CN107200713A - Antitumoral compounds and preparation method thereof and purposes - Google Patents

Abstract

The present invention relates to antitumoral compounds and preparation method thereof and purposes, the antitumoral compounds are specially the structure shown in Formulas I.The invention further relates to the compound shown in the Formulas I or its pharmaceutically acceptable salt, or containing its pharmaceutical composition by suppressing EGFR, mutant egf R^T790MSkin factor receptor protein tyrosine kinase and FAK focal adhesion kinases and then treatment tumor disease, the particularly purposes for treating non-small cell lung cancer, ED-SCLC；

Description

Antitumoral compounds and preparation method thereof and purposes

Technical field

The present invention relates to antitumoral compounds and preparation method thereof and purposes, specifically, the antitumoral compounds are phonetic Pyridine class compound, belongs to pharmaceutical technology field.

Background technology

Protein tyrosine kinase (protein tyrosine kinase, PTKs) is by controlling the signal transduction of cell to lead to A series of physiological and biochemical procedures such as growth, differentiation, the apoptosis of road regulation cell.Receptor type tyrosine kinase is a class across cell The relatively large kinases of film, its ectodomain with ligand binding, membrane spaning domain and a zymogenesis-in phosphorylation Specific tyrosine residue and the thus intracellular domain of influence cell propagation.(such as lung cancer, mammary gland in general human cancer Cancer, stomach cancer, oophoroma, lymthoma) have found the unconventionality expression of the kinases.Protein tyrosine kinase turns into antineoplastic One of important target spot of research and development.

Epidermal growth factor recipient tyrosine kinase (epidermal growth factor receptor tyrosine Kinase, EGFR) it is one of protein tyrosine kinase for finding earliest, EGFR intracellular region has ATP-binding site, and EGFR suppresses Agent competitive can be combined with ATP-binding site, so as to suppress EGFR Phosphorylation events, block the conduction of downstream signal, And then suppress the growth, differentiation and transfer (Yun, et al.Cancer Cell 2007,11,217-227) of tumour cell.EGFR It has been elucidated with as the biochemical process of anti-tumor target, its crystal structure and active site are also clearer, as target spot Medicine Gefitinib (gefitinib), Erlotinib (erlotinib), Afatinib (afatinib) etc. be applied to Clinic, and with the further investigation of EGFR structures and activity relationship, many better outstanding EGFR inhibitors (EP0566226、WO9961428、WO0051587、WO0375947、WO0132651、WO9633980、WO9630347、 US7709479, US6716847, US6593333, US6251912, CN201080060451.4, CN201110191525.4 etc.) Have been observed that.But these medicines are inevitably present the problem of anti-drug resistance is poor.Research shows：Soviet Union's ammonia in 790 sites The mutation (T790M) of acid to the methionine in 790 sites is the main inducing of such drug resistant.There is clinical case number According to display, about 60% patient's acquired resistance is all originated from caused by the mutation in T790M sites.Therefore anti-drug resistance is developed more By force, the new E GFR inhibitor that toxicity is smaller, activity is stronger has extremely important realistic price.

AZD9291 (Osimertinib) is oral irreversible, T790M Catastrophic selection EGFR inhibitors, thin in LoVo To the EGFR of 19 missings of extron in born of the same parents^L858R/T790MAnd EGFR^WTIC₅₀Respectively 11.44 and 493.8nM, in 2015 12 Being approved by the FDA in the United States listing (WO2013014448) is used to treat EGFR^T790MDrug-resistant type lung cancer.CO-1686(Rociletinib, AVL-301) it is another irreversible, Catastrophic selection EGFR^T790MInhibitor, acts on EGFR in Cell free assay^T790M And EGFR^WTIC₅₀Respectively 21.5nM and 303.3nM, is currently in clinical III phases research (WO2012061299).Its Its such as HM61713, EGF816, PF-06747775, Avitinib, ASP8273 are also the new E GFR suppression into clinical research Agent (Ma et al., J.Med.Chem., 2016, DOI:10.1021/acs.jmedchem.5b00840.), the patent being related to Such as：US20120157426、US8563568B2、CN102740847、CN102083800.

Focal adhesion kinase (focal adhesion kinase, FAK) is a kind of intracellular nonreceptor tyrosine kinase, FAK is a kind of intracellular nonreceptor tyrosine kinase, participates in the conduction of many signal paths of intracellular.FAK is polytype swollen All occur expression quantity and activity up-regulation phenomenon in oncocyte, relied on by kinases and nonkinase dependent mechanism participates in tumour cell and invaded Attack, shift, breeding, growing and multiple processes such as anti-apoptotic, being to study one of more anti-tumor target at present.

In view for the treatment of cancer is necessary to develop new mechanism of action and better good in the urgent need to, this area Antineoplastic.

The content of the invention

An object of the present invention is one antitumoral compounds of offer or its pharmaceutically acceptable salt, and this is antitumor Compound is specially the pyrimidines containing morpholine, and such compound has good antitumor activity.

Another object of the present invention is to provide the preparation method of foregoing antitumoral compounds.

Another object of the present invention is to provide to contain described containing the pyrimidines of morpholine or its is pharmaceutically acceptable Salt pharmaceutical composition.

It is still another object of the present invention to provide described containing the pyrimidines of morpholine or its is pharmaceutically acceptable Salt, or the composition purposes.

Therefore, on the one hand, the present invention provides a kind of antitumoral compounds or its pharmaceutically acceptable salt, antitumorization Compound has the structure shown in formula (I)：

Structural compounds shown in formula (I) are the pyrimidines containing morpholine, are named as DM3001, and the present invention is antitumor Screening active ingredients show that the compound has strong suppression non-small cell lung cancer (NSCLC) ability of cell proliferation, shows ratio Anti- EGFR more excellent Rociletinib^T790MActivity, and can act on FAK kinases.It is used as novel point of a class formation Research compound has the potentiality for developing into the double target spot inhibitor of new and effective EGFR and FAK in son, the present invention, related to treatment Tumor disease especially non-small cell lung cancer, ED-SCLC have larger application value.

Structure shown in foregoing formula (I) has following title：

(I) N- [1- [[the chloro- 2- of 5- [[4- [(4- morpholines base) methyl] phenyl] amine] -4- pyrimidine radicals] amine] phenyl] -2- Acrylamide.

On the other hand, the present invention provides the preparation method of foregoing antitumoral compounds, and the antitumoral compounds press following road It is prepared by line：

Compound of the present invention is due to their possibility purposes in medicine, the preferred medicine of salt of compound shown in formula (I) The acceptable salt of thing.The compound of the present invention is alkali, and salt form needed for it can pass through appropriate parties legal system known in the art It is standby, including mineral acid treatment free alkali is used, the inorganic acid is such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid；Or with organic Acid treatment free alkali, the organic acids for example acetic acid, trifluoroacetic acid, maleic acid, butanedioic acid, mandelic acid, fumaric acid, malonic acid, Pyruvic acid, oxalic acid, hydroxyacetic acid, salicylic acid, pyranose thuja acid (pyranosidyl acid), such as glucuronic acid or galactolipin Aldehydic acid, such as 'alpha '-hydroxy acids, citric acid or tartaric acid, such as amino acid, aspartic acid or glutamic acid, such as aromatic acid, benzene first Acid or cinnamic acid, sulfonic acid, such as p- toluenesulfonic acids, methanesulfonic acid, ethyl sulfonic acid.The embodiment of pharmaceutically acceptable salt includes sulphur Hydrochlorate, pyrosulfate, disulfate, sulphite, bisulfites, phosphate, chloride, bromide, iodide, acetic acid Salt, propionate, caprate, caprylate, acrylates, formates, isobutyrate, caproate, enanthate, propionate (propiolates), oxalates, malonate, benzoate, chloro benzoate, methyl benzoic acid salt, dinitrobenzoic acid Salt, hydroxy benzoate, methoxy benzoic acid salt, phthalate, phenyl acetate salt, phenylpropionic acid salt, PB (phenylbutrates), citrate, lactate, gamma hydroxybutyrate, hydroxyl acetate, tartrate, amygdalate With sulfonate, such as xylenesulfonate, mesylate, propane sulfonic acid salt, naphthalene -1- sulfonate and naphthalene-2-sulfonic acid salt.

On the other hand, the present invention provides a kind of pharmaceutical composition, and it contains shown in the formula of the present invention (I) of effective dose Compound or its pharmaceutically acceptable salt, and pharmaceutical carrier.

The pharmaceutical composition of the present invention usually contains a kind of the compounds of this invention.However, in some embodiments, this hair Bright pharmaceutical composition, which contains, has more than a kind of compound of the invention.In addition, the pharmaceutical composition of the present invention can also optionally include One or more other pharmaceutically active compounds.It is of the present invention containing the pyrimidines of morpholine or its is pharmaceutically acceptable Carrier there is the activity of good suppressions EGFR skin factor receptor tyrosine kinases, therefore, contain described in the present invention also offer The pyrimidines of morpholine or its pharmaceutically acceptable carrier, or described pharmaceutical composition are preparing EGFR skin factors Application in receptor tyrosine kinase inhibitors.

Present invention discover that described have well containing the pyrimidines of morpholine or its pharmaceutically acceptable carrier Suppress the activity of FAK focal adhesion kinases, therefore, the present invention also provide it is described containing the pyrimidines of morpholine or its pharmaceutically Acceptable carrier, or application of the described pharmaceutical composition in FAK focal adhesion kinase inhibitor is prepared.

The present invention also provides the compound or its pharmaceutically acceptable salt shown in the formula (I), or of the present invention Purposes of the pharmaceutical composition in the medicine for preparing treatment tumour.Preferably, the tumour is selected from non-small cell lung cancer, cellule Lung cancer, further preferred non-small cell lung cancer.It is highly preferred that the purposes is mainly by suppressing EGFR skin factor receptor proteins What EGFR-TK and FAK focal adhesion kinases were realized.

Brief description of the drawings

Fig. 1 is different disposal group H1975, A431 cell DAPI staining conditions.

Fig. 2 is different disposal group H1975, A431 cell AO/EB staining conditions.

Fig. 3 is different disposal group H1975, A431 cell cut and Cell migration assay result.

Fig. 4 be DM3001 in two kinds of cells of H1975 (A) and A431 (B) to EGFR downstreams coherent signal protein expression Situation.

Embodiment

The explanation present invention is further described below in conjunction with specific embodiment, but these embodiments are not meant as limiting this hair Bright scope.

The experimental method of unreceipted actual conditions in the embodiment of the present invention, generally according to normal condition, or according to raw material or Condition proposed by commodity manufacturer.The unreceipted reagent specifically originated, is the conventional reagent of market purchase.

The preparation of the target molecule of embodiment 1

Tlc silica gel plate uses Yantai Huanghai Sea GF254 or Qingdao GF254 silica gel plates, and thin-layered chromatography (TLC) is used The specification that uses of silicon amine plate be 0.15mm-0.2mm, the specification that thin-layer chromatography isolates and purifies product use is 0.4mm-0.5mm.

The raw material that the present invention is used is mainly purchased from commercially available from Chemical Reagent Co., Ltd., Sinopharm Group, Beijing coupling science and technology Co., Ltd, Aladdin chemical reagent Co., Ltd, up to companies such as auspicious chemicals.

Without specified otherwise in embodiment, solution refers to the aqueous solution.

Without specified otherwise in embodiment, the temperature of reaction is room temperature, is 20 DEG C -30 DEG C.

The technical solution adopted by the present invention is as follows：

The synthetic route reagents and condition of compound (I):(a) p-nitrophenyl ammonia, DIPEA, 1,4- dioxane, rt, 2h, 91%；B) 4- morpholines methyl aniline, DIPEA, 1,4- dioxane, 60 DEG C, 2h, 49-72%；(c)Fe-NH₄Cl, MeOH-H₂O, 80 DEG C to rt, 62-85%；(d) acryloyl chloride, NaHCO₃, acetone, 10min, 43-68%.

I-b synthesis

Take I-a (23.44mmol) and DIPEA (4.5g, 35.16mmol) in 50mL dioxane, be slowly added into 4- nitre Base aniline (3.8g, 23.44mmol), is heated up 60 DEG C, and after reacting 5 hours, reaction is finished, and cooling adds 400mL water, is separated out yellow White solid, suction filtration, drying obtains white-yellowish solid, and direct next step reaction is not purified.

I-c synthesis

Take I-b (23.44mmol) and trifluoroacetic acid (4.5g, 35.16mmol) in 2-BuOH (50ml), be slowly added into 4- Morpholine methyl aniline (23.44mmol), is heated up 100 DEG C, and after reacting 8 hours, reaction is finished, and unsaturated carbonate is poured into cooling In hydrogen sodium solution, solid is separated out, suction filtration, washing, drying silica gel column chromatography separation obtains brown solid.

I-d synthesis

I-c (1.9g, 6.8mmol) and ammonium chloride (0.73g, 13.6mmol) are taken in reaction bulb, add MeOH (15mL) and Water (15mL), stirring is lower to add iron powder (1.5g, 27.2mmol), heats up 60 DEG C and reacts 2 hours, while hot suction filtration, aqueous phase acetic acid Ethyl ester extracts (100mL × 3), and combined ethyl acetate layer, saturated common salt is washed once, and anhydrous sodium sulfate drying, evaporated under reduced pressure is obtained Yellow semisolid I-d.

The synthesis of object (I)

Take I-d (2.1g, 9.7mmol) to be dissolved in 20mL acetonitriles, add sodium acid carbonate (1.6g, 19.4mmol), and methyl Piperazine (1.2g, 11.64mmol), heats up 60 DEG C and reacts 5 hours, solvent is drained after completion of the reaction, add water 200mL, does not separate out solid Body, aqueous phase is extracted with ethyl acetate (100mL x 3), and combined ethyl acetate layer, saturated aqueous common salt washed once, anhydrous sodium sulfate Dry, evaporated under reduced pressure obtains target molecule (I).

Target molecule is synthesized according to above method, the physicochemical data of synthesized target molecule is as follows：

(I) N- [1- [[the chloro- 2- of 5- [[4- [(4- morpholines base) methyl] phenyl] amine] -4- pyrimidine radicals] amine] phenyl] -2- Acrylamide

Yield 40.8%；White-yellowish solid.¹H NMR(DMSO-d₆):δ10.20(s,1H),9.32(s,1H),8.96(s, 1H), 8.15 (s, 1H), 7.91 (s, 1H), 7.56 (d, J=8.4Hz, 3H), 7.31 (d, J=6.0Hz, 2H), 7.04 (d, J= 8.4Hz, 2H), 6.48 (dd, J=17.2,10.4Hz, 1H), 6.27 (dd, J=16.8,2.0Hz, 1H), 5.76 (dd, J= 10.0,2.0Hz,1H),3.55(br,4H),3.32(s,2H),2.29(s,4H).¹³C NMR(DMSO-d₆)δ163.6,158.2, 156.7,155.3,139.8,139.7,139.6,139.3,132.4,130.6,129.4(2C),129.0,127.3,119.8, 119.3(2C),115.8,104.2,66.7(2C),62.6,53.6(2C).HRMS(ESI)for C₂₄H₂₅ClN₆O₂,[M+H]⁺Reason By calculating:465.1800, actual measurement:465.1808.

Method of the target molecule into salt

The preparation method of inorganic acid salt：Take target molecule (1mmol) to be dissolved in 10mL absolute methanols, under ice bath, slowly drip Plus the 5mL absolute methanol solutions of inorganic acid (1mmol), completion of dropping, stirred 30 minutes at a temperature of this, then first is evaporated off in normal temperature Alcohol, produces the inorganic acid salt of target molecule.By this method be prepared for compound I hydrochloride (I-1), hydrobromate (I-2), Sulfate (I-3) and phosphate (I-4)；

The preparation method of acylate：Take target molecule (1mmol) to be dissolved in 10mL absolute methanols, under ice bath, slowly drip Plus the 5mL dry ethers of organic acid (1mmol), completion of dropping, stirred 30 minutes at a temperature of this, then solvent is evaporated off in normal temperature, Produce the acylate of target molecule.By this method be prepared for compound I maleate (I-5), succinate (I-6) and Fumarate (I-7).

The target molecule biological evaluation of embodiment 2

1st, it is external to receptor tyrosine kinase inhibitory activity method of testing

Prepare kinase assay buffer

1. melt kinase assay buffer (Kinase Detection Buffer) in room temperature, see whether precipitation.

2. if there is precipitation, just it is incubated kinase assay buffer (Kinase Detection Buffer) 15 at 37 DEG C Minute simultaneously often shakes, dissolving precipitation.Or, supernatant is carefully siphoned away, precipitation is removed.

Prepare kinase assay reagent

1. using preceding at equilibrium at room temperature kinase assay buffer (Kinase Detection Buffe) and kinase assay bottom Thing (Kinase Detection Substrate).

2. kinase assay buffer (Kinase Detection Buffer) is all poured into equipped with kinase assay substrate In the brown bottle of (Kinase Detection Substrate), freeze-dried powder substrate is dissolved, kinase assay has thus been made Reagent.

3. gently shake, be vortexed or reverse mixing, as homogeneous solution, substrate should dissolve in 1 minute.

4. it should be used immediately after kinase assay reagent is prepared, or packing is stored in -20 DEG C, the reagent prepared passes through freeze thawing several times Posterior circle signal activity is not all lost.

Make the standard curve that ATP changes into ADP

1. the Ultra that kit is provided is diluted with 1 × kinase reaction buffer solution (kinase reaction buffer) Pure ATP and ADP, are made 50 μM of ADP of 900 μ L 50 μM of ATP and 500 μ L.

2. 50 μM of ATP and 50 μM of ADP solution previous step prepared by table 1 Suo Shi in 384 orifice plate A1-A12 mix, The ATP and ADP of each conversion percentages concentration are simulated, is mixed.

Table 1. prepares 50 μM of series A TP+ADP standard items

3. 5 μ L ADP-Glo is added per hole^TMReagent terminates kinase reaction.In incubation at room temperature 40 minutes.

4. 10 μ L kinase assays reagents (Kinase Detection Reagent) are added per hole and ADP is changed into ATP, and Luciferase and luciferin is introduced to detect ATP.

5. in incubation at room temperature 30-60 minutes, measure fluorescent with multi-function microplate reader and record fluorescent value.

6. the standard curve that ATP changes into ADP is drawn.

Determine the IC of kinase inhibitor₅₀Value

1. 1 × kinase reaction buffer solution (kinase reaction are prepared according to promega kit specifications Buffer), 2.5 × 50ng/ μ L kinases and 2.5 × 0.5 μ g/ μ L substrates and 125 μM of ATP.

2. 3 μ 1 × kinase reactions of L buffer solutions (kinase reaction buffer), 2 μ are added in without enzyme control wells The μ g/ μ L substrates of L2.5 × 0.5 and 125 μM of ATP.1 μ L 1 × kinase reaction buffer solutions (kinase is added in negative control hole Reaction buffer), 2 μ L 2.5 × 50ng/ μ L kinases, the μ g/ μ L substrates of 2 μ L 2.5 × 0.5 and 125 μM of ATP.In test Add 1 μ L 5 × medicine to be measured in hole, 2 μ L 2.5 × 50ng/ μ L kinases, the μ g/ μ L substrates of 2 μ L2.5 × 0.5 and 125 μM of ATP.

3. flat board is mixed, is incubated 60 minutes.

4. 5 μ L ADP-Glo is added per hole^TMReagent terminates kinase reaction.In incubation at room temperature 40 minutes.

5. 10 μ L kinase assays reagents (Kinase Detection Reagent) are added per hole and ADP is changed into ATP, and Luciferase and luciferin is introduced to detect ATP.In incubation at room temperature 30-60 minutes, measure fluorescent with multi-function microplate reader and remember Record fluorescent value.

6. interpretation of result, result is shown in table 2.

2nd, cell growth assay (MTT detection methods)

Cell is inoculated with：Exponential phase cell is collected, concentration of cell suspension is adjusted, with every hole 7x10³Individual cell, per hole body 100 μ L of product are inoculated into 96 orifice plates, and every group sets 4 multiple holes (edge hole is filled with sterile PBS)；

Cell culture：After cell attachment, 0%FBS RPMI-1640 starvation 8h, control group 10%FBS RPMI-1640 Culture, 37 DEG C, 5%CO₂Continue to cultivate (empirically requiring to cultivate different time respectively) in incubator；

Colour generation：Three groups of cells are respectively at adding termination culture after 10 μ L MTT solution (5mg/mL), 4h after culture 72h, often Hole adds the liquid of 100 μ L tri-, in low-speed oscillation 10min on shaking table, crystallization is fully dissolved；

Colorimetric：Each hole shading value (OD values) is determined on enzyme-linked immunosorbent assay instrument, 570nm wavelength is selected, with acellular I.e. RPMl-1640 nutrient solutions blank well returns to zero, and surveys the absorbance in each hole.Experiment is in triplicate

Record result：Inhibitory rate of cell growth=(the experimental group absorbance of control group absorbance one)/control group extinction Angle value × 100%, cell proliferation rate=(experimental group absorbance/control group absorbance) × 100；

Draw cell growth curve：Using the time as abscissa, inhibiting rate/proliferation rate is that ordinate draws cell growth song Line.

Do figure for inhibitor concentration in GraphPad Prism mapping softwares in GraphPad softwares, so as to by Log [inhibitor] estimates IC relative to reaction, variable slope model₅₀。

As shown in table 3, table 2 and table 3 show that the activity in obtained compound antitumor cell propagation is imitated to test result Really^a。

Table 2

Table 3

a:H1975 is EGFR^T790MMutant clone, A431EGFR^WTCell, HCC827 is EGFR del E746_A750 Mutant clone, HBE is people's normal bronchial epithelial cell .b:IC₅₀：Half effective inhibition concentration

3rd, cell dyeing is tested

3.1 experiment materials and reagent

H1975 cells, A431 cells, RAPI1640 basal mediums, DMEM basal mediums (Hyclone companies, cat: SH30022.01B), FBS hyclones (GIBCO companies, cat:16400-044), dual anti-(Beijing summer limited public affairs of this biotechnology Department, prospec cat:SV30010), low speed centrifuge (upper sea cowry grace bio tech ltd, cat:TDZ4B-WS), CO₂ Incubator (the rich news in Shanghai, cat:BC-J160S), superclean bench (the rich news model SW-CJ-2FD in Shanghai), is inverted fluorescent electronic Microscope (Leica DMI3000B), the double transfection reagent boxes of AO/EB, DAPI kits (the green skies).

3.2 experimental method

3.2.1 cell culture：It is conventional to expand culture A431, H1975 cell, the cell in growth period of taking the logarithm with 2x10⁵The density in cells/ holes is inoculated in 6 orifice plates；

3.2.2 drug-treated：In the adherent good cell of medicine (inhibitor) processing 48 hours of secondary daily various concentrations； Specific medicine and concentration such as table 4 below：

Table 4

3.2.3DAPI dyeing：Nutrient solution is discarded, is washed with PBS twice, 15min is fixed with the paraformaldehydes of 1mL 4% per hole, Discard and washed once with PBS again after fixer；Add after 1 μ g/mL DAPI dyeing liquor 1mL/ holes, 37 DEG C of incubation 5min, PBS Observed, taken pictures with inverted fluorescence microscope.As a result it is as shown in Figure 1.

AO-EB is dyed

3.2.4 cell culture：It is conventional to expand culture A431, H1975 cell, the cell in growth period of taking the logarithm with 2x10⁵The density in cells/ holes is inoculated in 6 orifice plates；

3.2.5 drug-treated：In the adherent good cell of medicine (inhibitor) processing 48 hours of secondary daily various concentrations； Specific medicine and concentration such as table 5 below：

Table 5

3.2.6AO-EB dyeing：Nutrient solution is discarded, is washed with PBS twice, per hole with 20 μ LAO-EB solution (1 μ g/mL AO+1 μ g/mL EB in PBS) dye, the change such as each phase apoptosis of observation cell, death, takes pictures, as a result such as under inverted fluorescence microscope Shown in Fig. 2.

4th, Cell migration assay

4.1 experiment materials and reagent

H1975 cells, A431 cells, RAPI1640 basal mediums, DMEM basal mediums (Hyclone companies, cat: SH30022.01B), FBS hyclones (GIBCO companies, cat:16400-044), dual anti-(Beijing summer limited public affairs of this biotechnology Department, prospec cat:SV30010), low speed centrifuge (upper sea cowry grace bio tech ltd, cat:TDZ4B-WS), CO₂ Incubator (the rich news in Shanghai, cat:BC-J160S), superclean bench (the rich news model SW-CJ-2FD in Shanghai), is inverted fluorescent electronic Microscope (Leica DMI3000B), ruler, marking pen, matrigel (U.S. company BD), methanol, (China is military for crystal violet dye liquor Biotechnology company of Chinese Google)

4.2 experimental method

4.2.1 tumour cell is inoculated in 6 orifice plates with certain density, continues to cultivate 48 hours.

4.2.2 scratch experiment is carried out in single-layer culturing cell, cell fragment is washed away with isotonic PBS liquid, then it is dense with difference The inhibitor processing cell of degree 48 hours, specific medicine and concentration such as table 6 below, are washed away dead thin with isotonic PBS liquid afterwards Taken pictures under born of the same parents, microscope, graphical analysis, as a result as shown in Figure 3.

Table 6

The transfer ability of Transwell experiment detection cells

4.2.3 filter membrane is pre-processed：By matrigel (1 of the filter membrane with 60 μ L before experiment：8 dilutions) coating, then at the top of cell Serum free medium is added, 0.5 hour is stood in 37 DEG C of incubators, hydration process again is carried out；

4.2.4 cell culture：A431, H1975 cell use conventional digestion propagating method, and cell suspension is made, and count, and adjust Whole cell concentration is 1 × 10⁶/mL；

4.2.5 the upper chamber in cell adds 200 μ L above-mentioned cell suspension, is 2 × 10 per hole cell concentration⁵It is individual；

4.2.6 give cell drug processing：Cell is handled 24 hours with the medicine (inhibitor) of various concentrations, specific medicine And concentration such as table 7 below：

Table 7

4.2.7 the nutrient solution that 600-800 μ L contain 10% hyclone is added in the lower room (i.e. 24 orifice plate bottoms) of cell；

4.2.8 37 DEG C of incubator dosings were incubated processing cell after 24 hours altogether, carefully took out cell with tweezers, blotted Room liquid, is moved on in hole in advance added with about 800 μ L methanol (4% paraformaldehyde), and room temperature fixes 10-30 minutes；

4.2.9 cell is moved on to and be previously added in the hole of about 800 μ L Giemsa dye liquors or in 2% crystal violet solution, room temperature Dyeing 15-30 minutes；

4.2.10 PBS cell is used, the cell on upper chamber bottom film surface is carefully wiped with wet swab stick；

4.2.11 thoroughly dry behind cell, inverted microscope counts, taken pictures, as a result as shown in Figure 3.

5、Western Blotting

5.1 experiment materials and reagent

H1975, A431 cell, RAPI1640, DMEM basal mediums (Hyclone companies, cat:SH30022.01B), FBS hyclones (GIBCO companies, cat:16400-044), dual anti-(Beijing Xia Si bio tech ltd, prospec cat:SV30010), low speed centrifuge (upper sea cowry grace bio tech ltd, cat:TDZ4B-WS), CO₂Incubator (Shanghai Rich news, cat:BC-J160S), superclean bench (the rich news model SW-CJ-2FD in Shanghai) EGFR primary antibodies (Bioworld ), Technology p-EGFR primary antibodies (Bioworld Technology), AKT (Bioworld Technology), p-AKT mono- Anti- (Bioworld Technology), ERK (Bioworld Technology), p-ERK primary antibodies (Bioworld ), Technology β-actin (Hangzhou China Huaan is biological), protein lysate (the green skies biotech company of Chinese Shanghai), BCA determination of protein concentration kit (the green skies biotech company of Chinese Shanghai), skimmed milk power (Chinese company of Erie), BeyoECL Plus (super quick ECL chemical luminescence reagent kits) (the green skies biotech company of Chinese Shanghai), SDS-PAGE albumen Sample-loading buffer (5X) (the green skies biotech company of Chinese Shanghai), BeyoColor^TMColored pre-dyed Protein Marker (the green skies biotech company of Chinese Shanghai), PHS-3C types precision pH meter (the state-run testing equipment research institute of Changzhou), pure water Machine (U.S. Millipore), ice machine (U.S. Forma), gel-electrophoretic apparatus, wet type transferring film instrument and drying system (U.S. Bio- Rad), gel imaging system (UVP companies of the U.S.), decolorization swinging table (Zhuo Xin great achievements Science and Technology Ltd. of BeiJing, China) is inverted aobvious Micro mirror (Japanese OLYMPAS).

5.2 experimental method

5.2.1 total protein of cell is extracted

1st, after the culture of A431, H1975 cell expansion, give respectively such as the processing of table 8 below conventional medicine, processing 48 hours：

Table 8

2nd, PBS washes cell twice after 48 hours, adds vortex 8-10 times on 150ul protein lysates, vortex instrument, splits on ice Solve 35-40min；

3rd, after the completion of cracking, 15min is centrifuged in 12000rpm at 4 DEG C；

4th, protein concentration is determined：Using green skies Bradford determination of protein concentration kits；

5th, the supernatant after centrifugation is added into sample-loading buffer, then 100 DEG C of 5min are put in -80 DEG C of preservations, standby.

5.2.2 total protein of cell is quantitative (Bradford methods)

1st, standard curve is made

(1) it is standby after room temperature thawing from -20 DEG C of taking-up 1mg/ml BSA.

(2) 6 1.5ml centrifuge tubes are taken, 0 μ g, 2.5 μ g, 5.0 μ g, 10.0 μ g, 20.0 μ g, 40.0 μ g is respectively labeled as.

(3) according to the form below 9 adds various reagents in each pipe.

Table 9

Pipe is other

0μg

2.5μg

5.0μg

10.0μg

20.0μg

40.0μg

1mg/ml BSA

-

2.5μL

5.0μL

10.0μL

20.0μL

40.0μL

0.15mol/L NaCl

100μL

97.5μL

95.0μL

90.0μL

80.0μL

60.0μL

Coomassie Brillant Blue solution G250

1ml

1ml

1ml

1ml

1ml

1ml

(4) after mixing, room temperature places 2min.Every group of sample more than being added in 96 orifice plates, per hole 200ul, each concentration If three multiple holes, OD values are determined in ELIASA 595nm, standard curve is prepared.

2nd, sample protein content is detected

(1) the 1.5ml centrifuge tubes for the amount of taking fully, the Coomassie Brillant Blue solution 1ml of 4 DEG C of storages of often pipe addition.Room temperature is placed It can be used to survey albumen after 30min.

(2) a pipe Coomassie brilliant blue plus the μ L of 0.15mol/L NaCl solutions 100 are taken, mixing placement 2 minutes can be as blank Sample, ibid method measure OD values, test in triplicate, take average.

(3) a pipe Coomassie brilliant blue plus 95 μ L 0.15mol/L NaCl solutions and 5 μ L testing protein samples are taken, after mixing 2min is stood, ibid method determines OD values, experiment in triplicate, takes average.

(4) testing sample protein concentration is obtained according to standard curve and the conversion of sample absorbance.

5.2.3SDS-PAGE electrophoresis

Determine after protein content, it is applied sample amount to calculate the liquor capacity containing 100 μ g albumen.Take out loading sample extremely In 0.5ml centrifuge tubes, addition 5 × SDS sample-loading buffers to final concentration of 1 ×.(loading cumulative volume is usually no more than 15 μ L, plus Sample hole can add 20 μ L samples to greatest extent.) sample is boiled into 5min in boiling water before loading make albuminous degeneration, it is subsequently placed in ice On, it is standby.

1st, encapsulating：Clean glass plate first before encapsulating：One hand fastens glass plate, and it is light that another hand dips detergent Lightly scrub.Two sides is rinsed well to water is not hung repeatedly after all cleaning with running water, is finally totally stood afterwards with distilled water flushing Dry.

(1) it is put into clamping in gum-making rack after glass plate alignment.Then vertical card prepares encapsulating on the top of the shelf.(will during operation Make two glass alignments, in order to avoid leak adhesive.)

(2) being shaken up immediately after 10% separation gel of preparation, addition TEMED can encapsulating.During encapsulating, 10ml can be used

Liquid-transfering gun is drawn 5ml glue and released along glass with certain speed, when glue surface is raised to greenbelt medium line height.So Add one layer of water in glue surface afterwards, the gelation rate after fluid-tight is greatly improved.(starting during encapsulating can soon, and glue surface is near required To be slowed down during height.Glue must be flowed down along glass plate during operation, to avoid occurring bubble in glue.Add water fluid-tight when speed Slow, otherwise separation gel can be broken up, be deformed.)

(3) when there is fringence between Dang Shui and glue, separation gel is pointed out to coagulate.Wait 3min glue is fully solidified again, incline Glue upper liquid water shutoff, and carefully blotted water with filter paper, it is careful not to contact glue surface.

(4) being shaken up immediately after the concentration of preparation 4% glue, addition TEMED can encapsulating.Then remaining space is filled into concentration glue By in comb insertion concentration glue.Also glue is set to be flowed down along glass plate in order to avoid producing bubble during encapsulating.To make comb as far as possible when inserting comb Sub- holding level.Because volume can shrink reduction when gelling is solid, so that the loading volume of well reduces, so in concentration glue Will be often in both sides glue during solidification.Until after concentration gelling is solid, two hands pinch the both sides of comb vertically upward respectively Gently it is pulled out.

(5) rinsed with water and concentrate glue, put it into electrophoresis tank.(small glass is inward-facing, and big glass is faced out.)

2nd, it is loaded

(1) enough electrophoretic buffers are added into electrophoresis tank, prepare loading.(electrophoresis liquid will at least submerge the small glass in inner side Edge on glass.)

(2) protein sample pre-processed is taken out, with the adherent pipette samples of microsyringe, inspiration bubble is careful not to. Sample injector syringe needle is inserted in well and is slowly added to sample.(sample-adding can make sample go out well very much soon, if there is bubble also may be used Sample can be overflowed.When adding next sample, injector need to be washed 3 times in water jacket electrophoretic buffer, in order to avoid cross pollution.

3rd, electrophoresis

(1) power supply is plugged, terminal electrodes direction is noted

(2) setting constant pressure, 60V (6V/cm), after indicating dye bromophenol blue forward position enters separation gel, time about 30min, Voltage is brought up into 100-120V (15V/cm), continues electrophoresis until bromophenol blue reaches about 1cm above separation gel bottom, closing Power supply, terminates electrophoresis.

5.2.4 transferring film

1st, preparation

(1) 6 7.0-8.3cm filter paper and 1 7.3-8.6cm pvdf membrane need to be prepared by turning a film.Cut filter paper and film When must wear gloves, it is to avoid hand protein contamination film.The pvdf membrane cut is carried out into mark, immersion 2 hours is placed in water. (one side that film is pinched with tweezers is gently placed in the plate of ultra-pure water, floats on film waterborne, and only lower floor just connects with water Touch.So because capillarity can soak whole film.A layer of air film is formed if film is sunk in water, between film and water, this Sample can block film water suction.

(2) clip of transferring film, two pieces of foam-rubber cushions, glass bar, filter paper and the film soaked are put into slow added with transfer In the enamel tray of fliud flushing.

(3) opening clip makes black one side holding level.In one foam-rubber cushion of pad above, rolled back and forth with glass bar several times To roll away the bubble of the inside.(rolling another hand on the other hand and pushing down mat prevents it from will moving.) the metafiltration paper of pad three on mat (can three paper first stack be padded on mat), on the other hand fixed filter paper bubble therein is rolled with glass bar on the other hand.

2nd, glue is taken

(1) gently pried open on two sides of glass plate, carefully remove glass plate.Concentration glue is gently cut into (concentration Glue influence operation), it is to avoid separation gel is scratched, an oblique angle is cut in the upper left corner of separation gel as mark.

(2) separation gel is dipped in transfering buffering liquid and be balanced.

3rd, transferring film

(1) separation gel after balance is placed on filter paper, it is alignd with filter paper with hand adjustment is whole, gently rolled with glass bar Bubble.By membrane cover on glue, covering completely whole glue (can not be moved again after under membrane cover) and remove bubble.3 filter paper of lid on film And remove bubble.Finally cover another foam-rubber cushion, roll it is several under can close clip.Whole operation is carried out in transfer liquid, Constantly roll bubble.The filter paper on film both sides can not contact with each other, and short circuit can occur after contact.(transfer liquid contains methanol, during operation Gloves are worn, lab space maintains the circulation of air.)

(2) clip is put into transfer groove groove, makes the black flour of folder to the black flour of groove, the red face of the fine flour of folder to groove.Electricity Meeting heat production during transfer, can place ice block cooling (100V/100mA) on one side of groove.

(3) film is dyed into 5min with 1 × Ponceaux dye liquor after transferring film is finished (in being shaken on decolorization swinging table).Then rinsed with water Fall the dye liquor do not caught with regard to the albumen on observable film.Film is dried standby.

5.2.5 immune response

1st, close

(1) confining liquid (5% skim milk in TBST) is prepared

(2) after film is soaked from bottom to top with TBST, move in the plate containing confining liquid, delay at room temperature on decolorization swinging table Slow to shake, room temperature is closed 2 hours.

2nd, an anti-binding

(1) primary antibody dilution is prepared：5%BSAin TBST, adjustment pH is 7.4, to ensure antigen-antibody reaction in suitable PH under the conditions of carry out).

(2) film is taken out in confining liquid, after being rinsed one time with TBST, is immersed in the hybridizing box containing primary antibody (such as table 10 below) In, it is placed on 4 DEG C of shaking tables and slowly shakes, is incubated overnight.Phospho-AB is at least incubated 12 hours.Other antibody also can room temperature incubate Educate 2-3h.

Table 10

Primary antibody title	Source	Extension rate	Secondary antibody
				EGFR primary antibodies	Bioworld Technology	1:300	1:6000
P-EGFR primary antibodies	Bioworld Technology	1:300	1:6000
				AKT	Bioworld Technology	1:300	1:6000
P-AKT primary antibodies	Bioworld Technology	1:300	1:6000
				ERK primary antibodies	Bioworld Technology	1:300	1:6000
p-ERK	Bioworld Technology	1:300	1:6000

3rd, two anti-binding

(1) after primary antibody incubation terminates, primary antibody is reclaimed, and in -20 DEG C of preservations.Pvdf membrane is decolourized with TBST at room temperature to shake Washed three times on bed, each 10min.

(2) corresponding secondary antibody is selected according to the source of primary antibody, secondary antibody is subjected to a certain proportion of dilution (1 with confining liquid: 2000-1:5000), it is incubated at room temperature 1-2h.

4th, chemiluminescence, develops, and is fixed

(1) secondary antibody is incubated terminate after, the recycling of secondary antibody can be carried out, using discarding afterwards twice.Film is with TBST in room Washed three times on the lower decolorization swinging table of temperature, each 10min.

(2) exposure box, nitrite ion and preservative film etc. are got out.

(3) two kinds of reagents of ECL-A and ECL-B are mixed in equal volume on preservative film；After 1min, by pvdf membrane albumen Fully contacted with this mixed liquor down；After 1min, film is moved on another preservative film, residual liquid is removed, wraps, be put into In X- mating plates folder.

(4) in darkroom, 1 × developer solution and fixing solution are poured into vinyl disc respectively；X- mating plates are taken out under red light, are used Hand papercutter is cut out appropriately sized (long and wide than film is both needed to big 1cm)；X- mating plates folder is opened, X- mating plates are placed on film, once Put, just can not move again, close X- mating plates folder, start timing；According to the power of the signal appropriate adjustment time for exposure, it is generally 1min or 5min, also may be selected the multiple tabletting of different time, to obtain optimum efficiency；After the completion of exposure, X- mating plates folder is opened, is taken Go out X- mating plates, develop in rapid immersion developer solution, after obvious band to appear, development is terminated at once.Developing time is generally 1- 2min (20-25 DEG C), (being less than 16 DEG C) when temperature is too low needs proper extension developing time；After development terminates, at once by X- mating plates Immerse in fixing solution, fixing time is generally 5-10min, untill film is transparent；The fixing solution of residual is washed away with running water Afterwards, dry at room temperature.(note：Development and fixing when need to move film, one jiao of film of taking as far as possible, finger nail should not be scratched Film, otherwise will produce influence to result.)

5th, gel images are analyzed：Film is scanned or taken pictures, with gel images analysis software (Quantity One) The molecular weight and OD value of target stripe are analyzed, acquired results are as shown in Figure 4.

Above bioactivity result shows, in the present invention DM3001 inside and outside action effect significantly, be mainly manifested in Under several aspects:(1) DM3001 has strong inhibition effect to mutant egf R kinases (EGFR L858R/T790M), and IC50 is only For 0.71nM, hence it is evident that better than reference compound Rociletinib (21.5nM) and Gefitinib (823.3nM) and for wild type EGFR kinases (EGFR^wild-type) IC50 values up to 448.7nM.Compared with Rociletinib (SI=21.4), DM3001 choosing Select sex index (SI=632) and be higher by 21 times, as a result imply that compound DM3001 effect it is more single-minded, it is thus possible to toxic side effect Can be smaller, meanwhile, the compound is only 1.71nM to the inhibition of FAK focal adhesion kinases, imply that DM3001 may act on Double target spots play more preferable antitumor activity.(2) antiproliferation result is disclosed, and DM3001 is for carrying mutant egf R^T790MH1975 cells have a strong inhibitory action, IC50 values are only 0.037 μM, and for carrying Wild type EGFR^wild-type's Cell line A431 inhibitory action is very weak, and IC50 values are respectively 0.382 μM.It is substantially better than reference agent Rociletinib, Ji Fei For Buddhist nun.HCC827 (EGFR for carrying sensitizing mutation^{del E746_A750}), DM3001 and reference agent have good suppression Effect.In addition, DM3001 is weaker to people's normal bronchial epithelial cell HBE inhibitory action, H1975 cells are suppressed in its performance Valid density under, almost normal cell is not influenceed, it was demonstrated that the toxicity of such compound is smaller.(3) AO/EB double fluorescent stainings Method and DAPI decoration method results show that DM3001 acts on EGFR wild strain A431 cells and T790M mutant strain H1975 cells, The morphological change of Apoptosis can be caused.DM3001 is more notable for H1975 apoptosis-induced effect simultaneously, it was demonstrated that it is acted on It is more single-minded.(4) migration of cell and Matrigel show that DM3001 being capable of strong inhibition H1975 cells and A431 cells.With Rociletinib and Osimertinib compare, under same concentration DM3001 to the inhibitory action of H1975 cell invasion abilities more By force, DM3001 is illustrated while tumor cell proliferation is suppressed, the also migration of strong inhibition cell.(5)Western Blotting experiment shows, DM3001 can significantly inhibit the expression of GAP-associated protein GAP in the AKT signal paths of EGFR downstreams, with to The increase successively of concentration, p-EGFR, p-AKT, p-ERK expression is lowered successively, shows dose dependent；To H1975 The inhibitory action of cell is better than A431 cells, further higher in the selectivity that molecular level demonstrates DM3001.The compound With stronger suppression non-small cell lung cancer (NSCLC) ability of cell proliferation, more more excellent than Rociletinib resist is shown EGFR^T790MActivity, and can act on FAK kinases.As the novel molecule of a class formation, research compound has in the present invention The potentiality of the double target spot inhibitor of new and effective EGFR and FAK are developed into, to treatment-related tumor disease especially non-small cell Lung cancer, ED-SCLC have larger application value.

Described above is only the preferred embodiments of the present invention, it is noted that for the ordinary skill people of the art For member, without departing from the technical principles of the invention, some improvements and modifications can also be made, these improvements and modifications Also it should be regarded as protection scope of the present invention.

Claims (10)

1. a kind of antitumoral compounds or its pharmaceutically acceptable salt, the antitumoral compounds have the structure shown in formula (I)：

2. antitumoral compounds according to claim 1 or its pharmaceutically acceptable salt, wherein, described pharmaceutically may be used The salt of receiving includes sulfate, pyrosulfate, disulfate, sulphite, bisulfites, phosphate, chloride, bromination Thing, iodide, acetate, propionate, caprate, caprylate, acrylates, formates, isobutyrate, caproate, enanthate, Propionate, oxalates, malonate, benzoate, chloro benzoate, methyl benzoic acid salt, dinitro-benzoate, hydroxy benzenes Formates, methoxy benzoic acid salt, phthalate, phenyl acetate salt, phenylpropionic acid salt, PB, citrate, One or more in lactate, gamma hydroxybutyrate, hydroxyl acetate, tartrate, amygdalate and sulfonate.

3. the preparation method of the antitumoral compounds or its pharmaceutically acceptable salt described in claim 1 or 2, wherein, this resists Neoplastic compound is prepared by following route：

4. a kind of pharmaceutical composition, its contain antitumoral compounds described in the claim 1 or 2 of effective dose or its pharmaceutically Acceptable salt, and pharmaceutical carrier.

5. antitumoral compounds or its pharmaceutically acceptable salt described in claim 1 or 2, or the medicine described in claim 4 Compositions are preparing the application of EGFR skin factor receptor protein tyrosine kinase inhibitor.

6. antitumoral compounds or its pharmaceutically acceptable salt described in claim 1 or 2, or the medicine described in claim 4 Application of the compositions in FAK focal adhesion kinase inhibitor is prepared.

7. antitumoral compounds or its pharmaceutically acceptable salt described in claim 1 or 2, or the medicine described in claim 4 Purposes of the compositions in the medicine for preparing treatment tumour.

8. purposes according to claim 7, wherein, the tumour in non-small cell lung cancer, ED-SCLC one Plant or a variety of.

9. purposes according to claim 8, wherein, the tumour is non-small cell lung cancer.

10. the purposes according to claim 7 or 8, wherein, the purposes is mainly by suppressing EGFR, EGFR^T790MSaltant type What skin factor receptor protein tyrosine kinase and FAK focal adhesion kinases were realized.