CN106668008A - Target anti-cancer medicine based on STAT3 (Signal Transducers and Activators of Transcription type 3) protein target spot - Google Patents

Abstract

The invention relates to a target anti-cancer medicine based on an STAT3 (Signal Transducers and Activators of Transcription type 3) protein target spot. Cell lines of a plurality of types of breast cancers have an abnormal regulation and control mechanism of an intracellular STAT3 signal conduction system. The invention provides one type of compound, which is an effective regulator of STAT3 signal conduction, and can be applied to preparation of medicines for treating breast cancer.

Description

Invention based on the targeted anticancer medicine of STAT3 albumen target spots

Technical field

The present invention relates to a kind of application of signal transducting system inhibitor compound in terms for the treatment of cancer medicine is prepared.

Background technology

Cancer becomes the Chinese national health of impact and causes one of factor of heavy economic losses.Traditional treatment cancer The control of method-operation, radiotherapy, chemotherapy and biological therapy to the tumour patient state of an illness has certain limitation.Calendar year 2001, the first generation The appearance of targeted drug-Gleevec brings new favourable turn and hope to the clinical treatment of cancer patient, and aobvious with clinical efficacy The advantages of writing less with toxic and side effect.Target medicine-the Conmana of the treatment nasopharyngeal carcinoma of Chinese unique independent research is attacked Biology target spot be EGF-R ELISA (EGFR), this with treatment non-small cell lung cancer Gefitinib target spot thunder Together, the innovation of shortage target spot, indication are single, totally reflect the more American-European backwardness of R ＆ D Level of the original medicine of domestic targeting.Plus It, the patient's drug resistance and import targeted drug that the targeted drug for having come out causes in clinical Long-Time Service is expensive so that visiting The new biological targets of cancer and research and development novel targets targeted drug is asked to become the task of top priority.Signal Transducer and Activator of Transcription type3 are (referred to as：STAT3) it is a kind of signal transduction and activating transcription factor, its Albumen receives much concern at present as the novel targets of therapy-related cancer, STAT3 and head and neck cancer, breast cancer, lung cancer, prostate cancer and The generation of many cancers such as leukaemia, shift and go back to the nest closely related.The STAT3 inhibitor of international correlative study report also has： STAT 21 (biologically active of the compound of two generations optimization is not improved), STATTIC (interact without compound-protein Structural model, structure optimization is had no way of handling) and Hong Kong Chinese University research report suppression nasopharyngeal carcinoma cucurbitacin I (naturalization Compound modification is more difficult).In a word, the target spot now for the targeting antitumor medicine of clinical treatment tends to Cell signal propagation pathways Upstream, easily cause drug resistance, and be based on STAT3 target spots can effectively overcome this weakness grinding medicine.This project is adopted With the drug design theory and efficient drug screening flow process of international advanced protein molecular target, from nearly 1,000,000 compounds First generation compound is filtered out in the compound library of structure, according to 27 higher compound groups of biologically active compound is established Quantitative structure activity relationship model, the biologically active of the lead compound after optimization significantly improves compared with the first generation, is international STAT3 suppressions The latest developments in preparation research field.Project application people find third generation STAT3 inhibitor living body biological activity rating compared with More than the high twice of taxol, meet the pharmacokinetics standard and internal medication standard of oral drugs.This project relies on Henan big Huaihe River hospital is learned, using the organic synthesis ability and medicine vivo assessment system of pharmaceutical college in natural drug and immune engineering laboratory System, and by means of U.S. side up to the advanced preparation process of medical science Co., Ltd, medicine generation, Safety Assessment System and abundant medicine Thing managerial experiences etc., must produce a desired effect, and realize the key breakthrough of long-term program for the development of science and technology in country.

We intend to solve the Drug-resistant sex chromosome mosaicism occurred in clinical cancer targeted therapy.Introducing the advanced medicine of American side While appraisement system and preparation process, with its efficient medicine management regulation and the Chinese independent knowledge of clinical method for transformation exploitation are ground The targeting type new drug of property right, and build combination medicine research and development centre of Sino-U.S..The research and development of this project will successfully facilitate China's cancer to control The important breakthrough for the treatment of method, promotes Chinese Medical Industry's to be converted from imitated sexual norm to innovative pattern, accelerates long-term in country The realization of goal of science and technology development planning.Simultaneously we push away advanced drug screening and drug evaluation strategy with Chinese characteristics Guang Dao worlds pharmaceutical field, international sale is applied to the cancer clinical treatment in the U.S., brings up Chinese single variety target medicinal and exists Leading situation in international pharmaceutical industries like product, while declaring clinical trial and the production batch of Chinese CFDA and U.S. FDA Number, seek medicament research and development and declare the new international cooperation pattern of " two-orbit parallel propulsion ".The STAT3 of this Project-developing suppresses Agent breaches at present limitation of the targeting cancer therapy based on the target spot of the signal transduction such as EGFR, ABL upstream both at home and abroad, belongs to sulphonyl Amine, synthesizes with low cost, it is contemplated that price is only the 1/3 of the price of Conmana, and the 1/20 of Gleevec, research and develop and be successfully expected into The just novel targets targeting type new drug of Chinese first independent research, its feature be evident in efficacy, anticancer spectrum it is wider, cheap and The features such as replacement cycle is shorter, researches and develops and will successfully lift the R ＆ D Level of the original target medicinal of Chinese Medical Industry's, drives Chinese Medical Industry's are converted from imitated property Industry Model to innovation mode.This project meets《Long-term Science and Technology is sent out in country Exhibition planning outline (2006-2020)》The preferential problem of major fields "：The preventing and treating of tumour and great NCD.

STAT families and physiological mechanism Signal transducer and activator of transcription are simple Referred to as Stat, is signal transduction and activating transcription factor.Stat families include STAT1,2,3,4,5a, 5b and 6, are one group endogenous The cell transcription factor of property.Janus Kinase are referred to as Jak, including Jak1, and 2,3 and Tyk2, it is one group and ceases manner of breathing with Stats The non-receptor tyrosine kinase of pass.The organic cooperations of Jak-Stat build the cellular signal transduction of one group of important mammal Approach, and many important cytokine signalings can be transmitted in core from cells in vitro, so as to control the transcription of target gene With the expression of downstream protein.Multinomial related Research Literature report：It is normal thin that Jak-Stat signal transduction paths participate in regulation The breeding of born of the same parents, differentiation and apoptosis.Seven Stats protein of all mammals all have six structural regions：The first petiolareas of N (ND), composite screw area (CC), DNA lands (DBD), bonding pad (LD), Src homeodomains 2 (SH2), C termination environments turn Record activation domain (CD).Cell factor (Cytokine) triggers the Dimerized (dimerization) or many of acceptor with receptor binding Multimerizing (oligomerization) and allow Jaks phosphorylations (phosphorylation) to activate Jak-Stat signals, lead to Cross in recipient cell after birth the phosphorylation of the specific tyrosine of C termination environments many places and combined with the SH2 regions of Stat, so as to complete to receive The combination of body-Stat.The activation of Stat interacts the N sections of unphosphorylated hibernation state Stat the antiparallel dimerization to be formed Body Conformation transition is the parallel conformation of the bidirectional crossed interaction in the SH2 regions of activated state Stat of phosphorylation.Activation Stat3 dimers are transferred in nucleus by the assistance of related input carrier (importin-alpha), and with it is specific DNA target mark is combined (see figure one).Stat causes Chromosome recombination with other transcription factor interactions.Nucleoprotein tyrosine phosphorus Acidifying (N-PTPs) is assisted Stat to depart from DNA and Stat non-phosphorylatings is converted to hibernation state so as to the activated state for completing Stat, And it is finally completed the circulation of Stat hibernation state-activated state-hibernation state.Cytokine signaling albumen inhibiting factor (SOCS2) is formed Negative regulator is circulated, and cuts off the conduction of Jak-Stat signals.Activation Stat albumen inhibiting factors (PIAS) by blocking dna and The combination of Stat3 is suppressing the activity (1) of Stat.

The relation of Stat3 and Stat1 and cancer is in cell proliferation and breaks up, IL-s of the Stat3 in inhibited apoptosis Play a crucial role in 6-gp130 signal transduction paths, for example, (IL-6)-gp130-Stat3 signal transductions participate in regulation and control B cell Differentiation is shaped as the thick liquid cell of antibody.Stat3 participates in the signal transduction path of various IL-6 families, including IL-6, leukaemia suppression The factor (LIF) processed, ciliary nerve nutrition is because of (CNTF), tumour inhibitor-M (OSM), the myocardial nutrition factor 1 (CT1).Stat3 is various The downstream regulatory factor of kinases.Such as：V-abl, LMP1, v-eyk, HTLV-1, c-src and Jak family.Jak-Stat3 Non-intermittents It is excited cause many cancers, such as：Head and neck neoplasm (＞ 90%), breast cancer, acute myelogenous leukemia (AML) is chronic Lymphatic leukemia (CLL).Stat1 is uniquely to participate in the signal transduction of interferon gamma to suppress the Stat families of cell proliferation Member, although Stat1 can be activated together with Stat3 sometimes, but mainly Cell growth inhibition, Stat1 is considered as tumour suppression The factor processed.The tumour growth for rejecting the mouse of the carrying malignant tumour of Stat1 genes is swifter and more violent (2) than the development of wild type.

Stat3 SH2 regions are converted into activated state by hibernation state and are joined as the theoretical foundation Stat albumen of pharmacological target In the signal transduction of relevant cell factor, including two important steps：One is special by terminal many places in own cells film The tyrosine for determining phosphorylation completes the process that acceptor recruits Stat with Stat SH2 regional interactions；Second, passing through Stat The specific phosphorylation of C-terminal tyrosine (pTyr705) and the SH2 regions of corresponding Stat bidirectional crossed interaction, Stat- Stat forms the activated state dimer of parallel conformation.Block in the mechanism of pTyr-SH2 areas interaction using Stat3,5 Stat3,5 activity, to realize suppressing abnormal Stat3,5 signals to conduct in cancer cell, is a cancer cell specific induction of apoptosis and controls The more fabulous conceptual frame (1-4) of cancer.

Stat SH2 regions are the protein modules that a class possesses about 100 amino acid.The function in SH2 regions is specificity Identification phosphorylation tyrosine and position it.In Jak-Stat signal transduction paths, tyrosine phosphorylation cause albumen- Interaction between albumen, and activate with forming waterfall type.Receive with endochylema based intracellular cvtokine including Stat SH2 regions The tyrosine of SH2 regions and specific phosphorylation in the interaction of the tyrosine of the C- ends phosphorylation of body and dimer (pTyr705) intersect interact (3,4).

The main secondary structure in SH2 areas includes：Big β-the platy structure of two alpha-helixs and one.Cytokine receptor born of the same parents Stat3 C- ends phosphorylation in the feature that combined of peptide sequence pYXXQ and Stat3 SH2 region and activated state dimer of slurry C-terminal The features that combined with Stat3 SH2 regions of TYR peptide sequence PpYLKTK show Stat3 SH2 and other molecule phases jointly The characteristics of interaction.Positioned at the N- ends in β-platy structure central authorities, Arg β B5 (R609/Stat3, R618/Stat5) is and phosphorylation The key position that tyrosine is combined, two oxygen molecules of phosphorylated tyrosine side chain form pole with the side chain of Arg β B5 basic amine groups Property adds the interaction of electrostatic.The amino acid of the C-terminal of phosphotyrosine peptide and Stat SH2 mono- deep hydrophobic region (its Have W623/Stat3, W641/Stat5 including important characteristic amino acid) interact.So, the computer that we select is matched somebody with somebody The field of force region of position simulation (docking) mainly includes phosphorylated tyrosine action site Arg β B5 (R609/Stat3, R618/ Stat5) and hydrophobic effect site (W623/Stat3, W641/Stat5) (3,4).

The content of the invention

The invention mainly solves the technical problem of controlling in preparation there is provided a kind of signal transducting system inhibitor compound Treat the application in terms of cancer drug.

For achieving the above object, the invention provides the compound of general formula is preparing treating cancer medicine or controlling Treat the application in psoriasis drug object space face：

Molecular docking test result indicate that Cpd0460 can with Stat3 Protein Ss H2 interval (domain) crucial amino Acid-lysine K591 and arginine R609, and hydrophobic cave that SH2 is interval (mainly by valine V637, glutamine Q635, color Propylhomoserin W632, tyrosine Y640 and tyrosine Y657 constitute) produce strong interaction, so as to block polypeptide in phosphorylated tyrosine With the interaction in the interval, cause to block the Stat3 albumen attached collection and Stat3 protein activations on cytokine receptor pair The formation of body conformation, and then reach the effect for suppressing Stat3 signal transductions and cancer cell specific induction of apoptosis (see Fig. 2).BIAcore's Experimental evidence proves that Cpd0460 can form strong interaction (see figure in low concentration (in striving unexpectedly with MALDI-PSD) and Stat3 3).The experimental evidence of protein immunoblot (Westernblot) proves that Cpd0460 can effectively suppress at low concentrations Stat3 Phosphorylation and downstream signal transduction (see Fig. 4).MTT cell apoptosis assays evidence proves that Cpd0460 can be lured at low concentrations Lead the Apoptosis (see Fig. 5) of a series of breast cancer cancer cell strong to Stat3 signal transduction dependences.

Description of the drawings

Fig. 1 is Stat3 signal transducting system schematic diagrames；Cell factor (Cytokine) triggers the two of acceptor with receptor binding Multimerizing (dimerization) or polymer (oligomerization), and make Jaks phosphorylations (phosphorylation) so as to activate Jak-Stat signals, by the phosphorus of the specific tyrosine of C termination environments in recipient cell after birth Acidifying makes that Stat is attached to be combined on acceptor；After phosphoric acid, Stat forms the binary parallel conformation of bidirectional crossed interaction；It is transferred to In nucleus, and combined with specific DNA target mark, inducing DNA transcribes the expression with downstream albumen.

Fig. 2 is compound Cpd0460 and the experimental result of STAT3 Protein S H2 functional domain molecular dockings in embodiment 1, is changed Compound Cpd0460 is presented in green box, and backbone carbon atoms (C) and latter end hydrogen atom (H) are unmarked, remaining all kinds of atom With atomic symbol mark-oxygen (O), nitrogen (N) and sulphur (S)；On Stat3 Protein Ss H2 interval, with Stat3-I-Q molecule phases The key amino acid of interaction has：Lysine K591 and arginine R609 and tryptophan W632 use respectively K591, R609 and W632 Mark；The interval beta- of Stat3 Protein Ss H2 is folded, alpha spirals and random coil respectively with delaying straight band, hurricane band and thin Pipe is represented.

Fig. 3 is the experimental data with polypeptide BIAcore of compound Cpd0460 in embodiment 2, (a) in different concentration Under, BIAcore difference in signal strength [(Stat3-I-Q is combined with MALDI-PSD)-(Stat3-I-Q and non-phosphoric acid is measured for the first time Change polypeptide combination)] time dependent indicatrix；B the mean value of () normalized measurement result twice is with change in concentration Indicatrix, the italic numeral on right side represents the concentration of each indicatrix.

Fig. 4 is the test of the protein immunoblot (Western blot) of Cpd0460 in embodiment 3, upper glue-phosphorylation Stat3 albumen is measurement theme, and the next glue-total amount Stat3 albumen is measurement percentage positive control；According to the test, compound At concentration 5 (uM), Stat3 albumen is substantially reduced.

Fig. 5 is the test of phosphorylation Stat3 (Y705) protein immunoblot of the cellular level of Cpd0460 in embodiment 4. Abscissa is Log (compound concentration) (concentration unit：Mole (M))；Surveyed concentration is respectively：0.5,1,5,10,50,100 He 500 micro- rub (uM). ordinate for phosphorylation Stat3/ total amount Stat3 percentage. gained IC50 is 4.6 micro- to rub (uM).

Fig. 6 is the MTT cell apoptosis assays of compound Cpd0460 in embodiment 5

Fig. 7 is compound Cpd0460 organic synthesis route maps in embodiment 6

Specific embodiment

To describe technology contents of the invention, structural feature in detail, purpose and effect being realized, below in conjunction with embodiment And coordinate accompanying drawing to be explained in detail.

Embodiment 1：Molecular docking (docking) is tested

Method：In order to the selective target for providing one group of rational computer forecast attacks the chemical probe of Stat3, we It is main with phosphorylation TYR (pY-705) calmodulin binding domain CaM in Stat3 SH2 areas as computer coordination simulation (docking) site To include phosphorylated tyrosine action site R609 and hydrophobic effect site W623.The structure coordinate of Stat3 SH2 is taken at albumen Matter structural database (PDB data bank, ID：1BG1).Selected chemical molecular storehouse (Chemical compound Library) it is LIFE CHEMICALS.The method of molecular docking (docking)：All of computer coordination simulation (docking) experiment is completed on the operating platform of Schrodinger-Maestro, computer coordination simulation used (docking) instrument is Glide.First, by Stat3 SH2 from PDB data bank take out and under be loaded in Schrodinger Operating platform-Maestro.After removing water, it is plus hydrogen atom and electric charge and by PDB file translations by the amino acid of Stat3 SH2 Maestro files.(mainly include phosphorylated tyrosine action site R609 and hydrophobic effect site according to selected site W623) calculate, determine potential energy level (potential gradient) and make the experiment (3- of computer coordination simulation (docking) 5).Interpretation of result：Fraction (Score) and conformation and transactional analysis according to simulation (docking).We choose optimum 200 compounds make the experiment test in downstream.Cpd0460 conformations are shown in respectively Fig. 1.To verify the 200 of computer simulation prediction Whether remaining compound can suppress the interaction of Stat3 and the polypeptide of fixed phosphorylation in molecular level, and we apply SPR skills Art, on the instruments of BIAcore 3000, operates and completes this test.

Embodiment 2：BIAcore is tested

Method：Selected compounds are at 25 DEG C, by phosphoric acid with the experiment (BIAcore) of Stat3 intermolecular interactions Change polypeptide to be fixed on the biochip of the BIAcore 3000 of streptavidin attachments (biosensor), flowing used Buffer solution is the Tris buffer of the 20mM concentration of pH8 and contains water-soluble with the mercaptoethanol and 5%DMSO of 2mM concentration Liquid, the speed of its flowing is 10uL/min.Before test, Stat3 albumen and the selected compounds of 500nM concentration are mixed into determined dense Degree 0.1,0.5,1,5,10,50,100,500uM.Every time after test, rinsed with the glycine of the 100mM of the 10uL that pH is 1.5 Biochip (biosensor) is treating next test.The Stat3 albumen of the 500nM without medicine mixing is from initial to termination root point Do not test once to determine whole test system normal operation (6) all the time.Experiment (BIAcore) interpretation of result：Cpd0460 is not With the time dependent indicatrix of BIAcore signals visible (Fig. 3 (a)) of concentration, (abscissa numeral 0 in Fig. 3 (a), 50, 100th, different time points is represented respectively within 150,200,250,300,350,400 and 450 seconds.The experimental result of BIAcore is by table The indicatrix of the mean value with change in concentration of normalized measured value twice is shown as, normalization BIAcore signals are not assimilate The measured value of compound is multiplied by 100 (see Fig. 3 (b)) divided by the reference point without compound, abscissa numeral 0 in Fig. 3 (b), 0.1, 0.5th, 1,5,10,50 and 100uM compound concentration is represented respectively.(in indicatrix Fig. 3 (b), Diamond spot represents each concentration The measured value of BIAcore signals, the vertical short wires on Diamond spot represent the standard error for measuring twice).Cpd0460 suppresses 50% Stat3 and the concentration value (IC50) of compound of interaction of MALDI-PSD be all 1-5 (uM).Above-mentioned experimental evidence table It is bright：Cpd0460 is combined and can be suppressed the Stat3 of half and the phase of MALDI-PSD under the low concentration of sub-micromolar with Stat3 Interaction.

Embodiment 3：Protein immunoblot (Westernblot)

Method：The experimental technique of protein immunoblot (Western blot)：At the standard conditions, cultivate in six orifice plates People's liver HCC (HepG2).Respectively with variable concentrations -0.5,1,5,10 (uM), with compound treated cells and to cultivate 1 little When.Cell being processed with the IL-6 that concentration is 30ng/ml and cultivating half an hour go to stimulate Stat3 phosphorylations.After cell is collected, with height Salt buffer (high-salt buffer) liquid separates albumen.Albumen after separation mixes with SDS, at 100 DEG C, heats 5 minutes. Electrophoresis is done in SDS-PAGE polyacrylamide gels, the albumen of different quality is distinguished and fix, then by protein delivery to PVDF Film simultaneously is gone to cover pvdf membrane with BSA.Molecular weight marker is inserted in advance SDS-PAGE glue and is transferred to pvdf membrane.Use anti-phosphorus The first antibody of acidifying pY705-Stat3 and the first antibody of anti-total amount Stat3 go to detect phosphorylation pY705-Stat3 and total Amount Stat3；With the chimeric sheep-anti-mouse IgG of HRP as enzyme target SA.Detectable signal is removed with chemical photosensitive system is strengthened. Before the detection of total amount Stat3 antibody is transformed into, gone with buffer solution-Restore Western Blot Stripping Buffer Detectable signal (7).The experimental result of protein immunoblot (Western blot)：Upper glue is phosphorylation Stat3, and the next glue is Total amount Stat3, signal shows from left to right：First is classified as the double negative controls of signal factor-compound (stimulates and while nothing without IL-6 Compound is added), second is classified as the n- compound negative control of signal factor (IL-6 stimulates and adds without compound simultaneously), and the 3rd Arrange and be followed by, in the case where signal factor stimulates, the compound of variable concentrations (0.5,1,5,10 micromole) is to phosphorylation Stat3 Interference and result；Its result shows the ability that Cpd0460 has significant suppression Stat3 phosphorylations under sub- molar concentration.

Embodiment 4：Phosphorylation Stat3 (Y705) protein immunoblot (the Cell-based ELISA of cellular level Phospho-STAT3(Y705)immunoassay)

The experimental technique of phosphorylation Stat3 (Y705) protein immunoblot of cellular level：By about the 20000 of 100 microlitres Individual MBA-MD-231 population transplants are in each hole of 96 orifice plates (bottom hole is transparent and face is surveyed for black in hole).Cultivated 5% gas concentration lwevel, in the cell culture incubator (cell culture incubator) at a temperature of 37 DEG C overnight.Second My god, with changing and thing processes cell (compound concentration is respectively 0.5,1,5,10,50,100,500 micromole (uM)) and by chemical combination Cell after thing process places cell culture incubator culture one hour.Then, with cell factor-IL-6 cell is processed with hyperplasia Stat3 phosphorylations are simultaneously placed cell culture incubator culture half an hour, and IL-6 concentration is 30ng/ml.With 4% good fortune of 100 microlitres (ul) The fixed cell of your Malin (formaldehyde) is simultaneously cultivated 20 minutes at room temperature.After three times are rinsed, with the 0.6% of 100 microlitres H2O2 process cell and in incubated at room temperature 20 minutes.After three times are rinsed, processed with stop buffer solution (Blocking buffer) Cell is simultaneously cultivated 60 minutes at room temperature.Three times rinse after, with first antibody (Primary anti-body) mixed liquor (rabbit The antibody of the Stat3 antibody of 705 tyrosine of anti-phosphorylation and the anti-pervasive Stat3 of mouse) process cell and place 2 DEG C of refrigerators Overnight incubation.After three times are rinsed, with second antibody (Secondary anti-body) mixed liquor (sheep of HRP enzyme conjugations-anti- The sheep of rabbit igg antibody and AP enzyme conjugations-anti-mouse IgG antibody) cell is processed, and cultivate two hours at room temperature.Lavation buffer solution (Washing buffer) is rinsed twice and after 1X PBS rinse twice, is located with 75 microlitres of the first substrate (Substrate)-F1 Reason cell is simultaneously wrapped up with tin platinum paper, places room temperature 30 minutes.Cell is processed with 75 microlitres of the second substrate (Substrate)-F2 And wrapped up with tin platinum paper, place room temperature 30 minutes.Finally, solution device (fluorescence plate are read with fluorescence labeling Reader, infinite-200, Tecan) read solution data.The IgG antibody of HRP enzyme conjugations excitation state wavelength -540nm, radiation State wavelength -600nm is explaining；The IgG antibody of AP enzyme conjugations excitation state wavelength -360nm, radiant state wavelength -450nm are explaining (8)。

The experimental result of phosphorylation Stat3 (Y705) protein immunoblot of cellular level：Cell based ELISA The experimental result of immunoblot assay is shown in Fig. 5.Abscissa is the Log values of compound concentration, and compound concentration unit is to receive Mole (nM)；Surveyed compound concentration is respectively：0.5th, 1,5,10,50,100 and 500 are micro- rubs (uM).Ordinate is phosphorylation (Stat3P705-STAT3) divided by percentage (the Ratio of pYSTAT3/Total of total amount Stat3 (Total STAT3) STAT3).The test result of the Immunoblot-IC50 of gained Cpd0460 is respectively 4.6 (uM).Above the results show： Under the concentration of sub-micromolar, Cpd0460 can effectively suppress a kind of MBA-MD-231 (breasts of Stat3 signals Non-intermittent activation Gland cancer cancer cell) cell line Stat3 phosphorylations.

Embodiment 5：The experiment of induced breast cancer cancer cell-apoptosis MTT

Method：MTT is widely used in the detection of cell propagation and cytotoxicity.Our MTT experiment is mainly medicine Detection to the cytotoxicity of in vitro culture.Preparatory work of experiment：Stimulating factor：IL-6；Compound：Cpd0460, test cell： HepG2, MDA-MB-231 negative control cell：MCF-7, dry powder solvent：DMSO or sterilizing ddH20, medicine：WP1066、 F1566-0352、F1566-0424、F1566-0460.Experimental procedure：1st, first day by hepG2, MDA-MB-231, MCF-7 tri- Kind 5000/hole of cell count bed board in good condition is in 96 orifice plates.2nd, IL-6 is overnight added to stimulate cell, IL-6 final concentrations afterwards For 30ng/ml, stimulation time is 30min.Then several cells distinguish drug-treated, and dosing final concentration is with 1,3,10,30,100 (uM) several gradients carry out drug-treated, and continuously cultivate 48h.3rd, after drug-treated, MTT 10 microlitres of (reagents of reagent are added Box), 37 degree are incubated 4 hours, are subsequently adding 100 microlitres of Formanzan solution to being completely dissolved (4 hours or so), upper ELIASA Readings (570nm), records result.

Experimental result and analysis twice understands：The OD values that compound Cpd0460 is measured in MDA-MB-231 cells are big In the OD values of MCF-7 cells, the effect for illustrating this compound inducing mammary JEG-3 (MDA-MB-231) apoptosis is better than the moon Property control MCF-7 cells.

Embodiment 6：Organic synthesis

Method：The chloro- 1,4-naphthoquinones of 2- (0.5mmol) and 4- methoxybenzenesulphoismides (0.6mmol) are dissolved in into tetrahydrofuran (1.8ml) in, stirring adds titanium tetrachloride tetrahydrofuran compound (0.6mmol) at 0 DEG C, and adds triethylamine (1.3mmol). Mixture is stirred to room temperature in 80w heated under microwave conditions to 60 DEG C of reaction 1h.Reaction is cooled to room temperature and adds 20ml acetic acid Ethyl ester.Material is filtered by diatomite in reaction bulb, and ethyl acetate washing, filtrate is spin-dried for.10ml dichloros are added in thing is spin-dried for Ethane, is filtered by diatomite, is spin-dried for after filtrate that residue is dissolved in the tetrahydrofuran of 10ml.Add in tetrahydrofuran solution 3- sulfydryl -1 of 0.5mmol, 2,4- triazoles, stirring reaction 4h under room temperature condition, and thing is spin-dried for the dissolving of 10ml ethyl acetate.Plus Enter 5ml water and 2.5mmol sodium thiosulfate.Mix 1h.Layering extraction, water is washed through three 10ml ethyl acetate Afterwards, organic phase is used in mixed way into 25ml saturated common salt water washings, after anhydrous sodium sulfate drying, is filtered, be spin-dried for slightly being produced Thing, carries out crude product pillar layer separation and obtains target product.

Citation

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Claims (3)

1. the signal transducting system inhibitor compound of following chemical formula is preparing the application for the treatment of cancer medicine：

。

2. the application according to claim 1, it is characterised in that the medicine is oral drugs or intravenous (IV) drug.

3. the application according to claim 1, it is characterised in that the medicine is the medicine for treating breast cancer.