CN101781285A

CN101781285A - Quinazoline derivates, preparation method thereof and pharmaceutical application thereof

Info

Publication number: CN101781285A
Application number: CN200910045382A
Authority: CN
Inventors: 邓炳初; 李心; 王俊; 王斌; 金池琼
Original assignee: Shanghai Hengrui Pharmaceutical Co Ltd
Current assignee: Shanghai Hengrui Pharmaceutical Co Ltd
Priority date: 2009-01-15
Filing date: 2009-01-15
Publication date: 2010-07-21

Abstract

The invention relates to quinazoline derivates, a preparation method thereof and pharmaceutical application thereof, in particular to novel quinazoline compounds represented by a general formula (I), tautomers thereof, enantiomers thereof, diastereomers thereof, racemes thereof and pharmaceutically acceptable salts thereof, metabolins thereof and metabolic precursors thereof or prodrugs thereof, and application thereof as therapeutic agents, in particular as protein kinase inhibitors. Substituent groups are defined in the description.

Description

Quinazoline derivative, its preparation method and in pharmaceutically application

Technical field

The pharmaceutical composition that the present invention relates to a kind of new quinazoline derivative, its preparation method and contain this derivative with and as therapeutical agent particularly as the purposes of protein kinase inhibitors.

Background technology

The signal conduction of cell is a kind of mechanism of action of basis, in the signal conductive process, is passed to cell interior from extracellular stimulation, and then regulates the process of different cells.These signals can be regulated multiple physiological responses, comprise cell proliferation, differentiation, apoptosis and motion etc., and they exist with different sorts dissolution factor form, comprise the somatomedin based on paracrine factor, the autocrine factor and endocrine factor.By combining with specific transmembrane receptor, the somatomedin part is delivered to signal pathway in the cell with the extracellular signal, thereby causes the reaction of individual cells pair cell external signal.A lot of signal transduction process are reversing processes of utilizing protein phosphorylation, relate to specific protein kinases and phosphorylated enzyme.

Protein kinase (PKs) is the enzyme that the phosphorylation of the hydroxyl on proteinic tyrosine, Serine, the threonine residues is played katalysis.In the signal conductive process, the reverse mechanism of protein kinase and phosphorylated enzyme can balance and conditioning signal stream.A protein phosphorylation state can influence activity, the cellular localization of its conformation, enzyme; the respective action of protein kinase and Phosphoric acid esterase is modified; phosphorylated is an important regulation mechanism in the signal conduction, improper differentiation, conversion and the growth that can cause cell unusually in the signal conductive process.For example, cell can become cancer cells, the growth factor receptor protein that the oncogene that Tyrosylprotein kinase comes to this is coded by its a part of DNA is converted into oncogene; Tyrosylprotein kinase can also sport activated form and cause multiple human cell's variation, we can say that also the normal Tyrosylprotein kinase of overexpression can cause abnormal cellular proliferation.

Tyrosylprotein kinase (PKs) can be divided into two classes easily: protein tyrosine kinase (PTKs) and serine-threonine kinase (STKs).PTKs makes the tyrosine residues phosphorylation on the protein, and STKs makes Serine, the threonine residues phosphorylation on the protein.Tyrosylprotein kinase not only can be that receptor type (comprise cell foreign lands, cell internal area and stride the theca cell territory) can also be non-receptor type (comprising whole cell internal areas).The active main aspect of PTK is that they relate to as the cell surface protein growth factor receptors.Have the active growth factor receptors of PTK and be called as receptor tyrosine kinase (" RTKs "), 90 kinds of Tyrosylprotein kinases are identified in Human genome, wherein about 60 kinds is receptor type, about 30 kinds is non-receptor type, these growth factor receptors families can be further divided into 20 kinds of receptor tyrosine kinase subtribes and 10 kinds of nonreceptor tyrosine kinase subtribes (Robinson etc., Oncogene, 2000,19,5548-5557).

The RTKs subtribe comprises following several: (1) EGF family, and as EGF, TGF α, Neu and erbB etc.; (2) Regular Insulin family comprises insulin receptor, insulin-like growth factor I receptor (IGF1) and insulin receptor dependency acceptor (IRR)); (3) III type family, as platelet derived growth factor receptor (PDGF, comprise PDGF α and PDGF beta receptor), relevant Tyrosylprotein kinase 3 (Flt3) receptor tyrosine kinase of STEM CELL FACTOR RTKs (SCF RTK, so-called c-Kit), fms-and colony-stimulating factor 1 acceptor (CSF-1R) Tyrosylprotein kinase etc.They are playing a part crucially aspect control cell growth and the differentiation, also be the crucial transmitter (referring to Schlessinger and Ullrich, Neuron 1992,9,383) who causes producing the cell signal of somatomedin and cytokine.The non-limiting kinases of a part comprises Abl, ARaf, ATK, ATM, bcr-abl, Blk, BRaf, Brk, Btk, CDK 1, CDK2, CDK3, CDK4, CDK5, CDK6, CDK7, CDK8, CDK9, CHK, AuroraA, AuroraB, AuroraC, cfms, c-fms, c-Kit, c-Met, cRaf1, CSF1R, CSK, c-Src, EGFR, ErbB2, ErbB3, ErbB4, ERK, ERK1, ERK2, Fak, fes, FGFR1, FGFR2, FGFR3, FGFR4, FGFR5, Fgr, FLK-4, Fps, Frk, Fyn, GSK, gsk3a, gsk3b, Hck, Chk, Axl, Pim-1, Plh-1, IGF-1R, IKK, IKK1, IKK2, IKK3, INS-R, Integrin-linked kinase, Jak, JAK1, JAK2, JAK3, JNK, JNK, Lck, Lyn, MEK, MEK1, MEK2, p38, PDGFR, PIK, PKB1, PKB2, PKB3, PKC, PKCa, PKCb, PKCd, PKCe, PKCg, PKCl, PKCm, PKCz, PLK1, Polo-like kinase, PYK2, tie1, tie2, TrkA, TrkB, TrkC, UL13, UL97, VEGF-R1, VEGF-R2, Yes and Zap70 etc.It is believed that PKs and central nervous system disease such as senile dementia (referring to Mandelkow .FEBS Lett.1992 such as E.M., 314,315; Sengupta .Mol.Cell.Biochem.1997 such as A., 167,99), pain (referring to Yashpal, K.J.Neurosci.1995,15,3263-72), inflammation for example sacroiliitis (referring to Badger, J.Pharmn Exp.Ther.1996,279,1453), psoriasis (referring to Dvir, etc., J.Cell Biol.1991,113,857), skeletal diseases for example osteoporosis (referring to Tanaka etc., Nature, 1996,383,528), (referring to Hunter and Pines, Cell 1994,79 for cancer, 573), arteriosclerosis is (referring to Hajjar and Pomerantz, FASEB J.1992,6,2933), thrombosis is (referring to Salari, FEBS 1990,263,104), metabolism disorder such as diabetes (referring to Borthwick .Biochem.Biophys.Res.Commun.1995 such as A.C., 210,738), vascular proliferative disease such as vasculogenesis (referring to Cancer Res.1996 such as Strawn, 56,3540; J.Pharm.Exp.Ther.1998 such as Jackson, 284,687), autoimmune disease and graft-rejection are (referring to Bolenand Brugge, Ann.Rev.Immunol.1997,15,371), transmissible disease as virus (referring to Littler, E.Nature 1992,358,160) and fungi infestation (referring to Lum, R.T.PCT Int Appl., WO 9805335A1980212) etc. the target spot of disease has close contact.

In the PTKs signal conductive process, interact in the extracellular between the particular growth factor (part), receptor dimerization subsequently, the intrinsic activity of activated protein kinase in moment, and carry out phosphorylated.The binding site of internal signal transduction molecule produces, and has generated the mixture with cytoplasmic signal molecule, promotes for example cell fission of various cell responses (propagation), to born of the same parents' metabolic expression of microenvironment outward etc.

The binding site of receptor tyrosine kinase phosphorylated also is to have the binding site of high affinity with signal transduction molecule SH2 (with the src homology) territory.Substrate protein is determined in a lot of cells relevant with receptor tyrosine kinase, and can be divided into two classes: (1) has the no catalytic domain substrate of catalytic domain substrate (2), but can be used as combination, and has the molecule of catalytic activity relevant with some.The interactional specificity of acceptor or albumen and substrate SH2 territory is by determining near the aminoacid sequence of phosphorylated tyrosine residues, SH2 territory and phosphorylated tyrosine sequence on every side aminoacid sequence and the otherness of special receptor bonded otherness and substrate phosphorylated be consistent.The protein tyrosine kinase function can be determined by expression pattern and part operability, also can be determined by special receptor activated catchment signal conducting path.Therefore, phosphorylated provides an important adjustable step, and this step can be determined selectivity and the differentiation factor receptors by the conduction of special receptor activated signal.The improper expression of receptor tyrosine kinase or sudden change may cause the disappearance of uncontrollable cell proliferation (as malignant growth) or crucial evolution etc.

Tyrosylprotein kinase, in the human tumour of major part, in cancers such as leukemia, mammary cancer, prostate cancer, nonsmall-cell lung cancer (comprising gland cancer, lung squamous cell cancer), gastrointestinal cancer (comprising colorectal carcinoma, the rectum cancer and cancer of the stomach), bladder cancer, the esophageal carcinoma, ovarian cancer, carcinoma of the pancreas, sudden change or overexpression all can appear.By the human tumor cell is detected, Tyrosylprotein kinase popularity and cognation have further obtained affirmation.For example: comprise that at human cancer the EGFR Tyrosylprotein kinase can be undergone mutation and overexpression in lung cancer, the cancer of the brain, neck cancer, gastrointestinal cancer, mammary cancer, the esophageal carcinoma, ovarian cancer, uterus carcinoma, bladder cancer and the thyroid carcinoma.

" HER " or " Erb " receptor tyrosine kinase subtribe comprises EGFR, HER2, HER3 and HER4.These subtribes are made up of born of the same parents outer glycosylation ligand binding domain, membrane-spanning domain and born of the same parents' inner cell matter catalytic domain that the tyrosine sequence on the protein can be carried out phosphorylated.The receptor tyrosine kinase catalytic activity can be activated by acceptor overexpression or ligand-mediated two polymerizations.HER2 family polymer has homodimer and two kinds of forms of heterodimer.An example of homotype dimerization is that HER1 (EGFR) (comprises EGF with EGF family part, transforming growth factor-alpha, betacellulin, with Vitrum AB bonded EGF, epiregulin) abnormal shape two polymerizations between the polymerization, four kinds of HER Tyrosylprotein kinases can be by being accelerated with combining of family's part of heregulin (also being neuregulin).Though one of acceptor of HER3 does not have enzymic activity, HER2 and HER3, or the special-shaped dimerization of HER3 and HER4 also can stimulate tyrosine kinase receptor two polymerizations significantly.In all kinds cell, the acceptor overexpression can activate the kinase whose activity of HER2.The activation of acceptor homodimer and heterodimer can be carried out phosphorylated with acceptor and other intracellular protein tyrosine sequences; signal pathway such as microtubule-associated protein kinases (map kinase) and phosphatidylinositols (3) kinases (PI3 kinases) also are activated in the cell subsequently; the activation of these signal pathways impels cell proliferation, suppresses natural death of cerebral cells.

Another subtribe of RTK comprises insulin receptor (IR), IGF-1R (IGF-1R), insulin receptor associated receptor (IRR).IR, IGF-1R and Regular Insulin, IGF-I and IGF-II interact, and have generated by the outer glycosylation α subunit of two kinds of complete born of the same parents and two to pass cytolemma and contain the different tetramer that tyrosine kinase domain β subunit constitutes.

The 3rd subtribe of RTK is meant platelet derived growth factor acceptor (PDGFR) family, comprising PDGFR α, and PDGFR β, CSFIR, c-Kit and c-fms.These acceptors are formed by containing various immunoglobulin-like ring glycosylation extracellular domains and an extracellular domain, and wherein the Tyrosylprotein kinase district is blocked by incoherent aminoacid sequence in born of the same parents' internal area.

The platelet derived growth factor acceptor also is to stride the film tyrosine kinase receptor as PDGFR α and PDGFR β etc.When they combine with part, or formation homotype dipolymer (PDGF-AA, PDGF-BB), or special-shaped dipolymer (PDGF-AB).Receptor dimerization subsequently, Tyrosylprotein kinase is activated, and signal and promote tumor growth in the district downstream.Transgenation is that acceptor does not rely on the reason that combines with part and be activated, and also is the motivating force that tumour generates.In multiple different tumor cell line, particularly in the cell of mastocarcinoma, colorectal carcinoma, ovarian cancer, prostate cancer, sarcoma and glioma, all find to activate the expression of PDGFR somatomedin-PDGF, brain tumor wherein, the pernicious gliosis of prostate cancer (comprising gland cancer and metastatic carcinoma of bone) has researching value.

C-Kit is the member of pdgf receptor family, and when itself and ligand SCF (STEM CELL FACTOR) when combining, activity is activated.In various solid tumor, the c-Kit expression pattern is studied, in sarcoma, gi tract glioma (GIST), in spermocytoma and the carcinoid tumor, c-Kit has overexpression.[referring to Weber etc., J.Clin.Oncol.22 (14S), 9642 (2004)].GIST is a kind of non-epithelial tumor, and great majority are present in stomach, and minority is distributed in small intestine, exists seldom in esophagus, also is distributed in positions such as liver, peritoneal cavity.GIST comes from Caial mesenchymal cell (ICC), and ICC can partly form the intestines autonomic nervous system, participates in the control gastric motility.It is because the c-Kit gene is undergone mutation that great majority (50～80%) GIST produces, in digestive tube, the c-Kit/CD117 stained positive generally all be GIST, c-Kit sudden change can make it not rely on SCF and activate and just have the c-Kit function, thereby the cell fission rate is increased, cause genomic instability.In diseases such as distortion mastocytoma, mastocytosis, myeloproliferative syndrome, urticaria, also can detect the expression of c-Kit, the expression of c-Kit is also arranged in acute AML and malignant lymphoma, all there is c-Kit to express (referring to Sch ǔ tteet al., innovartis 3/2001) in SCBC, spermocytoma, dysgerminoma, testis, intraepithelial neoplasias, melanoma, mastocarcinoma, neuroblastoma, Ewing sarcoma.As everyone knows, RET (rearranged during transfection).Proto-oncogene point genetic mutation is a tumorigenesis, suffering from multiple endocrine adenomas 2 (MEN 2) patient may cause suffering from pheochromocytoma, medullary thyroid carcinoma and illnesss such as parathyroid adenoma and hyperplasia and (see Huang et al., Cancer Res.60,6223-6 (2000)).

Very similar because of tire liver kinases (Flk) acceptor subtribe to the PDGFR subtribe, be attributed to this family sometimes.This subtribe is inserted territory-acceptor tire liver kinases-1 (KDR/FLK-1, VEGFR2), Flk-1R, Flk-4 and fms sample Tyrosylprotein kinase 1 (Flt-1) is formed by containing kinases.

The another one member of Tyrosylprotein kinase growth factor receptors family is fibroblast growth factor (FGF) acceptor subtribe.This subtribe is by four acceptors, and FGFR1-4, seven parts and FGF1-7 form.Though determine as yet at present, these acceptors by comprise the glycosylated extracellular domain of various immunoglobulin-like rings and one wherein the cell internal area blocked by incoherent aminoacid sequence of tyrosine-kinase enzyme sequence form.

The another one member of Tyrosylprotein kinase growth factor receptors family is vascular endothelial growth factor (VEGF) acceptor subtribe.Similar to PDGF, VEGF is a biglycan albumen, but biological function is different with target cell specificity in the body.Particularly, VEGFR and associated angiogenesis suppress vasculogenesis by suppressing VEGFRs, just are being applied to clinical treatment tumour, and are obtaining better curative effect.VEGF is in various malignant entity tumors, as lung cancer, mammary cancer, non-Hodgkin malignant lymphoma, ovarian cancer, carcinoma of the pancreas, malignant pleural mesothelioma and melanoma strong expression is arranged, and relevant with the canceration process, expression is also arranged in leukocytosis and lymphoma.Except its angiogenic activity, VEGFR, VEGF part also can be by directly promoting tumor growth by pro-survival character in tumour cell, and PDGF also has angiogenic action.The process that new vessel generates continues growth for tumour and plays keying action, and under the normal circumstances, the physiological process that is created on the people of new vessel such as embryo growth, wound healing and female genital each process all are very important.Yet the vasculogenesis on non-expectation or the pathology but a series of states with disease is relevant, as diabetic retinopathy, psoriasis, cancer, rheumatoid arthritis, atheroma, Ka Boji (family name) sarcoma and vascular tumor etc.The generation of vascular endothelial cell activates vasculogenesis, and having stimulates more active polypeptide of generation of vascular endothelial cell in the body to be identified, and comprises the fibroblast growth factor (aFGF and bFGF) and the vascular endothelial growth factor of acidity, alkalescence.Because the limiting expression of vegf receptor, the activity of its somatomedin is compared with aFGF and bFGF activity, and endotheliocyte is had specificity comparatively speaking.Nearest evidence shows, in the vasculogenesis and blood vessel process of osmosis of VEGF under normal circumstances and pathology situation, all is very important stimulant.VEGF can induction of vascular rudiment phenotype, and the expression of its inducing endothelial cell propagation, proteolytic enzyme and migration promote capillary vessel to generate, thereby forms super infiltration, jejune blood vessel network, and this is the characteristic feature of typical pathology vasculogenesis.People expect that antagonism VEGF activity particularly suppresses the value that tumor growth can have application in treatment disease such as the tumour relevant with angiogenic action or vascular permeability.

FLT3 (Fms sample Tyrosylprotein kinase) is Tyrosylprotein kinase (PTK) Ill type family member, in leukocytosiss such as adult and child's acute myeloid leukemia (AML), acute myeloid leukemia, myelodysplastic syndrome, the improper expression of FLT3 gene.35% acute myeloid leukemia patient's FLT3 sudden change is activated and prognosis mala, most sudden change all has the phenomenon of duplicating in the structure in nearly film territory, patient's N 835 origination points sudden change of 5-10%, the tyrosine kinase activity of FLT3 is activated, and causing also has signal to exist under the situation of part disappearance and breed.According to the study, there is the probability of patient's healing of mutant form expression of receptor to reduce.In a word, in people's leukocytosis and myelodysplastic syndrome, all the generation with tumour is relevant in the FLT3 sudden change.

Verified pHGF (HGF) acceptor (c-MET or HGFR) Tyrosylprotein kinase and tumour generate, strengthen cell mobility, invasion and attack and transfer closely related (referring to Ma, P.C etc. (2003b) .Cancer MetastasisRev, 22,309-25; Maulik, G. etc. (2002b) .Cytokine Growth Factor Rev, 13,41-59).Various tumours comprise in the small cell lung cancer (SCLC) overexpression or the sudden change can activate c-MET (HGFR) (referring to Ma, P.C. etc. (2003a) .Cancer Res, 63,6272-6281).

The proto-oncogene c-Met hepatocyte growth factor receptor of encoding, be cytolemma glycoprotein with tyrosine kinase activity, various kinds of cell propagation, differentiation are had important physical regulating effect .c-met gene in many malignant tumours, cross expression, be the important factor of follicular epithelial cell canceration, and with pathological staging, the invasion and attack of thyroid carcinoma and shift closely related.

About the PKT subtribe, Plowman etc. are at DN﹠amp; P 7 (6): have among the 334-339 (1994) more and describe in detail, the document is attached to herein by reference as an integral body.

Except PTKs, also there is other cellular enzymes family, be called receptor tyrosine kinase inhibitors, and use back one title at this, be abbreviated as " CTK ".CTKs itself lacks cell foreign lands and membrane-spanning domain.At present, in 11 subtribes (Src, Frk, Btk, Csk, Abl, Zap70, Fes/Fps, Fak, Jak, Ack and LIMK), identified above 24 kinds of CTKs.As if so far, Src subtribe CTKs number at most, comprises Src, Yes, and Fyn, Lyn, Lck, Blk, Hck, Fgr and Yrk, and Src subtribe enzyme generates relevant with tumour.About the more detailed description of CTKs, can be referring to iBolen, 1993, Oncogen 8:2025-2031, it comprises that in full any accompanying drawing as whole a proposition, is attached to herein by reference.

Similar with CTKs, serine-threonine kinase or STKs, dominate in cell is though only there are several STK receptor kinases.STKs is the most general cytosol kinases, and promptly it brings into play its function in the parts of fine kytoplasm, rather than in cytoplasmic organelles.Cytosol is zone in the cell, in this most cells intermediary metabolism and the active generation of biosynthesizing; As protein is to carry out synthetic on the cytosol rrna.

With one of feature of hyper-proliferative relative disease such as cancer etc. be that the pair cell pathway destroys, the cell pathway is by cell cycle control process.In eukaryotic cell, the orderly cascade reaction of cell cycle and proteinic phosphorylated is closely related, and in the mechanism of signal conduction, as if a lot of families of PKs all play a part key in the cell division cycle cascade.

About cancer, propose two main hypothesis and explain excessive cell proliferation, this propagation drives and is and the known relevant tumor development of being regulated by PK of function.That is, people think that malignant growth is because machine-processed destroyed the causing of control cell fission or propagation.Proto-oncogene protein matter product can the interference adjustments cell signal transduction path of growth and propagation, the protein of these proto-oncogenes comprises extracellular discussed above somatomedin, transmembrane growth factor PTK acceptor (RTKs), tenuigenin PTKs (CTKs) and cytosol STKs.

People wait in expectation and can synthesize the inhibitor with anti-tumour cell proliferative activity, wish to suppress among PTKs, CTKs or the STKs one or more, treat effectively and improve super propagation physiologic derangement by PTKs, CTKs or STKs and angiogenic action mediation.

Summary of the invention

In order to overcome the deficiencies in the prior art part, the object of the present invention is to provide the new quinazoline compounds shown in a kind of general formula (I), and their tautomer, enantiomorph, diastereomer, raceme and pharmacy acceptable salt, and meta-bolites and metabolic precursor thereof or prodrug.

Wherein:

R ₁Be selected from hydrogen atom and alkyl, wherein alkyl randomly further by one or more be selected from hydroxyl ,-SO ₂R ₂Or-NR ₃R ₄Substituting group replace;

R ₂Be selected from alkyl;

R ₃And R ₄Be selected from alkyl separately, perhaps

R ₃And R ₄Can further form 5～6 yuan of heterocycles, wherein 5～6 yuan of heterocycles contain one or more N, O or S atom.

Preferred compound of the present invention includes, but are not limited to:

Wherein, described salt is above-claimed cpd and the salt that is selected from following acid formation: oxysuccinic acid, lactic acid, toxilic acid, hydrochloric acid, methylsulfonic acid, sulfuric acid, phosphoric acid, citric acid, tartrate, acetate or trifluoroacetic acid.

One aspect of the present invention provides a kind of pharmaceutical composition, contains the compound shown in the general formula (I), its pharmacy acceptable salt or its prodrug and pharmaceutically acceptable carrier or vehicle.The purposes of described pharmaceutical composition in the medicine of preparation treatment and protein kinase diseases associated.

Another aspect of the present invention provides a kind of control method of protein kinase catalytic activity, comprises protein kinase is contacted with compound or its pharmacy acceptable salt of general formula (I).Described protein kinase is selected from receptor tyrosine kinase, nonreceptor tyrosine kinase and serine-threonine kinase.

Another aspect of the present invention provides the compound of a kind of preparation general formula (I) or the method for its pharmaceutically-acceptable salts, said method comprising the steps of:

Iodo quinazoline compound and 1-(tri isopropyl silane base)-1H-pyrroles-3-ylboronic acid carries out the Suzuki coupling, the compound 1 that obtains under alkaline condition with halogenated compound R ₁X carries out substitution reaction, or under the sodium hydride condition, with the reaction of racemic epoxide chloropropane more further with NHR ₃R ₄Carry out ring-opening reaction, obtain general formula (I) compound.Obtain general formula (I) compound.

Another aspect of the present invention provides the compound of a kind of preparation general formula (I) or the method for salt, said method comprising the steps of:

Pyrroles's boric acid ester and halogenated compound R ₁X or with the methylsulfonyl ethylene reaction, pyrroles's boric acid ester of 1 replacement that obtains and iodo quinazoline compound carry out the Suzuki coupling and obtain general formula (I) compound.

Another aspect of the present invention provides general formula (I) compound or the purposes of its pharmacy acceptable salt in the medicine of preparation treatment and protein kinase diseases associated.Described protein kinase is selected from receptor tyrosine kinase, nonreceptor tyrosine kinase or serine-threonine kinase.

Another aspect of the present invention provides the purposes of composition in the medicine of preparation treatment and protein kinase diseases associated that contains general formula (I) compound or its pharmaceutically-acceptable salts.Described protein kinase is selected from receptor tyrosine kinase, nonreceptor tyrosine kinase or serine-threonine kinase.

Detailed description of the invention

Unless the phase counter-statement is arranged, the term that uses in specification sheets and claims has following implication.

" alkyl " refers to saturated aliphatic hydrocarbon group, comprises the straight chain and the branched group of 1 to 20 carbon atom.The alkyl that preferably contains 1 to 10 carbon atom, for example methyl, ethyl, propyl group, 2-propyl group, normal-butyl, isobutyl-, the tertiary butyl, amyl group etc.The low alkyl group that more preferably contains 1 to 4 carbon atom, for example methyl, ethyl, propyl group, 2-propyl group, normal-butyl, isobutyl-or the tertiary butyl etc.Alkyl can be to be substituted that base replaces or unsubstituted, and when being substituted base and replacing, substituting group is preferably one or more, be independently selected from hydroxyl ,-SO ₂R ₂Or-NR ₃R ₄

" choose wantonly " or " randomly " mean describe subsequently ground incident or environment can but needn't take place, this explanation comprises that this incident or environment take place or spot occasion not.For example, " the optional heterocyclic group that is replaced by alkyl " mean alkyl can but must not exist, this explanation comprises the situation that situation that heterocyclic group is replaced by alkyl and heterocyclic group are not replaced by alkyl.

" pharmaceutical composition " expression contains on one or more compounds described herein or its physiology/mixture of pharmacy acceptable salt or prodrug and other chemical compositions, and other components physiology/pharmaceutically acceptable carrier and vehicle for example.The purpose of pharmaceutical composition is the administration that promotes organism, is beneficial to the absorption and then the performance biological activity of activeconstituents.

The synthetic method of The compounds of this invention

In order to finish purpose of the present invention, the present invention adopts following technical scheme:

The preparation method of general formula of the present invention (I) compound or its salt may further comprise the steps:

Scheme I

Pyrroles 1a and tri isopropyl chlorosilane reaction, the 1-tri isopropyl silane base-1H-pyrroles 1b that obtains obtains 1c and obtains 1-(tri isopropyl silane base)-1H-pyrroles-3-ylboronic acid 1d with the trimethyl borate reaction again through bromo-reaction; Behind 6-iodo-3H-quinazoline-4-one 1e and the phosphorus oxychloride reaction, the compound 1g that obtains with 3-chloro-4-(pyridine-2-methoxyl group)-aniline 1f reaction and 1-(tri isopropyl silane base)-1H-pyrroles-3-ylboronic acid 1d carry out the Suzuki coupling and obtain compound 1 again; Compound 1 and halogenated compound R ₁X replaces under alkaline condition, or under the sodium hydride condition with racemic epoxide chloropropane reaction more further with NHR ₃R ₄Carry out ring-opening reaction, obtain general formula (I) compound.

Scheme II

3-pyrroles's boric acid ester and halogenated compound R ₁X or with the methylsulfonyl ethylene reaction, 1-position substituted azole boric acid ester that obtains and compound 1g carry out the Suzuki coupling and obtain general formula (I) compound.

Embodiment

The structure of compound is determined by nucleus magnetic resonance (NMR) or mass spectrum (MS).NMR displacement (δ) provides with 1,000,000/(ppm) unit.The mensuration of NMR is that measuring solvent is deuterochloroform (CDCl with Bruker AVANCE-400 nuclear magnetic resonance spectrometer ₃), deuterated methanol (CD3OD-d ₄) and deuterated dimethyl sulfoxide (DMSO-D ₆), in be designated as tetramethylsilane (TMS), chemical shift is with 10 ^-6(ppm) provide as unit.

The mensuration of MS FINNIGAN LCQAd (ESI) mass spectrograph.

The mensuration of the average inhibiting rate of kinases VEGFR is used HTScan microplate reader (Cell Signaling company).

The mensuration of the average inhibiting rate of kinases EGFR/HER-2 is with NovoStar microplate reader (German BMG company).

Thin layer silica gel uses Yantai Huanghai Sea HSGF254 or Qingdao GF254 silica-gel plate.

Column chromatography generally uses Huanghai Sea silica gel 200～300 order silica gel in Yantai to be carrier.

Do not have specified otherwise among the embodiment, reaction is all carried out under nitrogen atmosphere.

Nitrogen atmosphere is meant that reaction flask connects an about 1L volumetrical nitrogen balloon.

Do not have specified otherwise among the embodiment, the solution in the reaction is meant the aqueous solution.

Preparation embodiment:

Embodiment 1

[3-chloro-4-(pyridine-2-ylmethoxy)-phenyl]-[6-(1H-pyrroles-3-yl)-quinazoline-4-yl]-amine

The first step

1-tri isopropyl silane base-1H-pyrroles

In the 2L there-necked flask, add 60% sodium hydride and mineral oil mixture (33.6g, 0.84mol), normal hexane is extracted in a small amount of normal hexane washing back, to wherein adding new tetrahydrofuran (THF) and the 500mL exsiccant acetonitrile that steams of 500mL, be cooled to and be lower than 0 ℃, constant pressure funnel slowly drips pyrroles 1a, and (46.96g 0.7mol), has a large amount of gases to emit.After dropwising, at room temperature react and do not have the gas generation in 0.5 hour substantially.(134.97g 0.7mol), generates a small amount of tawny smog, ambient temperature overnight to add tri isopropyl chlorosilane under 0 ℃ in there-necked flask.Add the 50mL shrend reaction of going out in the reaction solution, outstanding steaming behind most of solvent with ethyl acetate extraction (200mL * 4), the organic phase saturated common salt water washing that merges, anhydrous sodium sulfate drying, the reddish-brown oily matter that concentrating under reduced pressure obtains is positioned over the refrigerator crystallization, with the crude product that obtains decompression column chromatography (eluent: normal hexane) purify, obtain 1-tri isopropyl silane base-1H-pyrroles 1b (125g, colourless transparent oil liquid), productive rate: 79.9%

Second step

3-bromo-1-tri isopropyl silane base-1H-pyrroles

In the 1L there-necked flask with 1-tri isopropyl silane base-1H-pyrroles 1b (60g, 0.268mol) be dissolved in the tetrahydrofuran (THF) of new steaming, the argon replaces air, be cooled to-78 ℃ in the dry ice-propanone bath, add N-bromosuccinimide (47.8g in batches, 0.268mol), keep this temperature to continue reaction 1 hour, at room temperature reacted then 1 hour.In reaction solution, add the 100mL shrend reaction of going out.Decompression is concentrated solvent down, the residue n-hexane extraction that obtains, the organic phase saturated common salt water washing that merges, anhydrous sodium sulfate drying, concentrating under reduced pressure is with the crude product that obtains decompression column chromatography (eluent: normal hexane) purify, obtain 3-bromine 1-tri isopropyl silane base-1H-pyrroles 1c (63g, colourless oil liquid), productive rate: 77.8%.

The 3rd step

1-(tri isopropyl silane base)-1H-pyrroles-3-ylboronic acid

In the 100mL there-necked flask with 3-bromo-1-tri isopropyl silane base-1H-pyrroles 1c (2.32g, 7.67mmol) be dissolved in the 40mL exsiccant tetrahydrofuran (THF), bathe under the cooling at dry ice-propanone, temperature is reduced to-70 ℃, and the dropping tert-butyl lithium (9.2mL, 19.18mmol), after dropwising, keep-70 ℃ to stir 30 minutes, and the dropping trimethyl borate (1.75mL, 15.37mmol), after dropwising, stirring is after 2 hours down in-10 ℃, and the mixed solution of adding 2mL methyl alcohol and 2mL water rises to room temperature in there-necked flask, pour 20mL water into, tell organic layer, water layer ethyl acetate extraction (20mL * 3), the organic phase of merging is used the saturated common salt water washing successively, anhydrous sodium sulfate drying, filter, concentrating under reduced pressure, the product 1-that obtains (tri isopropyl silane base)-1H-pyrroles-3-ylboronic acid 1d directly carries out next step reaction without separating.

The 4th step

[3-chloro-4-(pyridine-2-ylmethoxy)-phenyl]-(6-iodine quinazoline-4-yl)-amine

6-iodo-3H-quinazoline-4-one 1e (5g, 18.3mmol is according to the WO2002002552 preparation) is dissolved in 50mL thionyl chloride and 0.5mLN, and in the mixed solvent of dinethylformamide, reflux is transparent to reaction solution.Stir after 6 hours, tlc analysis is followed the tracks of raw material and disappeared, and steams thionyl chloride, and is standby.

Standby intermediate is dissolved in the 160mL Virahol, adds 3-chloro-4-(pyridine-2-ylmethoxy)-aniline 1f (4.25g, 18.2mmol is according to the WO2006127207 preparation), reflux is spent the night.Tlc analysis tracks to raw material and disappears, reaction solution is cooled to room temperature, decompress filter, the gained solid is added ammoniacal liquor (200mL, volume ratio is 1: 1) with 5mL dissolve with methanol dilution back, regulate pH value to 8～9, ice-water bath cooling back suction filtration, drying obtains [3-chloro-4-(pyridine-2-methoxyl group)-phenyl]-(6-iodine quinazoline-4-yl)-amine 1g (8g, off-white color solid).Productive rate: 89.6%.

MS?m/z(ESI)：489[M+1]

¹HNMR(400MHz，DMSO-d ₆)：δ9.78(s，1H)，8.60(d，3H)，8.16(d，1H，J＝9.2Hz)，8.04(m，1H)，7.89(m，2H)，7.76(d，1H，J＝9.2Hz)，7.59(m，3H)，7.38(t，1H)，7.31(d，1H，J＝8.8Hz)，6.38(s，2H)，5.31(s，2H)

The 5th step

In the 50mL eggplant-shape bottle with [3-chloro-4-(3-fluoro-benzyloxy)-phenyl]-(6-iodo-quinazoline-4-yl)-amine 1k (3g, 6.14mmol), 1-(triisopropyl silicon)-1H-pyrroles-3-boric acid 1d (1.64g, 6.14mmol), four triphenylphosphine palladiums (710mg, 0.614mmol) and salt of wormwood (2.54g, 18.42mmol) be dissolved in 55mLN, in the mixed solvent of dinethylformamide and 8mL water, the mixture heating up to 55 that obtains ℃, stirring is spent the night.Reaction solution is cooled to room temperature, pour in the 200mL frozen water, separate out white solid, stir after 5 minutes, with dichloromethane extraction reaction solution (80mL * 4), the organic phase that merges is used the saturated common salt water washing successively, anhydrous sodium sulfate drying, filter, decompression concentrates down, further (methylene dichloride: separation and purification methyl alcohol=80: 1) obtains title product [3-chloro-4-(pyridine-2-ylmethoxy)-phenyl]-[6-(1H-pyrroles-3-yl)-quinazoline-4-yl]-amine 1 (1.2g, light yellow solid) to the solid that obtains by column chromatography.Productive rate: 45.7%.

MS?m/z(ESI)：428[M+1]

¹HNMR(400MHz，DMSO-d ₆)：δ11.08(s，1H)，9.68(s，1H)，8.62(s，1H)，8.57(s，1H)，8.50(s，1H)，8.09(d，1H，J＝8.8Hz)，8.05(s，1H)，7.90(m，1H)，7.76(d，1H，J＝8.8Hz)，7.71(d，1H，J＝8.8Hz)，7.61(d，1H，J＝7.6Hz)，7.45(s，1H)，7.38(m，1H)，7.31(d，1H，J＝8.8Hz)，6.90(s，1H)，6.70(s，1H)，5.31(s，2H)

Embodiment 2

[3-chloro-4-(pyridine-2-ylmethoxy)-phenyl]-6-[1-(2-methylsulfonyl-ethyl)-1H-pyrroles-3-yl]-quinazoline-4- Base }-amine

The first step

3-(4,4,5,5-tetramethyl--[1,3,2] dioxane pentaborane-2-yl)-(10g 16.5mmol) is dissolved in the 100mL tetrahydrofuran (THF) 1-(tri isopropyl silane base)-1H-pyrroles, is cooled to-78 ℃ in dry ice-propanone is bathed with 3-bromine 1-tri isopropyl silane base-1H-pyrroles 1c of 50% in the 250mL there-necked flask, add n-Butyl Lithium (15.8g, 39.7mol), kept this thermotonus 0.5 hour, add 2-isopropoxy-4,4,5,5-tetramethyl--[1,3,2] dioxane pentaborane (4mL, 19.8mmol), continuing-78 ℃ of reactions 2 hours, tlc analysis tracks to raw material and disappears, the mixed solvent that adds 10mL methyl alcohol and 10mL water, reaction solution is poured in the 200mL frozen water, and with ethyl acetate extraction (100mL * 4), the organic phase of merging is used the saturated common salt water washing successively, anhydrous sodium sulfate drying, filter, concentrating under reduced pressure, the residue that obtains is by the further separation and purification of silica gel column chromatography (normal hexane: ethyl acetate=50: 1), obtain title product 3-(4,4,5,5-tetramethyl--[1,3,2] dioxane pentaborane-2-yl)-and 1-(tri isopropyl silane base)-1H-pyrroles 2a (1.7g, white solid), productive rate: 29.5%.

MS?m/z(ESI)：349.3[M+1]

Second step

3-(4,4,5,5-tetramethyl--[1,3,2] dioxane pentaborane-2-yl)-1H-pyrroles

In the 100mL eggplant-shape bottle with 3-(4,4,5,5-tetramethyl--[1,3,2] dioxane pentaborane-2-yl)-1-(tri isopropyl silane base)-1H-pyrroles 2a (500mg, 1.43mmol) be dissolved in the 10mL tetrahydrofuran (THF), ice bath is cooled to 0 ℃, and the tetrahydrofuran solution of adding 1M tetrabutyl ammonium fluoride (1.43mL, 1.43mmol), reaction solution rose to room temperature reaction 2 hours, tlc analysis tracks to raw material and disappears, and reaction solution is poured in the 50mL frozen water, with ethyl acetate extraction (50mL * 4), the organic phase that merges is used the saturated common salt water washing successively, anhydrous sodium sulfate drying filters concentrating under reduced pressure, the crude product 3-(4 that obtains, 4,5,5-tetramethyl--[1,3,2] dioxane pentaborane-2-yl)-and 1H-pyrroles 2b (600mg, dark oil liquid), productive rate: 100%.

MS?m/z(ESI)：193[M+1]

The 3rd step

1-(2-methylsulfonyl ethyl)-3-(4,4,5,5-tetramethyl--[1,3,2] dioxane pentaborane-2-yl)-1H-pyrroles

In the 100mL eggplant-shape bottle with 3-(4; 4,5,5-tetramethyl--[1; 3; 2] dioxane pentaborane-2-yl)-(276mg 1.43mmol) is dissolved in the 10mL acetonitrile 1H-pyrroles 2b, adds salt of wormwood (600mg successively; 4.3mmol); (228mg, 2.1mmol), reflux stirs spends the night methylsulfonyl ethene.Tlc analysis tracks to raw material and disappears, and filters, and filter cake is given a baby a bath on the third day after its birth inferior with ethyl acetate, and filtrate decompression concentrates; the crude product 1-that obtains (2-methylsulfonyl-ethyl)-3-(4,4,5; 5-tetramethyl--[1,3,2] dioxane pentaborane-2-yl)-1H-pyrroles 2c directly carries out next step reaction without separating.

MS?m/z(ESI)：299.18[M+]

The 4th step

[3-chloro-4-(pyridine-2-ylmethoxy)-phenyl]-6-[1-(2-methylsulfonyl-ethyl)-1H-pyrroles-3-yl]-quinazoline-4-yl }-amine

With 1-(2-methylsulfonyl ethyl)-3-(4,4,5; 5-tetramethyl--[1,3,2] dioxane pentaborane-2-yl)-1H-pyrroles 2c (299mg; 1mmol); [3-chloro-4-(pyridine-2-methoxyl group)-phenyl]-(6-iodine quinazoline-4-yl)-amine 1k (500mg, 1mmol), salt of wormwood (415mg; 3mmol) and tetra-triphenylphosphine palladium (116mg; 0.1mmol) being dissolved in 10mLN, in the mixed solvent of dinethylformamide and 2mL water, reaction solution is heated to 80 ℃ of stirrings and spends the night.Tlc analysis tracks to raw material and disappears; reaction solution is poured in the 75mL frozen water; with methylene chloride (10: 1)) extraction (50mL * 4); merging organic interdependent time washs with saturated nacl aqueous solution; anhydrous sodium sulfate drying; filter; decompression concentrates down; the residue that obtains is by the further separation and purification of silica gel column chromatography (methylene dichloride: methyl alcohol=30: 1); obtain title product [3-chloro-4-(pyridine-2-ylmethoxy)-phenyl]-{ 6-[1-(2-methylsulfonyl-ethyl)-1H-pyrroles-3-yl]-quinazoline-4-yl }-amine 2 (400mg, light yellow solid).Productive rate: 74.9%.

MS?m/z(ESI)：534.4[M+1]

¹HNMR(400MHz，DMSO-d ₆)：δ9.74(s，1H)，8.65(d，1H)，8.60(s，1H)，8.51(s，1H)，8.05(d，1H，J＝6.4Hz)，8.03(s，1H)，7.90(m，1H)，7.65(d，1H，J＝7.6Hz)，7.62(d，1H，J＝8.8Hz)，7.61(d，1H，J＝8.0Hz)，7.52(s，1H)，7.39(m，1H)，7.29(d，1H，J＝8.8Hz)，7.01(s，1H)，6.70(s，1H)，5.31(s，2H)，4.40(t，2H)，3.72(t，2H)，2.82(s，3H)

Embodiment 3

[3-chloro-4-(pyridine-2-ylmethoxy)-phenyl]-6-[1-(2-morpholine-4-base-ethyl)-1H-pyrroles-3-yl]-quinazoline -4-yl }-amine

The first step

4-(2-bromotrifluoromethane)-morpholine hydrobromate

Cryosel is bathed below 0 ℃, in the test tube that has the fluorinated ethylene propylene stopper, add successively 40% hydrobromic acid solution (3.2mL, 23.2mmol), phosphorus tribromide (1.7mL, 17.4mmol) and 2-morpholine-4-base-ethanol 3a (1.4mL, 11.6mmol), finish sealing, slowly be heated to 140 ℃ of stirrings and spend the night.Be cooled to room temperature, boil off most of solvent, add 15mL ebullient ethanol, stir and be cooled to room temperature, add the 25mL ether, stirred 30 minutes, filter, oven dry obtains 4-(2-bromotrifluoromethane)-morpholine hydrobromate 3b (8.1g, white solid), productive rate: 84%.MS?m/z(ESI)：194.2[M+1]

Second step

[3-chloro-4-(pyridine-2-ylmethoxy)-phenyl]-6-[1-(2-morpholine-4-base-ethyl)-1H-pyrroles-3-yl]-quinazoline-4-yl }-amine

In the 10mL eggplant-shape bottle with [3-chloro-4-(pyridine-2-ylmethoxy)-phenyl]-[6-(1H-pyrroles-3-yl)-quinazoline-4-yl]-amine 1 (85.6mg, 0.2mmol) be dissolved in 2mL N, in the dinethylformamide, ice bath is cooled to 0 ℃, (40mg 1mmol), stirs and adds 4-(2-bromotrifluoromethane)-morpholine hydrobromate 3b (66mg after 30 minutes to add 60% sodium hydride and mineral oil mixture, 0.24mmol), in 0 ℃ of reaction 3-4 hour.Tlc analysis tracks to raw material and disappears, pour into reaction solution in the 20mL frozen water and constantly stirring, with ethyl acetate (50mL * 3) extractive reaction liquid, the organic phase that merges is used saturated aqueous common salt (20mL * 2) washing successively, anhydrous sodium sulfate drying, filter, decompression concentrates down, the residue that obtains prepares the further separation and purification of plate (methylene dichloride: methyl alcohol=10: 1) by thin-layer chromatography, obtain title product [3-chloro-4-(pyridine-2-ylmethoxy)-phenyl]-{ 6-[1-(2-morpholine-4-base-ethyl)-1H-pyrroles-3-yl]-quinazoline-4-yl }-amine 3 (53mg, yellow solid).Productive rate: 49.0%.

MS?m/z(ESI)：541.6[M+1]

¹HNMR(400MHz，DMSO-d ₆)：δ9.74(s，1H)，8.65(d，1H)，8.54(s，1H)，8.49(s，1H)，8.04(m，2H)，7.88(m，1H)，7.77(d，1H，J＝2.4Hz)，7.75(d，1H，J＝2.4Hz)，7.65(d，1H，J＝8.4Hz)，7.45(s，1H)，7.35(m，1H)，7.31(d，1H，J＝9.2Hz)，6.93(s，1H)，6.66(s，1H)，5.31(s，2H)，4.08(m，2H)，3.59(m，4H)，2.73(m，2H)，2.48(m，4H)

Embodiment 4

[3-chloro-4-(pyridine-2-ylmethoxy)-phenyl]-6-[1-(2-diethyllaminoethyl)-1H-pyrroles-3-yl]-quinazoline-4- Base }-amine

In the 10mL eggplant-shape bottle with [3-chloro-4-(pyridine-2-ylmethoxy)-phenyl]-[6-(1H-pyrroles-3-yl)-quinazoline-4-yl]-amine 1 (85.6mg, 0.2mmol) be dissolved in 2mLN, in the dinethylformamide, ice bath is cooled to 0 ℃, (40mg 1mmol), stirs and adds (2-chloroethyl)-diethylamine hydrochloride (41.3mg after 30 minutes to add 60% sodium hydride and mineral oil mixture, 0.24mmol), in 0 ℃ of reaction 4～5 hours.Tlc analysis tracks to raw material and disappears, pour into reaction solution in the 20mL frozen water and constantly stirring, with ethyl acetate (50mL * 5) extractive reaction liquid, the organic phase that merges is used saturated aqueous common salt (20mL * 2) washing successively, anhydrous sodium sulfate drying, filter, decompression concentrates down, the residue that obtains prepares the further separation and purification of plate (methylene dichloride: methyl alcohol=10: 1) by thin-layer chromatography, obtain title product [3-chloro-4-(pyridine-2-ylmethoxy)-phenyl]-{ 6-[1-(2-diethyllaminoethyl)-1H-pyrroles-3-yl]-quinazoline-4-yl }-amine 4 (50mg, yellow solid).Productive rate: 47.4%.

MS?m/z(ESI)：527.4[M+1]

¹HNMR(400MHz，DMSO-d ₆)：δ9.74(s，1H)，8.62(d，2H)，8.51(s，1H)，8.06(m，2H)，7.92(m，1H)，7.79(d，1H，J＝2.8Hz)，7.72(d，1H，J＝8.8Hz)，7.61(d，1H，J＝7.6Hz)，7.48(s，1H)，7.38(m，1H)，7.31(d，1H，J＝8.8Hz)，6.95(s，1H)，6.68(s，1H)，5.31(s，2H)，4.08(m，2H)，2.81(m，2H)，2.56(m，4H)，0.85(s，6H)

Embodiment 5

1-(3-{4-[3-chloro-4-(pyridine-2-ylmethoxy)-phenylamino]-quinazoline-6-yl }-pyrroles-1-yl)-3-morpholine-4-base- Propan-2-ol

[3-chloro-4-(pyridine-2-ylmethoxy)-phenyl]-[6-(1-epoxy ethyl methyl isophthalic acid H-pyrroles-3-yl)-quinazoline-4-yl]-amine

In the 100mL eggplant-shape bottle with [3-chloro-4-(pyridine-2-ylmethoxy)-phenyl]-[6-(1H-pyrroles-3-yl)-quinazoline-4-yl]-amine 1 (1.09g, 2.34mmol) be dissolved in 15mL N, in the dinethylformamide, ice bath is cooled to 0 ℃, add 60% sodium hydride and mineral oil mixture (408mg, 9.36mmol), stir after 30 minutes, (325mg, 3.51mmol), afterreaction finished in 2 hours to add epoxy chloropropane under the room temperature.Reaction solution is poured in the 100mL frozen water, there is solid to separate out, obtain [3-chloro-4-(pyridine-2-ylmethoxy)-phenyl]-[6-(1-epoxy ethyl methyl isophthalic acid H-pyrroles-3-yl)-quinazoline-4-yl]-amine 5a (1.2g, yellow solid), product directly carries out next step reaction without separating.

MS?m/z(ESI)：484[M+1]

¹HNMR(400MHz，DMSO-d ₆)：δ9.717(s，1H)，8.609(d，1H，J＝4.8Hz)，8.555(d，1H，J＝1.6Hz)，8.499(s，1H)，8.603(m，2H)，7.910(m，1H)，7.762(m，2H)，7.607(d，1H，J＝8.0Hz)，7.442(s，1H)，7.388(m，1H)，7.308(d，1H，J＝9.2Hz)，6.932(s，1H)，6.707(s，1H)，5.306(s，2H)，4.320(dd，1H，J1＝3.2Hz，J2＝14.8Hz)，3.993(dd，1H，J1＝6.4Hz，J2＝14.8Hz)，3.323(m，1H)，2.841(t，1H，J＝5.2Hz)，2.595(t，1H，J＝5.2Hz)

Second step

1-(3-{4-[3-chloro-4-(pyridine-2-ylmethoxy)-phenylamino]-quinazoline-6-yl }-pyrroles-1-yl)-3-morpholine-4-base-propan-2-ol

In the 100mL eggplant-shape bottle with [3-chloro-4-(pyridine-2-ylmethoxy)-phenyl]-[6-(1-epoxy ethyl methyl isophthalic acid H-pyrroles-3-yl)-quinazoline-4-yl]-amine 5a (250mg, 0.52mmol) be dissolved in the 5mL methyl alcohol, stir and add morpholine (90mg down, 1.03mmol), the reaction solution reflux is spent the night.Reaction solution is under reduced pressure concentrated, the residue that obtains is by the further separation and purification of silica gel column chromatography, obtain this title product 1-(3-{4-[3-chloro-4-(pyridine-2-ylmethoxy)-phenylamino]-quinazoline-6-yl-pyrroles-1-yl)-3-morpholine-4-base-propan-2-ol 5 (100mg, yellow solid), productive rate: 33%.

MS?m/z(ESI)：571[M+1]

¹HNMR(400MHz，DMSO-d ₆)：δ9.702(s，1H)，8.610(d，1H，J＝5.2Hz)，8.531(s，1H)，8.487(s，1H)，8.038(m，2H)，7.906(m，1H)，7.761(dd，1H，J＝2.4Hz，8.8Hz)，7.705(d，1H，J＝8.4Hz)，7.606(d，1H，J＝8.0Hz)，7.414(s，1H)，7.388(m，1H)，7.306(d，1H，J＝9.2Hz)，6.880(s，1H)，6.648(s，1H)，5.305(s，2H)，4.955(d，1H，J＝4.8Hz)，3.956(m，1H)，3.886(m，1H)，3.652(m，1H)，3.607(m，4H)，2.418(m，4H)，2.258(m，2H)

Embodiment 6

1-(3-{4-[3-chloro-4-(pyridine-2-ylmethoxy)-phenylamino]-quinazoline-6-yl }-pyrroles-1-yl)-the 3-diethylamino Base-propan-2-ol

In the 100mL eggplant-shape bottle with [3-chloro-4-(pyridine-2-ylmethoxy)-phenyl]-[6-(1-epoxy ethyl methyl isophthalic acid H-pyrroles-3-yl)-quinazoline-4-yl]-amine 5a (250mg, 0.52mmol) be dissolved in the 40mL methyl alcohol, stir and add diethylamine (76mg down, 1.04mmol), the reaction solution reflux is spent the night.Reaction solution is under reduced pressure concentrated, the residue that obtains is by the further separation and purification of silica gel column chromatography, obtain this title product 1-(3-{4-[3-chloro-4-(pyridine-2-ylmethoxy)-phenylamino]-quinazoline-6-yl-pyrroles-1-yl)-3-diethylin-propan-2-ol 6 (92.5mg, yellow solid), productive rate: 32%.

MS?m/z(ESI)：557[M+1]

¹HNMR(400MHz，DMSO-d ₆)：δ9.900(s，1H)，8.682(s，1H)，8.606(d，1H，J＝4.4Hz)，8.491(s，1H)，8.095(s，1H)，8.040(d，1H，J＝7.2Hz)，7.901(m，1H)，7.822(d，1H，J＝6.8Hz)，7.708(d，1H，J＝8.8Hz)，7.604(d，1H，J＝8.0Hz)，7.504(s，1H)，7.382(m，1H)，7.292(d，1H，J＝9.2Hz)，6.900(s，1H)，6.726(s，1H)，5.301(s，2H)，4.099(m，2H)，3.938(m，1H)，2.886(m，4H)，2.700(m，2H)，1.091(m，6H)

Test case:

Biological assessment

Example 1EGFR suppresses the cell proliferation test

Following in vitro tests is to be used for measuring the cell strain inhibition proliferation activity of The compounds of this invention for human tumor cell A431EGFR high expression level.

Cell in vitro described below test can determine test-compound to the anti-angiogenesis activity of the tumour cell of high expression level EGFR and suppress proliferation activity, its active available IC ₅₀Value is represented.The general approach of this type of test is as follows: the human tumor cell who at first selects high expression level EGFR, be seeded on 96 well culture plates with (5000 cells of exp/mL medium) under the suitable cell concn, then cell is cultivated in carbon dioxide incubator, when growing to 85%, they converge, change substratum for being added with the substratum of a series of concentration degree of passing (general 6 to 7 concentration) test-compound solution, culture plate is put back to incubator again, cultured continuously 72 hours.After 72 hours, available sulphonyl rhodamine B (SRB) method is carried out test compounds for suppressing cell-proliferation activity.IC ₅₀Value can be by under a series of different concns, and test-compound calculates for cell inhibiting numerical value.

Material and method:

A. dimethyl sulfoxide (DMSO) (Sinophma chemical reagent company, catalogue T20050806 number)

B.A431 cell (purchasing biology) in Institute of biochemistry and cell

C.Falcon 100mm Tissue Culture Plate (Baton Dickison Labware, Baton Dickison andcompany, No. 18677, catalogue)

D. healthy and free from worry 96 well culture plates (healthy and free from worry Incorporated, No. 3599, catalogue)

E.Fisher transfer pipet (Fisher scientific, catalogue 03-692-164 number)

F.DMEM/F12 cell culture medium (Gibco, catalogue 12400-024 number)

G. Australian foetal calf serum (Gibco, catalogue 10099-141 number)

H. phosphate buffered saline (PBS) (Gibco, catalogue 10010-072 number)

I.0.25% Regular Insulin-EDTA (Gibco, catalogue 25200-056 number)

J. sulphonyl rhodamine B (Sigma, catalogue 3520-42-1 number)

K. acetic acid (Sinophma chemical reagent company, catalogue T20060508 number)

L. Tricholroacetic Acid (Sinophma chemical reagent company, catalogue T20060305 number)

M.Tris alkali (Amresco, No. 0826, catalogue)

N.II level A/B3 type biological safety cabinet (ThermoForma catalogue .HB0053-03 number)

O. serial II water-jacket typ CO2gas incubator (ThermoForma model: 3111)

P. whizzer (Fisher Scientific Marathon 8k, catalogue 0027-02 number)

Q.Novastar plate reader (BMG Labtech, catalogue 700-0081 number)

R. orbital shaker (Qilinbeier, catalogue TS-1 number)

Scheme:

Following scheme is used for testing test-compound of the present invention for A431 cell inhibiting cell proliferation IC ₅₀Value.

1. the A431 cell is grown in the healthy and free from worry culture plate of 100mm in growth substrate (is nutrient solution with the DMEM/F12+10% foetal calf serum), cultivate (37 ℃, 5%CO ₂), fully converge until cell;

In the 100mm culture plate with foetal calf serum washing A431 cell, behind the Tyrpsin peptic cell, again with cell inoculation on healthy and free from worry 96 porocyte culture plates, concentration is 50000cells/mL, each plate empty 6 holes, hole in contrast.;

3. at 37 ℃, 5%CO ₂Under the condition, cell is cultivated in 96 orifice plates, converged until reaching about 85%;

4. dissolve test-compound with DMSO, configuration 20mM mother liquor, mother liquor is diluted with DMSO in the back, obtains the solution of the test-compound of a series of concentration, i.e. 2mM, 1mM, 0.2mM, 20 μ M, 2 μ M, 0.2 μ M;

5. use the compound solution that is disposed above cell culture medium (the DMEM/F12+10% foetal calf serum the is nutrient solution) dilution.20 times of each DMSO series concentration compound solution dilutions add 5 μ L DMSO compound solutions and 95 μ L nutrient solutions at every turn in cell culture medium, guarantee that the concentration of A431 cellular exposure in DMSO solution is no more than 0.5%, use vortex mixed;

6. when the A431 cell attachment, growth reach 85% converge after, substratum is changed to the new substratum that is added with DMEM/F12+10% foetal calf serum nutrient solution, every Kong Zhongzai adds 180 μ L nutrient solutions and 20 μ L prepared test-compound solution in the 5th step.The negative control cell group adds the 20 μ L nutrient solutions that contain 0.5%DMSO, and the ultimate density of A431 cellular exposure in test-compound solution is 100 μ M like this, 10 μ M, 5 μ M, 1 μ M, 0.1 μ M, 0.01 μ M, and 0.001 μ M;

7. culture plate is put into thermostat container, at 37 ℃, 5%CO ₂Under the condition, cultured continuously 72 hours;

8.72 after hour, culture plate is transferred to aseptic working spaces from thermostat container;

9. reagent level pure water is joined prepare among the TCA fixing agent (50% Tricholroacetic Acid-TCA), with cell at leisure layering be placed in the cold TCA solution of 50 μ L;

10. under 4 ℃, cultivated 1 hour, after wash with water for several times to remove TCA, serum protein etc.Culture plate is stored stand-by at air drying.Blank is the numerical value that incubation is cultivated gained in the substratum that does not have the cell growth by the mensuration of scape optical density (OD) value.

11. prepare 0.4% sulphonyl rhodamine B solution with 10% acetum, and in every hole, add 50 μ L sulphonyl rhodamine B solution;

12. painted 30 minutes of cell;

13. prepare 10% acetic acid washing soln.When painted will finishing, discard tinting material, get cell express developed with 10% acetum.Repeat above-mentioned operation till tinting material is cleaned, reduce washing time as far as possible with the absorption of going of minimizing with protein bound tinting material.The flushing finish after, with culture plate at air drying;

14. the blended tinting material is dissolved in the sulphonyl rhodamine B of certain volume, (10mM Tris) is identical with the substratum original volume for solubilising solution, and culture plate was at room temperature placed 5 minutes, slowly stirs with shaking table and accelerates and the mixing of dyestuff;

15. use spectrophotometry, under wavelength 565nm, read absorbance.Absorbancy numerical value is that absorbancy deducts under the 690nn 96 orifice plates by the numerical value of scape absorbancy gained under the 565nm;

16. make and calculate inhibiting rate ratio with the following method:

IR=100 * (control group absorbance-medication group absorbance)/control group absorbance %.

IC ₅₀Value can obtain by compound inhibiting rate ratio calculation under the different concns.

The activity of The compounds of this invention

The biochemical activity of The compounds of this invention is measured by above test, the IC that records ₅₀Value sees the following form.

The embodiment numbering	??IC ₅₀(EGFR/A431)(μM)
The embodiment numbering	??IC ₅₀(EGFR/A431)(μM)	??1	??0.41
??2	??1.08	??1	??0.41
??2	??1.08	??3	??1.24
??4	??0.76	??3	??1.24

Example 2.EGFR kinase activity is measured

External EGFR kinase activity is tested by following method.

Material and reagent:

A. lavation buffer solution (PBS-T damping fluid): 1x PBS (137mM NaCl, 2.7mM KCl, 4.3mMNa ₂HPO ₄, 1.4mM KH ₂PO ₄, transfer pH to 7.2) and 0.05%Tween-20

B.1% bovine serum albumin (BSA, Calbiochem#136593) PBS-T damping fluid

C. stopping of reaction damping fluid: 50mM EDTA, pH 8.0.

The anti-mouse IgG of europium mark (PerkinElmer Life Sciences#AD0124)

Signal multiplication liquid (PerkinElmer Life Sciences#1244-105)

Streptavidin wraps the yellow plate (PerkinElmer Life Sciences#AAAND-0005) in 96 holes

G.EGFR kinases (50mM Tris-HCl (pH 8.0), 100mM NaCl, 5mM DTT, 15mM glutamylcystyl-glycine and 20% glycerine, Cell signaling technology#7908)

H.10mM ATP solution (Cell signaling technology#9804).

I.PTP1B (Tyr66) biotinylation albumen (Cell signaling technology#1325).

J.Phospho-tyrosine mouse mAb (P-Tyr-100) (Cell signaling technology#9411).

K.HTScan ^TMTyrosylprotein kinase damping fluid (4x)

1x kinase buffer liquid:

60mM?HEPES

5mM?MgCl ₂

5mM?MnCl ₂

3μMNa ₃VO ₄

(Cell?signaling?technology#9805).

1.1.25M?DTT(1000x)(Cell?signaling?technology).

Scheme:

Use following scheme to test:

1. reach the ultimate density value with DMSO dilution test-compound;

In each test, add 1 μ L test-compound, negative control and blank (not accepting any test-compound), only add 1 μ L DMSO;

2. use dH ₂O1: 1 dilution, 6 μ M substrate proteins (Tyr589), and add 15 μ L to each test;

3. enzyme is directly transferred on ice from-80 ℃, the EGFR enzymolysis freezes on ice;

4. get 3 μ g EGFR enzymes in each test;

5. add 10 μ L DTT (1.25M) to 2.5mL 4x HTScan ^TMTyrosylprotein kinase damping fluid (240mMHEPES pH 7.5,20mM MgCI ₂, 20mM MnCI ₂, 12 μ M Na ₃VO ₄) in, make DTT/ kinase buffer liquid;

6. transferase 10 .75mL DTT/ kinase buffer liquid makes the 4x reaction mixture in each test, and adds 7.5 μ L4x reaction solutions in each test;

7. add 2 μ LATP (10mM) to 496 μ L dH ₂Among the O, and in each test, add 7.5 μ L;

30 μ L reaction final test condition is:

60mM?HEPES?pH?7.5

5mM?MgCl ₂

5mM?MnCl ₂

3μM?Na ₃VO ₄

1.25mM?DTT

20μM?ATP

1.5 μ M peptide substrate

30ng EGFR kinases

8. under 25 ℃, with reaction tubes incubation 45 minutes;

9. add 30 μ L stopped reaction damping fluid (50mM EDTA, pH 8.0) stopped reactions in each test;

10. added 25 μ L reaction solutions and 75 μ L dH in every hole by culture plate at 96 hole streptavidin bag ₂O, at room temperature, and jolting 60 minutes;

11. pat on paper handkerchief to remove remaining liquid with 200 μ L PBS-T damping fluids washing 3 times in every hole;

12., in every hole, add the antibody of 100 μ L dilution with 1% bovine serum albumin PBS-T damping fluid dilution in 1: 1000 antibody Phospho-tyrosine mAb (P-Tyr-100);

13. at room temperature, the jolting incubation is 60 minutes;

14. by the described method washing of the 11st step;

15., and in every hole, add 100 μ L dilution antibody with the anti-mouse IgG of 1% bovine serum albumin PBS-T damping fluid dilution in 1: 500 europium mark;

16. at room temperature, the jolting incubation is 30 minutes;

17. pat on paper handkerchief to remove remaining liquid with PBS-T damping fluid 200 μ L washing 5 times in every hole;

18. add 100 μ D in every hole Signal multiplication liquid;

19. at room temperature, the jolting incubation is 5 minutes;

20., read fluorescence intensity with suitable temporal resolution plate reader at the 615nm place.

Calculate inhibiting rate ratio: IR (%)=100-100* (X-B)/(N-B)

X=test-compound fluorescent value

The N=negative control

The B=blank

IC ₅₀Value can obtain by the inhibiting rate ratio calculation under the test-compound different concns degree of passing.

The activity of The compounds of this invention

The embodiment numbering	??IC ₅₀(EGFR/BIO)(μM)
The embodiment numbering	??IC ₅₀(EGFR/BIO)(μM)	??1	??0.11
??2	??0.03	??1	??0.11
??2	??0.03	??3	??0.54
??4	??0.07	??3	??0.54

The determination of activity of example 3:HER-2 kinase inhibitor

This test is measured external HER-2 kinase inhibitor activity with enzyme-linked immunosorbent assay (ELISA).

Material and reagent:

A. lavation buffer solution (PBS-T damping fluid): 1x PBS (137mM NaCl, 2.7mM KCl, 4.3mMNa ₂HPO ₄, 1.4mM KH ₂PO ₄, regulate pH7.2) and 0.05%Tween-20

B.1% bovine serum albumin (BSA, Calbiochem#136593) PBS-T damping fluid

C. seal damping fluid: 50mM EDTA, pH 8.0

The anti-mouse IgG of europium mark (PerkinElmer Life Sciences#AD0124)

Signal multiplication liquid (PerkinElmer Life Sciences#1244-105)

Streptavidi wraps the yellow plate (PerkinElmer Life Sciences#AAAND-0005) in 96 holes.

G.HER-2/ErbB2 kinases (Invitrogen corporation#PV3366).

H.10mM ATP solution (Cell signaling technology#9804).

I.FLT3 (Tyr589) biotinylation protein (Cell signaling technology#1305).

J.Phospho-tyrosine mouse mAb (P-Tyr-100) (Cell signaling technology#9411).

K.HTScan ^TMTyrosylprotein kinase damping fluid (4x)

1x kinase buffer liquid

60mM?HEPES

5mM?MgCl ₂

5mM?MnCl ₂

3μMNa ₃VO ₄

(Cell?signaling?technology#9805).

1.1.25M?DTT(1000x)(Cell?signaling?technology).

Scheme:

Use following scheme to test:

1. reach the ultimate density value with DMSO dilution test-compound;

In each test, add 1 μ L test-compound, negative control (not accepting any test-compound) and 1 μ LDMSO;

2. use dH ₂O dilutes 1: 16 μ M substrate protein (Tyr87), and adds 15 μ L in each test;

3. the HER-2 enzyme is directly transferred on ice from-80 ℃, the HER-2 enzymolysis freezes on ice;

4. get 4.5 μ g HER-2 enzymes in the enzyme pipe;

5. add 10 μ L DTT (1.25M) to 2.5mL 4x HTScan ^TMTyrosylprotein kinase damping fluid (240mM

HEPES pH 7.5,20mM MgCI ₂, 20mM MnCI ₂, 12 μ M Na ₃VO ₄) in, make DTT/ kinase buffer liquid;

6. transferase 10 .75mL DTT/ kinase buffer liquid makes the 4x reaction mixture in each enzyme pipe, and adds 7.5 μ L 4x reaction solutions in each test;

30 μ L react final test conditions:

60mM?HEPES?pH?7.5

5mM?MgCl ₂

5mM?MnCl ₂

3μMNa ₃VO ₄

1.25mM?DTT

20μMATP

1.5 μ M substrate protein

45ng HER-2 kinases

8. under 25 ℃, with reaction tubes incubation 60 minutes;

9. add 30 μ L sealing damping fluid (50mM EDTA, pH 8.0) termination reaction in each test;

10. in the every hole of 96 hole streptavidin bag culture plate, add 25 μ L reaction solutions and 75 μ L dH ₂O at room temperature, and followed jolting 60 minutes;

11. pat on paper handkerchief to remove excessive liquid with 200 μ L PBS-T damping fluids washing 3 times in every hole;

12., and in every hole, add the main antibody that 100 μ L dilute with 1% bovine serum albumin PBS-T damping fluid dilution in 1: 1000 main antibody Phospho-tyrosine mAb (P-Tyr-100);

13. under the room temperature, followed under the jolting incubation 60 minutes;

14. wash by described method of the 11st step;

15., and in each hole, add the antibody that 100 μ L dilute with the anti-mouse IgG of 1% bovine serum albumin PBS-T damping fluid dilution in 1: 500 europium mark;

16. under the room temperature, followed under the jolting incubation 30 minutes;

17. pat on paper handkerchief to remove excessive liquid with PBS-T damping fluid 200 μ L washing 5 times in every hole;

18. add 100 μ D in every hole

Signal multiplication liquid;

19. under the room temperature, followed under the jolting incubation 5 minutes;

20. under wavelength 615nm, read absorbancy with suitable temporal resolution plate reader.

Calculate inhibiting rate ratio: IR (%)=100-100* (X-B)/(N-B)

X=test-compound fluorescent value

The N=negative control

The B=blank

IC ₅₀Value can obtain by the inhibiting rate ratio calculation under the different tonsure concentration of test-compound.

The activity of The compounds of this invention

The biochemical activity of The compounds of this invention is measured by above test, the IC that records ₅₀Value sees the following form

The embodiment numbering	??IC ₅₀(HER ₂/BIO)(μM)
The embodiment numbering	??IC ₅₀(HER ₂/BIO)(μM)	??1	??0.002
??2	??0.011	??1	??0.002
??2	??0.011	??3	??0.012
??4	??0.003	??3	??0.012

Example 4:HER-2 suppresses the cell proliferation test

Following in vitro tests is to be used for measuring the cell strain inhibition proliferation activity of The compounds of this invention for human tumor cell SK-BR-3HER-2 high expression level.

Cell in vitro described below test can determine test-compound to the anti-angiogenesis activity of the tumour cell of highly expressing HER-2 and suppress proliferation activity, its active available IC ₅₀Value is represented.The general approach of this type of test is as follows: the human tumor cell who at first selects highly to express HER-2, to be seeded on 96 well culture plates under the suitable cell concn, then cell is cultivated in carbon dioxide incubator, when growing to 60%, they converge, change substratum for being added with the substratum of a series of concentration degree of passing (general 6 to 7 concentration) test-compound solution, culture plate is put back to incubator again, cultured continuously 96 hours.After 96 hours, available sulphonyl rhodamine B (SRB) method is carried out test compounds for suppressing cell-proliferation activity.IC ₅₀Value can be by under a series of different concns, and test-compound calculates for cell inhibiting numerical value.

Material and method:

A dimethyl sulfoxide (DMSO) (Sinophma chemical reagent company, catalogue T20050806 number)

B.SK-BR-3 cell (purchasing biology) in Institute of biochemistry and cell

E.Fisher transfer pipet (Fisher scientific, catalogue 03-692-164 number)

F.RPMI1640 cell culture medium (Gibco, catalogue 12400-021 number)

G. Australian foetal calf serum (Gibco, catalogue 10099-141 number)

H. phosphate buffered saline (PBS) (Gibco, catalogue 10010-072 number)

I.0.25% Regular Insulin-EDTA (Gibco, catalogue 25200-056 number)

J. sulphonyl rhodamine B (Sigma, catalogue 3520-42-1 number)

K. acetic acid (Sinophma chemical reagent company, catalogue T20060508 number)

M.Tris alkali (Amresco, No. 0826, catalogue)

O. serial II water-jacket typ CO2gas incubator (ThermoForma model: 3111)

P. whizzer (Fisher Scientific Marathon 8k, catalogue 0027-02 number)

Q.Novastar plate reader (BMG Labtech, catalogue 700-0081 number)

R. orbital shaker (Qilinbeier, catalogue TS-1 number)

Scheme:

Following scheme is used for testing test-compound of the present invention for SK-BR-3 cell inhibiting cell proliferation IC ₅₀Value.

1. the SK-BR-3 cell is grown in the healthy and free from worry culture plate of 100mm in growth substrate (is nutrient solution with the RPMI1640+10% foetal calf serum), cultivate (37 ℃, 5%CO ₂), fully converge until cell;

In the 100mm culture plate with foetal calf serum washing SK-BR-3 cell, behind the Tyrpsin peptic cell, again with cell inoculation on healthy and free from worry 96 porocyte culture plates, concentration is 50000cells/mL, each plate empty 6 holes, hole in contrast.;

3. at 37 ℃, 5%CO ₂Under the condition, cell is cultivated in 96 orifice plates, converged until reaching about 60%;

5. use the compound solution that is disposed above cell culture medium (the RPMI1640+10% foetal calf serum the is nutrient solution) dilution.20 times of each DMSO series concentration compound solution dilutions add 5 μ L DMSO compound solutions and 95 μ L nutrient solutions at every turn in cell culture medium, guarantee that the concentration of SK-BR-3 cellular exposure in DMSO solution is no more than 0.5%, uses vortex mixed;

6. when the SK-BR-3 cell attachment, growth reach 60% converge after, substratum is changed to the new substratum that is added with RPMI1640+10% foetal calf serum nutrient solution, every Kong Zhongzai adds 180 μ L nutrient solutions and 20 μ L prepared test-compound solution in the 5th step.The negative control cell group adds the 20 μ L nutrient solutions that contain 0.5%DMSO, and the ultimate density of SK-BR-3 cellular exposure in test-compound solution is 100 μ M like this, 10 μ M, 5 μ M, 1 μ M, 0.1 μ M, 0.01 μ M, and 0.001 μ M;

7. culture plate is put into thermostat container, at 37 ℃, 5%CO ₂Under the condition, cultured continuously 96 hours;

8.96 after hour, culture plate is transferred to aseptic working spaces from thermostat container;

10. under 4 ℃, cultivated 1 hour, after wash with water for several times to remove TCA, serum protein etc.Culture plate is stored stand-by at air drying.Blank is the numerical value that incubation is cultivated gained in the substratum that does not have the cell growth by the mensuration of scape optical density (OD) value;

12. painted 30 minutes of cell;

15. use spectrophotometry, under wavelength 565nm, read absorbance.Absorbancy numerical value is that absorbancy deducts under the 690nm 96 orifice plates by the numerical value of scape absorbancy gained under the 565nm;

16. make and calculate inhibiting rate ratio with the following method:

IR=100 * (control group absorbance-medication group absorbance)/control group absorbance %

The activity of The compounds of this invention

The embodiment numbering	??IC ₅₀(HER ₂/SK-BR-3)(μM)
The embodiment numbering	??IC ₅₀(HER ₂/SK-BR-3)(μM)	??1	??0.283
??2	??0.439	??1	??0.283
??2	??0.439	??3	??0.212
??4	??0.263	??3	??0.212

Pharmacokinetics is estimated

The pharmacokinetics test of test case 1 embodiment of the invention 1 compound

1, summary

With the rat is animal subject, uses the LC/MS/MS method and has measured rat and irritate stomach respectively and give the drug level in the different blood plasma constantly behind embodiment 1 compound.The research The compounds of this invention is estimated its characteristics of pharmacokinetics in the pharmacokinetics in rats behavior.

2, testing program

2.1 test drug

Embodiment 1 compound

2.2 experimental animal

16 of healthy adult SD rats, male and female half and half are available from west, Shanghai pul-Bi Kai laboratory animal company limited, animal production licence number: SCXK (Shanghai) 2003-0002.

2.3 medicine preparation

Take by weighing appropriate amount of drug, add tween 80 and grind evenly, the back adds 1% Xylo-Mucine and is ground to the even suspendible of sample, and the tween 80 final concentration is 1%, and sample concentration is 2.5mg/mL, faces the time spent preparation.

2.4 administration

16 of healthy adult SD rats, male and female half and half, difference gastric infusion behind the overnight fasting, dosage is 25.0mg/kg, administration volume 10mL/kg.

2.5 sample collecting

16 of SD rats, male and female half and half are divided into 4 groups, gastric infusion after one night of fasting, dosage is 25mg/kg.Before administration and after the administration 0.5,1.0,2.0,3.0,4.0,5.0,7.0,9.0, placed the heparinization test tube by eye socket blood sampling 0.2mL in 12.0,24.0,30.0 hours, centrifugal 10 minutes separated plasmas of 3500rpm, in 20 ℃ of preservations, feed in 2 hours after the administration.

3. analytical procedure

3.1 plant and instrument

TSQ Quantum UltraAM triple quadrupole bar mass spectrograph, U.S. Thermo Finnigan company;

Agilent 1200 highly effective liquid phase chromatographic systems, U.S. Agilent company.

3.2 plasma sample pre-treatment

Draw each rat plasma 50 μ L constantly behind the medicine, add inner mark solution 50 μ L, add methyl alcohol 100 μ L, vortex mixed is 3 minutes behind the mixing, and centrifugal 10 minutes (13500r/min) gets 5 μ L and carry out LC/MS/MS and analyze.

3.3 surpass the mensuration of upper limit of quantification sample

The sample that measured concentration is higher than upper limit of quantification is rechecked, and dilution step is as follows: get plasma sample 10 μ L, add 90 μ L rat blank plasmas, and the eddy current mixing, this diluted sample factor is 10, by " plasma sample pre-treatment " operation down.

3.4 typical curve preparation

With rat blank plasma 50 μ L, add standard serial solution 50 μ L respectively, making Plasma Concentration is 50.0,100,200,500,1000,2000, and 5000ng/mL adds inner mark solution 50 μ L, and methyl alcohol 50 μ L are by " plasma sample pre-treatment " operation down.With the Plasma Concentration is X-coordinate, and sample is ordinate zou with interior mark chromatographic peak area ratio, with weighted least-squares method (w=1/x ²) carry out linear regression.

3.5 pharmacokinetic parameter calculates

The compartment model match is carried out in pharmacokinetics behavior to test-compound, and calculates main pharmacokinetic parameter, wherein C _Max, t _MaxAdopt measured value.

4, pharmacokinetic parameter result

The pharmacokinetic parameter of The compounds of this invention is as follows:

Claims

1. the compound shown in the general formula (I) or its pharmacy acceptable salt:

Wherein:

R ₂Be selected from alkyl;

R ₃And R ₄Be selected from alkyl separately, perhaps

2. compound according to claim 1 or its pharmacy acceptable salt, wherein this compound is selected from:

3. compound according to claim 1 and 2 or its pharmacy acceptable salt, wherein said salt are described compound and the salt that is selected from following acid formation: oxysuccinic acid, lactic acid, toxilic acid, hydrochloric acid, methylsulfonic acid, sulfuric acid, phosphoric acid, citric acid, tartrate, acetate or trifluoroacetic acid.

4. medicinal compositions, described pharmaceutical composition contain the treatment effective dose according to any one described compound or its pharmacy acceptable salt in the claim 1～3, and pharmaceutically acceptable carrier.

5. method for preparing general formula according to claim 1 (I) compound said method comprising the steps of:

Iodo quinazoline compound and 1-(tri isopropyl silane base)-1H-pyrroles-3-ylboronic acid carries out the Suzuki coupling, the compound 1 that obtains under alkaline condition with halogenated compound R ₁X carries out substitution reaction, or under the sodium hydride condition, with the reaction of racemic epoxide chloropropane more further with NHR ₃R ₄Carry out ring-opening reaction, obtain general formula (I) compound.

6. method for preparing general formula according to claim 1 (I) compound said method comprising the steps of:

7. method of regulating the protein kinase catalytic activity is comprising any one described compound in described protein kinase and the claim 1～3 or pharmacy acceptable salt are contacted.

8. method according to claim 7, wherein said protein kinase is selected from receptor tyrosine kinase, nonreceptor tyrosine kinase or serine-threonine kinase.

9. according to each described compound of claim 1～3 or its pharmacy acceptable salt purposes in the medicine of preparation treatment and protein kinase diseases associated.

10. purposes according to claim 9, wherein said protein kinase is selected from receptor tyrosine kinase, nonreceptor tyrosine kinase or serine-threonine kinase.

11. the purposes of composition according to claim 4 in the medicine of preparation treatment and protein kinase diseases associated.

12. purposes according to claim 11, wherein said protein kinase is selected from receptor tyrosine kinase, nonreceptor tyrosine kinase or serine-threonine kinase.