFR; The effect of autocrine loop strengthens; The destruction of receptor down-regulated mechanism; The activation of abnormal signal conduction path etc.The expression of crossing of EGFR plays an important role in the evolution of malignant tumour, has crossing of EGFR to express in the tissues such as spongiocyte, kidney, lung cancer, prostate cancer, carcinoma of the pancreas, mammary cancer.
Summary of the invention
The invention provides the compound of a kind of formula (I), or its medicinal acceptable salt:
Wherein, Y is fluorophore, is used as record cell,
W forms with L the link unit that is connected Btk inhibitor parent and fluorophore jointly,
X is selected from C
1-3alkyl, halogen, methoxyl group, and trifluoromethyl,
R is selected from trifluoromethyl, halogen, and methoxyl group, and N, N-dimethyl,
W is selected from
-NR
1-R
2-ZR
3-, and
Wherein
R
1, R
2and R
3independently of one another, in the same manner or be differently selected from key, H, and C
1-6alkyl,
Z is selected from C and N,
L is selected from key, C
1-10alkyl,
-OC (O)-,-NR
ac (O)-,-NR
a-,-O-,-S-,-S-S-,-O-NR
a-,-OC (O) O-,-OC (O) NR
a-,-NR
ac (O) NR
a-,-N=CR
a-,-SC (O)-,-S (O)-, and-S (O)
2-, or the combination between above unit,
Wherein
R
afor H or C
1-6alkyl,
Y is selected from BODIPY493/503, BODIPY FL, BODIPY R6G, BODIPY530/550, BODIPY TMR, BODIPY558/568, BODIPY564/570, BODIPY576/589, BODIPY581/591, BODIPY TR, Fluorescein, 5 (6)-Carboxyfluorescein, 2, 7-Dichlorofluorescein, N, N-Bis (2, 4, 6-trimethylphenyl), HPTS, Ethyl Eosin, DY-490XL MegaStokes, DY-485XL MegaStokes, Adirondack Green520, ATTP 465, ATTO488, ATTO495, YOYO-1, 5-FAM, BCECF, dichlorofluorescein, rhodamin110, rhodamine123, Rhodamine Green, YO-PRO-1, SYTOX Green, Sodium Green, SYBR Green I, Alexa Fluor500, Fluo-3, Fluo-4, fluoro-emerald, YoYo-1ssDNA, YoYo-1dsDNA, YoYo-1, SYTO RNASelect, Diversa Green-FP, Dragon Green, EvaGreen, Surf Green EX, Spectrum Green, Oregon Green488, Neuro Trace500525, NBD-X, MitoTracker Green FM, LysoTracker Green DND-26, CBQCA, PA-GFP (post-activation), FIASH-CCXXCC, Azami Green monomeric, Azami Green, EGFP (Campbell Tsien2003), EGFP (Patterson2001), Fluorescein, Kaede Green, 7-Benzylamino-4-Nitrobenz-2-Oxa-1, 3-Diazole, Bex1, Doxorubicin, Lumio Green, with SuperGlo GFP.
In a preferred embodiment,
X is selected from methyl, trifluoromethyl, and chlorine,
R is trifluoromethyl or chlorine,
W is selected from piperazine, piperidines, and-N (CH
3) (CH
2)
2nH-,
Be selected from-(CH of L
2)
mnHC (O) (CH
2)
n-,-(CH
2)
mnHC (O) NH (CH
2)
n-,
-(CH
2)
moC (O) (CH
2)
n-,-NHC (O) (CH
2)
mnHC (O) (CH
2)
n-, and
-C(O)NH(CH
2)
mNHC(O)(CH
2)
n-，
Wherein
M, n independently of one another, in the same manner or is differently selected from integer 0-10,
Y is selected from BODIPY493/503, BODIPY FL, BODIPY R6G, BODIPY530/550, BODIPY TMR, BODIPY558/568, BODIPY564/570, BODIPY576/589, BODIPY581/591, BODIPY TR.
In a kind of preferred embodiment of another kind,
X is methyl or chlorine,
R is trifluoromethyl or chlorine,
W is piperazine or piperidines,
L is-(CH
2)
mnHC (O) (CH
2)
n-or-NHC (O) (CH
2)
mnHC (O) (CH
2)
n-
Wherein
M, n independently of one another, in the same manner or is differently selected from integer 1-4,
Y is BODIPY FL,
The invention provides a kind of compound:
The invention provides a kind of pharmaceutical composition, comprising compound of the present invention and medicinal acceptable vehicle.
The invention provides and a kind ofly comprise that the compounds of this invention or medicinal compositions comprise Btk for external mark, separation, Blk, or the albumen of EGFR.
The invention provides a kind of the compounds of this invention or medicinal compositions of comprising for external marker expression Btk, Blk, or the cell or tissue of EGFR.
The invention provides and a kind ofly comprise that the compounds of this invention or medicinal compositions are for assessment of a kind of Btk inhibitor, or a kind of Blk inhibitor, or the drug effect of a kind of EGFR inhibitor in Mammals.
Described here is the active probe of bruton's tyrosine kinase (Bruton ' s tyrosine kinase, Btk).The active probe of the Btk that it is comprised of irreversible Btk inhibitor, link unit and record cell.This active probe can form covalent linkage with the cysteine residues at ATP binding pocket place with Btk kinase domain.This active probe also can be used as the probe of other Tyrosylprotein kinase, particularly have with Btk kinase domain in Cys481 identical bits be equipped with the kinases of halfcystine.For example, this active probe also can with B lymph Tyrosylprotein kinase (B lymphoid tyrosine kinase, Blk) halfcystine at ATP binding pocket place forms covalent attachment and in EGF-R ELISA (Epidermal Growth Factor Receptor, EGFR) kinase domain.The present invention has also described the method for synthetic this type of probe and this type of active probe has been applied to study Btk and other is as the method for the Tyrosylprotein kinases such as Blk, EGFR.
Link unit in this Btk active probe is covalently bound inhibitor unit and record cell, its link unit can be selected from key, the alternative alkyl replacing, tertiary amine unit, the alternative heterocycle replacing, the alternative acid amides replacing, ketone, alternative carbamate, the ester replacing, or the combination between above-mentioned these unit.In another embodiment, the link unit in this Btk active probe comprises the heterocyclic units that can be replaced by selectivity.In a further embodiment, the heterocyclic units that the alternative in this Btk active probe replaces comprises the unit based on piperazine or piperidines.
What be here described equally is that record cell in this Btk active probe can be selected from following component, comprise a kind of label, a kind of fuel, a kind of photocrosslinking agent, a kind of to the virose compound of cell, a kind of medicine, a kind of affinity tag, a kind of smooth affinity tag, a kind of active compound, a kind of antibody or antibody fragment, a kind of biomaterial, a kind of nanometer ball, a kind of spin label, a kind of fluorophore, the metallic unit of a kind of bag, a kind of radioactive cells, a kind of new functional group, a kind of can be covalently or non-covalently and the group of other molecularity, a kind of photon cage unit, a kind of actinic radiation excites unit, a kind of part, a kind of photon isomerization unit, vitamin H, vitamin H analogue, a kind of unit that comprises heavy atom, can be by the group of chemical cracking, a kind of reagent with redox active, a kind of isotope-labeled unit, a kind of physiology probe, a kind of phosphorescent group, a kind of chromophoric group, a kind of energy transfering reagent, a kind of bioactive agents, a kind of label that can be detected, or the combination between said components.In another embodiment, the fluorophore of this Btk active probe is BODIPY fluorophor.In embodiment further, the BODIPY fluorophore of this Btk active probe is BODIPY FL fluorophore.
The inhibitor unit of this Btk active probe of showing is here from a kind of Btk covalency irreversible inhibitor.In one embodiment, the chemical structure of the irreversible Btk inhibitor in this Btk active probe is:
This Btk active probe has following structure in another embodiment:
In a further embodiment, this Btk active probe phosphorylation conformation of mark Btk optionally.In another embodiment, the Btk conformation of this Btk active probe phosphorylation can be the active or nonactive conformation of Btk.In a further embodiment, the conformation of the Btk of this Btk active probe phosphorylation is the nonactive conformation of Btk.In one embodiment, this Btk active probe has permeates cell membranes ability.
On the one hand, this Btk active probe can be used to assess the effect of a kind of potential Btk inhibitor in Mammals, comprise to Mammals and take in a kind of potential Btk inhibitor, this Btk active probe of cellular uptake of giving Mammals or separating from Mammals; Measure the activity of the record cell of this Btk active probe, and the activity of record cell is compared with a kind of standard.
On the other hand, this active probe can be used to assess the drug effect of a kind of Btk inhibitor in Mammals, comprise to Mammals and take in a kind of Btk inhibitor, maybe this Btk active probe is taken in to Mammals or separated cell from Mammals, then after taking in a kind of Btk inhibitor, at different time points, detect the activity of the record cell on this Btk active probe.
Further, this invention can be used to external mark Btk enzyme.In one embodiment, can, by Btk enzyme is cultivated together with this Btk active probe, can reach the object of external mark Btk enzyme.
On the other hand, this active probe can be cultivated together with the cell or tissue with expression Btk, reaches the object of the cell or tissue of external marker expression Btk enzyme.
On the one hand, this Btk active probe can be used to the different albumen that mark separation comprises Btk enzyme, and the Btk enzyme in these albumen can, by this Btk active probe mark, can arrive by the Btk enzyme of this active probe mark with fluoroscopic examination by electrophoresis.
It is pointed out that method as described herein and component are not limited in certain specific method as described herein, operating process, cell strain, component and reagent, have corresponding variation in certain embodiments.It is pointed out that in addition terminology used here is only in order to describe specific embodiment, is not limited to method as described herein and compositional range, and only can be limited to separate statement.
Accompanying drawing explanation
Fig. 1 is that sample of the present invention carries out the fluorescent scanning figure after gel electrophoresis;
Wherein swimming lane b be compound 12 in conjunction with Blk, swimming lane c be compound 12 in conjunction with Btk, swimming lane f is that compound 12 is in conjunction with EGFR.
Embodiment
Concrete non-limiting example will be construed as merely illustrative below, and limit never in any form the disclosure.Although without describing in further detail, can believe that those skilled in the art can, based on description herein, utilize the disclosure completely.
Synthesizing of compound
Synthetic schemes I
Step 1:
By compound 1(0.270g, 1.69mmol) be mixed in 12mL acetonitrile with the chloro-5-nitro-pyrimidine of 2-(0.352g, 1.69mmol), add subsequently salt of wormwood (0.702g, 5.08mmol).Reaction solution at room temperature stirred after 3 hours, concentrating under reduced pressure, and in water and ethyl acetate separatory.Organic phase is yellow solid with using anhydrous magnesium sulfate drying, concentrating under reduced pressure to obtain thick product after saturated common salt water washing again.This thick product and 5%Pd/C are placed in to 10mL methyl alcohol, after system emptying air, under atmosphere of hydrogen, stirring at room is 5 hours, filters, column chromatography for separation after filtrate is concentrated (normal hexane: ethyl acetate=1:1) obtain compound 3(0.400g) is yellow solid, and two step productive rates are 78%.
1H?NMR(400MHz,DMSO-d
6):δ9.13(s,1H),8.86(s,1H),7.95(s,2H),7.77(s,1H),7.25(d,1H,J=8.1Hz),7.05(t,1H,J=8.1Hz),6.92(d,1H,J=7.8Hz),4.77(s,2H),1.47(s,9H);
13CNMR(125MHz,DMSO-d
6):δ152.8,152.7,143.6,141.9,139.4,135.5,128.0,111.7,110.5,107.9,78.5,28.0;HRMS(ESI)m/z?calculated?for?C
15H
20N
5O
2 +[M+H]
+:302.1617,found:302.1602.
Step 2:
By compound 4(0.500g, 2.63mmol) be placed in 5mL SOCl
2in, reaction solution is under agitation heated to reflux and keeps 1 hour, is naturally cooled to subsequently room temperature, adds after the dilution with toluene that 15mL is dry, and reaction solution vacuum concentration obtains oily residue.This residue is dissolved in after 20mL methylene dichloride, under agitation adds 5-amino-2-methyl phenylformic acid (0.478g, 3.16mmol), adds subsequently two triethylamines.After end reaction liquid at room temperature stirs and spends the night, concentrating under reduced pressure, with saturated ammonium chloride solution and ethyl acetate separatory, organic phase is with using anhydrous magnesium sulfate drying after saturated common salt water washing again.After final organic phase concentrating under reduced pressure, column chromatography for separation (normal hexane: ethyl acetate=1:1) obtain compound 5(0.70g) is white solid, and two step productive rates are 82%.
1H?NMR(400MHz,DMSO-d
6):δ12.84(br?s,1H),10.48(s,1H),8.27-8.21(m,3H),7.92(d,1H,J=7.8Hz),7.85(dd,1H,J=2.0Hz,J=8.3Hz),7.74(t,1H,J=7.8Hz),7.25(d,1H,J=8.3Hz),(3H?under?the?solvent?peak).
Step 3:
By compound 3(0.400g, 1.33mmol), 5(0.430g, 1.33mmol) and HATU(0.760g, 2.00mmol) be mixed in 8mL DMF, add subsequently DIEA(0.65mL, 4.0mmol).This reaction solution at room temperature stirred after 5 hours, added shrend and went out, with ethyl acetate and saturated sodium bicarbonate solution separatory.Organic phase is with after saturated common salt water washing, and with anhydrous magnesium sulfate drying, it is white solid that concentrating under reduced pressure obtains thick product.This thick product is dissolved in the mixing solutions of 3mL trifluoroacetic acid and 3mL methylene dichloride, stirring at room is concentrating under reduced pressure after 3 hours, with saturated sodium bicarbonate solution and ethyl acetate separatory, organic phase is with using anhydrous magnesium sulfate drying after the washing of saturation experiments chamber again, column chromatography for separation (normal hexane: ethyl acetate=1:1) obtain compound 7(0.48g) is yellow solid, and two step productive rates are 72%.
1H?NMR(500MHz,DMSO-d
6):δ10.56(s,1H),10.36(s,1H),9.30(s,1H),8.76(s,2H),8.33(s,1H),8.28(d,1H,J=7.9Hz),7.98(d,1H,J=7.8Hz),7.93(d,1H,J=2.0Hz),7.85(dd,1H,J=2.0Hz,J=8.3Hz),7.80(t,1H,J=7.9Hz),7.33(d,1H,J=8.4Hz),7.07(s,1H),6.89(t,1H,J=7.8Hz),6.84(d,1H,J=8.1Hz),6.17(d,1H,J=7.6Hz),4.94(s,2H),2.39(s,3H);
13C?NMR(100MHz,DMSO-d
6):δ167.5,163.9,156.8,149.8,148.8,141.1,136.5,136.4,135.5,131.8,130.9,130.8,129.8,129.3,129.0,128.6,128.2,125.8,124.1,121.7,119.2,107.7,107.0,104.2,18.8;HRMS(ESI)m/z?calculated?for?C
26H
20F
3N
6O
2 -[M-H]
-:505.1600,found:505.1591.
Step 4:
By compound 8(0.100g, 0.606mmol) be scattered in 2mL SOCl
2in, reaction solution is under agitation warming up to and reflux keeps, after 1 hour, naturally cooling to room temperature, add the toluene that 10mL is dry after concentrating under reduced pressure obtain oily residue.This oily matter is dissolved in to 5mL methylene dichloride, adds subsequently compound 7(0.304g, 0.600mmol) and DIEA(0.15mL, 0.90mmol).Reaction solution is in stirring at room after 1 hour, and concentrating under reduced pressure, uses saturated sodium bicarbonate solution and ethyl acetate separatory, and organic phase is with using anhydrous magnesium sulfate drying after saturated common salt water washing again.After final organic phase is concentrated, using column chromatography (normal hexane: ethyl acetate=2:1) obtain compound 9(0.32g) is white solid, and two step productive rates are 81%.
Step 5:
By compound 10(0.400g, 1.75mmol), 9(0.286g, 0.438mmol) be mixed in the THF that 5mL is dry, reaction solution is under agitation warming up to 50 ℃ and spends the night.Following reaction liquid temp is down to room temperature, with methylene dichloride and saturated sodium bicarbonate separatory.Organic phase is used after saturated common salt water washing, then uses anhydrous magnesium sulfate drying, and concentrated rear column chromatography for separation (methylene dichloride: methyl alcohol=10:1) obtain compound 11(0.24g) is yellow solid, and productive rate is 68%.
Step 6:
By compound 11(0.120g, 0.150mmol) be dissolved in 2mL methylene dichloride and 2mL trifluoroacetic acid mixing solutions, this reaction solution is in stirring at room after 1 hour, and concentrating under reduced pressure obtains brown oil.This oily matter is dissolved in 4mL DMF, adds subsequently BODIPY FL(0.044g, 0.15mmol) and DIEA(73 μ L, 0.45mmol).Reaction solution at room temperature stirred after 5 hours, with methylene dichloride and saturated sodium bicarbonate separatory, organic phase saturated common salt water washing, with anhydrous magnesium sulfate drying, after concentrating under reduced pressure, use column chromatography to obtain compound 12(0.089mg) be garnet solid, two step productive rates are 61%.
1H?NMR(400MHz,DMSO-d
6):δ10.69(s,1H),10.48(s,1H),10.15(s,1H),9.66(s,1H),8.82(s,2H),8.44(s,1H),8.33(d,2H,J=10.9Hz),8.09(s,1H),7.97(d,2H,J=8.2Hz),7.86(dd,1H,J=1.7Hz,J=8.2Hz),7.80(t,1H,J=7.8Hz),7.69(s,1H),7.38-7.32(m,3H),7.19(t,1H,J=8.0Hz),7.09(d,1H,J=3.8Hz),6.71-6.68(m,1H),6.50-6.37(m,2H),6.29(s,1H),3.48(s,6H),3.21-2.95(m,12H),2.46(s,3H),2.39(s,3H),2.25(s,3H).
13C?NMR(100MHz,DMSO-d
6):δ169.4,165.8,161.1,158.4,151.7,142.8,141.0,138.4,138.3,137.3,136.3,134.9,133.8,132.8,132.7,131.7,131.6,130.8,130.5,130.2,128.3,127.2,126.1,122.2,121.2,118.4,114.6,111.5,66.8,53.1,51.0,47.2,35.6,25.7,20.7,16.4,12.9,10.3.HRMS(ESI)m/z?calculated?for?C
50H
52BF
5N
11O
4 +[M+H]
+:976.4217,found:976.4194.
The external labelling experiment of kinase activity probe
In the external labelling experiment of protein kinase, can detect by method as described below or similar approach selectivity and the labeling effciency of kinase activity probe.
Prepare respectively Btk, Blk, and each 20 μ L of the HEPES buffered soln (PH=7.4) of EGFR, concentration is all adjusted to 1 μ M.In three kinds of kinase whose buffered soln, add respectively the DMSO solution of the compound 12 that 0.2 μ L concentration is 1mM, at 4 ℃, cultivate 3 hours.Sample and sample loading buffer are mixed, and 100 ℃ are heated 3 minutes.Get polyacrylamide precast glue (
12%), with being placed in electrophoresis chamber after electrophoretic buffer washing sample groove.Application of sample, adjusts voltage to 150V, deenergization when dyestuff arrives at separation gel bottom.Film, from electrophoresis chamber takes out, with deionized water wash, is placed in fluorescent scanning instrument, with the fluorescence exciting wavelength of BODIPY, excites, scans, detects, and with software Quantity One, carries out data results.
The foregoing is only preferred embodiment of the present invention; not in order to limit the present invention; anyly be familiar with those skilled in the art in the technical scope that the present invention discloses, the variation that can expect easily or replacement, all should be encompassed in protection scope of the present invention in.Therefore, protection scope of the present invention should be as the criterion with the protection domain that claim was defined.
Claims (8)
1. a compound for formula (I), or its medicinal acceptable salt:
Wherein, Y is fluorophore, is used as record cell,
W forms with L the link unit that is connected Btk inhibitor parent and fluorophore jointly,
X is selected from C
1-3alkyl, halogen, methoxyl group, and trifluoromethyl,
R is selected from trifluoromethyl, halogen, and methoxyl group, and N, N-dimethyl,
W is selected from
-NR
1-R
2-ZR
3-, and
Wherein
R
1, R
2and R
3independently of one another, in the same manner or be differently selected from key, H, and C
1-6alkyl,
Z is selected from C and N,
L is selected from key, C
1-10alkyl,
-OC (O)-,-NR
ac (O)-,-NR
a-,-O-,-S-,-S-S-,-O-NR
a-,-OC (O) O-,-OC (O) NR
a-,-NR
ac (O) NR
a-,-N=CR
a-,-SC (O)-,-S (O)-, and-S (O)
2-, or the combination between above unit,
Wherein
R
afor H or C
1-6alkyl,
Y is selected from BODIPY493/503, BODIPY FL, BODIPY R6G, BODIPY530/550, BODIPY TMR, BODIPY558/568, BODIPY564/570, BODIPY576/589, BODIPY581/591, BODIPY TR, Fluorescein, 5 (6)-Carboxyfluorescein, 2, 7-Dichlorofluorescein, N, N-Bis (2, 4, 6-trimethylphenyl), HPTS, Ethyl Eosin, DY-490XL MegaStokes, DY-485XL MegaStokes, Adirondack Green520, ATTP 465, ATTO488, ATTO495, YOYO-1, 5-FAM, BCECF, dichlorofluorescein, rhodamin110, rhodamine123, Rhodamine Green, YO-PRO-1, SYTOX Green, Sodium Green, SYBR Green I, Alexa Fluor500, Fluo-3, Fluo-4, fluoro-emerald, YoYo-1ssDNA, YoYo-1dsDNA, YoYo-1, SYTO RNASelect, Diversa Green-FP, Dragon Green, EvaGreen, Surf Green EX, Spectrum Green, Oregon Green488, Neuro Trace500525, NBD-X, MitoTracker Green FM, LysoTracker Green DND-26, CBQCA, PA-GFP (post-activation), FIASH-CCXXCC, Azami Green monomeric, Azami Green, EGFP (Campbell Tsien2003), EGFP (Patterson2001), Fluorescein, Kaede Green, 7-Benzylamino-4-Nitrobenz-2-Oxa-1, 3-Diazole, Bex1, Doxorubicin, Lumio Green, with SuperGlo GFP.
2. according to the compound of claim 1, or pharmaceutically acceptable acceptable salt, wherein
X is selected from methyl, trifluoromethyl, and chlorine,
R is trifluoromethyl or chlorine,
W is selected from piperazine, piperidines, and-N (CH
3) (CH
2)
2nH-,
Be selected from-(CH of L
2)
mnHC (O) (CH
2)
n-,-(CH
2)
mnHC (O) NH (CH
2)
n-,
-(CH
2)
moC (O) (CH
2)
n-,-NHC (O) (CH
2)
mnHC (O) (CH
2)
n-, and
-C(O)NH(CH
2)
mNHC(O)(CH
2)
n-，
Wherein
M, n independently of one another, in the same manner or is differently selected from integer 0-10,
Y is selected from BODIPY493/503, BODIPY FL, BODIPY R6G, BODIPY530/550, BODIPY TMR, BODIPY558/568, BODIPY564/570, BODIPY576/589, BODIPY581/591, BODIPY TR.
3. according to the compound of claim 1, or the salt of pharmaceutically acceptable acceptance, wherein
X is methyl or chlorine,
R is trifluoromethyl or chlorine,
W is piperazine or piperidines,
L is-(CH
2)
mnHC (O) (CH
2)
n-or-NHC (O) (CH
2)
mnHC (O) (CH
2)
n-
Wherein
M, n independently of one another, in the same manner or is differently selected from integer 1-4,
Y is BODIPY FL.
4. a compound:
5. a pharmaceutical composition, comprising compound and the medicinal acceptable vehicle of any one in claim 1-4.
6. in claim 1-4, the compound of any one or the medicinal compositions in claim 5 comprise Btk, Blk, or the albumen of EGFR for external mark, separation.
7. in claim 1-4, the compound of any one or the medicinal compositions in claim 5 are used for external marker expression Btk, Blk, or the cell or tissue of EGFR.
In claim 1-4 the compound of any one or the medicinal compositions in claim 5 for assessment of a kind of Btk inhibitor, or a kind of Blk inhibitor, or the drug effect of a kind of EGFR inhibitor in Mammals.
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Cited By (5)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017063103A1 (en) * | 2015-10-12 | 2017-04-20 | Peking University Shenzhen Graduate School | Novel inhibitors and probes for kinases and uses thereof |
| CN109305944A (en) * | 2017-07-28 | 2019-02-05 | 深圳睿熙生物科技有限公司 | The inhibitor of bruton's tyrosine kinase |
| CN109422696A (en) * | 2017-09-04 | 2019-03-05 | 北京睿熙生物科技有限公司 | The inhibitor of bruton's tyrosine kinase |
| CN110201192A (en) * | 2019-06-05 | 2019-09-06 | 东华大学 | A kind of dendrimer package nanogold particle probe and its preparation and application |
| CN113234026A (en) * | 2021-03-28 | 2021-08-10 | 北京大学深圳研究生院 | Compound with B lymphocyte tyrosine kinase inhibitory activity and application thereof |
-
2013
- 2013-12-16 CN CN201310689503.XA patent/CN103694268A/en not_active Withdrawn
Cited By (11)
| Publication number | Priority date | Publication date | Assignee | Title |
|---|---|---|---|---|
| WO2017063103A1 (en) * | 2015-10-12 | 2017-04-20 | Peking University Shenzhen Graduate School | Novel inhibitors and probes for kinases and uses thereof |
| CN108779078A (en) * | 2015-10-12 | 2018-11-09 | 北京大学深圳研究生院 | New inhibitor and probe of kinases and application thereof |
| CN108779078B (en) * | 2015-10-12 | 2021-12-31 | 北京大学深圳研究生院 | Inhibitors and probes of kinases and uses thereof |
| CN109305944A (en) * | 2017-07-28 | 2019-02-05 | 深圳睿熙生物科技有限公司 | The inhibitor of bruton's tyrosine kinase |
| CN109305944B (en) * | 2017-07-28 | 2022-09-02 | 深圳睿熙生物科技有限公司 | Inhibitors of bruton's tyrosine kinase |
| CN109422696A (en) * | 2017-09-04 | 2019-03-05 | 北京睿熙生物科技有限公司 | The inhibitor of bruton's tyrosine kinase |
| CN109422696B (en) * | 2017-09-04 | 2020-10-30 | 北京睿熙生物科技有限公司 | Inhibitors of bruton's tyrosine kinase |
| CN110201192A (en) * | 2019-06-05 | 2019-09-06 | 东华大学 | A kind of dendrimer package nanogold particle probe and its preparation and application |
| CN110201192B (en) * | 2019-06-05 | 2021-08-31 | 东华大学 | A dendrimer-encapsulated gold nanoparticle probe and its preparation and application |
| CN113234026A (en) * | 2021-03-28 | 2021-08-10 | 北京大学深圳研究生院 | Compound with B lymphocyte tyrosine kinase inhibitory activity and application thereof |
| CN113234026B (en) * | 2021-03-28 | 2024-04-30 | 北京大学深圳研究生院 | Compounds with B lymphocyte tyrosine kinase inhibitory activity and their use |
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