CN107235931B - New pyrimidine anti-tumor compounds and preparation method thereof and purposes - Google Patents

New pyrimidine anti-tumor compounds and preparation method thereof and purposes Download PDF

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Publication number
CN107235931B
CN107235931B CN201710563571.XA CN201710563571A CN107235931B CN 107235931 B CN107235931 B CN 107235931B CN 201710563571 A CN201710563571 A CN 201710563571A CN 107235931 B CN107235931 B CN 107235931B
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morpholine
acetamido
pyrimidine
dmso
400mhz
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CN107235931A (en
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马晓东
王璐红
袁宏
吴斌
赵婧媛
李艳霞
王长远
刘克辛
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Dalian Medical University
First Affiliated Hospital of Dalian Medical University
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Dalian Medical University
First Affiliated Hospital of Dalian Medical University
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/04—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms
    • C07D295/14—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
    • C07D295/145—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals with the ring nitrogen atoms and the carbon atoms with three bonds to hetero atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings
    • C07D295/15—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms with substituted hydrocarbon radicals attached to ring nitrogen atoms substituted by carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals with the ring nitrogen atoms and the carbon atoms with three bonds to hetero atoms attached to the same carbon chain, which is not interrupted by carbocyclic rings to an acyclic saturated chain

Abstract

The present invention relates to new pyrimidine anti-tumor compounds and preparation method thereof and purposes, the novel pyrimidines compound is specially logical formula (I) compound represented, and each substituent group of logical formula (I) is defined in the description.The invention further relates to the logical formula (I) compound represented or its pharmaceutically acceptable salts, or containing its pharmaceutical composition by inhibiting focal adhesion kinase, and then treat tumor disease, especially for treating cancer of pancreas, lung cancer, the purposes of breast cancer；

Description

New pyrimidine anti-tumor compounds and preparation method thereof and purposes
Technical field
The present invention relates to new pyrimidine anti-tumor compounds and preparation method thereof and purposes, belong to pharmaceutical technology field.
Background technique
Protein tyrosine kinase (protein tyrosine kinase, PTKs) is logical by the signal transduction of control cell Road adjusts a series of physiological and biochemical procedures such as growth, differentiation, the apoptosis of cell.Receptor type tyrosine kinase is one kind across cell The relatively large kinases of film, extracellular domain, transmembrane domain and zymogenesis-with ligand binding are in phosphorylation Specific tyrosine residue and the intracellular domain for thus influencing cell Proliferation.(such as lung cancer, mammary gland in general human cancer Cancer, gastric cancer, oophoroma, lymthoma) it has been found that the kinases unconventionality expression.Protein tyrosine kinase has become anti-tumor drug One of the important target spot of research and development.
Focal adhesion kinase (focal adhesion kinase, FAK) is a kind of positioned at focal adhension, by 1028 amino acid The cytoplasmic tyrosine kinase and skelemin of composition participate in a variety of receptors or nonreceptor tyrosine kinase signal path, including Oncoprotein Src, vascular endothelial growth factor -3 (VEGFR-3), p53, phosphatidylinositol 3-kinase (PI3K) and Insulin-Like The accesses such as epidermal growth factor -1 (IGF-1) have adjusting to make the biobehaviorals such as the survival of cell, proliferation, migration and invasion With.Existing research shows the overexpression for having FAK in many tumour cells.Therefore, it is novel to have become research and development by FAK The important target of anti-tumor drug.Studies have shown that all there is the table excessively of FAK in the tumor tissues of most Patients with Pancreatic Cancer It reaches, and Fak inhibitor can reduce the viability of pancreatic cancer cell through a variety of ways, inhibit its growth, promote its apoptosis, but Fak inhibitor only has little effect to normal cell.Currently, being almost into preclinical or clinical research Fak inhibitor Micromolecular inhibitor, since FAK and many cell signaling proteins have effect, according to the difference of mechanism of action, as FAK The FAK micromolecular inhibitor of anti-tumor drugs targeting is roughly divided into two major classes: ATP dependent form and ATP independent form.ATP is relied on Type FAK micromolecular inhibitor can interfere the activity of FAK catalysis region, may will affect multiple downstream signaling pathways, cause more Extensive side effect, and specific protein-can be blocked if non ATP dependent form FAK micromolecular inhibitor such as allosteric Fak inhibitor Protein interacts (interaction of such as p53 and FAK), to inhibit the activity of FAK.Have at present it is multiple have much treatment before The FAK micromolecular inhibitor of scape is developed successively into preclinical and clinical research, but such drug there is no to list.Wherein there is generation The FAK micromolecular inhibitor of table has: PF-00562271 is the benzene sulfonate of PF-562271, is a kind of effective, ATP competition Property, reversible Fak inhibitor, IC50For 1.5nM, acts on Pyk2 and be compared to be compared to for low 10 times of FAK effect or so for it His high 100 times of protein kinase (in addition to some CDKs) selectivity or more.The positive Phase 1 that carries out studies (Serrels A., et at present al.Int.J.Cancer,2012,131(2):287-97.).For another example, TAE226 (NVP-TAE226) is a kind of effective FAK suppression Preparation, IC50Most effective to act on Pyk2 for 5.5nM, to InsR, IGF-1R, ALK and c-Met function and effect weak 10 are arrived than its 100 times or so.Tae226 shows effective antiproliferative and tumor-inhibiting action to a series of malignant tumours in testing in vivo and in vitro, At present there is no in relation to its clinical test report (Schultze A., et al.Invest New Drugs, 2010,28 (6): 825-33.).In addition, Defactinib (VS-6063, PF-04554878) is a kind of selectivity, and orally active FAK inhibits Agent.The positive Phase 2 that carries out is studied at present.(Kang Y.,et al.J.Natl.Cancer Inst.,2013,105(19): 1485-95.)
In addition such as with the closely related patent of the present invention: US2013/0281438 A1, CN102264371 A, CN101830889A、CN103052627 A、CN103059030 A、CN103476776 A、CN103534240 A、 CN103534241 A。
In view for the treatment of cancer there is an urgent need to it is necessary to develop the more good drug of new effect for this field.
Summary of the invention
One of the objects of the present invention is to provide a kind of morpholine acetamido pyrimidines or its can pharmaceutically connect The salt received, such compound have good anti-tumor activity.
Another object of the present invention is to provide containing the morpholine acetamido pyrimidines or its pharmaceutically may be used The pharmaceutical composition of the salt of receiving.
A further object of the present invention is to provide the morpholine acetamido pyrimidines or its can pharmaceutically connect The purposes of the salt or the composition received.
On the one hand, the present invention provides a kind of logical formula (I) compound represented or its pharmaceutically acceptable salt, the general formula (I) compound represented has the following structure:
R is selected from methyl, ethyl, cyclopropyl, isopropyl or 2,2,2- trifluoroethyl；
R1Selected from hydrogen, chlorine, methyl or methoxy；
R2Selected from fluorine, chlorine.
As a kind of specific embodiment of the invention, logical formula (I) compound represented of the present invention has I-1~I- Structure shown in 36:
Preferably, the logical formula (I) compound represented is I-1.
Structural compounds as shown above are morpholine acetamido pyrimidines, and antitumor activity screening shows this hair Compound major part in bright all has stronger inhibition pancreatic cancer cell, and (Aspc-1 cell, Bxpc-3 cell and Panc-1 are thin Born of the same parents) proliferative capacity, inhibit lung carcinoma cell (H1975 cell) proliferative capacity, inhibits breast carcinoma resistance cell (Mcf-7/adr) proliferation Ability；Part of compounds is shown than referring to drug Tae226 (CAS:761437-28-9) unexpected more excellent anti- FAK kinase activity.As the molecule of a kind of structure novel, there is the compound in the present invention exploitation to inhibit at new and effective FAK The potentiality of agent have biggish application value to treatment-related tumor disease especially cancer of pancreas, lung cancer and breast cancer.
Structure shown in aforementioned I-1~I-36 is respectively provided with following title:
(I-1) N- [3- [[the chloro- 2- of 5- [4- [3- ((1- morpholine) acetamido) methyl] aniline] -4- pyrimidine] amino] Phenyl] -2- formamide
(I-2) N- [3- [[the chloro- 2- of 5- [4- [3- ((1- morpholine) acetamido) methoxyl group] aniline] -4- pyrimidine] ammonia Base] phenyl] -2- formamide
(I-3) N- [3- [[the chloro- 2- of 5- [4- [3- ((1- morpholine) acetamido) methyl] aniline] -4- pyrimidine] amino] Phenyl] -2- cyclopropyl amide
(I-4) N- [3- [[the chloro- 2- of 5- [4- [3- ((1- morpholine) acetamido) methoxyl group] aniline] -4- pyrimidine] ammonia Base] phenyl] -2- cyclopropyl amide
(I-5) N- [3- [[the chloro- 2- of 5- [4- ((1- morpholine) acetamido) aniline] -4- pyrimidine] amino] phenyl] -2- Acetamide
(I-6) N- [3- [[the chloro- 2- of 5- [4- [3- ((1- morpholine) acetamido) methyl] aniline] -4- pyrimidine] amino] Phenyl] -2- acetamide
(I-7) N- [3- [[the chloro- 2- of 5- [4- [3- ((1- morpholine) acetamido) methoxyl group] aniline] -4- pyrimidine] ammonia Base] phenyl] -2- acetamide
(I-8) N- [3- [[the chloro- 2- of 5- [4- [3- ((1- morpholine) acetamido) chlorine] aniline] -4- pyrimidine] amino] benzene Base] -2- acetamide
(I-9) N- [3- [[the chloro- 2- of 5- [4- ((1- morpholine) acetamido) aniline] -4- pyrimidine] amino] phenyl] -2- Isopropamide
(I-10) N- [3- [[the chloro- 2- of 5- [4- [3- ((1- morpholine) acetamido) methyl] aniline] -4- pyrimidine] amino] Phenyl] -2- Isopropamide
(I-11) N- [3- [[the chloro- 2- of 5- [4- [3- ((1- morpholine) acetamido) methoxyl group] aniline] -4- pyrimidine] ammonia Base] phenyl] -2- Isopropamide
(I-12) N- [3- [[the chloro- 2- of 5- [4- [3- ((1- morpholine) acetamido) chlorine] aniline] -4- pyrimidine] amino] benzene Base] -2- Isopropamide
(I-13) N- [3- [[the chloro- 2- of 5- [4- [3- ((1- morpholine) acetamido) chlorine] aniline] -4- pyrimidine] amino] benzene Base] -2- formamide
(I-14) N- [3- [[the chloro- 2- of 5- [4- [3- ((1- morpholine) acetamido) methyl] aniline] -4- pyrimidine] amino] Phenyl] -2- trifluoroacetamide
(I-15) N- [3- [[the chloro- 2- of 5- [4- [3- ((1- morpholine) acetamido) methoxyl group] aniline] -4- pyrimidine] ammonia Base] phenyl] -2- trifluoroacetamide
(I-16) N- [3- [[the chloro- 2- of 5- [4- [3- ((1- morpholine) acetamido) chlorine] aniline] -4- pyrimidine] amino] benzene Base] -2- trifluoroacetamide
(I-17) N- [3- [[the chloro- 2- of 5- [4- [3- ((1- morpholine) acetamido) chlorine] aniline] -4- pyrimidine] amino] benzene Base] -2- cyclopropyl amide
(I-18) N- [3- [[the fluoro- 2- of 5- [4- [3- ((1- morpholine) acetamido) methyl] aniline] -4- pyrimidine] amino] Phenyl] -2- formamide
(I-19) N- [3- [[the fluoro- 2- of 5- [4- [3- ((1- morpholine) acetamido) methoxyl group] aniline] -4- pyrimidine] ammonia Base] phenyl] -2- formamide
(I-20) N- [3- [[the fluoro- 2- of 5- [4- [3- ((1- morpholine) acetamido) methyl] aniline] -4- pyrimidine] amino] Phenyl] -2- cyclopropyl amide
(I-21) N- [3- [[the fluoro- 2- of 5- [4- [3- ((1- morpholine) acetamido) methoxyl group] aniline] -4- pyrimidine] ammonia Base] phenyl] -2- cyclopropyl amide
(I-22) N- [3- [[the fluoro- 2- of 5- [4- ((1- morpholine) acetamido) aniline] -4- pyrimidine] amino] phenyl] -2- Acetamide
(I-23) N- [3- [[the fluoro- 2- of 5- [4- [3- ((1- morpholine) acetamido) methyl] aniline] -4- pyrimidine] amino] Phenyl] -2- acetamide
(I-24) N- [3- [[the fluoro- 2- of 5- [4- [3- ((1- morpholine) acetamido) methoxyl group] aniline] -4- pyrimidine] ammonia Base] phenyl] -2- acetamide
(I-25) N- [3- [[the fluoro- 2- of 5- [4- [3- ((1- morpholine) acetamido) chlorine] aniline] -4- pyrimidine] amino] benzene Base] -2- acetamide
(I-26) N- [3- [[the fluoro- 2- of 5- [4- ((1- morpholine) acetamido) aniline] -4- pyrimidine] amino] phenyl] -2- Isopropamide
(I-27) N- [3- [[the fluoro- 2- of 5- [4- [3- ((1- morpholine) acetamido) methyl] aniline] -4- pyrimidine] amino] Phenyl] -2- Isopropamide
(I-28) N- [3- [[the fluoro- 2- of 5- [4- [3- ((1- morpholine) acetamido) methoxyl group] aniline] -4- pyrimidine] ammonia Base] phenyl] -2- Isopropamide
(I-29) N- [3- [[the fluoro- 2- of 5- [4- [3- ((1- morpholine) acetamido) chlorine] aniline] -4- pyrimidine] amino] benzene Base] -2- Isopropamide
(I-30) N- [3- [[the fluoro- 2- of 5- [4- [3- ((1- morpholine) acetamido) chlorine] aniline] -4- pyrimidine] amino] benzene Base] -2- cyclopropyl amide
(I-31) N- [3- [[the fluoro- 2- of 5- [4- [3- ((1- morpholine) acetamido) methyl] aniline] -4- pyrimidine] amino] Phenyl] -2- trifluoroacetamide
(I-32) N- [3- [[the fluoro- 2- of 5- [4- [3- ((1- morpholine) acetamido) methoxyl group] aniline] -4- pyrimidine] ammonia Base] phenyl] -2- trifluoroacetamide
(I-33) N- [3- [[the fluoro- 2- of 5- [4- [3- ((1- morpholine) acetamido) chlorine] aniline] -4- pyrimidine] amino] benzene Base] -2- trifluoroacetamide
(I-34) N- [3- [[the fluoro- 2- of 5- [4- [3- ((1- morpholine) acetamido) chlorine] aniline] -4- pyrimidine] amino] benzene Base] -2- formamide
(I-35) N- [3- [[the fluoro- 2- of 5- [4- ((1- morpholine) acetamido) aniline] -4- pyrimidine] amino] phenyl] -2- Formamide
(I-36) N- [3- [[the chloro- 2- of 5- [4- ((1- morpholine) acetamido) aniline] -4- pyrimidine] amino] phenyl] -2- Formamide
On the other hand, the present invention provides a kind of pharmaceutical composition, the logical formula (I) institute of the present invention containing effective dose The compound shown or its pharmaceutically acceptable salt and pharmaceutical carrier.
For compound of the present invention due to their possibility purposes in drug, the salt preferred agents of formula (I) compound can The salt of receiving.The compound of the present invention is alkali, wherein required salt form can be prepared by appropriate method known in the art, is wrapped It includes with mineral acid treatment free alkali, described inorganic acid such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid etc.；Or at organic acid Manage free alkali, the organic acids such as acetic acid, trifluoroacetic acid, maleic acid, succinic acid, mandelic acid, fumaric acid, malonic acid, acetone Acid, oxalic acid, hydroxyacetic acid, salicylic acid, pyranose thuja acid (pyranosidy1 acid), such as glucuronic acid or galacturonic Acid, 'alpha '-hydroxy acids, such as citric acid or tartaric acid, amino acid, such as aspartic acid or glutamic acid, aromatic acid, such as benzoic acid Or cinnamic acid, sulfonic acid, such as p- toluenesulfonic acid, methanesulfonic acid, ethanesulfonic acid etc..The embodiment of pharmaceutically acceptable salt includes sulfuric acid Salt, pyrosulfate, disulfate, sulphite, bisulfites, phosphate, chloride, bromide, iodide, acetate, 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, phenylbutyrate (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.
Pharmaceutical composition of the 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 the compound of the present invention.In addition, pharmaceutical composition of the invention can also optionally include One or more other pharmaceutically active compounds.
The present invention also provides the novel pyrimidines compound or its pharmaceutically acceptable carrier, described pharmaceutical compositions By inhibiting focal adhesion kinase, and then inhibit the purposes of tumor proliferation.Specifically, which predominantly prepares for treating pancreas Purposes in the drug of cancer, lung cancer and breast cancer.
The present invention provides compound represented or its pharmaceutically acceptable salt or pharmaceutical composition of the present invention exists Prepare the application in focal adhesion kinase inhibitor.
The present invention provides the logical formula (I) compound represented or its pharmaceutically acceptable salt or of the present invention Purposes of the pharmaceutical composition in the drug of preparation treatment tumour.Preferably, the tumour is selected from cancer of pancreas, lung cancer and breast cancer One or more, further preferred cancer of pancreas.It is highly preferred that the purposes, which mainly passes through, inhibits focal adhesion kinase realization.
Detailed description of the invention
Fig. 1 is that different disposal of embodiment of the present invention group changes over time situation to the inhibiting effect of Aspc-1.
Fig. 2 .1 is different disposal of embodiment of the present invention group Aspc-1 Apoptosis situation.
Fig. 2 .2 is different disposal of embodiment of the present invention group Aspc-1 cell cycle distribution.
Fig. 3 is influence of different disposal of the embodiment of the present invention group to nude mouse tumor volume change.Gross tumor volume is expressed as putting down Means standard deviation (n=5).
Fig. 4 is influence of different disposal of the embodiment of the present invention group to nude mouse tumor weight.Compared to the blank group * p < 0.05, * p < 0.01 * (n=5).
Specific 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 range.
Test method without specific conditions in the embodiment of the present invention, usually according to normal condition, or according to raw material or Condition proposed by commodity manufacturer.The reagent in specific source is not specified, for the conventional reagent of market purchase.
The preparation of 1 target molecule of embodiment
Tlc silica gel plate uses Yantai Huanghai Sea HSGF254 or Qingdao GF254 silica gel plate, and thin-layered chromatography (TLC) makes The specification that silicon amine plate uses is 0.15mm-0.2mm, and the specification that thin-layer chromatography isolates and purifies product use is 0.4mm- 0.5mm。
The raw material that the present invention uses mainly is purchased from commercially available from Sinopharm Chemical Reagent Co., Ltd., Beijing coupling science and technology Co., Ltd, reaches the companies such as auspicious chemicals at Aladdin chemical reagent Co., Ltd.
Without specified otherwise in embodiment, solution refers to aqueous solution.
Without specified otherwise in embodiment, it is 20 DEG C -30 DEG C that the temperature of reaction, which is room temperature,.
The technical solution adopted by the invention is as follows:
Synthetic route, reagent and the condition of compound I-i: (d) NaHCO3,CH3CN, 2h, 0 DEG C, 85%；(e) morpholine, K2CO3,KI,CH3CN, 4h, 80 DEG C, 75-80%；(b)Fe-NH4Cl,MeOH-H2O, 2h, 70 DEG C, 72%.
The synthesis of I-h
Take I-f (3.29mmol) and 4.929mmolNaHCO3In 15mL acetonitrile, it is slowly added into I-g (6.57mmol), 0 After DEG C reaction 2h, end of reaction drains solvent, and 80mL water is added, and solid is precipitated, and filters, and drying obtains solid I-h, does not purify straight It connects and reacts in next step.
The synthesis of I-i
Take I-h (2mmol) and K2CO3(2.4mmol), KI (0.2mmol) are slowly added dropwise under stirring in 10ml acetonitrile Coffee quinoline (3mmol), after being warming up to 60 DEG C, reaction 4 hours, end of reaction.Water is added in reaction solution evaporated under reduced pressure, and solid is precipitated, and takes out Next step reduction reaction is thrown in filter, drying.Previous step product (2mmol) and NH4MeOH-H is added in Cl (4mmol)2In O (1:1), stir It mixes down and is slowly added into iron powder (8mmol), heat up 60 DEG C, after reaction 2 hours, end of reaction filters while hot, and 200mL water, analysis is added Solid out filters, and drying obtains solid I-i, does not purify and directly reacts in next step.
Synthetic route, reagent and the condition of compound (I): (a) SOCl2,1h,60℃；100%；(b)RNH2, NaHCO3, CH3CN, 4h, 0 DEG C；95%；(c)Fe-NH4Cl,MeOH-H2O, 2h, 70 DEG C, 72%；(d) N, N- diisopropyl ethyl (DIPEA), isopropanol (IPA), 6h, 80 DEG C；85%；(e)NaHCO3,CH3CN, 2h, 0 DEG C, 85%；(f) morpholine, K2CO3, KI,CH3CN, 4h, 80C, 75-80%；(g) p-methyl benzenesulfonic acid, 2-BuOH, 100 DEG C, 12h, 10-20%.
The synthesis of I-b
It takes I-a (5.98mmol, 1.00g) in three-necked bottle, is added DMSO (7.176mmol), heats up 80 DEG C and react 1 hour Afterwards, end of reaction, reaction solution evaporating solvent under reduced pressure obtain liquid, directly throw in next step；Previous step product (3mmol) is slowly added dropwise Enter RNH2(2.94mmol), NaHCO3In the 15ml acetonitrile solution of (7.5mmol), 0 DEG C of reaction is reacted after 4 hours and is completed.Reaction Liquid evaporated under reduced pressure is poured into 80ml water and solid is precipitated, and filters, and drying obtains I-b, does not purify and directly reacts in next step.
The synthesis of I-c
Take I-b (2mmol) and NH4MeOH-H is added in Cl (4mmol)2In O (1:1), iron powder is slowly added under stirring (8mmol) heats up 60 DEG C, and after reaction 2 hours, reaction is completed, and is filtered while hot, and 200mL water is added, and solid is not precipitated, and water phase is used Ethyl acetate extracts (100mL × 3), combined ethyl acetate layer, and saturated common salt washing is primary, and anhydrous sodium sulfate is dry, and decompression is steamed White solid I-c is done to obtain, does not purify and directly reacts in next step.
The synthesis of I-e
I-c (2mmol) and DIPEA (3mmol) are taken, 8mL isopropanol is added, it is phonetic that 2,4,5- trichlorines are slowly added dropwise under stirring Pyridine (2mmol) or 2, bis- chloro- 5-FU (2mmol) of 4-, after being warming up to 80 DEG C of reactions 6 hours, reaction is completed.Reaction solution decompression It is evaporated, solid is added to 20mL saturation NaHCO3In aqueous solution, solid is precipitated.It filters, drying obtains white solid I-e, do not purify Directly react in next step.
The synthesis of object (I)
I-i (1.0mmol) and I-e (1.0mmol) are taken, is dissolved in 10mL 2-BuOH, p-methyl benzenesulfonic acid is added (1.5mmol) heats up 100 DEG C and reacts 12 hours, revolve solvent evaporated after completion of the reaction, adds MeOH 5mL, then plus unsaturated carbonate hydrogen Solid is precipitated in sodium water solution 50mL, and thin-layer chromatography isolates and purifies to obtain target compound (I).
Target molecule is synthesized according to above method, the physicochemical data of synthesized target molecule is as follows:
(I-1) N- [3- [[the chloro- 2- of 5- [4- [3- ((1- morpholine) acetamido) methyl] aniline] -4- pyrimidine] amino] Phenyl] -2- formamide
1H NMR (400MHz, DMSO-d6) δ 11.65 (s, 1H), 9.54 (s, 1H), 9.42 (s, 1H), 8.78 (d, J= 4.0Hz, 2H), 8.23 (s, 1H), 8.03 (d, J=8.0Hz, 1H), 7.76 (d, J=8.0Hz, 1H), 7.45 (t, J=8.0Hz, 1H), 7.38 (s, 1H), 7.29 (d, J=8.0Hz, 1H), 7.13 (d, J=8.0Hz, 1H), 3.68-3.65 (m, 4H), 3.12 (s, 2H), 2.81 (d, J=4.0Hz, 3H), 2.54 (d, J=4.0Hz, 4H), 2.50 (s, 3H)；13C NMR (400MHz, DMSO- d6) δ 169.41,167.62,158.15,155.39,155.04,148.91,139.85,137.02,131.91,128.50, 122.36,121.89,121.63,121.05,119.56,113.92,105.56,103.77,66.96 (2C), 62.14, 56.30,53.65 (2C), 26.79；HRMS (ESI), C25H28ClN7O3, [M+H]+theoretical calculation: 510.1942, actual measurement: 510.2025。
(I-2) N- [3- [[the chloro- 2- of 5- [4- [3- ((1- morpholine) acetamido) methoxyl group] aniline] -4- pyrimidine] ammonia Base] phenyl] -2- formamide
1H NMR (400MHz, DMSO-d6)δ11.65(s,1H),9.54(s,1H),9.43(s,1H),8.82–8.74(m, 2H), 8.23 (s, 1H), 8.03 (d, J=8.0Hz, 1H), 7.76 (dd, J=8.0,2.0Hz, 1H), 7.45 (t, J=8.0Hz, 1H), 7.38 (s, 1H), 7.28 (dd, J=8.0,2.0Hz, 1H), 7.17-7.11 (m, 1H), 3.69-3.63 (m, 4H), 3.12 (s, 2H), 2.81 (d, J=4.0Hz, 3H), 2.55-2.52 (m, 3H), 2.50 (dt, J=4.0,2.0Hz, 3H)；13C NMR (400MHz, DMSO-d6) δ 169.43,167.65,158.17,155.42,155.07,148.93,139.87,137.04, 131.94,128.52,122.38,121.91,121.65,121.07,119.58,119.52,111.92,103.79,66.96 (2C), 62.14,56.30,53.66 (2C), 26.79；HRMS (ESI), C25H28ClN7O4, [M+H]+theoretical calculation: 526.1891 actual measurement: 526.1978.
(I-3) N- [3- [[the chloro- 2- of 5- [4- [3- ((1- morpholine) acetamido) methyl] aniline] -4- pyrimidine] amino] Phenyl] -2- cyclopropyl amide
1H NMR (400MHz, DMSO-d6) δ 11.49 (s, 1H), 9.42 (s, 1H), 9.28 (s, 1H), 8.73 (d, J= 4.0Hz, 2H), 8.22 (s, 1H), 7.72 (dd, J=8.0,4.0Hz, 1H), 7.57-7.37 (m, 4H), 7.16-7.06 (m, 1H),3.71–3.55(m,4H),3.12(s,2H),2.93–2.84(m,1H),2.61–2.52(m,4H),2.17(s,3H), 0.77-0.66 (m, 2H), 0.61 (dd, J=8.0,4.0Hz, 2H)；13C NMR (400MHz, DMSO-d6)δ170.31, 168.11,158.20,155.42,155.17,139.61,137.40,132.06,130.72,130.66,128.81,123.86, 122.32,121.72,121.62,121.32,118.00,105.32,66.77(2C),62.24,53.77(2C),23.58, 18.39,6.20(2C)；HRMS (ESI), C27H30ClN7O3, [M+H]+theoretical calculation: 536.2099, actual measurement: 536.2191.
(I-4) N- [3- [[the chloro- 2- of 5- [4- [3- ((1- morpholine) acetamido) methoxyl group] aniline] -4- pyrimidine] ammonia Base] phenyl] -2- cyclopropyl amide
1H NMR (400MHz, DMSO-d6) δ 11.53 (s, 1H), 9.54 (s, 1H), 9.42 (s, 1H), 8.76 (d, J= 4.0Hz, 2H), 8.23 (s, 1H), 8.03 (d, J=8.0Hz, 1H), 7.72 (dd, J=8.0,4.0Hz, 1H), 7.43 (dd, J= 8.0,4.0Hz, 2H), 7.28 (dd, J=8.0,4.0Hz, 1H), 7.12 (t, J=8.0Hz, 1H), 3.77 (s, 3H), 3.71- 3.60 (m, 4H), 3.11 (s, 2H), 2.93-2.85 (m, 1H), 2.57-2.51 (m, 4H), 0.73 (td, J=8.0,4.0Hz, 2H),0.66–0.57(m,2H)；13C NMR (400MHz, DMSO-d6)δ170.33,167.61,158.14,155.40, 155.06,148.90,139.67,137.02,131.95,128.82,122.31,121.87,121.65,121.18,119.56, 111.87,105.55,103.73,66.95(2C),62.14,56.30,53.65(2C),23.58,6.19(2C)；HRMS (ESI), C27H30ClN7O4, [M+H]+theoretical calculation: 552.2048, actual measurement: 552.2136.
(I-5) N- [3- [[the chloro- 2- of 5- [4- ((1- morpholine) acetamido) aniline] -4- pyrimidine] amino] phenyl] -2- Acetamide
1H NMR (400MHz, DMSO-d6) δ 11.52 (s, 1H), 9.64 (s, 1H), 9.42 (s, 1H), 8.76 (dd, J= 12.0,8.0Hz, 2H), 8.20 (s, 1H), 7.76 (dd, J=8.0,4.0Hz, 1H), 7.63-7.44 (m, 5H), 7.23-7.09 (m, 1H), 3.69-3.60 (m, 4H), 3.30 (dt, J=12.0,8.0Hz, 2H), 3.11 (s, 2H), 2.56-2.50 (m, 4H), 1.14 (t, J=8.0Hz, 3H)；13C NMR (400MHz, DMSO-d6)δ168.72,168.07,158.22,155.43, 155.10,139.72,136.35,133.36,131.80,128.55,122.39,121.91,121.58,120.41,120.33 (2C),105.29,66.56(2C),62.53,53.69(2C),34.59,14.97(2C)；HRMS (ESI), C25H28ClN7O3, [M + H]+theoretical calculation: 510.1942, actual measurement: 510.2042.
(I-6) N- [3- [[the chloro- 2- of 5- [4- [3- ((1- morpholine) acetamido) methyl] aniline] -4- pyrimidine] amino] Phenyl] -2- acetamide
1H NMR (400MHz, DMSO-d6) δ 11.53 (s, 1H), 9.42 (s, 1H), 9.28 (s, 1H), 8.76 (dd, J= 32.0,8.0Hz, 2H), 8.21 (s, 1H), 7.76 (dd, J=8.0,2.0Hz, 1H), 7.57-7.34 (m, 4H), 7.21-7.07 (m, 1H), 3.70-3.61 (m, 4H), 3.30 (dt, J=12.0,8.0Hz, 2H), 3.12 (s, 2H), 2.59-2.52 (m, 4H), 2.17 (s, 3H), 1.14 (t, J=8.0Hz, 3H)；13C NMR (400MHz, DMSO-d6)δ168.74,168.11,158.20, 155.41,155.15,139.72,137.41,131.96,130.71,130.67,128.58,123.86,122.37,121.70, 121.64,121.58,118.01,105.31,66.77(2C),62.25,53.77(2C),34.59,18.39,14.97；HRMS (ESI), C26H30ClN7O3, [M+H]+theoretical calculation: 524.2099, actual measurement: 524.2190.
(I-7) N- [3- [[the chloro- 2- of 5- [4- [3- ((1- morpholine) acetamido) methoxyl group] aniline] -4- pyrimidine] ammonia Base] phenyl] -2- acetamide
1H NMR (400MHz, DMSO-d6) δ 11.57 (s, 1H), 9.54 (s, 1H), 9.42 (s, 1H), 8.78 (dd, J= 20.0,8.0Hz, 2H), 8.22 (s, 1H), 8.03 (d, J=8.0Hz, 1H), 7.76 (dd, J=8.0,2.0Hz, 1H), 7.50- 7.05 (m, 4H), 3.78 (s, 3H), 3.71-3.61 (m, 4H), 3.31 (dd, J=8.0,4.0Hz, 2H), 3.12 (s, 2H), 2.54 (m, 4H), 1.14 (t, J=8.0Hz, 3H)；13C NMR (400MHz, DMSO-d6)δ168.75,167.62,158.15, 155.40,155.04,148.91,139.78,137.03,131.85,128.59,122.36,121.88,121.64,121.44, 119.56,111.89,103.75,66.96(2C),62.14,56.30,53.65(2C),34.59,14.97(2C)；HRMS (ESI), C26H30ClN7O4, [M+H]+theoretical calculation: 540.2048, actual measurement: 540.2149.
(I-8) N- [3- [[the chloro- 2- of 5- [4- [3- ((1- morpholine) acetamido) chlorine] aniline] -4- pyrimidine] amino] benzene Base] -2- acetamide
1H NMR (400MHz, DMSO-d6) δ 11.56 (s, 1H), 9.72 (s, 1H), 9.65 (s, 1H), 8.74 (dd, J= 24.0,8.0Hz, 2H), 8.26 (s, 1H), 8.01 (d, J=8.0Hz, 2H), 7.77 (dd, J=8.0,2.0Hz, 1H), 7.58- 7.46 (m, 2H), 7.17 (t, J=8.0Hz, 1H), 3.72-3.64 (m, 4H), 3.30 (dt, J=12.0,8.0Hz, 2H), 3.16 (s, 2H), 2.57 (m, 4H), 1.14 (t, J=8.0Hz, 3H)；13C NMR (400MHz, DMSO-d6)δ168.69,168.30, 157.77,155.47,155.06,139.53,137.91,132.09,128.62,124.05,122.73,122.54,121.74, 121.65,119.46,118.88,106.06,66.87(2C),62.01,53.64(2C),34.59,14.96(2C)；HRMS (ESI), C25H27Cl2N7O3, [M+H]+theoretical calculation: 544.1552, actual measurement: 544.1640.
(I-9) N- [3- [[the chloro- 2- of 5- [4- ((1- morpholine) acetamido) aniline] -4- pyrimidine] amino] phenyl] -2- Isopropamide
1H NMR (400MHz, DMSO-d6) δ 11.37 (s, 1H), 9.64 (s, 1H), 9.42 (s, 1H), 8.62 (dd, J= 24.0,8.0Hz, 2H), 8.20 (s, 1H), 7.76 (dd, J=8.0,2.0Hz, 1H), 7.59-7.45 (m, 5H), 7.15 (t, J= 8.0Hz, 1H), 4.12 (dq, J=12.0,8.0Hz, 1H), 3.67-3.60 (m, 4H), 3.11 (s, 2H), 2.55-2.51 (m, 4H), 1.17 (d, J=8.0Hz, 6H)；13C NMR (400MHz, DMSO-d6)δ168.07(2C),158.22,155.45, 155.10,139.51,136.36,133.34,131.68,128.76,122.40,122.15,121.97,120.33(2C), 105.23,66.56(2C),62.53,53.69(3C),41.59,22.55(3C)；HRMS (ESI), C26H30ClN7O3, [M+H]+ Theoretical calculation: 524.2099, actual measurement: 524.2189.
(I-10) N- [3- [[the chloro- 2- of 5- [4- [3- ((1- morpholine) acetamido) methyl] aniline] -4- pyrimidine] amino] Phenyl] -2- Isopropamide
1H NMR (400MHz, DMSO-d6) δ 11.39 (s, 1H), 9.42 (s, 1H), 9.28 (s, 1H), 8.62 (dd, J= 16.0,8.0Hz, 2H), 8.21 (s, 1H), 7.76 (dd, J=8.0,2.0Hz, 1H), 7.60-7.37 (m, 4H), 7.20-7.10 (m, 1H), 4.12 (dq, J=12.0,8.0Hz, 1H), 3.72-3.61 (m, 4H), 3.12 (s, 2H), 2.63-2.53 (m, 4H), 2.17 (s, 3H), 1.18 (d, J=4.0Hz, 6H)；13C NMR (400MHz, DMSO-d6)δ168.09(2C),158.19, 155.41,155.13,139.50,137.40,131.83,130.70,130.63,128.79,123.85,122.37,122.13, 121.74,121.58,117.96,105.25,66.76(2C),62.24,53.76(2C),41.58,22.55(2C),18.39； HRMS (ESI), C27H32ClN7O3, [M+H]+theoretical calculation: 538.2255, actual measurement: 538.2348.
(I-11) N- [3- [[the chloro- 2- of 5- [4- [3- ((1- morpholine) acetamido) methoxyl group] aniline] -4- pyrimidine] ammonia Base] phenyl] -2- Isopropamide
1H NMR (400MHz, DMSO-d6) δ 11.44 (s, 1H), 9.54 (s, 1H), 9.42 (s, 1H), 8.64 (dd, J= 16.0,8.0Hz, 2H), 8.22 (s, 1H), 8.03 (d, J=8.0Hz, 1H), 7.76 (dd, J=8.0,2.0Hz, 1H), 7.48- 7.11(m,4H),4.19–4.03(m,1H),3.78(s,3H),3.70–3.58(m,4H),3.11(s,2H),2.58–2.52(m, 4H), 1.18 (d, J=4.0Hz, 6H)；13C NMR (400MHz, DMSO-d6)δ168.11,167.62,158.16,155.41, 155.05,148.91,139.59,137.04,131.75,128.81,122.36,121.97,121.87,121.67,119.56, 111.86,105.49,103.73,66.96(2C),62.14,56.31,53.66(2C),41.59,22.55(2C)；HRMS (ESI), C27H32ClN7O4, [M+H]+theoretical calculation: 554.2204, actual measurement: 554.2285.
(I-12) N- [3- [[the chloro- 2- of 5- [4- [3- ((1- morpholine) acetamido) chlorine] aniline] -4- pyrimidine] amino] benzene Base] -2- Isopropamide
1H NMR (400MHz, DMSO-d6) δ 11.41 (s, 1H), 9.72 (s, 1H), 9.65 (s, 1H), 8.60 (dd, J= 24.0,8.0Hz, 2H), 8.26 (s, 1H), 8.01 (d, J=8.0Hz, 2H), 7.76 (d, J=8.0Hz, 1H), 7.51 (dd, J= 12.0,4.0Hz, 2H), 7.17 (m, 1H), 4.12 (dd, J=12.0,8.0Hz, 1H), 3.73-3.64 (m, 4H), 3.16 (s, 2H), 2.57 (s, 4H), 1.18 (d, J=4.0Hz, 6H)；13C NMR (400MHz, DMSO-d6)δ168.30,168.04, 157.77,155.49,155.06,139.31,137.91,131.98,128.83,128.61,124.05,122.73,122.56, 122.31,121.71,119.43,118.85,106.01,66.87(2C),62.01,53.64(2C),41.59,22.54(2C)； HRMS (ESI), C26H29Cl2N7O3, [M+H]+theoretical calculation: 558.1709, actual measurement: 558.1799.
(I-13) N- [3- [[the chloro- 2- of 5- [4- [3- ((1- morpholine) acetamido) chlorine] aniline] -4- pyrimidine] amino] benzene Base] -2- formamide
1H NMR (400MHz, DMSO-d6) δ 11.65 (s, 1H), 9.72 (s, 1H), 9.65 (s, 1H), 8.74 (dd, J= 32.0,4.0Hz, 2H), 8.27 (s, 1H), 8.01 (dd, J=8.0,4.0Hz, 2H), 7.76 (dd, J=8.0,2.0Hz, 1H), 7.51 (d, J=8.0Hz, 2H), 7.16 (s, 1H), 3.69-3.65 (m, 4H), 3.16 (s, 2H), 2.82 (s, 3H), 2.57 (d, J =4.0Hz, 4H)；13C NMR (400MHz, DMSO-d6) δ 169.37,168.33,157.78,155.48,155.07,139.63, 137.92,132.18,128.65,128.54,124.07,122.75,122.55,121.63,121.34,119.49,118.91, 106.11,66.88 (2C), 62.02,53.65 (2C), 26.79；HRMS (ESI), C24H25Cl2N7O3, [M+H]+theoretical calculation: 530.1396 actual measurement: 530.1476.
(I-14) N- [3- [[the chloro- 2- of 5- [4- [3- ((1- morpholine) acetamido) methyl] aniline] -4- pyrimidine] amino] Phenyl] -2- trifluoroacetamide
1H NMR (400MHz, DMSO-d6) δ 11.61 (s, 1H), 9.43 (s, 1H), 9.26 (s, 1H), 8.77 (dd, J= 32.0,8.0Hz, 2H), 8.25 (s, 1H), 7.67 (dd, J=8.0,2.0Hz, 1H), 7.53-7.32 (m, 4H), 7.19-7.05 (m,1H),3.67–3.61(m,4H),3.30(s,2H),3.12(s,2H),2.41–2.37(m,4H),2.27(s,3H)；13CNMR (400MHz, DMSO-d6)δ170.82,168.11,158.20,155.41,155.15,139.72,137.41,131.96, 130.71,130.67,128.58,123.86,122.37,121.70,121.64,121.58,113.01,105.31,66.17 (2C),62.25,52.77(2C),34.59,18.39,14.97；HRMS (ESI), C26H27ClF3N7O3, [M+H]+theoretical calculation: 578.1816 actual measurement: 578.1889.
(I-15) N- [3- [[the chloro- 2- of 5- [4- [3- ((1- morpholine) acetamido) methoxyl group] aniline] -4- pyrimidine] ammonia Base] phenyl] -2- trifluoroacetamide
1H NMR (400MHz, DMSO-d6) δ 11.57 (s, 1H), 9.41 (s, 1H), 9.23 (s, 1H), 8.72 (dd, J= 32.0,8.0Hz, 2H), 8.22 (s, 1H), 7.65 (dd, J=8.0,2.0Hz, 1H), 7.51-7.33 (m, 4H), 7.19-7.05 (m,1H),3.67–3.61(m,4H),3.30(s,2H),3.12(s,2H),2.41–2.37(m,4H),2.27(s,3H)；13CNMR (400MHz, DMSO-d6)δ171.62,169.11,159.20,156.47,155.15,139.73,137.41,131.96, 130.72,130.65,128.56,123.83,122.35,121.71,121.64,121.58,113.01,105.31,66.19 (2C),62.25,52.75(2C),34.59,18.39,14.96；HRMS (ESI), C26H27ClF3N7O4, [M+H]+theoretical calculation: 594.1765 actual measurement: 594.1823.
(I-16) N- [3- [[the chloro- 2- of 5- [4- [3- ((1- morpholine) acetamido) chlorine] aniline] -4- pyrimidine] amino] benzene Base] -2- trifluoroacetamide
1H NMR (400MHz, DMSO-d6) δ 11.62 (s, 1H), 9.42 (s, 1H), 9.23 (s, 1H), 8.77 (dd, J= 32.0,8.0Hz, 2H), 8.25 (s, 1H), 7.64 (dd, J=8.0,2.0Hz, 1H), 7.53-7.32 (m, 4H), 7.19-7.05 (m,1H),3.67–3.61(m,4H),3.30(s,2H),3.12(s,2H),2.41–2.37(m,4H),2.27(s,3H)；13CNMR (400MHz, DMSO-d6)δ170.82,168.11,158.20,155.41,155.15,139.72,137.41,131.96, 130.71,130.67,128.58,123.86,122.37,121.70,121.64,121.58,113.01,105.31,66.15 (2C),62.25,52.75(2C),34.59,18.39,14.97；HRMS (ESI), C25H24Cl2F3N7O3, [M+H]+theoretical calculation: 598.1270 actual measurement: 598.1289.
(I-17) N- [3- [[the chloro- 2- of 5- [4- [3- ((1- morpholine) acetamido) chlorine] aniline] -4- pyrimidine] amino] benzene Base] -2- cyclopropyl amide
1H NMR (400MHz, DMSO-d6) δ 11.53 (s, 1H), 9.52 (s, 1H), 9.42 (s, 1H), 8.76 (d, J= 4.0Hz, 2H), 8.23 (s, 1H), 8.03 (d, J=8.0Hz, 1H), 7.72 (dd, J=8.0,4.0Hz, 1H), 7.43 (dd, J= 8.0,4.0Hz, 2H), 7.28 (dd, J=8.0,4.0Hz, 1H), 7.12 (t, J=8.0Hz, 1H), 3.71-3.60 (m, 4H), 3.11 (s, 2H), 2.93-2.85 (m, 1H), 2.57-2.51 (m, 4H), 0.73 (td, J=8.0,4.0Hz, 2H), 0.66-0.57 (m,2H)；13C NMR (400MHz, DMSO-d6)δ170.33,167.61,158.14,155.40,148.90,139.67, 137.02,131.95,128.82,122.31,121.87,121.65,121.18,119.56,111.87,105.55,103.73, 66.95(2C),62.14,56.30,53.65(2C),23.58,6.19(2C)；HRMS (ESI), C26H27Cl2N7O3, [M+H]+reason By calculating: 556.1552, actual measurement: 556.1578.
(I-18) N- [3- [[the fluoro- 2- of 5- [4- [3- ((1- morpholine) acetamido) methyl] aniline] -4- pyrimidine] amino] Phenyl] -2- formamide
1H NMR (400MHz, DMSO-d6) δ 11.65 (s, 1H), 9.54 (s, 1H), 9.42 (s, 1H), 8.78 (d, J= 4.0Hz, 2H), 8.23 (s, 1H), 8.03 (d, J=8.0Hz, 1H), 7.76 (d, J=8.0Hz, 1H), 7.45 (t, J=8.0Hz, 1H), 7.38 (s, 1H), 7.29 (d, J=8.0Hz, 1H), 7.13 (d, J=8.0Hz, 1H), 3.68-3.65 (m, 4H), 3.12 (s, 2H), 2.81 (d, J=4.0Hz, 3H), 2.54 (d, J=4.0Hz, 4H), 2.50 (s, 3H)；13C NMR (400MHz, DMSO- d6) δ 169.41,167.62,158.15,155.39,155.04,148.91,139.85,137.02,131.91,128.50, 122.36,121.89,121.63,121.05,119.56,113.92,105.56,103.77,66.96 (2C), 62.14, 56.30,53.65 (2C), 26.79；HRMS (ESI), C25H28FN7O3, [M+H]+theoretical calculation: 493.5333, actual measurement: 493.5427。
(I-19) N- [3- [[the fluoro- 2- of 5- [4- [3- ((1- morpholine) acetamido) methoxyl group] aniline] -4- pyrimidine] ammonia Base] phenyl] -2- formamide
1H NMR (400MHz, DMSO-d6)δ11.65(s,1H),9.54(s,1H),9.43(s,1H),8.82–8.74(m, 2H), 8.23 (s, 1H), 8.03 (d, J=8.0Hz, 1H), 7.76 (dd, J=8.0,2.0Hz, 1H), 7.45 (t, J=8.0Hz, 1H), 7.38 (s, 1H), 7.28 (dd, J=8.0,2.0Hz, 1H), 7.17-7.11 (m, 1H), 3.69-3.63 (m, 4H), 3.12 (s, 2H), 2.81 (d, J=4.0Hz, 3H), 2.55-2.52 (m, 3H), 2.50 (dt, J=4.0,2.0Hz, 3H)；13C NMR (400MHz, DMSO-d6) δ 169.43,167.65,158.17,155.42,155.07,148.93,139.87,137.04, 131.94,128.52,122.38,121.91,121.65,121.07,119.58,119.52,111.92,103.79,66.96 (2C), 62.14,56.30,53.66 (2C), 26.79；HRMS (ESI), C25H28FN7O4, [M+H]+theoretical calculation: 509.5327, Actual measurement: 509.5433.
(I-20) N- [3- [[the fluoro- 2- of 5- [4- [3- ((1- morpholine) acetamido) methyl] aniline] -4- pyrimidine] amino] Phenyl] -2- cyclopropyl amide
1H NMR (400MHz, DMSO-d6) δ 11.49 (s, 1H), 9.42 (s, 1H), 9.28 (s, 1H), 8.73 (d, J= 4.0Hz 2H), 8.22 (s, 1H), 7.72 (dd, J=8.0,4.0Hz, 1H), 7.57-7.37 (m, 4H), 7.16-7.06 (m, 1H),3.71–3.55(m,4H),3.12(s,2H),2.93–2.84(m,1H),2.61–2.52(m,4H),2.17(s,3H), 0.77-0.66 (m, 2H), 0.61 (dd, J=8.0,4.0Hz, 2H)；13C NMR (400MHz, DMSO-d6)δ170.31, 168.11,158.20,155.42,155.17,139.61,137.40,132.06,130.72,130.66,128.81,123.86, 122.32,121.72,121.62,121.32,118.00,105.32,66.77(2C),62.24,53.77(2C),23.58, 18.39,6.20(2C)；HRMS (ESI), C27H30FN7O3, [M+H]+theoretical calculation: 519.5706, actual measurement: 579.5811.
(I-21) N- [3- [[the fluoro- 2- of 5- [4- [3- ((1- morpholine) acetamido) methoxyl group] aniline] -4- pyrimidine] ammonia Base] phenyl] -2- cyclopropyl amide
1H NMR (400MHz, DMSO-d6) δ 11.53 (s, 1H), 9.54 (s, 1H), 9.42 (s, 1H), 8.76 (d, J= 4.0Hz, 2H), 8.23 (s, 1H), 8.03 (d, J=8.0Hz, 1H), 7.72 (dd, J=8.0,4.0Hz, 1H), 7.43 (dd, J= 8.0,4.0Hz, 2H), 7.28 (dd, J=8.0,4.0Hz, 1H), 7.12 (t, J=8.0Hz, 1H), 3.77 (s, 3H), 3.71- 3.60 (m, 4H), 3.11 (s, 2H), 2.93-2.85 (m, 1H), 2.57-2.51 (m, 4H), 0.73 (td, J=8.0,4.0Hz, 2H),0.66–0.57(m,2H)；13C NMR (400MHz, DMSO-d6)δ170.33,167.61,158.14,155.40, 155.06,148.90,139.67,137.02,131.95,128.82,122.31,121.87,121.65,121.18,119.56, 111.87,105.55,103.73,66.95(2C),62.14,56.30,53.65(2C),23.58,6.19(2C)；HRMS (ESI), C27H30FN7O4, [M+H]+theoretical calculation: 535.5700, actual measurement: 535.5716.
(I-22) N- [3- [[the fluoro- 2- of 5- [4- ((1- morpholine) acetamido) aniline] -4- pyrimidine] amino] phenyl] -2- Acetamide
1H NMR (400MHz, DMSO-d6) δ 11.52 (s, 1H), 9.64 (s, 1H), 9.42 (s, 1H), 8.76 (dd, J= 12.0,8.0Hz, 2H), 8.20 (s, 1H), 7.76 (dd, J=8.0,4.0Hz, 1H), 7.63-7.44 (m, 5H), 7.23-7.09 (m, 1H), 3.69-3.60 (m, 4H), 3.30 (dt, J=12.0,8.0Hz, 2H), 3.11 (s, 2H), 2.56-2.50 (m, 4H), 1.14 (t, J=8.0Hz, 3H)；13C NMR (400MHz, DMSO-d6)δ168.72,168.07,158.22,155.43, 155.10,139.72,136.35,133.36,131.80,128.55,122.39,121.91,121.58,120.41,120.33 (2C),105.29,66.56(2C),62.53,53.69(2C),34.59,14.97(2C)；HRMS (ESI), C25H28FN7O3, [M+ H]+theoretical calculation: 493.5333, actual measurement: 493.5418.
(I-23) N- [3- [[the fluoro- 2- of 5- [4- [3- ((1- morpholine) acetamido) methyl] aniline] -4- pyrimidine] amino] Phenyl] -2- acetamide
1H NMR (400MHz, DMSO-d6) δ 11.53 (s, 1H), 9.42 (s, 1H), 9.28 (s, 1H), 8.76 (dd, J= 32.0,8.0Hz, 2H), 8.21 (s, 1H), 7.76 (dd, J=8.0,2.0Hz, 1H), 7.57-7.34 (m, 4H), 7.21-7.07 (m, 1H), 3.70-3.61 (m, 4H), 3.30 (dt, J=12.0,8.0Hz, 2H), 3.12 (s, 2H), 2.59-2.52 (m, 4H), 2.17 (s, 3H), 1.14 (t, J=8.0Hz, 3H)；13C NMR (400MHz, DMSO-d6)δ168.74,168.11,158.20, 155.41,155.15,139.72,137.41,131.96,130.71,130.67,128.58,123.86,122.37,121.70, 121.64,121.58,118.01,105.31,66.77(2C),62.25,53.77(2C),34.59,18.39,14.97；HRMS (ESI), C26H30FN7O3, [M+H]+theoretical calculation: 507.5599, actual measurement: 507.4012.
(I-24) N- [3- [[the fluoro- 2- of 5- [4- [3- ((1- morpholine) acetamido) methoxyl group] aniline] -4- pyrimidine] ammonia Base] phenyl] -2- acetamide
1H NMR (400MHz, DMSO-d6) δ 11.57 (s, 1H), 9.54 (s, 1H), 9.42 (s, 1H), 8.78 (dd, J= 20.0,8.0Hz, 2H), 8.22 (s, 1H), 8.03 (d, J=8.0Hz, 1H), 7.76 (dd, J=8.0,2.0Hz, 1H), 7.50- 7.05 (m, 4H), 3.78 (s, 3H), 3.71-3.61 (m, 4H), 3.31 (dd, J=8.0,4.0Hz, 2H), 3.12 (s, 2H), 2.54 (m, 4H), 1.14 (t, J=8.0Hz, 3H)；13C NMR (400MHz, DMSO-d6)δ168.75,167.62,158.15, 155.40,155.04,148.91,139.78,137.03,131.85,128.59,122.36,121.88,121.64,121.44, 119.56,111.89,103.75,66.96(2C),62.14,56.30,53.65(2C),34.59,14.97(2C)；HRMS (ESI), C26H30FN7O4, [M+H]+theoretical calculation: 523.5593, actual measurement: 523.6541.
(I-25) N- [3- [[the fluoro- 2- of 5- [4- [3- ((1- morpholine) acetamido) chlorine] aniline] -4- pyrimidine] amino] benzene Base] -2- acetamide
1H NMR (400MHz, DMSO-d6) δ 11.56 (s, 1H), 9.72 (s, 1H), 9.65 (s, 1H), 8.74 (dd, J= 24.0,8.0Hz, 2H), 8.26 (s, 1H), 8.01 (d, J=8.0Hz, 2H), 7.77 (dd, J=8.0,2.0Hz, 1H), 7.58- 7.46 (m, 2H), 7.17 (t, J=8.0Hz, 1H), 3.72-3.64 (m, 4H), 3.30 (dt, J=12.0,8.0Hz, 2H), 3.16 (s, 2H), 2.57 (m, 4H), 1.14 (t, J=8.0Hz, 3H)；13C NMR (400MHz, DMSO-d6)δ168.69,168.30, 157.77,155.47,155.06,139.53,137.91,132.09,128.62,124.05,122.73,122.54,121.74, 121.65,119.46,118.88,106.06,66.87(2C),62.01,53.64(2C),34.59,14.96(2C)；HRMS (ESI), C25H27ClFN7O3, [M+H]+theoretical calculation: 527.9784, actual measurement: 527.9803.
(I-26) N- [3- [[the fluoro- 2- of 5- [4- ((1- morpholine) acetamido) aniline] -4- pyrimidine] amino] phenyl] -2- Isopropamide
1H NMR (400MHz, DMSO-d6) δ 11.37 (s, 1H), 9.64 (s, 1H), 9.42 (s, 1H), 8.62 (dd, J= 24.0,8.0Hz, 2H), 8.20 (s, 1H), 7.76 (dd, J=8.0,2.0Hz, 1H), 7.59-7.45 (m, 5H), 7.15 (t, J= 8.0Hz, 1H), 4.12 (dq, J=12.0,8.0Hz, 1H), 3.67-3.60 (m, 4H), 3.11 (s, 2H), 2.55-2.51 (m, 4H), 1.17 (d, J=8.0Hz, 6H)；13C NMR (400MHz, DMSO-d6)δ168.07(2C),158.22,155.45, 155.10,139.51,136.36,133.34,131.68,128.76,122.40,122.15,121.97,120.33(2C), 105.23,66.56(2C),62.53,53.69(3C),41.59,22.55(3C)；HRMS (ESI), C26H30FN7O3, [M+H]+reason By calculating: 507.5599, actual measurement: 507.6028.
(I-27) N- [3- [[the fluoro- 2- of 5- [4- [3- ((1- morpholine) acetamido) methyl] aniline] -4- pyrimidine] amino] Phenyl] -2- Isopropamide
1H NMR (400MHz, DMSO-d6) δ 11.39 (s, 1H), 9.42 (s, 1H), 9.28 (s, 1H), 8.62 (dd, J= 16.0,8.0Hz, 2H), 8.21 (s, 1H), 7.76 (dd, J=8.0,2.0Hz, 1H), 7.60-7.37 (m, 4H), 7.20-7.10 (m, 1H), 4.12 (dq, J=12.0,8.0Hz, 1H), 3.72-3.61 (m, 4H), 3.12 (s, 2H), 2.63-2.53 (m, 4H), 2.17 (s, 3H), 1.18 (d, J=4.0Hz, 6H)；13C NMR (400MHz, DMSO-d6)δ168.09(2C),158.19, 155.41,155.13,139.50,137.40,131.83,130.70,130.63,128.79,123.85,122.37,122.13, 121.74,121.58,117.96,105.25,66.76(2C),62.24,53.76(2C),41.58,22.55(2C),18.39； HRMS (ESI), C27H32FN7O3, [M+H]+theoretical calculation: 521.5865, actual measurement: 521.6232.
(I-28) N- [3- [[the fluoro- 2- of 5- [4- [3- ((1- morpholine) acetamido) methoxyl group] aniline] -4- pyrimidine] ammonia Base] phenyl] -2- Isopropamide
1H NMR (400MHz, DMSO-d6) δ 11.44 (s, 1H), 9.54 (s, 1H), 9.42 (s, 1H), 8.64 (dd, J= 16.0,8.0Hz, 2H), 8.22 (s, 1H), 8.03 (d, J=8.0Hz, 1H), 7.76 (dd, J=8.0,2.0Hz, 1H), 7.48- 7.11(m,4H),4.19–4.03(m,1H),3.78(s,3H),3.70–3.58(m,4H),3.11(s,2H),2.58–2.52(m, 4H), 1.18 (d, J=4.0Hz, 6H)；13C NMR (400MHz, DMSO-d6)δ168.11,167.62,158.16,155.41, 155.05,148.91,139.59,137.04,131.75,128.81,122.36,121.97,121.87,121.67,119.56, 111.86,105.49,103.73,66.96(2C),62.14,56.31,53.66(2C),41.59,22.55(2C)；HRMS (ESI), C27H32FN7O4, [M+H]+theoretical calculation: 537.5859, actual measurement: 537.6035.
(I-29) N- [3- [[the fluoro- 2- of 5- [4- [3- ((1- morpholine) acetamido) chlorine] aniline] -4- pyrimidine] amino] benzene Base] -2- Isopropamide
1H NMR (400MHz, DMSO-d6) δ 11.41 (s, 1H), 9.72 (s, 1H), 9.65 (s, 1H), 8.60 (dd, J= 24.0,8.0Hz, 2H), 8.26 (s, 1H), 8.01 (d, J=8.0Hz, 2H), 7.76 (d, J=8.0Hz, 1H), 7.51 (dd, J= 12.0,4.0Hz, 2H), 7.17 (m, 1H), 4.12 (dd, J=12.0,8.0Hz, 1H), 3.73-3.64 (m, 4H), 3.16 (s, 2H), 2.57 (s, 4H), 1.18 (d, J=4.0Hz, 6H)；13C NMR (400MHz, DMSO-d6)δ168.30,168.04, 157.77,155.49,155.06,139.31,137.91,131.98,128.83,128.61,124.05,122.73,122.56, 122.31,121.71,119.43,118.85,106.01,66.87(2C),62.01,53.64(2C),41.59,22.54(2C)； HRMS (ESI), C26H29ClFN7O3, [M+H]+theoretical calculation: 542.0500, actual measurement: 542.0507.
(I-30) N- [3- [[the fluoro- 2- of 5- [4- [3- ((1- morpholine) acetamido) chlorine] aniline] -4- pyrimidine] amino] benzene Base] -2- cyclopropyl amide
1H NMR (400MHz, DMSO-d6) δ 11.65 (s, 1H), 9.72 (s, 1H), 9.65 (s, 1H), 8.74 (dd, J= 32.0,4.0Hz, 2H), 8.27 (s, 1H), 8.01 (dd, J=8.0,4.0Hz, 2H), 7.76 (dd, J=8.0,2.0Hz, 1H), 7.51 (d, J=8.0Hz, 2H), 7.16 (s, 1H), 3.69-3.65 (m, 4H), 3.16 (s, 2H), 2.82 (s, 3H), 2.57 (d, J =4.0Hz, 4H)；13C NMR (400MHz, DMSO-d6) δ 169.37,168.33,157.78,155.48,155.07,139.63, 137.92,132.18,128.65,128.54,124.07,122.75,122.55,121.63,121.34,119.49,118.91, 106.11,66.88 (2C), 62.02,53.65 (2C), 26.79；HRMS (ESI), C24H25ClFN7O3, [M+H]+theoretical calculation: 513.9518 actual measurement: 513.8745.
(I-31) N- [3- [[the fluoro- 2- of 5- [4- [3- ((1- morpholine) acetamido) methyl] aniline] -4- pyrimidine] amino] Phenyl] -2- trifluoroacetamide
1H NMR (400MHz, DMSO-d6) δ 11.61 (s, 1H), 9.43 (s, 1H), 9.26 (s, 1H), 8.77 (dd, J= 32.0,8.0Hz, 2H), 8.25 (s, 1H), 7.67 (dd, J=8.0,2.0Hz, 1H), 7.53-7.32 (m, 4H), 7.19-7.05 (m,1H),3.67–3.61(m,4H),3.30(s,2H),3.12(s,2H),2.41–2.37(m,4H),2.27(s,3H)；13CNMR (400MHz, DMSO-d6)δ170.82,168.11,158.20,155.41,155.15,139.72,137.41,131.96, 130.71,130.67,128.58,123.86,122.37,121.70,121.64,121.58,113.01,105.31,66.17 (2C),62.25,52.77(2C),34.59,18.39,14.97；HRMS (ESI), C26H27F4N7O3, [M+H]+theoretical calculation: 561.5313 actual measurement: 561.5423.
(I-32) N- [3- [[the fluoro- 2- of 5- [4- [3- ((1- morpholine) acetamido) methoxyl group] aniline] -4- pyrimidine] ammonia Base] phenyl] -2- trifluoroacetamide
1H NMR (400MHz, DMSO-d6) δ 11.61 (s, 1H), 9.43 (s, 1H), 9.26 (s, 1H), 8.77 (dd, J= 32.0,8.0Hz, 2H), 8.25 (s, 1H), 7.67 (dd, J=8.0,2.0Hz, 1H), 7.53-7.32 (m, 4H), 7.19-7.05 (m,1H),3.67–3.61(m,4H),3.30(s,2H),3.12(s,2H),2.41–2.37(m,4H),2.27(s,3H)；13CNMR (400MHz, DMSO-d6)δ170.82,168.11,158.20,155.41,155.15,139.72,137.41,131.96, 130.71,130.67,128.58,123.86,122.37,121.70,121.64,121.58,113.01,105.31,66.17 (2C),62.25,52.77(2C),34.59,18.39,14.97；HRMS (ESI), C26H27F4N7O4, [M+H]+theoretical calculation: 577.5307 actual measurement: 577.6234.
(I-33) N- [3- [[the fluoro- 2- of 5- [4- [3- ((1- morpholine) acetamido) chlorine] aniline] -4- pyrimidine] amino] benzene Base] -2- trifluoroacetamide
1H NMR (400MHz, DMSO-d6) δ 11.61 (s, 1H), 9.43 (s, 1H), 9.26 (s, 1H), 8.77 (dd, J= 32.0,8.0Hz, 2H), 8.25 (s, 1H), 7.67 (dd, J=8.0,2.0Hz, 1H), 7.53-7.32 (m, 4H), 7.19-7.05 (m,1H),3.67–3.61(m,4H),3.30(s,2H),3.12(s,2H),2.41–2.37(m,4H),2.27(s,3H)；13CNMR (400MHz, DMSO-d6)δ170.82,168.11,158.20,155.41,155.15,139.72,137.41,131.96, 130.71,130.67,128.58,123.86,122.37,121.70,121.64,121.58,113.01,105.31,66.17 (2C),62.25,52.77(2C),34.59,18.39,14.97；HRMS (ESI), C25H24ClF4N7O3, [M+H]+theoretical calculation: 581.9498 actual measurement: 582.0236.
(I-34) N- [3- [[the fluoro- 2- of 5- [4- [3- ((1- morpholine) acetamido) chlorine] aniline] -4- pyrimidine] amino] benzene Base] -2- formamide
1H NMR (400MHz, DMSO-d6) δ 11.65 (s, 1H), 9.72 (s, 1H), 9.65 (s, 1H), 8.74 (dd, J= 32.0,4.0Hz, 2H), 8.27 (s, 1H), 8.01 (dd, J=8.0,4.0Hz, 2H), 7.76 (dd, J=8.0,2.0Hz, 1H), 7.51 (d, J=8.0Hz, 2H), 7.16 (s, 1H), 3.69-3.65 (m, 4H), 3.16 (s, 2H), 2.82 (s, 3H), 2.57 (d, J =4.0Hz, 4H)；13C NMR (400MHz, DMSO-d6) δ 169.37,168.33,157.78,155.48,155.07,139.63, 137.92,132.18,128.65,128.54,124.07,122.75,122.55,121.63,121.34,119.49,118.91, 106.11,66.88 (2C), 62.02,53.65 (2C), 26.79；HRMS (ESI), C24H25ClFN7O3, [M+H]+theoretical calculation: 513.9518 actual measurement: 513.9478.
(I-35) N- [3- [[the fluoro- 2- of 5- [4- ((1- morpholine) acetamido) aniline] -4- pyrimidine] amino] phenyl] -2- Formamide
1H NMR (400MHz, DMSO-d6) δ 11.54 (s, 1H), 9.64 (s, 1H), 9.43 (s, 1H), 8.75 (dd, J= 12.0,8.0Hz, 2H), 8.19 (s, 1H), 7.76 (dd, J=8.0,4.0Hz, 1H), 7.61-7.44 (m, 5H), 7.23-7.09 (m, 1H), 3.69-3.60 (m, 4H), 3.11 (s, 2H), 2.56-2.50 (m, 4H), 1.14 (t, J=8.0Hz, 3H)；13C NMR (400MHz, DMSO-d6)δ169.72,168.07,159.22,155.43,154.10,139.72,137.35,133.36, 131.80,128.55,122.39,121.91,120.41,120.31(2C),105.29,66.57(2C),62.53,53.70 (2C),34.59,14.97(2C)；HRMS (ESI), C24H26FN7O3, [M+H]+theoretical calculation: 479.5067, actual measurement: 479.5235。
(I-36) N- [3- [[the chloro- 2- of 5- [4- ((1- morpholine) acetamido) aniline] -4- pyrimidine] amino] phenyl] -2- Formamide
1H NMR (400MHz, DMSO-d6) δ 11.52 (s, 1H), 9.64 (s, 1H), 9.42 (s, 1H), 8.76 (dd, J= 12.0,8.0Hz, 2H), 8.20 (s, 1H), 7.76 (dd, J=8.0,4.0Hz, 1H), 7.63-7.44 (m, 5H), 7.23-7.09 (m, 1H), 3.69-3.60 (m, 4H), 3.11 (s, 2H), 2.56-2.50 (m, 4H), 1.14 (t, J=8.0Hz, 3H)；13C NMR (400MHz, DMSO-d6)δ168.72,168.07,158.22,155.43,155.10,139.72,136.35,133.36, 131.80,128.55,122.39,121.91,120.41,120.33(2C),105.29,66.56(2C),62.53,53.69 (2C),34.59,14.97(2C)；HRMS (ESI), C24H26ClN7O3, [M+H]+theoretical calculation: 495.9613, actual measurement: 495.9728。
Target molecule at salt method
The preparation method of inorganic acid salt: it takes target molecule (1mmol) to be dissolved in 10mL anhydrous methanol, under ice bath, slowly drips The 5mL absolute methanol solution for adding inorganic acid (1mmol), is added dropwise, and stirs 30 minutes at a temperature of this, then first is evaporated off in room temperature Alcohol to get target molecule inorganic acid salt.
The preparation method of acylate: it takes target molecule (1mmol) to be dissolved in 10mL anhydrous methanol, under ice bath, slowly drips The 5mL dry ether for adding organic acid (1mmol), is added dropwise, and stirs 30 minutes at a temperature of this, and then solvent is evaporated off in room temperature, Up to the acylate of target molecule.
The preparation of two target molecule mixtures
Above-mentioned two target molecule of equimolar amounts (1mmol) (5mL) in anhydrous methanol is taken, is stirred at room temperature 10 minutes, Solvent is evaporated off to get the mixture of target molecule in room temperature.
2 target molecule biological evaluation of embodiment
1, in vitro to receptor tyrosine kinase inhibitory activity test method
(1) kinase assay buffer is prepared
1. melting kinase assay buffer (Kinase Detection Buffer) in room temperature, precipitating has been seen whether.
2. it is just shaken in 37 DEG C of incubations (Kinase Detection Buffer) 15 minutes and often if there is precipitating, Dissolution precipitating.Alternatively, supernatant is carefully siphoned away, removal precipitating.
(2) kinase assay reagent is prepared
1. using preceding at equilibrium at room temperature kinase assay buffer (Kinase Detection Buffe) and kinase assay bottom Object (Kinase Detection Substrate).
2. all pouring into kinase assay buffer (Kinase Detection Buffer) 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 shaking, being vortexed or being mixed by inversion, become homogeneous solution, substrate should dissolve in 1 minute.
4. kinase assay reagent should use immediately after preparing, or packing is stored in -20 DEG C, it is believed that the reagent prepared passes through Cycle signal activity is not all lost after freeze thawing several times.
(3) production ATP is converted to the standard curve of ADP
1. the Ultra provided with 1 × kinase reaction buffer (kinase reaction buffer) dilution kit 50 μM of ADP of 900 μ L 50 μM of ATP and 500 μ L are made in Pure ATP and ADP.
2. by 50 μM of ATP and 50 μM of ADP solution that previous step prepares by being mixed in 384 orifice plate A1-A12 shown in table 1, The concentration for simulating the ATP and ADP of each conversion percentages, mixes.
Table 1. prepares 50 μM of series A TP+ADP standard items
3. the ADP-Glo of 5 μ L is added in every holeTMReagent terminates kinase reaction.In incubation at room temperature 40 minutes.
4. 10 μ L kinase assay reagents (Kinase Detection Reagent), which are added, in every hole is converted to ATP for ADP, and Luciferase and luciferin are introduced to detect ATP.
5. measuring fluorescent in incubation at room temperature 30-60 minutes with multi-function microplate reader and recording fluorescent value.
6. drawing the standard curve that ATP is converted to ADP.
(4) IC of kinase inhibitor is determined50Value
1. preparing 1 × kinase reaction buffer (kinase reaction according to promega kit specification 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 reaction of L buffers (kinase reaction buffer), 2 μ L are added in no enzyme control wells 2.5 × 0.5 μ g/ μ L substrates and 125 μM of ATP.1 μ L 1 × kinase reaction buffer (kinase is added in negative control hole Reaction buffer), 2 μ L 2.5 × 50ng/ μ L kinases, 2 μ L, 2.5 × 0.5 μ g/ μ L substrate and 125 μM of ATP.It is testing It is added 1 μ L 5 × drug to be measured in hole, 2 μ L 2.5 × 50ng/ μ L kinases, 2 μ L, 2.5 × 0.5 μ g/ μ L substrate and 125 μM ATP。
3. mixing plate, it is incubated for 60 minutes.
4. the ADP-Glo of 5 μ L is added in every holeTMReagent terminates kinase reaction.In incubation at room temperature 40 minutes.
5. 10 μ L kinase assay reagents (Kinase Detection Reagent), which are added, in every hole is converted to ATP for ADP, and Luciferase and luciferin are introduced to detect ATP.In incubation at room temperature 30-60 minutes, fluorescent was measured with multi-function microplate reader and is remembered Record fluorescent value.
6. interpretation of result, the results are shown in Table 2.
2, inhibit BTK high expressing cell growth experiment (CCK-8 detection method)
(1) cell type and selection: Aspc-1 cell, Bxpc-3 cell and Panc-1 cell (human pancreatic cancer cell, FAK Kinases height expression)；H1975 (Non-small cell lung carcinoma cell, the expression of FAK kinases height)；(human breast cancer cell is resistance to by Mcf-7/adr Medicine strain, the expression of FAK kinases height).
(2) cell inoculation: collecting logarithmic growth phase cell, concentration of cell suspension is adjusted, with every hole 4 × 103A cell, often 100 μ L of pore volume is inoculated into 96 orifice plates, and every group sets 3 multiple holes (edge hole is filled with sterile PBS)；
(3) cell culture: after cell inoculation, control group is cultivated with 10%FBS RPMI-1640, and experimental group uses 10 μ L respectively Tae226 (1.25-40 μm of ol/L), the variant drug (1.25-40 μm of ol/L) of various concentration gradient are intervened, and 37 DEG C, 5%CO2 Continue to cultivate (cultivating different time respectively by requirement of experiment) in incubator；
(4) colour generation: 10 μ L CCK-8 solution (5mg/ml) are added in two groups of cells after cultivating 48h, terminate training after 4h It supports, in low-speed oscillation 10min on shaking table, makes to crystallize abundant dissolution；
(5) colorimetric: measuring each hole shading value (OD value) on enzyme-linked immunosorbent assay instrument, 450nm wavelength is selected, with cell-free I.e. RPMl-1640 culture solution blank well zeroing, survey the absorbance value in each hole.Experiment is in triplicate；
(6) record result: inhibitory rate of cell growth=(one experimental group absorbance value of control group absorbance value)/control group is inhaled Shading value × 100%, cell proliferation rate=(experimental group absorbance value/control group absorbance value) × 100；
(7) draw cell growth curve: using the time as abscissa, inhibiting rate/proliferation rate is that ordinate draws cell growth Curve.Figure is done for inhibitor concentration in the GraphPad Prism mapping software in GraphPad software, so as to by log [inhibitor] estimates IC relative to reaction, variable slope model50。
Test result is as shown in table 2, and table 2 shows that compound obtained is inhibiting FAK kinases and anti-tumour cell proliferative In active effect.
2. compound of table inhibits kinase activity and antiproliferation
a:IC50: half effective inhibition concentration .b:Aspc-1, Bxpc-3, Panc-1 are typical pancreatic cancer cell, FAK kinases Height is expressed；H1975 is human lung adenocarcinoma cell；Mcf-7/adr is human breast cancer cell (adriamycin-resistant).
Meanwhile the Aspc-1 of this experiment discovery compound I-1, Panc-1 cell activity and time and concentration have very big Relationship, as shown in Figure 1, cell survival rate reduces with the increase of concentration, the Aspc-1 after especially 72h, Panc-1 cell is in medicine It when object concentration is up to 8 μm of ol/L, can hardly survive, hence it is evident that be higher than 48h, thus provable drug belongs to concentration and Time Dependent Property drug.And its inhibitory effect is substantially better than referring to drug Tae226.
3, Apoptosis and cell cycle (flow cytometry)
(1) experimental material and reagent
Aspc-1 cell, RAPIDMEM basal medium (Hyclone company, cat:SH30022.01B), FBS tire ox blood (GIBCO company, cat:16400-044) clearly, dual anti-(Beijing Xia Si Biotechnology Co., Ltd, prospec cat: SV30010), low speed centrifuge (upper sea cowry grace Biotechnology Co., Ltd, cat:TDZ4B-WS), CO2(Shanghai is rich for incubator News, cat:BC-J160S), superclean bench (the rich news model SW-CJ-2FD in Shanghai) is inverted fluorescent electronic microscope (Leica DMI3000B)。
(2) experimental method
1. expanding culture Aspc-1 cell；
2. drug-treated: using various concentration drug-treated cell 48 hours.
3. collected by trypsinisation of the cell without EDTA, PBS wash cell twice after drug-treated, cell 1~5 is collected ×105Cell；
4. the AnnexinV Binding Buffer suspension cell of 500uL is added；
5. after 5uL AnnexinV-FITC mixing is added, 5uLPropidium Iodide is added, and (cycle detection only adds 5ul Propidium Iodide), it mixes；
6. being protected from light, reacting at room temperature 10min；
7. with flow cytomery (Ex=488nm；Em=530nm) Apoptosis and the case where the period.
Test result is as follows shown in Fig. 2 .1 and Fig. 2 .2, and the following figure 2.1 and Fig. 2 .2 show compound I-1 obtained not With the result for causing Aspc-1 Apoptosis under concentration.
4, interior animal experiment (experiment of nude mouse)
(1) experimental material and reagent
Aspc-1 cell, RAPIDMEM basal medium (Hyclone company, cat:SH30022.01B), FBS tire ox blood (GIBCO company, cat:16400-044) clearly, dual anti-(Beijing Xia Si Biotechnology Co., Ltd, prospec cat: SV30010), low speed centrifuge (upper sea cowry grace Biotechnology Co., Ltd, cat:TDZ4B-WS), CO2(Shanghai is rich for incubator News, cat:BC-J160S), superclean bench (the rich news model SW-CJ-2FD in Shanghai) 1ml syringe, gastric perfusion needle, STOCK- Foxn1nu/NjuNude mouse (model animal research institute, Nanjing University, quality certification number: SCXK:2015-0001)
(2) experimental method
1. cell culture: expanding the Aspc-1 cell of culture logarithmic growth phase, 1000rpm/min centrifugation is received after pancreatin digestion Collect cell, is washed twice with cold PBS, be finally made 3 × 10 with PBS7The cell suspension of a/ml；
2. inoculation: the STOCK-Foxn1 of 5-6 week oldnu/NjuNude mouse obtains from model animal research institute, Nanjing University and (closes Lattice card number: SCXK:2015-0001), zoopery obtains experimental animal use and maintenance in strict accordance with the People's Republic of China (PRC) Regulation carries out.Aspc-1 cell suspension is suspended in 0.1ml PBS, is subcutaneously injected into mouse underarm areas；
3. grouping: the 5th day after inoculation, tumor formation rate 98%, mouse is randomly divided into 3 groups of (n=5) blank groups, two I-1 Processing group.30,60mg/kg, the two dosage processing of I-1 processing group；
4. administration: when gross tumor volume reaches 300mm3When left and right, continuous gavage is administered 11 days, once a day, is surveyed within every two days A gross tumor volume is measured, and is calculated with following formula: V=A × B2/ 2, wherein A and B respectively indicates minimum and maximum diameter.Tumour suppression Rate processed is calculated with following formula；Gross tumor volume × 100% of (gross tumor volume-treatment group gross tumor volume of blank group)/blank group.
5. after experiment, putting to death mouse, anatomic entities tumor, and carry out weighing measurement.
Experimental result such as Fig. 3, shown in Fig. 4:
The above bioactivity the result shows that, the present invention in I-1 inside and outside function and effect it is significant, be mainly manifested in following several A aspect: (1) I-1 has strong inhibition effect, IC to FAK kinases50Only 1.03nM, hence it is evident that be better than reference compound Tae226 (6.79nM).(2) antiproliferation result discloses, to the IC of Aspc-1, Bxpc-3 and Panc-1 cell50Respectively 0.909 μM, 0.761 μM and 1.218 μM, hence it is evident that better than reference compound Tae226 (6.73 μM, 12.33 μM, > 20.00 μM), it is right The IC of H1975 and Mcf-7/adr50Respectively 0.333 μM and 0.892 μM, it is superior to reference compound Tae226.(3) streaming point I-1 can make the apoptosis rate of Aspc-1 cell obviously increase (90.4%) and by cell-cycle arrest in the G0/G1 phase as the result is shown for analysis, In dose dependent.(4) the internal anti-tumor activity of Xenografts in nude mice experimental verification I-1, the results showed that I-1 can inhibit The growth of Aspc-1 cell in vivo, the tumor control rate (TGI) for giving 30,60mg/kg/day dosage are respectively 44.38% With 28.58%.It proves that I-1 can inhibit growth of tumour cell, and dose dependent is presented.Indicating that such molecule has may greatly open The potentiality for sending out into new and effective Fak inhibitor have biggish application value to treatment-related tumor disease especially cancer of pancreas.
The above is only preferred embodiments of the invention, it is noted that for the ordinary skill people of the art For member, without departing from the technical principles of the invention, several improvements and modifications can also be made, these improvements and modifications Also it should be regarded as protection scope of the present invention.

Claims (5)

1. a kind of -1 compound represented of Formulas I or its pharmaceutically acceptable salt, -1 compound represented of Formulas I has as follows Structure:
2. a kind of pharmaceutical composition, -1 compound represented of Formulas I described in the claim 1 containing effective dose or its pharmaceutically Acceptable salt and pharmaceutical carrier.
3. -1 compound represented of Formulas I or its pharmaceutically acceptable salt or drug as claimed in claim 2 described in claim 1 Composition is preparing the application in focal adhesion kinase inhibitor.
4. -1 compound represented of Formulas I or its pharmaceutically acceptable salt or drug as claimed in claim 2 described in claim 1 Purposes of the composition in the drug of preparation treatment cancer of pancreas.
5. purposes according to claim 4, wherein the purposes, which mainly passes through, inhibits focal adhesion kinase realization.
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