CN103893182A

CN103893182A - SET protein small-molecule inhibitor and application thereof

Info

Publication number: CN103893182A
Application number: CN201410119076.6A
Authority: CN
Inventors: 陈依军; 谢伟全; 彭志刚; 王淑珍
Original assignee: China Pharmaceutical University
Current assignee: China Pharmaceutical University
Priority date: 2014-03-27
Filing date: 2014-03-27
Publication date: 2014-07-02

Abstract

The invention discloses an SET protein small-molecule inhibitor and an application of the SET protein small-molecule inhibitor in treatment of SET protein abnormal expression related diseases. SET protein is also known as a template activation factor-1(emplate activating factor, TAF-1), or protein phosphatise 2A inhibitor-2(inhibitor of protein phosphatase2A-2, I2PP2A)]. The SET protein small-molecule inhibitor disclosed by the invention is used for treating SET protein abnormal expression inflammation, neurodegenerative diseases, malignant tumours and autoimmune diseases, etc.

Description

One class SET protein micromolecular inhibitor and uses thereof

Technical field

The present invention relates to a class SET protein micromolecular inhibitor and the application in treatment SET protein abnormal expression relevant disease thereof.

Background technology

Along with human living standard's raising and the improvement of medical condition, the whole world average life span is greatly improved.But due to environmental pollution, the impact of the factors such as the prevailing and aged tendency of population of bad life, dietary habit, the sickness rate of the diseases such as cancer, senile dementia is soaring year by year trend.World Health Organization (WHO) report shows, within 2008, approximately there are 1,270 ten thousand newly-increased cancer patients in the whole world, and 760 die ten thousand deaths in cancer (account for all death tolls 13%).Before the year two thousand twenty, the whole world will increase 1,500 ten thousand cancer patients every year newly by inference, and to the year two thousand thirty, the death toll of cancer may increase to 1,310 ten thousand.Oneself becomes the second largest cause of the death that is only second to cardiovascular disease cancer.The chemotherapeutic agent for the treatment of cancer is mainly alkylating agent at present, cisplatin, methotrexate, 5-fluorouracil, gemcitabine, the cytotoxic drugs such as oxaliplatin, these antitumor drug are all nonspecific cell growth inhibitings and cause cell death, in killing tumor cell, to human normal cell, the normal structures such as bone marrow, digestive tract, liver, kidney are also brought infringement, and side effect is obvious, and therapeutic effect is limited.In recent years, along with the development of the cross disciplines such as molecular weight tumor, molecular pharmacology, made the mankind to the understanding of tumor gradually deeply.The research of antitumor drug is from traditional cytotoxic drug, turns to the molecular targeted antitumor drug for a certain important target (certain protein kinase often) in the generation evolution of the different subtype of different tumors and same tumor.Oncotherapy has entered targeted therapy and individualized treatment new stage.Tumor molecular targeted therapy is with its specificity, with strong points, and patient tolerability is better, successful, and the advantage such as toxicity is relatively low, has obtained immense success at therapeutic field of tumor, progressively becomes the new trend of domestic and international therapeutic field of tumor.The molecular targeted antitumor drug having gone on the market at present mainly comprises small molecular protein inhibitors of kinases and monoclonal antibody two classes.And all obtain proud military success, for R & D Enterprises has been created tremendous economic interests, wherein imatinib, Rituximab, bevacizumab, Herceptins etc. have become " cookle " that global annual sales amount is greater than 1,000,000,000 dollars.Protein kinase is the important action target spot of a class in current neoplasm targeted therapy.Distinguish from the site of their phosphorylations, they can be divided into tyrosine, silk, Serineprotein kinase two classes, and they can make respectively the tyrosine in protein, the hydroxyl phosphorylation on silk, threonine.Protein kinase is being played the part of very important effect in the growth course of cell, particularly participates in the transmission of cell signal.In a lot of diseases, the activity of protein kinase raises extremely.Corresponding with protein kinase is phosphoprotein phosphatase.Phosphoprotein phosphatase is also divided into tyrosine, silk, serine/threonine protein phosphatase.They can make the tyrosine of corresponding substrate (protein kinase), silk, threonine site dephosphorylation and inactivation.Can say that the balance of protein kinase/protein phosphatase enzymatic activity is to keeping the normally most important of cell function.A lot of diseases comprise cancer, inflammation, neurodegenerative disease, protein kinase activity extremely raise or protein phosphatase enzymatic activity extremely on the low side.Protein kinase/phosphoprotein phosphatase can be used as the important target spot of disease treatment.The main target spot of target therapeutic agent having gone on the market is at present exactly protein kinase.Although for the easily drug resistance because of the sudden change of protein kinase of medicine of protein kinase, be still at present a line targeted drug of the various malignant diseases for the treatment of.Chinese invention patent (patent No. 01138070.5) discloses a class 2-phenoxy pyrimidine derivates, and this compounds is exactly the kinase whose inhibitor of an albuminoid, can by Profilin kinases particularly tyrosine protein kinase treat relevant various diseases.This patent discloses the inhibition of 17 kinds of compounds to Abl protein kinase, and half-inhibition concentration (IC50) is at 0.025-2.0 μ M.Although the compound in foregoing invention has obvious inhibitory action to tyrosine protein kinase, in this patent documentation, the proliferation activity of these compound inhibition tumor cells is not yet verified, practical use the unknown.Present patent application inventor finds that they do not demonstrate stronger inhibition in the test proliferative activity o f tumor experiment of after this carrying out.For improving foregoing invention, present patent application inventor has carried out structure of modification to 2-phenoxy pyrimidine derivates, has obtained a kind of phenoxy pyrimidine derivates and has applied for Chinese invention patent (application number 200810244308.5).Find that this compounds has stronger inhibitory action to different tumor cells, the active patent No. that is obviously better than is 01138070.5 the disclosed 2-phenoxy pyrimidine derivates of Chinese patent.Further vitro kinase experiment finds that phenoxy pyrimidine derivates is very weak to the inhibitory action of 64 kinds of different protein kinases, on the contrary not as 2-phenoxy pyrimidine derivates.The action target spot that its inhibition tumor cell is described may not be protein kinase but other target spot.In order to illustrate the mechanism of action of phenoxy pyrimidine compounds for treating relevant disease, need to find the direct target of its effect.Find at present drug target calibration method a lot, comprise computer virtual screening, gene chip, protein chip, two-dimensional protein electrophoretic, display technique of bacteriophage, yeast two-hybrid, quantitative proteomics and chemical proteomics (affinity chromatography etc.) etc.Wherein affinity chromatography is a kind of low cost and effective method.Successfully apply at present FK506, cyclosporin, Actinodephnine Acuminatin Acuminatoside Adenanthin, the identification of the drug targets such as colchicine.Common affinity chromatography has solid phase elution method and medicine competition law.Solid phase elution method is easily subject to the impact of speed of dissociating of " affinity column-target protein " complex, but also must prepare the contrast post of non-activity analog.Medicine competition law exists drug solubility is required to the shortcomings such as high.Series affinity chromatography is neoteric a kind of affinity chromatography that can overcome above two kinds of affinity chromatography shortcomings, has the advantages such as practical, reliable results.Based on above understanding, the present invention determines to adopt the serial affinity chromatography identification direct acting target of phenoxy pyrimidine compounds and carries out target confirmation.Find new drug effect target, for the treatment of relevant disease provides new therapeutic scheme.

Summary of the invention

The present invention is based on the discovery of phenoxy pyrimidine derivates action target, a kind of therapeutic scheme of high-efficiency low-toxicity is provided targetedly.For achieving the above object, use by suffering from the patient of SET protein abnormal expression disease the SET protein micromolecular inhibitor that contains phenoxy pyrimidine mother nucleus structure, to reach the object of targeted therapy disease.

The concrete technical scheme of the present invention is as follows:

Phenoxy pyrimidine compounds is the application in preparation and SET protein abnormal expression relevant disease medicine as SET protein inhibitor.Suppress the interaction between SET albumen and PP2A by phenoxy pyrimidine compounds, improve the activity of PP2A in cell, reduce and the phosphorylation level of the interactional protein kinase of PP2A, promote apoptosis, change cell cycle and bring into play corresponding biological action.

Phenoxy pyrimidine derivates action target described in such scheme is SET albumen, have another name called template activation factor-1 (emplate activating factor, TAF-1), PP2A inhibitor-2 (inhibitor of protein phosphatase2A-2, I2PP2A), the II of histocompatibility leukocyte antigen associated protein (PHAP II), DNA enzyme activation inhibitor (the inhibitor of GzmA Activated Dnase that granzyme A activates, IGAAD) etc., the albumen by SET gene code, comprise two kinds of main spliced bodies: TAF-α and SET/TAF-β.SET/TAF-β hypotype is being brought into play the main biological action of SET albumen.SET albumen is a kind of multifunctional protein, and its function relates to multiple biological processes such as DNA replication dna, nucleosome assembling, chromosome depolymerization, apoptosis, inhibition cell protein phosphatase 2A activity, is the important regulatory factor of cell physiological activity.In some digestive system and hematological system tumor, reproductive system, neural disease, express with SET or Subcellular Localization abnormal.It is 2 times of related normal tissue or higher such as SET/TAF-β in uterus, stomach, colon, rectal neoplasm tissue expresses.In the leukemia such as chronic marrow sexual cell leukemia, Acute myeloid leukemia, B cell non-Hodgkin's, all there is the abnormal phenomenon raising of SET complex expressing quantity.And the grade malignancy height correlation of its expression and tumor.In normal neurons cell, the main Asia of SET albumen is positioned nucleus, and at Alzheimer's disease patient brain, SET albumen increases in cytoplasm, makes the active Hyperphosphorylationof that causes Protein tau on the low side of PP2A in cell, causes the deposition of amyloid (APP) at brain.It is closely related that SET protein abnormal expression causes major reason of tumor and its to suppress the activity of PP2A (PP2A) in cell.PP2A is important silk in cell, Threonine Phosphatases, can, by regulating the kinase whose activity of substrate protein regulate cell various biological process, comprise cell proliferation, differentiation, apoptosis and migration.Generally believe that at present PP2A is the important cancer factor that presses down.The balance of PP2A and corresponding protein kinase activity is most important to maintaining stablizing of intracellular environment.The forfeiture of PP2A activity or inhibition can cause abnormal rising of the kinase whose activity of corresponding protein and lead oncogenic generation.PP2A is a heterogeneous triplet, comprises two common components, and a catalytic subunit (PP2A-C) and a support subunit (PP2A-A) composition catalytic center dimer are connecting an adjusting B subunit by four kinds of different family gene codings simultaneously.SET albumen can with PP2A-C combination, thereby suppress PP2A Phosphatase Activity.Often overexpression of SET albumen in many tumor cells, causes PP2A activity on the low side, thereby causes the protein kinase that cancer is relevant to cause cell carcinogenesis in sustained activation state.In the technical background of this patent, mention, although that kinases inhibitor has therapeutic effect is good,, also there is the shortcoming because of mutant protein kinase drug resistance in the advantage that toxic and side effects is low.Such as chronic myelocytic leukemia (CML) is the malignant tumor that a kind of size of being expressed by Philadelphia chromosome bcr-abl fusion gene protein kinase B CR-ABL1 that is 210KD causes.BCR-ABL1 albumen energy autophosphorylation is p-BCR-ABL1, and then the protein kinase of activation signal passage downstream, causes quantity of leucocyte to roll up, thereby causes chronic myelocytic leukemia.Because BCR-ABL1 is " arch-criminal " who causes CML, in order to treat CML, various countries scientist develops for the kinases inhibitor of BCR-ABL1 to have entered unremitting effort, calendar year 2001 is first BCR-ABL1 kinases inhibitor Imatinib(imatinib in the world) official listing, significant contribution has been made in the treatment that Imatinib is CML.But in its clinical use procedure, the problems such as after drug resistance, drug withdrawal, recur and also manifest gradually.The point mutation of BCR-ABL1 kinases is to cause the modal reason of Imatinib drug resistance, these kinases aminoacid point mutation cause the amino acid substitution in BCR-ABL1 protein kinase domain, destroy the combination of Imatinib and BCR-ABL1 albumen, the direct or indirect Imatinib drug resistance that causes.For overcoming Imatinib drug resistance, Dasatinib, the second filial generation BCR-ABL1 kinases inhibitors such as Nilotinib go on the market in succession, and they can treat the drug resistance CML that the nearly all kinase mutant except T315I sudden change causes.2012, the unique also listing finally of inhibitor Ponatinb that can treat T315I sudden change drug resistance CML, still, mixed sudden change such as the two sudden changes of E255V/T315I for some, and Ponatinb is still helpless.Have the active compared with normal cell of PP2A (PP2A) in a lot of tumor cells of bibliographical information including CML obviously on the low side, and it active on the low sidely increase relevant with its endogenous inhibitor SET (I2PP2A) expressing quantity extremely.Suppress PP2A Phosphatase Activity.Often overexpression of SET albumen in CML cell, causes PP2A activity on the low side, and the protein kinases such as BCR-ABL1 are in sustained activation state.Adopt PP2A activator (for example, Forskolin, FTY720, Ceramide) to make p-BCR-ABL1 dephosphorylation treat the various tumor diseases including drug resistance CML by improving PP2A activity.Illustrate that affecting SET/PP2A path can be used as one and effectively overcome the target spot that causes the disease of drug resistance because of mutant protein kinase.The mechanism of action of current existing PP2A activator is all by the activity of PP2A in direct and PP2A interaction and then raising cell.And there is not yet report for the micromolecular inhibitor of SET.The present invention is in order to find the target of phenoxy pyrimidine derivates effect, built phenoxy pyrimidine derivates-TGI1002(N-[4-methyl-3-[4-(3-pyridine radicals)-2-2-pyrimidinyl oxy]-phenyl]-3-(4-methyl piperazine methyl)-benzamide) and Small-molecule probe, and adopt serial affinity chromatography from chronic myelocytic leukemia K562 cell pyrolysis liquid " fishing " to go out the protein with TGI1002 with affinity interaction.By SDS-PAGE separate, cut glue enzymolysis, MALDI-TOF-peptide fingerprint Mass Spectrometric Identification is SET albumen.And by PP2A determination of activity, co-immunoprecipitation, immunofluorescence, it is by disturbing the interaction between SET/PP2A that the methods such as RNAi have been confirmed phenoxy pyrimidine derivates-TGI1002, improve the activity of PP2A in cell, change cell cycle, promote apoptosis to treat the relevant disease of the drug resistance particularly causing because of mutant protein kinase with SET protein abnormal expression.

The relevant disease of SET protein abnormal expression described in scheme mainly includes but not limited to: inflammation, neurodegenerative disease, malignant tumor and autoimmune disease etc.Described inflammation includes but not limited to central nervous system (CNS) inflammation, enteritis, arthritis etc.; Neurodegenerative disease includes but not limited to senile dementia, alzheimer's disease and other cerebral nerve disorder diseases; Malignant tumor includes but not limited to leukemia, hepatocarcinoma, colon cancer, carcinoma of prostate, breast carcinoma, pulmonary carcinoma, osteosarcoma, choriocarcinoma, nephroblastoma etc.; Autoimmune disease includes but not limited to systemic lupus erythematosus (sle), rheumatoid arthritis, scleroderma, autoimmune hemolytic anemia, chronic lymphocytic thyroiditis, hyperthyroidism, insulin dependent diabetes mellitus (IDDM), myasthenia gravis, chronic ulcerative colitis, pernicious anemia companion chronic atrophic gastritis, goodpasture syndrome, primary biliary cirrhosis, multiple sclerosis, acute idiopathic polyneuritis etc.

The preferred chronic myelogenous leukemia of disease (CML) that above-mentioned SET protein abnormal expression is relevant, chronic lymphocytic leukemia acute myeloid leukemia (CLL), acute lymphoblastic leukemia (ALL) or B cell non-Hodgkin′s lymphoma.

Above-mentioned chronic myelogenous leukemia disease comprises but does not limit BCR-ABL positive chronic myelogenous leukemia.

Above-mentioned BCR-ABL positive chronic myelogenous leukemia includes but not limited to imatinib drug resistance chronic myelogenous leukemia.

Above-mentioned imatinib drug resistance chronic myelogenous leukemia includes but not limited to the imatinib drug resistance chronic myelogenous leukemia causing that suddenlys change by BCR-ABL kinases territory T315I.

According to above summary of the invention, as long as the compound with following general formula phenoxy pyrimidine parent nucleus all can be brought into play various biological functions by mechanism of the present invention (that is: in conjunction with SET albumen, the interaction of dissociate itself and PP2A, raising PP2A activity),

Wherein, R1 and R2 are identical or different, be selected from following group: alkyl, halogen, cycloalkyl, alkylene, alkynyl, benzyl, heterocyclic radical, aryl, heteroaryl, phenyl (oxo or sulfo-) methyl, phenyl amino oxo (or sulfo-) methyl, phenylethyl, the fragrant heterocycle of 5-or 6-unit, 5-or 6-unit fragrant heterocycle (oxo or sulfo-) methyl, the fragrant heterocycle ethyl of 5-or 6-unit, phenyl-undersaturated or saturated 5-or the 6-unit carbocyclic ring condensing, phenyl-(undersaturated or saturated 5-or 6-unit carbocyclic ring) (oxo or the sulfo-) methyl condensing, phenyl-(undersaturated or saturated 5-or the 6-unit carbocyclic ring) ethyl condensing, the fragrant heterocycle of the phenyl-5-condensing or 6-unit, phenyl-the 5-condensing or 6-unit fragrant heterocycle (oxo or sulfo-) methyl, the fragrant heterocycle ethyl of the phenyl 5-condensing or 6-unit.

Above-mentioned phenoxy pyrimidine compounds is preferably N-[4-methyl-3-[4-(3-pyridine radicals)-2-2-pyrimidinyl oxy]-phenyl]-3-(4-methyl piperazine methyl)-benzamide, chemical formula is as follows:

Can application reference number be that 200810244308.5 the disclosed method of Chinese patent is prepared.

Above-mentioned benzene oxygen pyrimidines is except compound itself, also comprise medicinal salt and/or with the complex of drug administration carrier composition and by different approaches to human body administration.

Due to the utilization of technique scheme, the present invention compared with prior art has following advantages:

By catching and confirming phenoxy pyrimidine derivates action target spot, disclosed first SET albumen and can be used as the drug targets for the treatment of relevant disease and be used for screening relevant inhibitor, the disease that the micromolecular inhibitor that phenoxy pyrimidine derivates can be used as SET albumen is simultaneously treated SET abnormal expression targetedly particularly can overcome the disease that causes drug resistance because of mutant protein kinase.

Accompanying drawing explanation

Fig. 1: A figure be phenoxy pyrimidine derivates TGI1002 to 20 kinds of tumor cell effects the cell growth curve after 72 hours, IC50 represents half-inhibition concentration; B figure is that TGI1002 is to BaF3-p210(WT), BaF3-p210(Y253F), BaF3-p210(T315I) and the effect curve of three strain cytosiies, EC50 represents half effective concentration.

Fig. 2: A figure is phenoxy pyrimidine derivates TGI1002 Small-molecule probe synthetic route chart; B figure is that serial affinity chromatography is caught the SDS-PAGE analysis chart after specific adsorption albumen in K562 cell pyrolysis liquid (S3 is respectively the 1st for S1, S2, the sample of hatching for 2,3 times); C figure is Mass Spectrometric Identification and data base's comparison result of the TGI1002 specific adsorption Proteolytic enzyme peptide section of catching.

Fig. 3: A figure is the cell cycle flow cytometer showed figure (PI mono-dyes) of phenoxy pyrimidine derivates TGI1002 to K562 cell; B figure is cell cycle distribution statistics; C figure is apoptosis flow cytometer showed figure (PI-FITC is two to be dyed); D figure is apoptosis cartogram.

Fig. 4: A figure, B figure is respectively phenoxy pyrimidine derivates TGI1002 to K562, impact (the * P<0.05 of BaF3-p210 (T315I) cell PP2A activity, * P<0.01 is TGI1002 independent role group and matched group ratio, and #P<0.05 is TGI1002+OA coupling group and TGI1002 independent role group ratio).

Fig. 5: A figure is that co-immunoprecipitation method detects phenoxy pyrimidine derivates TGI1002 to the interactional impact of SET/PP2A in K562 cell; B figure is that immuno-fluorescence assay phenoxy pyrimidine derivates TGI1002 is on the interactional impact of SET/PP2A in K562 cell.

Fig. 6: A figure is the content of SET mRNA in the K562 cell infecting after chronic malicious 72h; B figure is the content of SET albumen in the K562 cell infecting after chronic malicious 72h; C figure is the growth rate figure that infects the K562 cell after chronic malicious 72h.

Fig. 7: A, B, C figure is respectively phenoxy pyrimidine derivates TGI1002 to K562, BaF3-p210(WT), the impact of the cell correlative protein expression such as BaF3-p210 (T315I).

The specific embodiment

Embodiment 1: phenoxy pyrimidine derivates-TGI1002(N-[4-methyl-3-[4-(3-pyridine radicals)-2-2-pyrimidinyl oxy]-phenyl]-3-(4-methyl piperazine methyl)-benzamide) SET abnormal protein is expressed and the toxicity screening of the tumor cell of drug resistance

1 experiment material

1.1 cell strains: 20 strain tumor cells, comprise A549, PC-3, SK-OV-3, HCT116, DU145, U251, MDA-MB-231, MCF7, SK-MEL-28, Colo205, HL-60, CCRF-CEM, K562, MOLT4, ACHN, HS578T, HCT-15, OVCAR-3,786-O, BaF3-p210(WT), BaF3-p210(Y253F), BaF3-p210(T315I) all pass through detection of mycoplasma.

1.2 reagent and culture medium: TGI1002 (purity is greater than 99.5%), F12K culture fluid, RPMI1640 culture fluid, MyCoys5A culture fluid, DMEM culture fluid, adds the streptomycin of 10% hyclone, 100 μ g/mL, the penicillin of 100U/mL before use.

2 experimental techniques: cytoactive analysis

Adopt CellTiter Glo method to analyze the growth inhibited effect of TGI1002 to tumor cells such as K562.Concrete operation step is as follows:

(1) compound treatment the previous day by tumor cell inoculation in 96 porocyte culture plates.Inoculum density is: 2000 μ l/ holes, cell/1000 or 4000 μ l/ holes, cell/100, concrete quantity determines according to cell growth rate.

(2) second days, the 2X compound solution preparing by culture medium is arranged and requires to join in Tissue Culture Plate according to experiment, every hole adds 100 μ l.

(3) light shaking cell plates, place it in 37 ℃ of incubators and continue to cultivate 72 hours.

(4) hatch after end and require in cell version, to add the reagent preparing according to CellTiter Glo reagent description, fully mix rear room temperature lucifuge and hatch 10 minutes.

(5) cell plates are put into and read plate instrument and analyze, set and read chemiluminescence record data.

(6) reading in every hole need to be converted into cell survival rate.Cell survival rate can use following formula to calculate:

Data after treatment will be used to do nonlinear regression analysis, obtain dose effect curve, and calculate the half-inhibition concentration (IC50) of TGI1002 to every kind of cell.

Experimental result: first screened the cytotoxicity of phenoxy pyrimidine derivates TGI1002 to 20 kinds of different tumor cells, find that TGI1002 has certain targeting, the tumor cell K562 that it is high to SET expressing quantity, PC-3, the activity of HT-29 is (Figure 1A) better, half-inhibition concentration (IC50) is respectively: 14.0 μ M, 15.7 μ M, 17.6 μ M.Further tested the chronic myeloid leukemia cell strain BaF3-p210(WT of TGI1002 to Mus source) and suddenly change two strain cell strain BaF3-p210(Y253F of the drug resistance causing of kinase b CR-ABL), BaF3-p210(T315I) cytotoxicity.Find that TGI1002 to the EC50 of three strain cells is not: 25.9 μ M, 20.1 μ M, 32.9 μ M(Figure 1B).Not obviously difference.Illustrate that TGI1002 can effectively resist the drug-resistant tumor that kinase mutant causes.

Preparation and the catching and Mass Spectrometric Identification target protein in K562 cell pyrolysis liquid thereof of embodiment 2:EAH Sepharose4B-TGI1002 small molecule active probe.

1 experiment material: TGI1002, monobromo-acetic acid, EAH Sepharose4B, SDS-PAGE, mass spectrograph etc.

2 experimental techniques:

(1) TGI1002 Small-molecule probe is synthetic: demethyl TGI1002 is reacted to 24h in 60 ℃ with monobromo-acetic acid in DMF, dry and get pressed powder and react under EDC catalysis with EAH Sepharose4B and obtain EAH Sepharose4B-TGI1002 Small-molecule probe (reaction scheme road is as Fig. 2 A).Adopt HPLC method to calculate coupling rate.

(2) serial affinity chromatography is caught TGI1002 specific adsorption albumen.In the K562 cell pyrolysis liquid of 0.2mL, (the about 5mg of total protein concentration) adds the EAH Sepharose4B-TGI1002 Small-molecule probe of 60 μ L, mix homogeneously, and ice bath jolting reaction 40min, centrifugal (2000rpm, 1min, 4 ℃), get precipitation and identify.Supernatant adds in 60 μ L Small-molecule probes again, mixes, ice bath jolting reaction 40min is centrifugal.Using such method is carried out compatible reaction continuously 3 times, and the precipitation of 3 secondary response gained adds respectively cell lysis buffer solution 0.2mL, shake well, and centrifugal (2000rpm, 1min, 4 ℃), suck supernatant, Using such method continuous washing 5 times.Wash in backward precipitation and add 30 μ L SDS-PAGE sample-loading buffers (reduced form), 95 ℃ of heating 5min, fully discharge the albumen being combined on post material.Centrifugal (10000rpm, 2min), supernatant carries out SDS-PAGE evaluation (silver dyes).

(3) Mass Spectrometric Identification of target protein: dye result according to silver, the protein band that in 3 series, band color depth reduces is successively the destination protein of specific binding.After this band is cut to enzymolysis, range of hydrolysed peptides section is carried out to MALDI-TOF analysis, and adopt peptide fingerprinting spectrometry to carry out data base's comparison, determine this albumen.

Experimental result: successfully synthesize EAH Sepharose4B-TGI1002 Small-molecule probe, coupling rate is 35.5%(Fig. 2 A), and adopt serial affinity chromatography to catch specific adsorption albumen in K562 cell pyrolysis liquid (Fig. 2 B) and be SET albumen (Fig. 2 C) by Mass Spectrometric Identification.

Embodiment 3: phenoxy pyrimidine derivates TGI1002 has cell cycle arrest (G0/G1), the effect of cell death inducing

1 experiment material: TGI1002, cell cycle detection kit (PI), cell apoptosis detection kit (Annexin-FITC), flow cytometer etc.

2 experimental techniques: K562 cell is inoculated in to (2-3 × 10, every hole in 6 orifice plates ⁵individual/hole), after 12h, add the TGI1002 mother solution of certain volume, collecting cell carry out the analysis of cell cycle and apoptosis according to the explanation of test kit after effect 24-48h.

3 experimental results: TGI1002 has obvious G0/G1 phase retardation (Fig. 3 A) and can promote apoptosis (Fig. 3 B).

Embodiment 4: phenoxy pyrimidine derivates TGI1002 has the active effect that improves PP2A in cell

Experimental technique: the PP2A immunoprecipitation determination of activity test kit that adopts Merck & Co., Inc. to produce is measured.

Experimental procedure:

1. the preparation of cell pyrolysis liquid for experiment: get one bottle of (25cm ²) in exponential phase and K562 and drug-resistant cell strain BaF3-p210(T315I in good condition) cell blows down by 3ml culture medium, get 3ml and add the culture medium of certain volume to be diluted to 28-30ml(cell concentration to be diluted to about 2.5-3 × 10 ⁵individual/ml), point get 2ml cell diluent and in 6 orifice plates, continues cultivation 18-24h, according to the TGI1002(10mM that adds respectively certain volume that arranges of table 1), okadaic acid (OA), mother solution makes TGI1002, the final concentration of OA meet table 1 require after continue cultivate (TGI1002, OA coupling hole adds TGI1002 effect 12h after first adding OA effect 12h again, TGI1002 independent role 12h, OA independent role 24h).

Table 1:TGI1002, OA concentration arranges table

TGI1002

	0,10μM,20μM,40μM
		OA	5nM
TGI1002+OA	10μM+5nM，20μM+5nM，40μM+5nM

2. drug effect is complete blows down cell afterwards by culture medium in hole (discarding 0.5mL), moves in the EP pipe of 1.5mL 4 ℃, the centrifugal 5min of 1200g, supernatant discarded, with TBS cleaning cell 2 times (each 1.0ml), the centrifugal 5min of 1200g again, after supernatant discarded, add cell pyrolysis liquid (20mM Tris-HCl, 150mM NaCl, the 1mM EDTA of 120 μ L, 1mM MgCl, 0.5%NP-40, protease inhibitor cocktail, pH7.5) in cell lysis 30min on ice.4 ℃, centrifugal 8min under 14500rpm.Supernatant proceeded in the EP pipe of an other 0.5mL and adopt BCA method to carry out the mensuration of protein concentration.

3. toward the PP2A-C antibody containing respectively adding 2 μ g in the lysis supernatant of total protein 100 μ g.And add that to add after the protein A agarose serosity of 25 μ L the TBS of certain volume to make its cumulative volume be that 500 μ L(protein concentrations are 0.2 μ g/ μ L again).At 4 ℃, on shaking table, hatching 2h(can be placed on sample in the ice chest being placed on shaking table and hatch).

4. hatched rear centrifugal (the instantaneous centrifugal 10s of 14000rpm under room temperature), first cleaned pearl 3 times with TBS, each 800 μ L, then measure buffer solution for cleaning once with 400 μ L pNPP Ser/Thr, carry out immediately phosphate radical detection.If do not polluted the Phosphorylated Peptide (final reaction density is 642 μ M) after the dilution that adds 45 μ L by phosphate radical.Add again the pNPP Ser/Thr of 25 μ L to measure buffer.At 30 ℃, on shaking table, hatch 15min.

5. hatch rear recentrifuge (the instantaneous centrifugal 10s of 14000rpm under room temperature), got 25 μ L supernatant in 96 hole microwell plates.Every hole adds the peacock green of 100 μ L to measure buffer.Under room temperature, place 15min.650nm measures absorbance.Obtain corresponding phosphate concentration according to standard curve.

Experimental result: the be significantly improved effect of cell PP2A activity of TGI1002 tool, and this interaction energy specific inhibitor OA institute antagonism (accompanying drawing 4A) that is PP2A.To drug-resistant tumor cell strain BaF3-p210(T315I) there is same effect (Fig. 4 B), illustrate that TGI1002 can treat the tumor disease of drug resistance.

Embodiment 5: phenoxy pyrimidine derivates TGI1002 is to the interactional interference of SET/PP2A

1 co-immunoprecipitation (Co-IP) detects TGI1002 to the interactional impact of SET/PP2A

Experimental procedure: the preparation of cell pyrolysis liquid for (1) experiment: get one bottle of (25cm ²) blow down by 3ml culture medium in the K562 of exponential phase cell, add complete medium to be diluted to 28ml(cell concentration and be diluted to approximately 4.5 × 10 ⁵) point get 2ml cell diluent and in 6 orifice plates, continues to cultivate 18h, add respectively the TGI1002(10mM of certain volume, deionized water is prepared) mother solution, make the final concentration of TGI1002 be: 0,10 μ M, 20 μ M, 30 μ M, 40 μ M.

(2) after drug effect 12h, by culture medium in hole (discarding 0.5mL culture medium), cell is blown down, moved in the EP pipe of 1.5mL the centrifugal 5min of 1200g, supernatant discarded, clean cell once with the PBS of 1ml, add cell pyrolysis liquid (20mM Tris-HCl, the 150mM NaCl of 100 μ L, 1mM EDTA, 1mM MgCl, 0.5%NP-40, protease inhibitor cocktail) in cell lysis 30min on ice.Centrifugal 8min under 14500rpm.Supernatant is transferred in another 1.5mL EP pipe and adopts BCA method carry out the mensuration of protein concentration and carry out WB and detect PP2A-C or SET (I2PP2A) expression whether change (guarantee expression does not change carry out again next step experiment).

(3) respectively add the PP2A-C antibody of 1 μ L or the I2PP2A of 1 μ g (SET, 5 μ L) toward containing in total protein approximately 200 μ g lysis supernatant (adjusting protein concentration with TBS is 5 μ g/ μ L, cumulative volume 40 μ L).At 4 ℃, on shaking table, hatching 10h(can be placed on sample in the ice chest being placed on shaking table and hatch).After antibody incubation is complete, add after protein A/G agarose serosity of 40 μ L at 4 ℃, on shaking table, hatch and after 12h, (sample can be placed in the ice chest being placed on shaking table and hatch) the instantaneous centrifugal 10s of 14500rpm, collect sepharose 4B-antigen antibody complex, remove supernatant, wash 2 times 600 μ L/ time with the PBS of pre-cooling.

(4) with 40 μ L2 × sample-loading buffers, complex is hanged, mix gently, 100 ℃ are boiled 5min, centrifugal, get supernatant and carry out WB, detect SET or the situation of change of PP2A-C under TGI1002 effect.

Experimental result: TGI1002 can disturb the interaction (Fig. 5 A) between SET/PP2A, along with the increase of TGI1002 concentration and the PP2A-C of SET protein-interacting reduce gradually.

2 immuno-fluorescence assay TGI1002 are on the interactional impact of SET/PP2A

Experimental procedure:

1) get one bottle of (25cm ²) blow down by 3ml culture medium in the K562 of exponential phase cell, get 0.6ml and add complete medium to be diluted to 16ml(cell concentration to be diluted to approximately 1.5 × 10 ⁵individual/ml) divide and get 2ml cell diluent continuation cultivation 18h in laser co-focusing culture dish (Φ 15mm at the bottom of glass, coated with poly-D-lysine in advance), add respectively the TGI1002(10mM of certain volume, deionized water preparation) mother solution, make the final concentration of TGI1002 be: 0,20 μ M, 40 μ M;

2) after drug effect 12h, suck culture medium, the TBS/PBS rinsing 1 time (1ml/ time) of 4 ℃, the fixing 30min of fixative (1ml) room temperature or longer time (can 4 ℃ fixedly spend the night);

3) suck fixative, the TBS/PBS rinsing 1 time (1ml/ time) of 4 ℃, each 2.5min(jiggles), add the TBS/PBS rinsing 1 time (1ml/ time) with 4 ℃ after incubated at room 5-10min after antigen retrieval liquid, 2.5min(jiggles at every turn);

4) 0.5%Triton-100(1ml) 4 ℃ of perforation 30min;

5) the TBS/PBS rinsing 1 time (1ml/ time) of 4 ℃, each 2.5min;

6) 5%BSA/TBST(1ml) room temperature sealing 30min (being preferably used in the two identical animal serums in anti-source seals);

7) add the primary antibodie (each 0.2ml, PP2A-C, 1:50 dilution, SET, 1:25 dilution) containing the TBST dilution of 0.5%BSA, hatch 16-24h in 4 ℃;

8) the TBST rinsing 3 times (2ml/ time) of 4 ℃, each 5min;

9) add the fluorescence two diluting containing the TBST of 0.5%BSA anti-(each 0.2ml, goat anti-mouse IgG-TRITC, 1:25 dilution, goat anti-rabbit IgG-FITC, 1:25 dilution) in incubated at room 2-3h;

10) the TBST rinsing 3 times (2ml/ time) of 4 ℃, each 5min;

11) after DAPI dyeing liquor (0.25ml) dyeing 5min, suck;

12) 3 times (2ml/ time) of the TBST of 4 ℃ washing, each 5min;

13) anti-fluorescent quenching mounting liquid (0.25ml) mounting;

14) under laser confocal microscope, observe coloration result.

Experimental result: TGI1002 can, by the SET albumen retardance in K562 cell and nucleus, not affect and the subcellular fraction of PP2A-C is distributed, thereby removes the inhibitory action (Fig. 5 B) of SET albumen to cytoplasmic PP2A-C.

In embodiment 6:RNA interference (RNAi) K562 cell, after the expression of SET albumen, the speed of Growth of Cells obviously reduces

1.RNA disturbs the expression (slow virus infection method) of SET albumen in K562 cell

Experiment material: can disturb the slow virus (LV-SET-RNAi) of SET protein expression, negative control slow virus (LV-RNAi), puromycin etc.

Experimental procedure:

(1) get 1 bottle of (25cm ²) K562 cell blows down by 3ml culture medium, get 0.25ml Cell sap culture medium be diluted to 10ml(now cell concentration be about 1 × 10 ⁵individual/ml), to get 100 μ l cell diluents and join in 96 orifice plates, hole inner cell number is 0.75 × 10 ⁴individual/hole.

(2) after cultivation 32h, (now cell number is about 4 × 10 ⁴individual/hole), suck the culture medium of 20 μ l in hole, adding 10 μ l titres is 1 × 10 ⁸(virus quantity adding is 1 × 10 to the virus liquid of TU/ml ⁶and the Polybrene of the 50 μ g/ml of 10 μ l TU).

(3) mix rear continuation and cultivate, observation of cell state after 12h, and change fresh complete medium.

(4) infect after 72h, observe luciferase expression situation.

(5) by high to control wells and fluorescence intensity, infect (96 orifice plate) cell in good hole and proceed to respectively in 6 orifice bores, after cell attachment, adding certain volume concentration is the puromycin mother solution of 10mg/ml, making its final concentration is 5 μ g/ml.Continue to cultivate 2-3 days.

(6), after the degrees of fusion of cell in 6 orifice plates is about 80%, cell is blown down and is proceeded to 25cm ²in Tissue Culture Flask, continue to cultivate (5 μ g/ml puromycin), after 2-3 days, go down to posterity (one passes three), continue to cultivate (5 μ g/ml puromycin), after the degrees of fusion of three bottles of cells that go down to posterity is about 80%, wherein one bottle frozen, one bottle goes down to posterity, one bottle of collecting cell carries out q-PCR, Western bloting verifies the disturbed condition to SET gene, and carries out the mensuration of cell growth rate.

Experimental result: SET mRNA and all obviously downwards (Fig. 6 A, B) of protein content in K562 cell after chronic poison infection 72h.

The mensuration of 2 cell growth rates

Experimental procedure:

1) get one bottle of (25cm ²) A/B/C in exponential phase blows down by 3ml culture medium, draws 65 μ l and adds the dilution of 10ml culture medium, counting is about 2.5 × 10 ⁴individual/ml adds 400 μ l diluent (every Kong Yuehan 1 × 10 in 48 orifice plates ⁴individual cell, the every hole of periphery hole adds 400 μ l PBS).Culture plate is put to 37 ℃ to 5%CO ₂in incubator, cultivate.Respectively at 24h, 48h, 72h, 96h, 120h, 144h carries out cell counting.

2) according to cell counting result, take cell number as vertical coordinate, the time (d) is abscissa, draws cell growth curve.According to formula T _d=Δ t*Lg2/ (LgNt ₂– LgNt ₁) try to achieve the population doubling time of cell.

Experimental result: the growth rate of the K562 cell after SET protein expression is lowered obviously reduces, and mean doubling time is 46h, and the mean doubling time of normal K562 cell is 22.7h(Fig. 6 C).

Embodiment 7: phenoxy pyrimidine derivates TGI1002 has the suddenly change effect of the imatinib drug-resistant leukemia causing of antagonism BCR-ABL1 kinases territory T315I

Experimental technique: Western blotting detects TGI1002 to K562, BaF3-P210 (WT), the impact of correlative protein expression in BaF3-P210 (T315I) cell

Experimental procedure:

1) in six orifice plates, inoculate K562, BaF3-P210 (WT), BaF3-P210 (T315I) cell (2-3 × 10, every hole ⁵individual cell, culture medium 2ml), after 12-18h, add the TGI1002 mother solution of certain volume, make the final concentration of compound be respectively 10,5,2.5 μ M move into cell in 1.5ml EP pipe after compound effects 24h, 4 ℃, the centrifugal 5min of 1200g, supernatant discarded, cleans cell precipitation with PBS and once discards afterwards PBS, add the cell pyrolysis liquid that has added protease or inhibitors of phosphatases of 120-150 μ l, high speed thermal agitation 5 seconds, allows cell precipitation suspend completely and to scatter, 4 ℃ of centrifugal 5min of 12000-16000g after ice bath 15-30min.Draw immediately supernatant (can use immediately also can-70 ℃ frozen) to the EP pipe of pre-cooling.

2) adopt BCA method to measure the protein concentration of cell pyrolysis liquid.

3) SDS-PAGE electrophoresis (the every hole of total protein applied sample amount 20-40ug, pre-prepared colloid 4-12%), 150V.

4) transferring film (300-350mA, 50-55min).

5) sealing (5%BSA), hatches primary antibodie (self-control sealing bag is hatched, the TBST preparation containing 1-5%BSA for primary antibodie), and 4 ℃ are cleaned film 3 times, each 10min with TBST after spending the night.Hatch two and resist, room temperature 1-2h.Again clean film 3 times with TBST, each 10min.

6) exposure colour developing.

Experimental result: TGI1002 can make K562, BaF3-P210 (WT), p-BCR-ABL albumen dephosphorylation (Fig. 7 A in BaF3-P210 (T315I) cell, B, C), illustrate that it can be to imatinib resistant drug resistance chronic myelocytic leukemia, this is because TGI1002 action target is not BCR-ABL, is not subject to the impact of BCR-ABL sudden change.It can suppress SET, thereby improves the activity of PP2A in cell and then make p-BCR-ABL albumen dephosphorylation.

In sum, inventor is by building the Small-molecule probe of phenoxy pyrimidine derivates TGI1002 and adopting the method for serial affinity chromatography to find target-SET albumen of phenoxy pyrimidine derivates performance pharmacological action and passed through co-immunoprecipitation, immunofluorescence, the methods such as RNAi are confirmed.Of the present invention completing particularly provides a kind of targeted therapy scheme of brand-new high-efficiency low-toxicity because mutant protein kinase causes the treatment of the malignant disease of drug resistance for the disease of SET protein abnormal expression.

Claims

1. a class SET protein micromolecular inhibitor and the application in treatment SET protein abnormal expression relevant disease thereof.

2. application according to claim 1, described in it is characterized in that, being applied as SET protein micromolecular inhibitor can be by suppressing the interaction between SET albumen and PP2A, improve the activity of PP2A in cell, reduce the phosphorylation level with the interactional protein kinase of PP2A, promote apoptosis, change cell cycle and bring into play corresponding biological action.

3. application according to claim 1, is characterized in that described SET protein micromolecular inhibitor has following general structure:

4. application according to claim 3, is characterized in that described SET protein micromolecular inhibitor is N-[4-methyl-3-[4-(3-pyridine radicals)-2-2-pyrimidinyl oxy]-phenyl]-3-(4-methyl piperazine methyl)-benzamide.

5. application according to claim 1, wherein said SET protein micromolecular inhibitor be its medicinal salt and/or with the complex of drug administration carrier composition and by different approaches to human body administration.

6. application according to claim 1, is characterized in that the disease of described SET protein abnormal expression includes but not limited to inflammation, neurodegenerative disease, malignant tumor and autoimmune disease etc.

7. according to the application described in claims 6, wherein said inflammation includes but not limited to central nervous system (CNS) inflammation, enteritis, arthritis etc.

8. according to the application described in claims 6, wherein said neurodegenerative disease includes but not limited to senile dementia, alzheimer's disease and other cerebral nerve disorder diseases.

9. according to the application described in claims 6, wherein said malignant tumor includes but not limited to leukemia, hepatocarcinoma, colon cancer, carcinoma of prostate, breast carcinoma, pulmonary carcinoma, osteosarcoma, choriocarcinoma and nephroblastoma etc.

10. according to the application described in claims 6, wherein said autoimmune disease includes but not limited to systemic lupus erythematosus (sle), rheumatoid arthritis, scleroderma, autoimmune hemolytic anemia, chronic lymphocytic thyroiditis, hyperthyroidism, insulin dependent diabetes mellitus (IDDM), myasthenia gravis, chronic ulcerative colitis, pernicious anemia companion chronic atrophic gastritis, goodpasture syndrome, primary biliary cirrhosis, multiple sclerosis, acute idiopathic polyneuritis etc.