CN104761544A

CN104761544A - Selectivity inhibitor of EGFR tyrosine kinase clinic important mutant

Info

Publication number: CN104761544A
Application number: CN201410444568.2A
Authority: CN
Inventors: 宋运涛; A.J.布里奇
Original assignee: Safe Medicine Co Science And Technology Ltd Of Nanjing Bohr
Current assignee: Jilin Huikang Pharmaceutical Co., Ltd.
Priority date: 2014-01-03
Filing date: 2014-09-03
Publication date: 2015-07-08
Anticipated expiration: 2034-09-03
Also published as: CN104761585A; CN104761585B; CN104761544B

Abstract

The invention relates to a selectivity inhibitor of an EGFR tyrosine kinase clinic important mutant. The invention relates to formular A compound and medical salt and comprises medical composition having the compound and the salt, as well as a purpose.Kinase can be prevented by the compound and the salt, especially epidermal growth factor receptor (EGFR,erbB-2,erbB-4) and kinase clinica important gatekeeper 'mutant'; and abnormal cell profilerative diseases such as cancers can be treated or improved.

Description

The selective depressant of the clinical important mutant of EGFR Tyrosylprotein kinase

background of invention

1. invention field

The present invention relates to compound and their pharmacologically acceptable salt of formula I-III, relate to the pharmaceutical composition comprising this compounds and salt, and relate to and uses thereof.Compound of the present invention and salt suppress suppressing kinases important in the resistance development of therapy for treating to EGFR, especially EGF-R ELISA EGFR, particularly its mutant, and can be used for treatment or improve aberrant cell proliferation illness, as cancer.

2. background information

The present invention relates to the biaryl aminocompound of the inhibitor that can be used as protein kinase PKs.PKs is very important intracellular signaling entity in communication in cell, the hydroxyl in the amino acid side chain that they to be transferred to protein from the ATP serving as phosphodonor (phosphodonor) by catalytic phosphatase group at this and modify numerous protein.Phosphate group is transferred to Serine on protein or Threonine hydroxyl by PKs usually, and they are serine/threonine kinase (S/TKs) in this case.On the phenolic hydroxyl that phosphoric acid is transferred to the tyrosine side chain on target protein by next modal kinases type and be protein tyrosine kinase (PTKs).Usually, Tyrosylprotein kinase is incorporated in the intracellular domain of great transmembrane protein, and this transmembrane protein has cognate ligand binding domains in ectodomain, and ligand binding activates Tyrosylprotein kinase thus.This quasi-molecule is receptor tyrosine kinase (RTKs).The hydroxyl that a more rare class target and various lipid are associated as phosphatidylinositols and sphingosine is also referred to as lipid kinase.Some of them are structurally closely related with some PKs, and due to this and the activity often needed in the approach identical with PKs due to lipid kinase, they consider usually together with PKs.The most rare classification is can by the dual-specificity kinase (DSKs) of serine/threonine and tyrosine hydroxyl all phosphorylation.Generally speaking, in cell normal amount PK classification none seem can by the substrate phosphorylation of other kind, although specific certain loss can be brought out in laboratory conditions, in some tumours same so, wherein express PK mutant forms or wherein may a large amount of process LAN PK.Structurally, kinases is understood quite fully.There is kinase domain, it can be whole protein or be only the sub-fraction of much bigger modularization albumen, this structural domain has the basic conserved structure of about 35 kD, be made up of two leaves (lobe), N-terminal structural domain is formed primarily of beta sheet, and larger C-terminal structural domain is formed primarily of alpha-helix.The drastic crack gap in conjunction with ATP and substrate is there is between these two leaves.Binding Capacity territory is quite large and quite variable, and for distinguishing the specificity of different protein substrates and maintenance phosphorylation.This species specificity may be very variable, and some enzymes are a kind of known substrate as MEK only has, and other enzymes can by the hundreds of different di in protein.

Phosphorylation can change the conformation of protein, usually enzyme is changed into activity form from inactive form, or vice versa, or protein and binding partners are associated closely, to cause the assembling of cellular localization or function multiprotein complex or the change in decomposing.Many transduttants that signal neutralizes in from cell surface to core to cell are PKs or control by PKs.Therefore, the inhibitor of the kinase activity of PKs has the impact of highly significant on cell signaling, to decay to the normal response of external signal and the unsuitable excessive response that usually caused by the sudden change of signal transduction molecule itself.The path of even now distributes widely and the disease participating in most of body function in such or such mode and may be caused by their dysfunction in vivo, the inhibitor of PKs is particularly useful for Therapeutic cancer and immune disorders, all extensively confirms the overactivity of PKs and they play a key effect usually in driving lysis itself in these two kinds of kinds of Diseases.

Many different types of kinase inhibitor are developed, some successful granted and listings.Seem to suppress one of many kinase whose molecular scaffold to be a series of threefold rings effectively, wherein two, usually all three rings are aromatics, and it may form U-shaped structure when being attached on kinases.Two distal loops are directly connected on center ring by key or by the various connection base be made up of 1-3 atomchain.Center ring---it is almost nitrogenous heteroaromatic system all the time---forms the hydrogen bond on the backbone residue in the kinases hinge area between 1-3 to N-and the C-end leaf just before so-called DFG ring (the not structure changes in kinases, it must correctly locate the activity conformation that could realize this enzyme).This end of this inhibitor also occupies a part for kinase whose VITAMIN B4 land (it is often very hydrophobic), and two rings forming " dry (stems) " of this U occupy the fat pipe of a part of often filling the space generally occupied by the remainder of ATP molecule.Although considerable, unique structural determinant that is expected to that specific kinase whose affinity comes from being conducive to target kinase is interacted and/or is unfavorable for decorating these center rings with substituting group selected by the kinase interactions of not wishing to suppress, but various torsion between these three rings and bending angle are come to various kinase whose many affinities and selectivity, and the direct and protein interaction of some substituting groups possibilities itself optimized the affinity of target kinase, but these three rings the most stable conformation relative to each other may be controlled.Therefore some substituent effects are the whole interior energy affecting this Inhibitory molecules, to stablize the conformation and non-immediate and kinase interactions that are conducive to combining.

Kinases has been shown to be the very important effector of numerous disease process, especially in cancer.Control cell proliferation by kinases with many different levelss and under the home of cell proliferation, must send signal from extracellular, at this, they to be attached on acceptor and activated receptor.Many important acceptor in cell signaling is kinases, especially RTKs, or is directly coupled on kinases---this kinases itself is activated by activated receptor.Once these kinases are activated, they are activation signal transduction cascade again, and this is usually directed to other kinases some in the amplification ripple of phosphorylation, and this finally causes the activation of the transcription factor in core.The activation of transcription factor causes producing the protein performing this intracellular various program (comprising those programs making this cell start to enter proliferating cycle).Usually, once this process continues a few hours, the protein of new synthesis can continue this process, and does not need further extracellular to input.If proliferative cell cycle starts, the first histone matter of synthesis comprises further transcription factor and drives their activator of the later phases of cell cycle and active cell to copy the effector with fission process.Kinases is the major control person of each step in this process.When this process do not have suitably controlled and cell performs the cell cycle when not having suitable external control time, and if they are converted immunity system do not eliminate them, then may form tumour.

When checking transformant, one of feature that they are constant is Hyperphosphorylationof, shows that too many kinases activates inadequately.This may be caused by sudden change extremely diversified in cell.Such as by cell produce inadequately its oneself acceptor connected to the part of one of kinases.Or owing to suitably not controlling its expression or the multiple additional copies due to the gene in cell, one of these kinases may serious process LAN.Another very common genetic flaw is the sudden change in kinases coding region, and this causes in formation, being activity and not needing proper signal to activate its kinases.Sometimes this kinases is not inadequately in active, but makes Phosphoric acid esterase (this is considered to limit its intracellular signaling by removing phosphoric acid from target molecule) inactivation by suddenling change or deleting.Cell cultures tumour and all almost always find this kind of defect in the phosphorylation of tumour cell from the inspection of the isolate of clinical tumor.

In the later stage eighties 20th century, find several small molecule kinase inhibitors.These molecules to be almost always combined in kinase whose catalysis crack and to compete its binding site with ATP.Therefore they are that ATP is emulative, and the most of inhibitor found since then belong to this class.But, found once in a while to compete with protein substrate, the kinase inhibitor of substrate competitive or the kinase inhibitor (double inhibitor) of more generally all competing with ATP and substrate or neither with Receptor Competition, also not with the kinase inhibitor (noncompetitive inhibitor) of substrate competition.After taking into account Premeabilisation of cells difference, find these compounds be separated kinase inhibition detect in effect and the kinase inhibition in cell between there is very good dependency.For many kinases, between the phosphorylation loss and the suppression of cell proliferation of downstream target, also there is excellent dependency.Owing to showing this dependency thousands of times with tens kinds of different kinases, clearly confirm that abnormal kinase signal transduction can cause the not controlled propagation of transformant, in many cases, block overactive kinases and can stop this propagation.In many cases, in fact kinase inhibitor can bring out alone the apoptosis of transformant, so that tumor regression.This may be owing to usually activating several short Apoptosis mechanism and Abnormal Phosphorylation may fully participate in apoptosis inhibit process and occur in tumour cell.Although these abilities in cell can prevent the good proof as the tumour of xenograft growth in nude mice late at the beginning, but along with reagent improves, conventionally confirm that kinase inhibitor can slow down the growth of tumour of the kinases oncogene expressing institute's target, better reagent causes tumor size usually to degenerate to immeasurablel degree, and the little regrowth of tumour after stopping administration, shows the tumour may curing animal.In addition, after exposing with tumour and being associated, in body, effect is associated with cytoactive and enzymic activity.

Clinical proof is come slower, may partly because clinical tumor be usually much complicated than the tumour grown under the careful condition controlled, part is because mouse is how strongly than people and can tolerate larger medicine relative dosage in biological chemistry, and main because be usually difficult to know which is the kinases being adapted at suppressing in any given tumour.But, imatinib---there is the quite potent inhibitor of the carcinogenic TK BCR-ABL of the fusion of really outstanding pharmacokinetic property, be approved for chronic myelocytic leukemia (CML) in 2000.This kinase inhibitor provides very compellent learning concept clinical proof, because the CML patient of about 2/3 (its tumour is almost by one of definition BCR-ABL containing two kinds of forms) responds treatment and leukemia cell's almost completely dissolve from circulation usually admirably.Surprisingly, this sudden change blocked around seems slowly, even treatment after 10 years this medicine still effective in 80% patient.This not yet confirms it is generalized case, may part because most of tumour to find more late than CMLs in their biohistory many and have the much longer time to become allelic heterogeneity, and part is because few tumour depends on a kind of oncogene as CML depends on BCR-ABL.

Urogastron RTK(EGFR, erbB-1) two kinds of inhibitor Gefitinib and erlotinib before about 10 years, be approved for lung cancer.EGFR is one of kinases of the most often lacking of proper care seen in solid tumor, usually in the tumor type of 50% or more, finds process LAN or sudden change, comprises nonsmall-cell lung cancer (NSCLC).Although the heterograft of these inhibitor to varied process LAN EGFR has excellent activity, but in NSCLC, observe very limited activity, the patient of about 10% is only had to respond this medicine and average response only continues about 1 year, although find much lasting respondent once in a while.When carrying out the careful inspection of respondent, find that most of good response person has one of several sudden changes in EGFR, the respondent containing wild-type (wt) EGFR does not have good response usually.When these mutant of analysis, especially during EGFR L858R and EGFR del746-750, finding that they all have the character of intrinsic activation, this means that they drive propagation when not having external signal, also than wt EGFR weaker in conjunction with the higher K of ATP( _m), there is the affinity to this inhibitor similar with wt EGFR simultaneously.This means, because these inhibitor are that ATP is emulative, from enzyme, compete ATP than easier in wt and cut off the kinase activity in susceptible mutant, promote the inhibitor effectiveness in mutant with reality.Meanwhile, the propagation of these tumours and the most of tumour of survival ratio more depend on EGFR intracellular signaling because since original mutation event signal reliably overacfivity.

As mentioned above, if lung cancer is to rather late usual when finding, may there is rear on average 6-12 at the initial initiator cell (founder cell) transformed in solid tumor.One of character of transformant is that they lose the control of the DNA replication dna quality control to them, and therefore their spontaneous mutation rate is more much higher than unconverted cell.Due to sudden change the easiest in DNA replication dna process occur and these cellular replications obtain quickly, this increases mutation rate further.Result is, along with tumour is aging, it obtains ever-increasing sudden change number, and this occurs in a random basis, so that in time through tumorigenic subclone---there is the genetics slightly different and slightly different each other with primary tumor.These subclones not only participate in the struggle for existence with health itself, also mutual struggle for existence, because compete their obtainable limited resources between them.If change the environment of advantage tumor colonies, becoming to make it relatively so do not shake down (such as by adding its effective inhibitor), if before much unsuccessful that secondary clone does not affect by described inhibitor, then can take over the ecological niche vacated.Or, if thoroughly do not kill this clone or cut off propagation completely, it can continue to produce sudden change, and if this suppression is avoided in sudden change, this subclone can freely be bred now, and the obstruction of the parental clone of not suppressed preparation or suppression (parental clone).Therefore natural selection prediction, cancer, just as transmissible disease, should produce resistance, and greatly drives this chosen process due to the competition between the tumour subclone in single host, whole structure promotes to have more aggressive subclone, and tumour becomes more fatal with its evolution usually.

When following the trail of the respondent to Gefitinib and erlotinib, find that the generation of resistance is associated from several different gene variation.When extremely rare, tumour seems to adopt diverse signal transducting system to drive tumour, but resistance is usually directed to the adjustment (tweaking) of primal system.The member of the erbB subfamily of RTKs is formed together with EGFR and erbB-2, erbB-3 with erbB-4.These acceptors are initiated the ligand activation of their dimerization, although EGFR-EGFR homodimer is quite usually used in intracellular signaling, but the more common process in this family makes part cause Heterodimerization, so that intracellular signaling entity is such as EGFR:erbB-2 or erb-B2:erbB-3 and suitable part.The simplest mode reactivating this system is the expression improving one of other erbBs, and this is also often visible even before the treatment, and the tumour contributing to being interpreted as what many process LAN wt EGFR does not respond EGFR suppression.Rather relevant mechanism relates to RTK HGFR, although it is not erbB family member, when being presented at process LAN and the carcinogenic heterodimer of erbB-3 height of formation, and the process LAN of HGFR is the common mechanism of the resistance to EGFR inhibitor.At least in laboratory is arranged, HGFR inhibitor is added in these cells to the susceptibility recovered EGFR inhibitor.The third and modal resistance pattern are the further sudden changes of EGFR, and this reduces its susceptibility to EGFR inhibitor.Modal in these is so-called " gatekeeper " mutation T 790M, and normal discovery has the NSCLCs of double mutant (as L858R T790M) in the initial response person subsequently EGFR inhibitor being produced to resistance.This type of subclone is that to exist all the time or only occur after the treatment be unknown, but seems that this sudden change most probable has existed and occurred as fresh mutation in the long-term respondent producing resistance after a while in short term response person.

Believe at first, these sudden changes spatially hinder inhibitor to be attached on mutant enzyme, therefore reduce their affinity and effect.But, nearer research shows, modal sudden change has extremely low impact to inhibitor affinity, but causes ATP to the binding affinity recovery or possible high to 10 times of wt EGFR, and the attainable inhibitor concentration of result is no longer high enough to shutoff signal conduction in treatment degree of functioning.In principle, only need the affinity fully improving inhibitor to overcome the ATP affinity of raising, but be difficult in practice do like this, because Gefitinib and erlotinib are the EGFR inhibitor with good PK character of very potent, secondary nmole, but active is only had to the tumour that wt EGFR drives.In addition, although T790M mutant does not reduce the affinity of EGFR to erlotinib and Gefitinib, its really restriction can improve the approach of affinity in the anilinoquinazoline chemotype of these two kinds of inhibitor.Therefore, in order to find, to the larger affinity of T790M type mutant, to have checked new chemical template, and some, the U-shaped inhibitor of type especially discussed above seems quite promising in this field.

EGFR acceptor plays a significant role at whole body, especially in whole GI epithelium and skin (being all tissue very active in propagation).Two kinds of major dose-limiting toxicity due to EGFR inhibitor are that fash and serious GI are disorderly, and these are almost certainly to a great extent based on (the mechanistically based) of mechanism.As long as tumour is driven by wt EGFR, this is just difficult to be avoided by appropriate design, especially oral disposition agent, wherein gi tract expose and are inevitable, if but tumour is driven by the EGFR that suddenlys change, then can alleviate the toxicity that disclosed clinical material standed for is observed, described material standed for all in anilinoquinazoline template and undoubtedly to 790 wt(T) mutant receptors (they are the M in position 790) of this family of EGFR comparison is obviously more potent.When treating the tumour having become and depended on the driving of T790M mutant, this does not reach optimal mode undoubtedly.Can suppose that several inhibitor pattern is more suitable for treating the tumour that this resistance process occurs in expection, EGFR inhibitor has the affinity to T790M mutant of raising thus, and all in the literature, some are nowadays in clinical trial for the example of all these.This patent application describes the compound meeting one of these standards.

The EGFR inhibitor being significantly higher than wt EGFR to the affinity of sudden change EGFR should be able to suppress the propagation in the tumour driven by this mutant under optimal dose, has low (if any) impact of antipode to the EGFR intracellular signaling in unconverted tissue (wherein wt EGFR is responsible for EGFR intracellular signaling) simultaneously.This should allow the mutant selectivity EGFR inhibitor giving obvious larger dose, to improve the effect of tumour and therapeutic index that drive mutant.It should be noted that, due to the impact that mutant combines ATP, this is situation about having occurred in erlotinib and Gefitinib respondent substantially, and the mutant wherein mainly due to response reduces the affinity of competitive part ATP, and they are in fact more responsive to inhibitor than wt EGFR.Disclosed several third generation EGFR inhibitor now, some of them are in clinical.These compounds normally irreversible inhibitor, it is at first based on U-shaped pentanoic yl pyrimidines support, but this has extended to several associated supports, but is all attached on pentanoic yl pyrimidines with icotype.Generally speaking, these compounds are the very potent inhibitor of sudden change EGFRs containing T790M sudden change, and to wt EGFR and some other sudden changes so not potent.Due to this pattern, believe (mechanism-based) toxicity based on mechanism that significantly can reduce wt EGFR and suppress, keep the extremely strong suppression effect to the tumour driven of being suddenlyd change by suitable EGFR simultaneously.Therefore such compound especially can be used as two roentgenism ies after becoming resistance to the patient of a line erlotinib or Gefitinib therapy sensitivity before.These inhibitor can not only suppress suitable mutant receptors as before by force, also should realize this point while itself can not be suppressed to cause significant machine-processed (mechanism-induced) toxicity of inducing by EGFR.Large cyclammonium inhibitor of the present invention is irreversible EFGR inhibitor, have and similar with these reagent mutant EGFR is suppressed to be better than selective mode that wt EGFR suppresses and excellent pharmacokinetic property, therefore confirm it is for NSCLC and the excellent reagent of the second line treatment of other tumour any that driven by the kinase whose this subfamily of sudden change EGFR.

The another kind of method improving inhibitor effectiveness is developed in the mid-90 in 20th century.Recognize that a part of TKs utilizes the cysteine residues on ATP engagement groove edge to form the hydrogen bond with the ribose of ATP, and major part uses Threonine for this reason.This halfcystine (the C in EGFR is all contained in EGFR family ⁷⁹⁷).Infer that this halfcystine may be connected the allcylating moiety alkylation on inhibitor, described inhibitor to be combined in ATP-binding site and to provide electrophile near hcy thiolactone.For making this concept effective, allcylating moiety must have low inherent reactivity, because do not wish that viewed from potential PK and toxicity reason it does not react with a large amount of nucleophile in body with making any distinction between.For making alkylating agent with highly selective and this inevitable quite weak electrophile reaction, according to the show, this compound itself must have height (non-covalent) affinity to binding site and must preferentially to make this weak electrophile near the combination of electrophile conformation.Finally, also find, this reaction needed was carried out fast relative to the plasma half-life of inhibitor, otherwise it did not react will wash out in body with the halfcystine of key.Find so irreversible Inhibitor, and find that they are not only EGFR inhibitor in much more potent than the reversible inhibitor of equivalence in theory body, as added advantage, erbB-2 and erbB-4 of difference that they also make (at least when anilinoquinazoline and relevant 3-cyano quinolines) suppresses template to become the very potent inhibitor of all erbBs, if prove that binding pattern is really good, may not be so most important to the high non-covalent affinity of target.Enter the irreversible inhibitor that clinical most of s-generation EGFR inhibitor are EGFR, use acrylamide derivative as electrophile, and seem general clinical middle more effective than reversible inhibitor.Clinical trial is tended to that what is paid close attention to and is regarded as the simplest registration policy (registration strategy), its receive Gefitinib/erlotinib respondent and formation resistance after second line treatment they.Owing to being frequently observed response in this set, these compounds obviously can overcome the T790M resistance of many patients, and it is not necessarily so, because great majority were designed before this catastrophe of discovery.But, this clinical strategy also means, not yet answer about irreversible inhibitor whether by wt EGFR(and may wt erbB-2/4) there is in the tumour that drives of process LAN the problem noticeable equally of useful clinical activity, but consider the similarity of ATP affinity between wt EGFR and double mutant, seem really likely like this.

WO 2013/014448 discloses a series of 4-(bicyclic aryl)-2-(the 3-acrylamido anilino) pyrimidine as potent and mutant optionally irreversible EGFR inhibitor, and they have low affinity to wt acceptor usually.This compound often has the quite similar effect being low to moderate time nanomolar range to EGFRdel746-750 and L858RT790M double mutant, although in fact the former is susceptibility mutations body, the latter is the mutant of the inhibitor of resistance to anilinoquinazoline.Poor efficiency 40 to >100 is tended to doubly to the activity of wt EGFT.Early clinic data presentation has the responsiveness (actual shrinkage) in more than half patients of T790M sudden change in its NSCLSs, observes very slight fash in many patients, shows DLTs no longer by the control that wt EGFR suppresses.

As discussed above, the activity of the allcylating moiety (such as michael acceptor) in irreversible inhibitor is verified extremely important.If it is too strong to the reactivity of nucleophile, then seldom arrive expection target, and the macromole that may produce a large amount of electrophilic modification is to cause extra risk of toxicity.In addition, plasma half-life is extremely short, and this throws into question in many tumours.The tumour driven by the oncogene had enough to meet the need slowly by be exposed to short pulse irreversible inhibitor under and long-term Inhibit proliferaton intracellular signaling because this oncogene permanent deactivation subsequently.But if tumour constantly produces new carcinogenic protein, plasma drug level rapidly drops to below effective concentration, and no longer suppress the acceptor of new synthesis, until this signal transduction pathway of next administration inhibitor just activity recovery.Even if tumour does not have enough to meet the need oncogene fast, this pulse administration also provides strong selective pressure to remove the control generated oncogene as obvious resistance approach.

If check the acrylamide core of irreversible inhibitor, several factor can be used for affecting its reactivity as michael acceptor; More be far more than and use most of allcylating moiety, because michael reaction is intrinsic reversible.They control the electron density of alkene usually, and the double bond of electron deficiency is more conducive to michael reaction in kinetics and thermodynamics.But the polarity degree of olefinic double bonds is also important, more important than the low electron density on α carbon compared with low electron density on β-carbon.Finally, this double bond is stablized relative to Michael-adduct by space crowded (steric crowding), because Michael addition makes the ligancy of two plane triangles (or being line style when alkynes) atom improve 1, and becomes tetrahedron (plane triangle).

The electron density of Michael's system depends in part on the character of carbonyl.Although carbonyl is acid amides all the time in acrylamide, its ability to whole system donates electrons can be affected with the group of this amide nitrogen bonding, and therefore affect the overall electron density of this michael acceptor π system quite significantly.In WO 2013/014448, described most of acrylamides are had two and are replaced to the benzyl ring of the substituting group (ether and tertiary amine) of electronics and a substituting group closer to neutrality (2-aminopyrimidine) by force, but with it is almost attainable to pass through to control acrylamide nitrogen-atoms, this substitute mode may make this π system to Michael addition inactivation generally.Extreme at another, 6-acrylamido Pyridopyrimidine has been described to irreversible EGFR inhibitor, but with regard to simple acrylamide, chemical reactivity is really too strong so that cannot treat effectively.By providing intramolecular hydrogen bond to body, also can indirectly control carbonyl itself.This hydrogen bond has the population effect one of lone electron pair of ketonic oxygen and normal phase ratio being pulled away from slightly Sauerstoffatom, so that a little more electron deficiency and the polarization of whole π system.But although this strategy is effective, it relates to the loss building and be enough to chemical scaffold (chemical scaffold) and the conformational flexibility allowing hydrogen bond to be formed, because the formation of ring-type hydrogen bond relates to serious space constraint all the time.If this implements the conformation being more conducive to goal response on molecule, this is more important more than electronic effect, if but it implements disadvantageous conformation, then may hinder the generation of goal response completely.

The reactive modal mode affecting michael acceptor arranges functional group on olefinic carbon.Usually, at alpha position, unique significant effect is electro---electron-withdrawing group makes this π system more electron deficiency improve reactivity, and increases π-electron density to electron substituent group and make the reactivity of this system to Michael addition lower.Although the character of electron-withdrawing group removes from β carbon integral polarity and its overall electron density that therefore larger electron density also changes this system by inductive effect in theory from alpha-carbon or by mesomeric effect, but seem in practice to have no difference and the group of all scarce α-electronics all tends to activate michael acceptor by force, make michael acceptor inactivation to the substituting group of α-electronics.At β-carbon place, for electronics and space reasons, substituting effect is often more complicated.For acrylamide, β atom must hold the volume of michael acceptor, if it has had the substituting group being greater than hydrogen, this can cause the initial non-existent space constraint in adducts.Many substituting groups, such as alkyl, can improve the electron density of whole π-system and improve its thermodynamic stability, the michael reaction and these effects can slow down to reduce the overall thermal mechanics of this reaction profitable, so that it more easily occurs against michael reaction.The electron-withdrawing group of β position accelerates michael reaction by them to the impact of the overall electron density of π-system usually, but the character of their electron attraction also becomes important now.Simple inducibility electron-withdrawing group is (as halogen or CF ₃) the further electron density reducing β atom of meeting, to make π-system more polarity, but intermediary electron-withdrawing group (as carbonyl) can draw more polyelectrons density from alpha position, so that olefinic double bonds is electron deficiency and more hypopolarization more, and with do not have compared with substituting group completely, fully can improve the thermodynamic driving force of this reaction, slow down integral power simultaneously.

When carrying out simple enzymatic determination with irreversible inhibitor, unlikely truly suppressed constant.If this compound has good non-covalent affinity, the target response of irreversible warhead only with it on enzyme and this enzyme are pure with 100% activity, and the overall thermal mechanics of Cheng Jian has the Δ G being greater than about 3 kcals, then IC ₅₀should be the half of active enzyme concentration in theory, but the IC recorded in practice ₅₀usually much higher.This may owing to the kinetics of alkylated reaction slowly, but situation is not like this usually, and regardless of the speed of alkylated reaction, IC ₅₀more much bigger than enzyme concn.

As the situation of most pharmaceutical agents, the cellular potency of irreversible EGFR inhibitor is often less than their separation enzymatic determination effect.Medicine must permeation cell film and not pumped by resistance protein.Have other potential targets many may detain (sequester) medicine invalidly in cell, and it may stand the metabolism in cell.For irreversible inhibitor, the quantity of possible emulative good nucleophile greatly improves in raji cell assay Raji, mean that indiscriminate michael acceptor has many potential targets to supply reaction now, and along with michael acceptor reactivity improves, only due to this reason, cellular potency just reduces all the time.

In whole animal detects, situation becomes obviously more complicated.Be not added in static system by inhibitor, but add in dynamic system, wherein many processes are carried out simultaneously.Even for the medicine (wherein mainly ignoring the Absorption Characteristics of medicine) of administered parenterally, this medicine fully must be distributed in target tissue and also stop long enough around to have useful effect.The recycle system be combined with excretion and metabolic process means, the medicine being even delivered to target also tends to only of short duration existence.For quick response inhibitor, short-time contact target may be enough to make it irreversibly suppress target, but for the inhibitor of deferred reaction, may be too short so that reaction cannot be made to proceed to completely on most of target molecule with the contact of target, slow alkylation inhibitor may show be very similar to (usually unoptimizable) reversible inhibitor.Therefore, start to realize, the michael acceptor only preparing pole low reactivity is not enough to prepare acceptable medicament.But, as discussed above, most of real tumour often at a good pace has enough to meet the need their oncogene, as sudden change and/or the EGFR protein of process LAN, therefore the michael acceptor that the reactivity of Rapid Alkylation Under target protein is stronger needs to have sizable transformation period, to make the major part between administration or whole period, there is sufficient inhibitor around with the acceptor alkylation of will newly synthesize.In practice, accomplish that the long-time plasma exposure needed for this point can only be realized by the compound providing the inherent reactivity of michael acceptor low, so that its major part keeps many hours of circulation, and the many nucleophiles do not touched in vivo with it (comprise a large amount of circulations and glutathion inside cell, one of its main application catches electrophilic xenobiotics (xenochemicals)) irreversibly react.

At J Med Chem 56, the intrinsic gsh describing a series of irreversible EGFR inhibitor in 7025 (2013) is reactive.Under a set condition of standard, describe 23->10, the transformation period of the gsh addition of 000 minute, and describe the useful range of 25-400 minute.Because Ah method replaces the t1/2 of Buddhist nun in human body to be 37 hours (Clin Pharmacokinet 52,1101 (2013)), how card replaces Buddhist nun to be about 3.5 hours (Clin Cancer Res, 12,4645 (2006)), but they have the transformation period of 25 and 23 minutes respectively in gsh measures, play a role at this without being suspected to have other metabolic factor.Due to dacomitinib---it replaces Buddhist nun the same for Buddhist nun with Ka Nai as Ah method is 4-anilino-7-alkoxyl group quinazoline-6-acrylamide derivative, but as Ah method for there is amino crotonamide side chain Buddhist nun but not card how for the non-substituted acrylamide of Buddhist nun---there is the plasma half-life of about 50 hours in human body and be 49 hours in this mensuration, this effectively shows that removing terminal acrylic acid amides can provide the advantage of the PK in human body, and the speed of this and michael reaction does not matter.But it is noted that the aniline of the very electron rich being different from WO 2013/014448 compound, these inhibitor acrylamide nitrogen is separately bonded on the 7-alkoxyl group-4-anilinoquinazoline of appropriate electron deficiency.This application does not propose any alkylation side chain longer than acryl, is very likely that this is at J Med Chem 56 because the intrinsic michael acceptor character of this compounds is too weak so that invalid, emphasize in 7025 (2013) a bit.But, as just discussed above, this compounds estimates to have the pharmacokinetics advantage being better than corresponding acrylamide, and the anilino-pyrimidine template can discussed in WO 2013/014448 when suitably regulating their electron density produces irreversible inhibitor.Object of the present disclosure be disclosed in those similar anilino-pyrimidines of disclosing in WO 2013/014448 on there is the compound of crotonamide (or larger) side chain of prolongation, the electron density wherein systematically reducing michael acceptor is enough to provide the treatment of the irreversible suppression of selectivity and the reactivity of pharmacodynamics advantage of T790M sudden change EGFR inhibitor with maintenance, keep the pharmacokinetic property with the excellence not having terminal double link to be associated on michael acceptor simultaneously.

summary of the invention

Part of the present invention provides alternative and regulates the protein kinase causing the resistance existing EGFR-base being suppressed to therapy, especially the novel cpd of the protein kinase, the most particularly activity of some mutant form of EGFR acceptor of I receptor Tyrosylprotein kinase (RTK) family or erbB family and pharmacologically acceptable salt thereof.This inhibit activities affects biological function, includes but not limited to cell proliferation and cell invasion, suppresses transfer, inducing apoptosis or inhibiting angiogenesis.Additionally provide pharmaceutical composition and medicine, described pharmaceutical composition and medicine only comprise compound of the present invention or salt or comprise the compound of the present invention or salt that combine with other therapeutical agent or negative catalyst.

The present invention relates to the compound of formula A

Or its pharmacologically acceptable salt, wherein

X ¹cH or N;

Y is or

R ¹be selected from hydrogen, fluorine, chlorine, methyl, CF ₃, CHF ₂and cyano group;

R ²be selected from methoxyl group, oxyethyl group, isopropoxy, ring propoxy-, methyl, ethyl, sec.-propyl and cyclopropyl;

R ³be selected from H, halogen, C _1-6alkyl, C _1-6haloalkyl, C _2-6halo alkynyl, optionally by R ⁷and C _1-6the C that alkyl replaces _2-6alkynyl, R ⁴n-C _2-6alkyl-NR ⁴r ⁴, R ⁷;

R ⁴be H, C independently _1-6alkyl, C _2-6hydroxyalkyl, C _1-6alkoxyalkyl or C _2-6alkyl NR ⁸r ⁹, or two R ⁴the carbon atom that group is connected with them forms 3-12 unit's monocycle or bicyclic ring systems, and wherein maximum two carbon atoms are by N, NR ⁴, O, S (O) _xwith S (O) (NR ¹⁴) substitute;

R ⁵h, F, Cl, CF ₃, CHF ₂, CF ₂c _1-6alkyl, CF ₂cH ₂nR ⁸r ⁹, CH ₂nR ⁸r ⁹or C _1-6alkyl;

R ^6eand R ^6zbe H, F, Cl, CF independently ₃, CHF ₂, C _1-6alkyl, aryl, heteroaryl, cycloalkyl, heterocyclic radical, (CH ₂) _mcHR ⁴r ⁷, CF ₂c _1-6alkyl, CF ₂(CH ₂) _mcHR ⁴r ⁷or C (R ₄) ₂r ⁷;

R ^6tc _1-6alkyl, C _3-6cycloalkyl, aryl, heteroaryl, heterocyclic radical, (CH ₂) _mcHR ⁴r ⁷, C (R ₄) ₂r ⁷;

R ⁷oH, NR ⁸r ⁹, OCH ₂(CH ₂) _mnR ⁸r ⁹, C _1-6alkoxyl group, C _1-6alkoxy-C _1-6alkoxyl group, C _2-6hydroxyl alkoxyl group, oxetanyl, oxetanyl oxygen base, oxetanyl is amino, tetrahydrofuran base, tetrahydrofuran base oxygen base, tetrahydrofuran base is amino, Oxyranyle, Oxyranyle oxygen base, Oxyranyle is amino, oxepane alkyl, oxepane alkyl oxygen base, oxepane alkyl is amino, azetidinyl, azetidinyl oxygen base, azetidinyl is amino, pyrrolidyl, pyrrolidyl oxygen base, pyrrolidyl is amino, piperidyl, piperidyl oxygen base, piperidyl amino, azepan base, azepan base oxygen base, azepan base is amino, dioxolane base, dioxane base, morpholino, thiomorpholine generation, thiomorpholine generation-S, S-dioxide, Piperazino (piperazino), Dioxepane base, Dioxepane base oxygen base, Dioxepane base is amino, oxaza heptane base, oxaza alkyl oxy in heptan, oxaza alkylamino in heptan, Diazesuberane base, Diazesuberane base oxygen base, Diazesuberane base is amino, and as R ⁷all NH of a part can optionally by R ⁴replace,

R ⁸and R ⁹be H, C independently _1-6alkyl, C _3-6thiazolinyl, C _3-6alkynyl, C _3-7cycloalkyl, C _3-7cycloalkenyl group, C ₁-C ₆acyl group, 4-12 unit heterocyclic radical, C ₆-C ₁₂aryl or 5-12 unit heteroaryl; Or R ⁸and R ⁹the atom be connected with them forms 4-12 unit's monocycle or bicyclic ring systems, and wherein maximum two carbon atoms are by N, NR ⁴, O, S (O) _xwith S (O) (NR ¹⁴) substitute; And R ⁸and R ⁹can separately or together by maximum three independently selected from hydroxyl, C _1-6alkoxyl group, C _1-6hydroxyalkyl, C _1-6alkoxy-C _1-6alkyl, C _1-6alkoxy-C _1-6alkoxyl group, C _2-6the substituting group of hydroxyl alkoxyl group replaces;

R ¹⁰h, C _1-6alkyl;

X ²c, CH or N;

X ³cH or N,

X ⁴cR ⁴or N or NR ¹⁰;

X ⁵and X ⁶be all C or can be N;

Two keys in a-e be double bond and other three be singly-bound, to make atom X ²-X ⁶all do not have two double bonds coupled;

Key f normally double bond, just works as X ⁴and X ⁵, or X ⁴and X ⁶be nitrogen and X ²-X ⁶in other three be C, CH or CR ⁴time, two key f can be also singly-bounds;

M is 0-4;

Condition is as follows:

X ²-X ⁶in at least one and no more than three be N or NR ⁴;

R ⁵, R ^6eand R ^6zin only one can be halogen or containing with the halogen of the carbon atom Direct Bonding be bonded directly in described alkene acid amides system itself.

dESCRIPTION OF THE PREFERRED

Term used herein " halogen (halogen) " or " halo (halo) " refer to fluorine, chlorine, bromine or iodine (F, Cl, Br, I).Halo preferably refers to fluorine or chlorine.

Term " alkyl " refers to saturated monovalent aliphatic alkyl, comprises the straight chain and branched group that have and specify carbonatoms.Term " C _1-6alkyl " or " C ₁-C ₆alkyl " refer to straight or branched alkyl containing 1 to 6 carbon atom, as methyl, ethyl, n-propyl, sec.-propyl, normal-butyl, isobutyl-, sec-butyl, the tertiary butyl, amyl group, hexyl etc.Similarly, term " C _1-4alkyl " or " C ₁-C ₄alkyl " refer to straight or branched alkyl containing 1 to 4 carbon atom, as methyl, ethyl, n-propyl, sec.-propyl, normal-butyl, isobutyl-, sec-butyl, the tertiary butyl etc.

Term used herein " halogen (halogen) " or " halo (halo) " refer to fluorine, chlorine, bromine or in some cases, substituted alkyl can specifically with reference to substituting group name.Such as, " haloalkyl " refers to the alkyl with appointment carbonatoms replaced by one or more halogenic substituent, and usually containing 1-6 carbon atom and 1,2 or 3 halogen atom (i.e. " C ₁-C ₆haloalkyl ").Therefore, C ₁-C ₆haloalkyl comprises trifluoromethyl (-CF ₃) and difluoromethyl (-CF ₂h).

Similarly, " hydroxyalkyl " refers to the alkyl with appointment carbonatoms being optionally substituted with one or more hydroxyl substituting group and replacing, and usually containing 1-6 carbon atom and 1,2 or 3 hydroxyl (i.e. " C ₁-C ₆hydroxyalkyl ").Therefore, C ₁-C ₆hydroxyalkyl comprises methylol (-CH ₂and 2-hydroxyethyl (-CH OH) ₂cH ₂oH).

Term " C _1-6alkoxyl group ", " C ₁-C ₆alkoxyl group " or " OC _1-6alkyl " refer to straight or branched alkoxyl group containing 1 to 6 carbon atom, as methoxyl group, oxyethyl group, positive propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert.-butoxy, pentyloxy, hexyloxy etc.Term " C _1-4alkoxyl group ", " C ₁-C ₄alkoxyl group ", " OC _1-4alkyl " refer to straight or branched alkoxyl group containing 1 to 4 carbon atom, as methoxyl group, oxyethyl group, positive propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tert.-butoxy etc.

Term " C _3-6cycloalkyloxy ", " C ₃-C ₆cycloalkyloxy " or " OC _3-6cycloalkyl " refer to cyclic alkoxy containing 3 to 6 carbon atoms, as ring propoxy-, cyclobutoxy group, cyclopentyloxy etc.

" alkoxyalkyl " refers to the alkyl with appointment carbonatoms replaced by one or more alkoxy substituent.Alkoxyalkyl usually in moieties containing 1-6 carbon atom by 1,2 or 3 C ₁-C ₄alkoxy substituent replaces.Such group is described to C in this article sometimes ₁-C ₄alkoxy-C ₁-C ₆alkyl." aminoalkyl group " refers to the alkyl with appointment carbonatoms replaced by one or more substituted or unsubstituted amino, further defines this type of group in this article.

Aminoalkyl group usually contains 1-6 carbon atom and is replaced by 1,2 or 3 amino-substituent in moieties.Therefore, C ₁-C ₆aminoalkyl group comprises such as amino methyl (-CH ₂nH ₂), N, N-dimethylarnino-ethyl (-CH ₂cH ₂n (CH ₃) ₂, 3-(N-cyclopropylamino) propyl group (-CH ₂cH ₂cH ₂nH- ^cpr) and n-pyrrolidyl ethyl (-CH ₂cH ₂ n-pyrrolidyl).

" thiazolinyl " refers to the alkyl be as defined herein made up of at least two carbon atoms and at least one carbon-to-carbon double bond.Thiazolinyl usually have 2 to 20 carbon atoms ( "c ₂-C ₂₀thiazolinyl "), preferably 2 to 12 carbon atoms ( "c ₂-C ₁₂thiazolinyl "), more preferably 2 to 8 carbon atom (" C ₂-C ₈thiazolinyl ") or 2 to 6 carbon atom (" C ₂-C ₆thiazolinyl ") or 2 to 4 carbon atoms ( "c ₂-C ₄thiazolinyl ").Representative example comprises vinyl, 1-propenyl, 2-propenyl, 1-, 2-or 3-butenyl etc." C ₂-C ₆thiazolinyl " refer to containing 2 to 6 carbon atoms and at least one at two sp ²the straight or branched group of the double bond between hydbridized carbon atoms.If they occur, such as, at O-(C with substituting group or as the substituting group of other group ₂-C ₆) in thiazolinyl, above-mentioned definition is also suitable for.Suitable C ₂-C ₆the example of thiazolinyl is positive propenyl, pseudoallyl, n-butene base, isobutenyl, positive pentenyl, secondary pentenyl, n-hexylene base, secondary hexenyl etc.Thiazolinyl can not be substituted or replaced by the identical group being described as being applicable to alkyl herein.

" alkynyl " refers to the alkyl be as defined herein made up of at least two carbon atoms and at least one carbon-to-carbon triple bond.Alkynyl has 2 to 20 carbon atom (" C ₂-C ₂₀alkynyl "), preferably 2 to 12 carbon atom (" C ₂-C ₁₂alkynyl "), more preferably 2 to 8 carbon atom (" C ₂-C ₈alkynyl ") or 2 to 6 carbon atom (" C ₂-C ₆alkynyl ") or 2 to 4 carbon atom (" C ₂-C ₄alkynyl ").Representative example includes, but not limited to ethynyl, 1-proyl, 2-propynyl, 1-, 2-or 3-butynyl etc.Alkynyl can not be substituted or replaced by the identical group being described as being applicable to alkyl herein." C ₂-C ₆alkynyl " refer to straight or branched group containing 2 to 6 carbon atoms and at least one triple bond between two sp hydbridized carbon atoms.If they occur, such as, at O-(C with substituting group or as the substituting group of other group ₂-C ₆) in alkynyl, above-mentioned definition is also suitable for.Suitable C ₂-C ₆the example of alkynyl is proyl, butynyl, pentynyl, hexin base etc.

" alkylidene group " used herein refers to the bivalent hydrocarbon radical with appointment carbonatoms that two other groups can be linked together.It refers to-(CH sometimes ₂) n-, wherein n is that 1-8, n are preferably 1-4.When specifying, alkylidene group also can be replaced by other group and can comprise one or more degree of unsaturation (i.e. alkenylene or alkynylene part) or ring.The open valency of alkylidene group does not need the end opposite at chain.Therefore ,-CH (Me)-and-C (Me) ₂-be also included within the scope of term " alkylidene group ", cyclic group is if cyclopropane-1,1-bis-base and unsaturated group are as vinylidene (-CH=CH-) or propenylidene (-CH ₂cH=CH-) be included in equally in the scope of term " alkylidene group ".When alkylidene group is described to optionally be substituted, substituting group comprise usually be present in as described herein on alkyl those.

" sub-assorted alkyl " refers to the alkylidene group as above that the one or more non-conterminous carbon atom of wherein alkylidene chain is substituted by-N-,-O-,-P-or-S-, be presented as such as-N (R)-,-P (=O) (R)-,-S (O) _x-Huo – S (=O) (=NR)-, wherein R is H or C ₁-C ₄alkyl and x is 0-2.Such as, group-O-(CH ₂) _1-4-be ' C ₂-C ₅'-Ya mixes alkyl, wherein one of the carbon atom of corresponding alkylidene group is substituted by O.

" aryl " or " aromatics " refers to the π-electron system with total conjugated and has full carbon monocycle or the many rings of condensed ring of aromaticity.Term " C ₆-C ₁₂aryl " and " C ₆- ₁₂aryl " to be included in this term and comprise there are 6 to 12 carbon atoms and in ring system not containing heteroatomic aromatic ring system.The example of aryl is phenyl and naphthyl.Aryl can be substituted or not be substituted.C ₆-C ₁₂substituting group on the adjacent ring carbon atom of aryl can in conjunction with formation optionally by one or more substituting group, as oxo, C ₁-C ₆5-or the 6-unit carbocyclic ring ring of alkyl, hydroxyl, amino and halogen substiuted, or optionally by one or more substituting group, as oxo, C ₁-C ₆alkyl, hydroxyl, amino and halogen substiuted be selected from N, O and S (O) containing 1,2 or 3 _x5-or the 6-unit heterocyclic ring of the ring hetero atom of (wherein x is 0,1 or 2).The example of aryl comprises phenyl, xenyl (biphenyl), naphthyl, anthryl, phenanthryl, indanyl, indenyl and tetralyl.Aryl can not be substituted or is substituted like that as further described herein.

" heteroaryl " or " heteroaromatic " refers to containing specifying annular atoms number and comprising at least one heteroatoms being selected from N, O and S as the monocycle with known aromaticity of ring members or condensed-bicyclic or many rings ring system at aromatic ring.Heteroatomicly add the aromaticity realized in 5-ring and 6-ring.Usually, heteroaryl contains 5 to 20 annular atomses (" 5-20 unit heteroaryl "), preferably 5 to 14 annular atomses (" 5-14 unit heteroaryl "), more preferably 5 to 12 annular atomses (" 5-12 unit heteroaryl ") or 5 to 6 annular atomses (" 5-6 unit heteroaryl ").Heteroaryl ring is connected in base molecule, to keep aromaticity via the annular atoms of hetero-aromatic ring.Therefore, 6-unit heteroaryl ring can be connected in base molecule via ring C atom, and 5-unit heteroaryl ring can be connected in base molecule via ring C or atom N.Heteroaryl can not be substituted or is substituted like that as further described herein.As used herein, " 5-6 unit heteroaryl " refers to the monocyclic groups with 5 or 6 annular atomses being selected from the ring hetero atom of N, O and S containing 1,2 or 3, but comprises and have 4 nitrogen, all the other annular atomses are C and have the tetrazyl of π-electron system of total conjugated.Substituting group on the adjacent cyclic atom of 5-or 6-unit heteroaryl can in conjunction with formation optionally by one or more substituting group, as oxo, C ₁-C ₆alkyl, hydroxyl, amino and halogen substiuted condense 5-or 6-unit carbocyclic ring ring, or optionally by one or more substituting group, as oxo, C ₁-C ₆alkyl, hydroxyl, amino and halogen substiuted be selected from N, O and S (O) containing 1,2 or 3 _xthe ring hetero atom of (wherein x is 0,1 or 2) condense 5-or 6-unit heterocyclic ring.If described fused rings itself is aromatics, it is referred to as and condenses (dicyclo) heteroaromatic class, and no matter whether second ring be containing heteroatoms.Pharmaceutically acceptable heteroaryl enough stablizes to be connected on compound of the present invention, is formulated into the heteroaryl also delivering medicine to the patient needing it in pharmaceutical composition subsequently.

Pyrryl, thienyl, furyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, tetrazyl, oxadiazolyl and thiadiazolyl group is comprised containing 1,2 or 3 example independently selected from the heteroatomic 5-unit heteroaryl ring of O, N and S.Preferred 6-unit heteroaryl ring contains 1 or 2 nitrogen-atoms.The example of 6-unit heteroaryl is pyridyl, pyridazinyl, pyrimidyl and pyrazinyl.The example of condensed heteroaryl ring comprises cumarone, thionaphthene, indoles, benzoglyoxaline, indazole, quinolone, isoquinoline 99.9, purine, pyrrolopyrimidine, naphthyridines and carbazole.

" arylidene " used herein refers to the divalent group obtained by removing hydrogen atom separately from two carbon atoms of core by aromatic hydrocarbons.In common embodiment, arylidene ring is that 1,2-bis-replaces or the dibasic arylidene of 1,3-.The aryl rings of arylidene part can optionally on open valency position by the group of applicable aryl rings replace to this replacement the degree that indicates.Arylidene ring is preferably C ₆-C ₁₂arylidene ring, such as 1,2-phenylene or 1,3-phenylen moiety.

Similarly, " inferior heteroaryl " used herein refers to and removes hydrogen atom and the divalent group that obtains by hetero-aromatic ring by respective from two carbon atoms of core or a carbon atom and a nitrogen-atoms.In common embodiment, inferior heteroaryl ring is that 1,2-bis-replaces or the dibasic inferior heteroaryl of 1,3-.The heteroaryl ring of heteroarylene moieties optionally by the group of applicable heteroaryl ring replace to this replacement the degree that indicates.The inferior heteroaryl ring that inferior heteroaryl ring is preferably 5-12 unit, may condenses, more preferably 5-6 unit inferior heteroaryl ring, they can optionally be substituted separately.

Term " assorted alicyclic ", " heterocyclic radical " or " heterocycle " are used interchangeably to represent that wherein this heterocyclic ring is connected in base molecule via annular atoms (it can be C or N) containing specifying annular atoms number, comprising at least one heteroatoms being selected from N, O and S as the non-aromatic, saturated of ring members or the undersaturated member ring systems of part in this article.Assorted alicyclic ring can be fused on other assorted alicyclic or carbocyclic ring ring one or more, this condensed ring can be saturated, part is undersaturated or aromatics.Assorted alicyclic ring preferably containing 1 to 4 heteroatoms being selected from N, O and S as ring members, more preferably 1 to 2 ring hetero atom, condition is that this type of assorted alicyclic ring is containing two adjacent Sauerstoffatoms.Heteroalicyclyl can not be substituted or replaced by the identical group being described as being applicable to alkyl, aryl or heteroaryl herein.

Preferred heteroalicyclyl comprises 3-12 unit heteroalicyclyl, the assorted alicyclic dicyclo of the assorted alicyclic monocycle of 5-8 unit's heterocyclic radical (or heteroalicyclyl), 4-12 unit and 6-12 unit according to definition herein." 3-12 unit is assorted alicyclic " used herein refers to the monocycle or bicyclic radicals with 3 to 12 annular atomses, and wherein 1,2,3 or 4 annular atoms is selected from N, O, P (O), S (O) _xthe heteroatoms of (wherein x is 0,1,2) and S (=O) (=NR), all the other annular atomses are C.This ring also can have one or more double bond.But this ring does not have the π-electron system of total conjugated.Substituting group on two ring carbon atoms can be selected from N, O and S (O) in conjunction with formation containing 1,2 or 3 _xthe carbocyclic ring of the ring hetero atom of (wherein x is 0,1 or 2) or assorted alicyclic 5-or 6-unit bridged ring.This heteroalicyclyl is optionally by oxo, hydroxyl, amino, C ₁-C ₆the replacements such as-alkyl.

In common embodiment, heteroalicyclyl contains 3-12 ring members, comprises carbon and non-heteroatoms, a preferred 4-6 ring members.In some preferred embodiment, the substituting group comprising 3-12 unit heteroalicyclyl is selected from azetidinyl, pyrrolidyl, piperidyl, piperazinyl, morpholinyl and thiomorpholine basic ring, and they can optionally be substituted separately in the degree that such replacement has chemical sense.

Should be understood that; except oxo or azepine group are connected on N, P or S to be formed such as but not limited to the group of nitro, phosphinyl (phosphinyl), phosphono amido (phosphinamido), sulfo group oximido (sulfoximino) and alkylsulfonyl and so on or at some hetero-aromatic ring; as the situation China and foreign countries of triazine, triazole, tetrazolium, oxadiazole, thiadiazoles etc., no more than two N, O or S atoms connect continuously usually.

" cycloalkyl " refers to containing specifying non-aromatic, the saturated or undersaturated carbocyclic ring member ring systems of part of carbonatoms, and it can be the monocycle be connected to via the carbon atom of cycloalkyl ring in base molecule, bridging or condensed-bicyclic or many rings member ring systems.Cycloalkyl of the present invention is usually containing 3 to 12 carbon atom (" C ₃-C ₁₂cycloalkyl "), preferably 3 to 8 carbon atom (" C ₃-C ₈cycloalkyl ").Other cycloalkyl comprise unsaturated the part (" C of the part with 4 to 7 carbon ₄-C ₇cycloalkenyl group ").Representative example comprises such as cyclopropane, tetramethylene, pentamethylene, cyclopentenes, hexanaphthene, tetrahydrobenzene, cyclohexadiene, suberane, cycloheptatriene, diamantane etc.Cycloalkyl can not be substituted or replaced by the identical group being described as being applicable to alkyl herein." C used herein ₃-C ₆cycloalkyl " refer to the full carbon monocycle or condensed ring polycyclic moiety with 3 to 6 carbon atoms.

" cycloalkylalkyl " can be used for description and (is generally C via alkylidene group connection base (linker) ₁-C ₄alkylidene group) cycloalkyl ring be connected in base molecule (is generally C ₃-C ₈cycloalkyl).Cycloalkylalkyl is described also usually containing 4-12 carbon atom (" C by carbocyclic ring ring and the total number of carbon atoms be connected in base ₄-C ₁₂cycloalkylalkyl ").Therefore, Cvclopropvlmethvl is C ₄-cycloalkylalkyl, and cyclohexyl-ethyl is C ₈-cycloalkylalkyl.Cycloalkylalkyl can not be substituted or replaced by the identical group being described as being applicable to alkyl herein in cycloalkyl and/or alkylene moiety.

" arylalkyl " refers to the aryl as described herein be connected to via alkylidene group or similar connection base in base molecule.Arylalkyl is described with the total number of carbon atoms be connected in base by this ring.Therefore benzyl is C ₇-arylalkyl, phenylethyl is C ₈-arylalkyl.Arylalkyl is usually containing 7-16 carbon atom (" C ₇-C ₁₆arylalkyl "), wherein aryl moiety contains 6-12 carbon atom, and alkylene moiety contains 1-4 carbon atom.This type of group is Ke Yi Biao Shi Wei – C also ₁-C ₄alkylidene group-C ₆-C ₁₂aryl.

" heteroarylalkyl " refers to the difference of " arylalkyl ", connecting base via alkylidene group is connected to heteroaryl as above in base molecule, is that at least one annular atoms of aromatic fractions is the heteroatoms being selected from N, O and S.Sometimes according to the sum of the non-hydrogen atom (i.e. C, N, S and O atom) of (not comprising substituting group) in the connection base of this ring and combination, heteroarylalkyl is described in this article.Therefore, such as, pyridylmethyl can be referred to as " C ₇"-heteroarylalkyl.Usually, unsubstituted heteroarylalkyl contains 6-20 non-hydrogen atom (comprising C, N, S and O atom), and wherein heteroaryl moieties is usually containing 5-12 atom, and alkylene moiety is usually containing 1-4 carbon atom.Such group also can be expressed as-C ₁-C ₄alkylidene group-5-12 unit heteroaryl.

Similarly, " alkoxy aryl " and " heteroarylalkoxy " refer to and are connected base (i.e.-O-alkylidene group-) are connected to aryl in base molecule and heteroaryl via Asia alkyl of mixing, and wherein describe these groups according to the sum of the non-hydrogen atom (i.e. C, N, S and O atom) in the connection base of this ring and combination.Therefore ,-O-CH ₂-phenyl and-O-CH ₂-pyridyl is referred to as C respectively ₈-alkoxy aryl and C ₈-heteroarylalkoxy.

When arylalkyl, alkoxy aryl, heteroarylalkyl or heteroarylalkoxy are described to optionally be substituted, substituting group can in divalent linker part or on the aryl or heteroaryl moieties of this group.Optionally be present in substituting group on alkylidene group or sub-assorted moieties identical with those usually description alkyl or alkoxyl group above, and it is identical with those usually description aryl or heteroaryl to be optionally present in substituting group on aryl or heteroaryl moieties above.

" hydroxyl " refers to-OH group.

" acyl group " refers to univalent perssad-C (O) alkyl, and wherein moieties has appointment carbonatoms (usual C ₁-C ₈, preferred C ₁-C ₆or C ₁-C ₄) and can be applied to alkyl group replace.Therefore, C ₁-C ₄acyl group comprises-C (O) C ₁-C ₄alkyl substituent, such as-C (O) CH ₃.Similarly, " acyloxy " refers to univalent perssad-OC (O) alkyl, and wherein moieties has appointment carbonatoms (usual C ₁-C ₈, preferred C ₁-C ₆or C ₁-C ₄) and can be applied to alkyl group replace.Therefore, C ₁-C ₄acyloxy comprises-OC (O) C ₁-C ₄alkyl substituent, such as-OC (O) CH ₃.

Term " monocycle or bicyclic ring systems " refers to that wherein heterocyclic ring is connected in base molecule via the annular atoms that can be C or N containing the aromatics of specifying annular atoms number, the saturated or undersaturated member ring systems of part optionally can comprise one or more heteroatoms being selected from N, O and S as ring members.Term " cycloalkyl ", " aryl ", " heterocyclic radical " and " heteroaryl " is comprised at this term.Monocycle of the present invention or bicyclic ring systems are usually containing 4 to 12 member atoms (" 4-12 unit's monocycle or bicyclic ring systems ").Bicyclic system can condense via 1,1-(spiral shell), 1,2-condense (condensing) or 1, >2-condense (end of the bridge) connect.Representative example comprises pentamethylene, cyclopentenes, hexanaphthene, norcamphyl (norbornyl), spiral shell [2.3] hexane, phenyl, xenyl, naphthyl, anthryl, phenanthryl, pyrryl, thienyl, furyl, pyrazolyl, imidazolyl, oxazolyl, isoxazolyl, thiazolyl, azetidinyl, pyrrolidyl, piperidyl, piperazinyl, benzothienyl, indyl etc.

All alkyl, thiazolinyl, alkynyl, cycloalkyl, cycloalkenyl group, monocycle and bicyclic heterocycle, aryl (monocycle and dicyclo), heteroaryl (monocycle and dicyclo), cycloalkylalkyl, arylalkyl, alkoxy aryl, heteroarylalkyl or heteroarylalkoxy (comprise any C _1-6alkyl, C _2-6thiazolinyl, C _2-6alkynyl, C _3-6cycloalkyl, C _4-6cycloalkenyl group, C _6-12the saturated monocyclic heterocycles of bicyclic alkyl, a 4-12 atom or the saturated bicyclic heterocycle of 6-12 atom, all C _6-12the heteroaryl monocycle of aryl monocycle or dicyclo and 6-12 atom or dicyclo) can optionally be replaced by multiple substituting group, described substituting group independent selected from halo, hydroxyl, oxo, hydroxyl amino, oximido, diazanyl, hydrazono-, cyano group, nitro, azido-, NR ⁸r ⁹, OC _1-6alkyl, OC _3-6thiazolinyl, OC _3-6alkynyl, C _1-6alkyl, OC _3-6cycloalkyl, OC _3-7cycloalkenyl group, C _1-6acyl group, C _1-6acyloxy, N (R ⁸) COR ⁴, CO ₂r ⁴, CONR ⁸r ⁹, NR ⁸cONR ⁸r ⁹, NR ⁸cO ₂r ⁴, OCO ₂r ⁴, OCONR ⁸r ⁹, S (O) _xr ⁴, S (R ⁴) (=O)=NR ⁸, S (=O) (=NR ⁸) NR ⁸r ⁹, SO ₂nR ⁸r ⁹, NR ⁸sO ₂r ⁴, NR ⁸sO ₂nR ⁸r ⁹,-NR ⁸s (=O) (=NR ⁸) R ⁴,-N=S (=O) (R ⁴) R ⁴,-N=S (=O) (NR ⁸r ⁹) R ⁴, ONR ⁸r ⁹, ON (R ⁸) COR ⁴, ONR ⁸cONR ⁸r ⁹, ONR ⁸cO ₂r ⁴, ONR ⁸sO ₂r ⁴, ONR ⁸sO ₂nR ⁸r ⁹.

As used in preparation and embodiment, following term has shown implication: " ng " refers to nanogram; " μ g " refers to microgram; " mg " refers to milligram; " g " refers to gram; " kg " refers to kilogram; " nmole " or " nmol " refers to nmole; " mmol " refers to mmole; " mol " refers to mole; " M " refers to volumetric molar concentration, and " mM " refers to millimolar concentration, and " μM " refers to micro-molar concentration, and " nM " refers to nanomolar concentration, and " L " refers to liter, and " mL " refers to milliliter, and " μ L " refers to microlitre.

The pharmacologically acceptable salt of compound of the present invention comprises its acid salt and alkali salt (comprising disalt).

Suitable acid salt is formed by the acid forming non-toxic salt.Example comprises acetate, aspartate, benzoate, benzene sulfonate, bicarbonate/carbonate, hydrosulfate/vitriol, borate, camsilate, Citrate trianion, ethanedisulphonate, esilate, formate, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, hybenzate (hibenzate), hydrochloride/muriate, hydrobromate/bromide, hydriodate/iodide, isethionate, lactic acid salt, malate, maleate, malonate, mesylate, Methylsulfate, naphthoate, 2-naphthalenesulfonate, nicotinate, nitrate, Orotate, oxalate, palmitate, embonate, phosphate/phosphor acid hydrogen salt/dihydrogen phosphate, sucrose hydrochlorate, stearate, succinate, tartrate, tosylate and trifluoroacetate.

Suitable alkali salt is formed by the alkali forming non-toxic salt.Example comprises aluminium salt, arginic acid salt, benzyl star (benzathine) salt, calcium salt, choline salt, diethylamine salt, diethanolamine salt, glycinate, lysine salt, magnesium salts, meglumine salt, ethanolamine salt, sylvite, sodium salt, trometamol salt and zinc salt.

About the summary of suitable salt, see " Handbook of Pharmaceutical Salts:Properties, Selection, and Use " (Wiley-VCH, Weinheim, Germany, 2002) of Stahl and Wermuth.

By optionally being mixed with required acid or alkali by the solution of this compound, the pharmacologically acceptable salt of compound of the present invention easily can be prepared.Salt can be precipitated and maybe can be reclaimed by evaporating solvent by collecting by filtration from solution.Degree of ionization in this salt can change from complete ionization to almost unionization.

Compound of the present invention containing one or more unsymmetrical carbon can exist with the form of two or more steric isomers.If compound of the present invention contains thiazolinyl or alkenylene, geometry cis/trans (or Z/E) isomer may be there is.If this compound contains such as ketone group or oximido or aromatic fractions, tautomerism (" tautomerism ") can be there is.Result is that single compound may show more than a kind of isomery.

Comprise all steric isomers of compound of the present invention, geometrical isomer and tautomeric form in the scope of claimed compound of the present invention, comprise the compound shown more than a kind of isomery, and one or more mixture.Also comprise acid salt or alkali salt, wherein gegenion is opticity, such as D-lactate or 1B salt, or gegenion is racemic, such as DL-tartrate or DL-arginic acid salt.

Cis/trans isomer is by well known to a person skilled in the art conventional art, and such as chromatography is separated with Steppecd crystallization.

Compound of the present invention also may show atropisomerism, and wherein limited rotation (especially around the key of two aryl rings connected in biaryl) causes different rotational isomers can not transform mutually under Normal Environmental Temperature and also very likely can not transform mutually under whole molecule keeps heat-staple temperature.In this case, also claimed different steric isomers produced by atropisomerism.

Comprise by suitable optical purity precursor chiral synthesize or use such as Chiral high pressure liquid phase chromatography (HPLC) for the preparation of/conventional art that is separated single enantiomer, especially resolution of racemic thing (or racemoid of salt or derivative) in simulation moving-bed (SMB) structure.

Such as, or racemoid (or racemize precursor) can with suitable optically-active compound, alcohol reaction, or when the compound of formula (I) contains acidity or basic moiety, with acid or alkali, such as tartrate or 1-phenyl-ethyl amine react.Gained diastereomeric mixtures is separated by chromatography and/or Steppecd crystallization and by the known method of technician, one or both diastereomers is changed into corresponding pure enantiomer.

Chipal compounds of the present invention (and chiral precurser) can utilize chromatography (usual HPLC) on asymmetric resin, use the moving phase be made up of hydrocarbon to obtain with enantiomer enriched form, described hydrocarbon is generally heptane or hexane, the Virahol (usual 2 to 20%) containing 0 to 50% and 0 to 5% alkylamine (usual 0.1% diethylamine).The concentrated of eluate provides enriched Mixture.

The mixture of steric isomer can be separated by conventional art well known by persons skilled in the art.[" Stereochemistry of Organic Compounds " (Wiley, New York, 1994) see such as E L Eliel].

The present invention includes all pharmaceutically useful isotope-labeled compounds of the present invention, wherein one or more atoms are had same atoms ordinal number but the atom that atomic mass or total mass number are different from atomic mass that occurring in nature finds usually or total mass number substitutes.

Be applicable to the isotropic substance that the isotopic example be included in compound of the present invention comprises hydrogen, as ²h and ³h, the isotropic substance of carbon, as ¹¹c, ¹³c and ¹⁴c, the isotropic substance of chlorine, as ³⁶cl, the isotropic substance of fluorine, as ¹⁸f, the isotropic substance of iodine, as ¹²³i and ¹²⁵i, the isotropic substance of nitrogen, as ¹³n and ¹⁵n, the isotropic substance of oxygen, as ¹⁵o, ¹⁷o and ¹⁸o, the isotropic substance of phosphorus, as ³²p, and the isotropic substance of sulphur, as ³⁵s.

Some isotope-labeled compound of the present invention, such as comprise radioisotopic those, can be used for medicine and/or substrate tissue distribution research.Consider their being easily incorporated to property and ready-made detection means, radio isotope tritium (namely ³h) and carbon-14 (namely ¹⁴c) this purposes is particularly useful for.

With higher isotope, such as deuterium, namely ²h substitutes some treatment benefit that can provide and be brought by higher metabolic stability, and therefore the Half-life in vivo such as increased or the dose requirements of reduction are preferred in some cases.

With Positron emitting isotopes, such as ¹¹c, ¹⁸f, ¹⁵o and ¹³n substitutes and can be used for positron emission computerized tomography (PET) research with detection substrate receptor share.

Usually by conventional art well known by persons skilled in the art or by with appended embodiment and those the similar methods described in preparing, use suitable isotope-labeled reagent replace before the unlabelled reagent that uses prepare isotope-labeled compound of the present invention.

Compound of the present invention can with the form administration of prodrug.Therefore, some derivative of the compound of the present invention of pharmacologically active itself almost or completely may do not had can such as to be changed into by hydrolytic rupture when delivering medicine in health or on health there is required active formula 1(or other formula disclosed herein) compound.Such derivative is referred to as " prodrug ".Further information about prodrug application is found in `Pro-drugs as Novel Delivery Systems, Vol. 14, ACS Symposium Series (T Higuchi and W Stella) and `Bioreversible Carriers in Drug Design`, Pergamon Press, 1987 (ed. E B Roche, American Pharmaceutical Association).

Can such as by being known as the suitable functional group that exists in some Some substitute compound of the present invention of " precursor portions (pro-moieties) " to prepare prodrug with the those skilled in the art as described in " Design of Prodrugs " (Elsevier, 1985) of such as H. Bundgaard.

Some examples of this type of prodrug comprise:

When this compound contains carboxylic acid functional (--COOH), prodrug comprises its ester, such as, use C ₁-C ₆alkyl substitutes hydrogen;

When this compound contains alcohol functional group (--OH), prodrug comprises its ether, such as, use C ₁-C ₆alkanoyloxymethyl (-C ₁-C ₆pivaloyloxymethyl) substitute hydrogen; With

When this compound contains primary amino or secondary amino functionalities (-NH ₂or-NHR, wherein R is not H) time, prodrug comprises its acid amides, such as, use (C ₁-C ₁₀) alkyloyl (-C ₁-C ₁₀acyl group) substitute one or two hydrogen.

Above-mentioned reference is found according to previous examples and the further example of the alternative group of the example of other prodrug types.

Finally, some compound of formula A itself can serve as the prodrug of other compound of formula A.

Other embodiments of the present invention are illustrated in formula (I) to (III) or its pharmacologically acceptable salt:

Wherein

R ^6ec _1-6alkyl, aryl, heteroaryl, cycloalkyl, heterocyclic radical, (CH ₂) _mcHR ⁴r ⁷, CF ₂(CH ₂) _mcHR ⁴r ⁷or C (R ₄) ₂r ⁷;

R ^6zh, F, Cl, CF ₃, CHF ₂, CF ₂c _1-6alkyl or C _1-6alkyl;

R ⁷oH, NR ⁸r ⁹, OCH ₂(CH ₂) _mnR ⁸r ⁹, C _1-6alkoxyl group, C _1-6alkoxy-C _1-6alkoxyl group, C _2-6hydroxyl alkoxyl group, oxetanyl, oxetanyl oxygen base, oxetanyl is amino, tetrahydrofuran base, tetrahydrofuran base oxygen base, tetrahydrofuran base is amino, Oxyranyle, Oxyranyle oxygen base, Oxyranyle is amino, oxepane alkyl, oxepane alkyl oxygen base, oxepane alkyl is amino, azetidinyl, azetidinyl oxygen base, azetidinyl is amino, pyrrolidyl, pyrrolidyl oxygen base, pyrrolidyl is amino, piperidyl, piperidyl oxygen base, piperidyl amino, azepan base, azepan base oxygen base, azepan base is amino, dioxolane base, dioxane base, morpholino, thiomorpholine generation, thiomorpholine generation-S, S-dioxide, Piperazino, Dioxepane base, Dioxepane base oxygen base, Dioxepane base is amino, oxaza heptane base, oxaza alkyl oxy in heptan, oxaza alkylamino in heptan, Diazesuberane base, Diazesuberane base oxygen base, Diazesuberane base is amino, and as R ⁷all NH of a part can optionally by R ⁴replace,

R ⁸and R ⁹be H, C independently _1-6alkyl, C _3-6thiazolinyl, C _3-6alkynyl, C _3-7cycloalkyl, C _3-7cycloalkenyl group, C _1-c ₆acyl group, 4-12 unit heterocyclic radical, C _6-c ₁₂aryl or 5-12 unit heteroaryl; Or R ⁸and R ⁹the atom be connected with them forms 4-12 unit's monocycle or bicyclic ring systems, and wherein maximum two carbon atoms are by N, NR ⁴, O, S (O) _xwith S (O) (NR ¹⁴) substitute; And R ⁸and R ⁹can separately or together by maximum three independently selected from hydroxyl, C _1-6alkoxyl group, C _1-6hydroxyalkyl, C _1-6alkoxy-C _1-6alkyl, C _1-6alkoxy-C _1-6alkoxyl group, C _2-6the substituting group of hydroxyl alkoxyl group replaces;

R ¹⁰h, C _1-6alkyl;

X ²c, CH or N;

X ³cH or N,

X ⁴cR ⁴or N or NR ¹⁰;

X ⁵and X ⁶be all C or can be N;

M is 0-4;

Condition is as follows:

X ²-X ⁶in at least one and no more than three be N or NR ⁴;

If R ^6ecF ₂(CH ₂) _mcHR ⁴r ⁷, then R ⁵and R ^6znot F, Cl, CF ₃, CHF ₂or CF ₂c _1-6alkyl, if but R ^6enot CF ₂(CH ₂) _mcHR ⁴r ⁷, then R ⁵and R ^6zone of be F, Cl, CF ₃, CHF ₂or CF ₂c _1-6alkyl;

Or the compound of structure (II)

Or its pharmacologically acceptable salt, wherein

R ⁵h, CH ₂nR ⁸r ⁹or C _1-6alkyl;

R ^6zh or C _1-6alkyl;

R ¹⁰h, C _1-6alkyl;

X ²c, CH or N;

X ³cH or N,

X ⁴cR ⁴or N or NR ¹⁰;

X ⁵and X ⁶be all C or can be N;

M is 0-4;

Condition is as follows:

X ²-X ⁶in at least one and no more than three be N or NR ⁴;

If R ^6ecF ₂(CH ₂) _mcHR ⁴r ⁷, then R ⁵can not be CF ₂c _1-6alkyl;

Or the compound of structure (III)

Or its pharmacologically acceptable salt, wherein

R ¹⁰h, C _1-6alkyl;

X ²c, CH or N;

X ³cH or N,

X ⁴cR ⁴or N or NR ¹⁰;

X ⁵and X ⁶be all C or can be N;

M is 0-4;

Condition is as follows:

X ²-X ⁶in at least one and no more than three be N or NR ⁴.

Compound of the present invention comprises:

。

synthetic method

Compound of the present invention and intermediate thereof can be prepared in many modes that the those of ordinary skill in organic synthesis field is known.Reagent and starting material are that those of ordinary skill in the art easily obtain.

The useful especially bibliography being applicable to the synthetic method preparing compound of the present invention is found in Larock, R. C. Comprehensive Organic Transformations, VCH:New York, and 1989, it is incorporated herein by this reference.Greene and Wuts about selecting for the protection of another useful bibliography of the suitable protecting group of the reactive functional groups existed in compound of the present invention; Protective Groups In Organic Synthesis; Wiley & Sons; 1991, it is also incorporated herein by this reference as fully setting forth.

Compound of the present invention is prepared by various technology, sets forth some of them below.Skilled person in the art will appreciate that these methods are representative and nonrestrictive.The many reactions that can be used for building compound of the present invention are described in WO 2013/014448, WO 2013/169401 and J Med Chem 56,7025 (2013) (all the full text is incorporated herein by reference).

The overall assembling of method A. compound of the present invention

。

scheme 1. fragment I, II and III Overall Group dress up final product

The overall synthesis of these compounds comprises the step of the suitable fragment I of structure, II and III, then usually follows thereafter the final package program of scheme 1.2-chlorine on required 4-(bicyclic aryl)-2-chloropyrimide (I) well known to a person skilled in the art 3-N-methyl-p-nitroaniline (or 3-amino-5-nitro-pyrimidine) (II) coupling under condition with suitable.This can be acid catalyzed process or as above be the coupling of Buchwald type shown in flow process, but it also can be the coupling that alkali causes, thus has several different approach to form the key of this key.3-nitroreduction is become corresponding amine, and this is the reaction can carried out in diversified mode.Some reducible group, the existence as alkynyl may make this reaction complicated, but the diversity that can be used for the reagent of this conversion makes to be sure of that it can complete.If R ⁴not H, R ⁴can introduce in this step.Exemplify standard reductive alkylation at this, but other technology can be used, if such as do not disturb group in this molecule, use alkylogen direct alkylation.But, in majority of case, R ⁴h.By with the acid derivative of chloride of acid or other suitable (III) by aromatic amine acidylate, complete the assembling of molecule, this may require use peptide coupling agent, wherein many is known.In some cases, this synthesis may not complete in this step, and may need further chemical process.Such as, from (III), remove protecting group possibly, or the amine that (III) may not install in this stage, this can install subsequently, such as, by the displacement of allyl halide---there is greatly the governed technology of precedent in this area.

Although do not illustrate at this, it being understood that has many possible variants in this approach, and they are verified favourable in some cases, can not materially affect whole strategy and be that those of ordinary skill in the art are apparent.Discuss several possible variant below, but these are intended to illustrate possible variant, and are not intended to contain the apparent all possibilities of those of ordinary skill in the art.

The example discussed is if 5-substituting group makes 4-position inactivation, before installation biaryl base key by aniline coupling on the 2-position of 2,4-dichloro pyrimidine, or use the 5-substituting group of ortho positioned, then convert it into required substituting group.

Second example is the protection form that nitro need not be used when coupling as the final 3-amine on aniline.With the process of 2-chloropyrimide base biaryl coupling in such as can use 3-halo aniline, and use currently known methods to convert it into amine.Or 3-amido functional group can be introduced before coupling, as long as it can suitably and optionally protected.This even relate to fragment (I) coupling before with fragment (III) coupling, although the suitability of this method is probably limited.

In some cases, can advantageously by fragment (II) aniline and the coupling of fragment (I) biaryl, R thus ²and/or R ³group is not last group, but its useful synthon, then can after this coupling or even after the step next described (if those skilled in the art think the most suitable) carry out chemistry and manipulate to prepare final R ²and/or R ³substituting group.

the alternative assembling of scheme 2. diaryl-amine core

Due to the broad applicability of Buchwald coupling, aryl halide (preferred aryl groups chlorine or aryl bromide) can be used 2-amino-4-bicyclic heteroaryl pyrimidine arylation.2-amino-4-chloropyrimide is all easy to get; and 2-amino-4-chloropyrimide itself has shown and to diversified heteroaromatic boronic acid, Suzuki occurs and react the biaryl connected to form corresponding 4-pyrimidyl; and do not need protection 2-amino; such as (Org Biomol Chem 9; 3139 (2011), Bioorg Med Chem 19,5756 (2011), Eur J Org Chem 4532 (2011)).Or, can by under high pressure intermediate muriate (II) being changed into corresponding 2-aminocompound simply with ammonia treatment.Larger difference can be there is in the potential chemistry of aryl because prepare aryl halide now but not aniline, below discuss as the alternative synthesis method of (II) fragment some the different chemistry brought by this change.

Above-mentioned change does not all change the elementary tactics of this synthesis, but changes the arrangement of time of some basic step simply and may specify some the clear and definite change in starting material.The selection of final approach depends on which kind of passes through avoid the most inconsistent conversion and provide the easiest chemistry.

The synthesis of method B. dibenzyl chlorine tablets section (II)

。

the synthesis of scheme 3. biaryl fragment (I) a-e.

The synthesis of the most of 4-bicyclic aryl-2-chloropyrimide described in present patent application has greatly precedent to follow.In scheme 2, all these approach all utilize 2,4-dihalopyrimidines in 4-position prior to the known priority that 2-position reacts, so that it is monosubstituted with high yield, 4-to occur.Various technology can be used, utilize indoles and imidazoles A, B, C, under alkali (base) condition of classics, realize the direct nucleophilic displacement of chlorine.But, if needed, this type of reaction also can run as Buchwald or Suzuki coupling as shown in G, the widespread commercial availability of heteroaromatic boronic acid and ester allows Suzuki reaction optionally for C, although and D utilizes Sonagashira to react as shown, then nucleophilic addition(Adn) is on the acetylene of electron deficiency, but this reaction also can optionally be carried out as Suzuki reaction.E and F utilizes Suzuki to react assembling, and required boric acid ester can be buied.In all these situations, if need further to replace, it is directly and greatly have precedent governed that corresponding heteroaryl bromine changes into required boric acid.

Such scheme 2 does not comprise f(R ¹=CHF ₂) or g(R ¹=CF ₃) subbreed.In f series, corresponding dichloro pyrimidine is lost, and trifluoromethyl is known makes adjacent halogen inactivation, so that first 2,4-bis-chloro-5-trifluoromethyl pyrimidine reacts in 2-position and most of nucleophile---difluoromethyl also may show this effect.

2,4-dichloro pyrimidine-5-formaldehyde is known preferentially to be changed in 4-position.Therefore Suzuki or Buchwald coupling can produce corresponding aldehyde in these series, and these aldehyde are easily by DAST, SF ₄etc. the difluoromethyl derivative changing into corresponding (I), the relatively low functionality of these molecules causes unlikely serious side reaction occurring.Similar program can be used for (I) g, and thus (I) e cyano compound or another suitable acid derivative are changed into 5-carboxylic acid, it is subsequently by HF/SF ₄or other technical transform becomes trifluoromethyl derivative (I) g.This acid also can from the oxidation of corresponding alcohol.But, in many cases, simpler solution may be reverse the first two step in final assembling simply---make 2, first 4-bis-chloro-5-trifluoromethyl pyrimidine reacts with aniline/aminopyridine (II) replaces to realize required 2-, then carries out required biaryl coupling in 4-position.

If be coupled to by aryl halide on aminopyrimidine, similar reverse can be realized.The chloro-5-trifluoromethyl pyrimidine of 2,4-bis-can not very optionally with ammonia react and product be 2 and 4-displacement 1:1 mixture, show CF ₃ortho position inactivation has large space components (steric component).If need the 2-selectivity improving this method; namely 2-that may be lower selects and more difficult acquisitions 5-difluoromethyl analogue, then use large volume (bulky) amine as dibenzylamine, then the productive rate of this approach can be made higher and avoid may difficult being separated at the Suzuki coupling of 4-position and amine deprotection.

In some cases, can advantageously at pyrimidine ring place by the bracket assembled of anilino biaryl pyrimidine together, especially when 6,5-bicyclic heteroaryl is connected on pyrimidine via carbon.As shown in following scheme 3b, if heteroaryl is replaced by ethanoyl in its 3-position, with DMF aldolization, then can make compound of the present invention with the condensation of N-aryl guanidine whole bracket assembled together with.Because many modes can by aniline amidineization (amidinate) used in the present invention, this approach provides feasible alternatives for several classes of compounds described in the present invention.Because 3-acetyl compounds is known in indoles, N-skatole, pyrazolo [1,5-a] pyridine and imidazo [1,2-a] pyridine series, the pyrimidine serial that this alternative synthesis method connects all 4-carbon is feasible.

scheme 3B. assembles the alternative route of multi-joint three aromatic backbone.

The synthesis of method C. 1,3-diaminobenzene fragment

1. check anilino compounds of the present invention, they require 2,4,5-substituted anilines of several different series of preparation.In 2-position, R ²can be one of several low alkyl group or lower alkoxy.This group may exist, as in 3-benzyl halide or 3-halogeno-benzene methyl ether, or its can be in or be not in after a while transition metal-catalyzed under added by the displacement of the halide group suitably activated.4-bit substituent R ³more various.It can be H, F, alkoxyl group, alkyl, alkynyl or tertiary amine, several structurally quite complicated in these groups, it has additional polar functional group, to help the physical chemistry and the pharmacokinetic property that improve this inhibitor.As described in above-mentioned overall plan 1,1-position is fixed as amino, and 3-position is fixed as nitro.But if utilize the Buchwald coupling of aminopyrimidine, then 1-position must be halogen, and time most of, Cl or Br is preferred.3-position is nitro expediently, but it can be amino, and most probable has protecting group, as Boc, if but electronics or steric effect (sterics) make it relative to 1-position amine inactivation, and may be not protected.It also can be halogen, and it is amination in transition metal-catalyzed reaction subsequently, or it can be carbonyl substituted base, and it resets subsequently in Curtius or Beckman reaction.Therefore, R is worked as ³when not being hydrogen, a large amount of 1,2,4,5-quaternary benzene is had to can be used as the synthon of amino aniline ring.Work as R ³when being hydrogen; have commercially available 1; the benzene synthon that 2,4-replaces can be used for all corresponding compound claimed in the application, and the chemistry needed for any example they being changed into fragment (II) is completely in the limit of power of those of ordinary skill in the art.

Provide some documents preparation of 1,2,4,5-quaternary benzene derivative of the synthon be applicable in this patent below.As long as there is halogen at the ortho position of nitro or contraposition, amino or alkoxyl group can be introduced via the nucleophilic displacement of halogen.Even if there is electron-donating group in this ring, these displacements also can be there is, the especially displacement of fluorine (fluoride).Muriate or bromide can be used even to introduce amino or alkyloxy side chain after removing all nitros in the reaction of Buchwald type, also can be used for as discussed above aromatic ring being coupled on aminopyrimidine.Several synthon there is 1,3-dinitrobenzene substituting group and have some documents to point out a nitro is better than another selective reduction.Although be unfavorable for productive rate, non-selective nitroreduction is not large problem, as long as can separating isomers.Because these two nitros finally all need reduction; do not have " mistake " isomer to be reduced; as long as can carry out for build fragment (II) another needed for chemistry because the first reduction of " unacceptable " " 3-nitro " can by protecting it, then reducing " 1-nitro " and be coupled on chloropyrimide and solve simply with Boc.Work as R ³when being carbon back substituting group, can Suzuki or Sonagashira be utilized to react on 4-halogen (preferably bromine or iodine).If there is ortho position or contraposition nitrogen, Finkelstein can be utilized to react muriate is changed into corresponding iodide.

scheme 4. is prepared for some documents of 1,2,4,5-quaternary benzene synthon of compound of the present invention

By correct R ²---need to do like this---if group inserts above-mentioned synthon and can realize with commercial reagent and not need to synthesize any such R in each case ²part.The R that great majority propose ³group also can be buied with the form that can incorporate them in aniline ring.The very diversified amine of commercially available acquisition, comprise diamines and triamine, this is equally applicable to alcohol, alkoxyl alcohol and amino alcohol, and wherein many have the ring texture be building up to wherein, and SAR unlikely requires the novel synthesis of these parts any.But this is not suitable for the R that more complicated carbon connects ³part, especially propargylamine.Two kinds of general preparations of this compounds are presented in scheme 5.

the preparation of scheme 5. 3-aminobutyne derivative

The quite easy and amine of propargyl chloride (or be acetic ester under copper catalyst exists) is replaced, even if leaving group is tertiary amine (tertiary).Or, under the condition forming imonium ion, directly can react to produce required compound as shown in the second reaction formula with various metallic acetylide with secondary amine process ketone.In this case, acetylene is successfully used to highly selective Sonagashira reaction subsequently, to form the 2-alkynyl arylamines of kind needed for compound of the present invention.

The preparation of method D. enoyl-and alkynes acyl moieties (III)

Some alkylation side chains in the present invention are simple crotonic acid derivates, quite in 4-position or may may have solubilising amine farther.Such side chain has greatly precedent can follow and not need further discussion in the literature.Many in them have too low as the reactive of michael acceptor so that cannot play a role on the aniline being rich in electronics, for these side chains, must make N-aryl substituent not so electron rich therefore lower inactivation a little.Other michael acceptor side chains contain alkynes, and this makes them be the michael acceptor more active than corresponding alkene inherently.Operable much alkynes has greatly precedent to follow in the literature, but describes the synthesis of some specific alkynes in this joint.When not changing electronic property (electronics) of N-aryl moiety, the main policies of activation michael acceptor adds electrophilic halogen atom further.Due to relatively high molecular weight and the lipotropy of the compound based on this support, electrophilic strategy is confined to fluorine atom to be incorporated in michael acceptor, and discuss hereinafter various list-, two-and borontrifluoride α, beta-unsaturated acid is as the synthesis of fragment (III) michael acceptor.The problem done like this is that fluorine is often quite good at activation Michael systems, and in many cases, this michael acceptor must be substituted to be improved reactive by electronics or Space design further and N-aryl must suitable electron rich.Another problem is that these compounds can be E or Z steric isomer mostly, and some preparations can produce more undesired isomer, and great majority can produce E/Z mixture.But usually can manipulate reaction conditions to improve the per-cent of required isomer, and various isolation technique can be used to be separated required isomer, these manipulations are all that those skilled in the art are familiar with.

the document synthesis of scheme 6. 2-and 3-fluoroolefins acid

The most direct method activating this olefin(e) acid ester system for Michael addition is directly placed on alkene carbon by fluorine atom.A fluorine atom on 2-or 3-carbon atom just provides sufficient activation, therefore adds alkyl and double bond is wise slightly to reduce overall activation.Scheme 6 shows some documents preparation of fluoridizing alkene of compound used in the present invention.

Prepare all quite generally for shown first four kinds, and the many different alkyl of tolerable, because they all depend on the reaction of alpha-fluoro ester precursor and carbonyl.Therefore one or two alkyl can be introduced the 3-position of final alkene amide system, significantly to regulate it reactive.In the reaction of top, also illustrate the allylic bromination tendency of these compounds.Acid/ester stage or be coupled to after on aniline with amine displacement allyl bromide 98 have good grounds in this field.In existing literature synthesis, there is not the special preparation of 2-alkyl-3-fluoro laurate or crotonate, but as shown in the reaction of three on the lower, 3,3-bis-substitutional crylic acid derivative has several general synthesis, and a large amount of precedent shows these ester products fully (cleanly) can be hydrolyzed into required carboxylic acid.Triphen phosphino-fluoromethane (triphenylphosphanylfluoromethylidene) (J Org Chem 40,2796 (1975), its seemingly uniquely known in its family member) may be able to methylate with Methyl triflate C-, then for Wittig reaction, although even root (parent) seems also quite to be a problem.But it is reported, with SF ₄in complex mixture, 2 are obtained with appropriate productive rate during process 2-alkyl 'beta '-ketoester, E/Z-mixture (the Zhurnal Organicheskoi Khimii of 3-dialkyl group-3-fluoro acrylic ester, 18,782 (1982)), consider that starting material are cheap, even if do not attempt improving this reaction completely, also can obtain the required quaternary alkene of sufficient quantity, this may be very general.2-alkyl-3-fluoro laurate and crotonate can be prepared as shown in scheme 7.Be separated 1-phenyl-iodide-2-fluoro alkene intermediate and with LDA and boron alkyl acid esters as during the process of catecholborane derivative by Methyl transporters to alpha-carbon, to form vinyl borine.These can use carbon monoxide carboxymethylation to produce a kind of isomer of required compound again under Pd catalysis.

the synthesis of scheme 7. 2-alkyl-3-fluoro laurate and unsaturated ester

。

some preparations of scheme 8. 2-trifluoromethyl chain acid ester

。

some preparations of the 3-trifluoromethyl acrylate ester of scheme 9. monoalkylation

There is several method for the synthesis of 2-trifluoromethyl crotonate and more senior analogue.Some of them are illustrated in scheme 8.First reaction is very meaningful, and because introduces 2,3-double bond when this end of synthesis, by addition or hydrogenation modification 4,5-double bond before carrying out sulfoxide oxidation-elimination, to obtain diversified 3,3-dialkyl group-2-trifluoromethyl acrylamides.

Horner-Wadsworth-Emmons reaction confirmation on trifluorumethylketone is the quite general preparation of this compounds, and this reaction is that main or complete E-is stereoselective.Across acetylenic acid ester (ynoate) addition HI can be complete Z-optionally and do not rely on trifluoromethyl (Tet Letters 36,2469 (1995)), therefore by as shown in the 3rd reaction HI to be added in 3-alkyl propynoic acid, then with trifluoromethyl cuprate (trifluoromethyl cuprate) reagent coupling, the stereoselectivity of second reaction can be reversed simply.By using the Stille coupling of Pd catalysis but not the copper coupling that describes of author, the 4th reaction can be made much general.

Although document seems well not prepare the method for 2-difluoromethyl crotonamide and more higher homologue, owing to may change into very reactive 3,3-difluoropropenes amide isomers by tautomerism, this compounds may not used.But the recent synthesis (J Fluorine Chem 130,682 (2009)) of 3,3-difluoro pyruvate allows to obtain these compounds (J Fluorine Chem 113,177 (2002)) by the approach based on phosphonic acid ester.Difluoromethyl (the J Amer Chem Soc 134 of vinyl halide, 5524 (2012), Angew Chem Int Edn 51,12090 (2012), with this type of reagent by 1, the ability (J Fluorine Chem 111,85 (2001)) of 1-dibromoalkene list difluoromethyl combines and allows their preparations as follows.

the preparation of scheme 10. 2-difluoromethyl chain-2-olefin(e) acid ester derivative

If tautomerization is a problem, crystalline structure shows to have living space in this position to leave 1 for, 1-bis-fluoro ethyl, although this functional group seems that it is not known for being set up at the alpha-position of chain-2-olefin(e) acid ester, but 3,3-bis-fluoro-2-Oxobutyric acid ester is known (J Org Chem 60,5174 (1999)) and can to form these compounds with mode like corresponding trifluoroacetone esters of gallic acid (J Fluorine Chem 113,177 (2002)) and Wittig reagent react.

the synthesis of scheme 11 4,4-difluoro crotonate and squaw weed acid esters (senecoate)

Scheme 11 shows the document synthesis of 4,4-difluoro crotonate and 4,4-difluoro squaw weed acid esters (any one all has the sufficient activity as side chain).But, at least with regard to crotonate, this is too active so that cannot use, this activity can be improved by extending alkyl chain, this can use be easy to get 2 as shown in scheme 12,2-difluoro alkanoates and Claisen condensation, then multistep reduction, or realized by the reaction of formation 2,2-bis-fluoro aldehyde, then Horner-Wadsworth-Emmons.Because Bromodifluoroacetic acid ethyl ester and oxalic acid diethyl ester all easily change into 2,2-bis-fluoroester and aldehyde of more long-chain, easily with alkylogen C-alkylation, the slight modifications of Utilization plan 11 Program can obtain diversified 4,4-difluoro chain acid ester side chains.

some preparations of scheme 12. 4,4-difluoro alkenoic acid

Scheme 13 shows has greatly the governed approach of precedent for the formation of E-3-methyl-4,4-difluoro chain-2-olefin(e) acid, and it is very similar to some chemistry in scheme 9.Such compound is also valuable, because be that disclose in the application all fluoridize minimum activation in michael acceptor.

the preparation of scheme 13. E-3-methyl-4,4-difluoro chain-2-olefin(e) acid

Scheme 14 (a) shows a kind of noticeable reaction, thus two-or three-fluorine crotonate can at low temperatures with 2-Methylpropanedioic acid transesterify 1-and 2-carbon atom to form 2-methyl analogue, this midparent had between crotonate and difluoro crotonate is electrical, but sterically hindered lower than difluoro squaw weed acid esters.Although also non-immediate has precedent to follow in the literature, but pass through at molecule, those molecules as preparation in scheme 12 carry out this conversion, the alkyl group side chain extended is estimated to produce E-2-methyl-4 by Stereoselective, 4-difluoro chain-2-olefin(e) acid ester, this can produce all the other the trisubstituted isomer in this series, and this method is illustrated in scheme 14 (b).

the preparation of scheme 14. 4,4-difluoro tiglate and more higher homologue

At J Med Chem 49, discuss the synthesis of the acetylenic acid side chain that much amino replaces in 1475 (2006), these are incorporated herein by this reference.Described the preparation of amino-3, the 3-dialkyl group propyne derivative of many 3-in this application before.These compounds can as J Med Chem 49, described in 1475 (2006) on acetylene 1-carbon carboxylation to be obstructed but the aminoalkynyl side chain of lyotropy for the preparation of the more space of compound of the present invention.

In scheme 15, describe the synthesis of bromo-2, the 6-bis-fluoro-3-nitropyridines of 5-in two steps.This novel cpd is the very general synthon of the diamino-pyridine of formula (II), and in this scenario exemplified with several different conversion of the pyridine ring of the compound for the preparation of formula (II).Base (J Org Chem 77 is connected with the amine of biaryl part because nitro and bromine can change into respectively by the amination of reduction or copper catalysis; 6908; 7471 (2012); Eur J Org Chem 1854 (2010)), then optionally carry out protection step, the displacement of arbitrary fluorine can be utilized to implant required substituting group.First nucleophile can optionally replace 2-fluorine, but under slightly more powerful condition (forcing conditions), replaces 6-fluorine subsequently, and therefore by selective cementation order, can build this molecule becomes the connection base with biaryl core to make bromine or nitro.By utilize nickel phosphine and phosphine oxide chemistry of complex Selective activation C-F key with various alkyl/aryl metal derivative cross-coupling, further enhancing 5-bromo-2, versatility (the J Org Chem 67 of the fluoro-3-nitropyridine of 6-bis-, 8991 (2002), Org Letters, 14,3316 (2012)).

scheme 15. 3,5-diamino-pyridine connects the synthesis of base.

Except the known compound described above, the starting material during other compounds many as known in the art can be used as synthesizing, and provide in table 1 below and be not intended to form a series of such compound of enumerating of exclusiveness and the reference about them.In many cases, the reference taking passages this compound show except clearly contain those except the also clear chemical conversion being applicable to other compound of the present invention, and skilled person in the art will appreciate that they are broadly applicable to other side of the present invention.

table 1. for the synthesis of other known compounds.

methods for the treatment of

The invention still further relates to methods for the treatment of and purposes, comprise and give compound or pharmaceutically acceptable salt thereof of the present invention that is independent or that combine with other therapeutical agent or negative catalyst.

In one embodiment, the present invention relates to treatment or suppress the method for mammiferous cell proliferation, cell invasion, transfer, apoptosis or vasculogenesis, comprise the compound or pharmaceutically acceptable salt thereof of the present invention giving to treat significant quantity to described Mammals.

In another embodiment, the present invention relates to treatment or suppress the method for mammiferous cell proliferation, cell invasion, transfer, apoptosis or vasculogenesis, comprise and give the compound or pharmaceutically acceptable salt thereof of the present invention with the treatment significant quantity of the second therapeutic combination to described Mammals, wherein the total amount of compound of the present invention and described second therapeutical agent is effectively treated or is suppressed described cell proliferation, cell invasion, transfer, apoptosis or vasculogenesis.

In one embodiment, described second therapeutical agent is the antineoplastic agent being selected from mitotic inhibitor, alkylating agent, metabolic antagonist, embedding microbiotic, growth factor receptor inhibitors, radiation, cell cycle inhibitor, enzyme, topoisomerase enzyme inhibitor, biological response properties-correcting agent, antibody, cytotoxin, antihormone and antiandrogen.

In other embodiments, described cell proliferation, cell invasion, transfer, apoptosis or vasculogenesis are by the member of the erbB family of RTKs, and mainly EGFR, most likely the T790M mutant form of EGFR mediates.

In another embodiment, described cell proliferation, cell invasion, transfer, apoptosis or vasculogenesis be selected from following cancer and be associated: glioblastoma, lung cancer (such as squamous cell carcinoma, nonsmall-cell lung cancer, gland cancer, bronchioalveolar carcinoma (BAC), there is the BAC of focal infiltration, there is the gland cancer of BAC feature, and large cell carcinoma), carcinoma of the pancreas, head and neck cancer (such as squamous cell carcinoma), mammary cancer, colorectal carcinoma, epithelial cancer (such as squamous cell carcinoma), other cancer any of ovarian cancer and prostate cancer and process LAN erbB family member or the carcinogenic Activating mutations body containing erbB family (no matter in tumour whether these protein of process LAN).

Another embodiment of the present invention relates to compound of the present invention, and it is used as medicine, and the suppression being used in particular for treating wherein EGFR and/or sudden change EGFR albumen (such as L858R/T790M EGFR) activity can cause the disease of benefit, as cancer.A further embodiment of the invention relates to the purposes with the medicine of EGFR inhibit activities of compound or pharmaceutically acceptable salt thereof of the present invention for the preparation of the disease and/or the patient's condition, particularly above-listed disease and/or the patient's condition that are used for the treatment of EGFR mediation.

Term " treatment significant quantity " refers to the dosage of the compound of one or more symptoms alleviating subject illness to a certain extent.About cancer therapy, treatment significant quantity refers to the amount with the effect reducing tumor size, suppression (namely slow down or stop) metastases, suppression (namely slow down or stop) tumor growth or tumor invasion and/or alleviate one or more symptom relevant to cancer or symptom to a certain extent.

Cure mainly diagnostician (as those skilled in the art) utilize routine techniques and by observing the result obtained in a similar situation, can easily determine to treat significant quantity.When determining treatment significant quantity (dosage), curing mainly diagnostician and considering many factors, including but not limited to: mammiferous species; Its build, age and general health situation; The disease specific related to; The infringement degree of disease or seriousness; The response of individual patient; The specific compound of administration; Mode of administration; The distinctive bioavailability of drug-delivery preparation; Selected dosage regimen; Concomitant medication; With other correlation circumstance.

Unless otherwise specified, term used herein " treatment " refer to one or more symptoms of the illness that this term is suitable for or the patient's condition or this type of illness or the patient's condition reverse, alleviate, process suppress or prevention.Term " treatment " also refers to the treatment behavior as " treatment " just defined above.Term " treatment " also comprises mammiferous assisting therapy.

" cancer " used herein refers to any pernicious and/or invasive growth or tumour that are caused by abnormal cell growth, comprises the solid tumor of the cell type name forming them, leukemia, bone marrow cancer or lymphsystem cancer.The example of solid tumor includes but not limited to sarcoma and cancer.The example of leukemia includes but not limited to leukemia, lymphoma and myelomatosis.Term " cancer " include but not limited to come from privileged site in body primary carcinoma, be diffused into the recurrence after alleviation of the metastatic carcinoma at other position of health, original primary carcinoma and Second primary tumors from its zero position---have the new of the people of dissimilar cancer history to send out primary carcinoma before.

In another embodiment, the invention provides the method for antiproliferative effect, comprise and cell is contacted with the compound or pharmaceutically acceptable salt thereof of the present invention of the amount of effective antiproliferative effect.In another embodiment, the invention provides and bring out apoptotic method, comprise and cell is contacted with the compound as herein described effectively bringing out apoptotic amount.

" contact " instigates compound of the present invention or pharmacologically acceptable salt and expression sudden change EGFR or the cell of one of other target kinase playing transformation puts together to make this compound can affect EGFR or other kinase whose activity directly or indirectly in particular cell types.Contact (namely at live body, such as but not limited in mouse, rat or rabbit) can realize in external (namely in artificial environment, such as but not limited in test tube or substratum) or body.

In some embodiments, described cell in clone, as in cancerous cell line.In other embodiments, described cell is in tissue or tumour, and this tissue or tumour can in Mammals (comprising people) bodies.

Can by described compound delivery to any method of action site being realized the administration of compound of the present invention.These methods comprise oral route, intraduodenal route, parenteral injection (comprising vein, subcutaneous, intramuscular, Ink vessel transfusing or infusion), local and rectal administration.

Dosage regimen can be regulated to provide response needed for the best.Such as, single bolus (bolus) can be given, can in time through giving several divided dose or can as treat situation emergency indicated by proportional reduction or increasing dose.Consistent with dosage for ease of administration, especially favourable with unit dosage preparation parenteral compositions.

Unit dosage used herein refers to the discrete unit of the physics of the single dose being suitable as Mammals to be treated; Constituent parts contains active compound that be combined with required pharmaceutical carrier, that produce the predetermined amount of required therapeutic action as calculated.The specification of unit dosage of the present invention depends on and the unique property directly depending on (a) this chemotherapeutics and the particular treatment that will realize or prophylactic effect, and (b) is used for the treatment of the inherent limitations in the compounding art of this type of active compound of individual sensitivity.

Suitable dosage becomes with the type of the patient's condition for the treatment of and severity, and can comprise single dose or multi-agent.Cure mainly diagnostician to understand any specific Mammals; should adjust concrete dosage regimen in time according to the professional judgement of the personnel of individual need and execution or the administration of supervision group compound, the dosage range is herein only exemplary and is not intended to the scope or the practice that limit composition required for protection.Such as, dosage can be adjusted according to pharmacokinetics or pharmacodynamic parameter (clinical effect may be comprised, as toxic effect and/or laboratory evaluation).Therefore, the present invention includes the Intra-patient dose escalation as technician determines.The suitable dosage of chemotherapeutics and the determination of dosage regimen are known in association area and are understood to be in the limit of power of the technician learning instruction content disclosed herein.

The effective dose of compound of the present invention can be determined by comparing their external activity and the activity in vivo in animal model.Need the amount of the compound that is used for the treatment of or its active salt or derivative not only to become with selected specific salts, also become, finally by attending doctor or clinicist's tailoring with the character of the patient's condition of route of administration, treatment and the age of patient and physical appearance.

Compound of the present invention can deliver medicine to patient with the dosage level of every day about 0.1 to about 2,000 milligrams.For the normal adult of about 70 kilograms of body weight, every day, the dosage of per kilogram of body weight about 0.01 to about 10 milligrams was preferred.But concrete dosage used can change.Such as, dosage can be depending on many factors, comprises the requirement of patient, the severity of the patient's condition for the treatment of and the pharmacologically active of compound used therefor.Well known to a person skilled in the art to the determination of the optimal dose of particular patient.In some cases, dosage level lower than above-mentioned range lower limit may be more than sufficient, and in other situations, can larger dose be used and harmful side effect can not be caused, precondition is that such larger dose is first divided into several smaller dose for whole day administration.

Pharmaceutical composition of the present invention can be prepared, pack or sell with the form of in bulk, single unitary dose or multiple unitary dose." unitary dose " used herein is the discrete amount of the pharmaceutical composition of the activeconstituents comprising predetermined amount.The amount of activeconstituents is generally equal to and gives the individual dosage of activeconstituents or the mark easily of this dosage, the half or 1/3 of such as this dosage.

Activeconstituents, pharmaceutically acceptable carrier and the relative quantity of any supplementary component in pharmaceutical composition of the present invention can change, and depend on that the identity of the individuality for the treatment of, build and physical appearance also depend on the route of administration of said composition further.Such as, said composition can comprise the activeconstituents of 0.1% to 100% (w/w).

Being applicable to pharmaceutical composition sending compound of the present invention and preparation method thereof is that those skilled in the art are apparent.Such composition and method of making the same is found in such as ' Remington's Pharmaceutical Sciences', the 19th edition (Mack Publishing Company, 1995), the full text is incorporated herein by reference for its disclosure.

Compound of the present invention can be taken orally.Oral administration may relate to be swallowed, and to make this compound enter gi tract, maybe can adopt oral cavity or sublingual administration, this compound directly enters blood flow from oral cavity thus.Be applicable to the preparation of oral administration and comprise solid preparation, as tablet, capsule containing particle, liquid or powder, lozenge (comprising liquid filling), masticatory, many-and nanometer-microgranules, gelifying agent, sosoloid, liposome, film (comprising mucous membrane paster), ovum pill (ovules), sprays and liquid preparation.

Liquid preparation comprises suspension, solution, syrup and elixir.This type of preparation can be used as the filler of soft capsule or hard capsule, and generally includes carrier, such as water, ethanol, polyoxyethylene glycol, propylene glycol, methylcellulose gum or suitable oil, and one or more emulsifying agents and/or suspending agent.Also liquid preparation can be prepared by the reconstruct of solid.

In the formulation that instant, the speed of also can be used on compound of the present invention collapses, the Expert Opinion in Therapeutic Patents of such as Liang and Chen, 11 (6), describe in 981986 (2001) those, the full text is incorporated herein by reference for its disclosure.

For Tabules, according to dosage, medicine can form 1 % by weight to 80 % by weight of formulation, more generally forms 5 % by weight to 60 % by weight of formulation.Except medicine, tablet is usually also containing disintegrating agent.The example of disintegrating agent comprises primojel, Xylo-Mucine, calcium carboxymethylcellulose, cross-linked carboxymethyl cellulose is received, Crospovidone, polyvinylpyrrolidone, methylcellulose gum, Microcrystalline Cellulose, low alkyl group replace hydroxypropylcellulose, starch, pregelatinized starch and sodium alginate.Usually, disintegrating agent accounts for 1 % by weight to 25 % by weight of formulation, and preferably 5 % by weight to 20 % by weight.

Usual use tackiness agent is to give tablet formulation cohesion.Suitable tackiness agent comprises Microcrystalline Cellulose, gelatin, sugar, polyoxyethylene glycol, natural and synthetic gum, polyvinylpyrrolidone, pregelatinized starch, hydroxypropylcellulose and Vltra tears.Tablet also can contain thinner, as lactose (monohydrate, spray-dired monohydrate, anhydrous etc.), N.F,USP MANNITOL, Xylitol, dextrose, sucrose, Sorbitol Powder, Microcrystalline Cellulose, starch and calcium phosphate dibasic dihydrate.

Tablet also optionally can comprise tensio-active agent, as sodium lauryl sulphate and tween 80, and glidant, such as silicon-dioxide and talcum.When it is present, tensio-active agent can account for 0.2 % by weight to 5 % by weight of tablet, and glidant can account for 0.2 % by weight to 1 % by weight of tablet.

Tablet usually also containing lubricant, as the mixture of Magnesium Stearate, calcium stearate, Zinic stearas, sodium stearyl fumarate and Magnesium Stearate and sodium lauryl sulphate.Lubricant accounts for 0.25 % by weight to 10 % by weight of tablet usually, preferably accounts for 0.5 % by weight to 3 % by weight of tablet.

Other possible composition comprises antioxidant, tinting material, seasonings, sanitas and odor mask.

Tablet mixture can direct pressing or by roll-in system to form tablet.Or, the part of tablet mixture or mixture can before compressing tablet wet granulation, non-slurry pelletizing or melt pelletization, melt solidifying or extrude.Final preparation may comprise one or more layer, and can be dressing or non-dressing; It even can incapsulate.

H. Lieberman's and L. Lachman, " Pharmaceutical Dosage Forms:Tablets, Vol. 1'', Marcel Dekker, N.Y., N.Y. discuss the preparation of tablet in 1980 (ISBN 0-8247-6918-X).

The above-mentioned preparation of various administration fashion discussed above can be mixed with quick-release and/or tune releases (modified release).Release formulation is adjusted to comprise delayed release, sustained release, pulse release, controlled release, Targeting delivery and sequencing release.In U.S. Patent No. 6,106, describe the suitable tune release formulation for the object of the invention in 864.People such as Verma, Pharmaceutical Technology On-line, the details of other suitable release tech visible in 25 (2), 1-14 (2001), such as high energy dispersions and infiltration and coated bead.Describe in WO 00/35298 and use Chewing gum (chewing gum) to realize controlled release.

Compound of the present invention also can directly be administered in blood flow, in muscle or in internal organs.Suitable Parenteral administration mode comprise intravenously, intra-arterial, intraperitoneal, in sheath, in ventricle, in urethra, in breastbone, encephalic, intramuscular and subcutaneous.Suitable Parenteral administration device comprises pin (comprising micropin) syringe, needleless injector and infusion techniques.

Parenteral formulation is the aqueous solution normally, it can contain vehicle, as salt, carbohydrate and the buffer reagent pH value of 3 to 9 (preferably to), but, for some purposes, they are more suitable for being mixed with sterile non-aqueous solution or will with suitable vehicle, as the dried forms that aseptic apirogen water is combined.

Use well known to a person skilled in the art standard pharmaceutical techniques, easily can realize parenteral formulation preparation aseptically (such as passing through freeze-drying).

Suitable compounding process can be used, such as, add solubilizing agent to improve the solubleness of the compound of formula (I) used in parenteral solutions preparation.

Parenteral administration preparation can be mixed with that quick-release and/or tune release.Therefore, compound of the present invention can be mixed with solid, semisolid or thixotropic fluid using as implanted bank agent (depot) administration providing the tune of active compound to release.The example of this type of preparation comprises the support and poly-(glycollide-dl-rac-Lactide) or PGLA microballoon that scribble medicine.

Compound of the present invention can with solvable macromole body, combine as cyclodextrin and suitable derivative thereof or containing the polymkeric substance of polyoxyethylene glycol with solubleness, dissolution rate, taste masking effect, bioavailability and/or the stability improved when they use with any above-mentioned mode of administration.Such as, Drug-cyclodextrin complexes it is found that and usually can be used for most of formulation and route of administration.Inclusion and non-inclusion complex can be used.As the replacement scheme with medicine direct combination, cyclodextrin can be used as supplementary additive, namely as carrier, thinner or solubilizing agent.Be most commonly used to these purposes be α-, β-and γ-cyclodextrin, the example is found in international patent application Nos. WO 91/11172, WO 94/02518 and WO 98/55148.

Term " combination therapy " refers to compound of the present invention together with at least one medication or medicament in succession or simultaneously administration.Combination therapy is included in formulation separately or in same medicine preparation, uses compound of the present invention and other therapeutical agent.Compound of the present invention can use with one or more therapeutic agent (simultaneously, in succession or respectively administration).

In one embodiment of the invention, it is anti-angiogenic agent (such as stoping tumour to form the reagent of new blood vessel) with the carcinostatic agent of compound of the present invention and pharmaceutical composition conbined usage as herein described.The example of anti-angiogenic agent comprises such as VEGF inhibitor, VEGFR inhibitor, TIE-2 inhibitor, PDGFR inhibitor, angiogenin inhibitor, PKCI3 inhibitor, CQX-2(cyclo-oxygenase II) inhibitor, integrin (α-v/ β-3), MMP-2(MMP2) inhibitor and MMP-9(GELB) inhibitor.Preferred anti-angiogenic agent comprises Sutent (SutenFM), rhuMAb-VEGF (Avastin ^tM) and Axitinib (AG 13736).

Additional anti-angiogenic agent comprises PTK787 (CGP 79787), Xarelto (Nexavar ^tM), Macugen (Macugen ^tM), ZD6474 (Zactima ^tM), PF-0337210(Pfizer), SU 14843(Pfizer), AZD 2171(AstraZeneca), Lucentis (Lucentis TM), Neovastat ^tM(AE 941), tetrathiomolybdate (tetrathiomolybdata) (Coprexa ^tM), AMG 706(Amgen), VEGF Trap(AVE 0005), CEP 7055(Sanofi-Aventis), XL 880(Exelixis), Telatinib (BAY 57-9352) and CP-868,596(Pfizer).

Celecoxib (Celebrex can be comprised with other example of the anti-angiogenic agent of compound of the present invention and pharmaceutical composition conbined usage as herein described ^tM), Parecoxib (Dynastat ^tM), deracoxib (deracoxib) (SC 59046), lumiracoxib (Preige ^tM), Valdecoxib (Bextra ^tM), rofecoxib (Vioxx ^tM), Ailamode ( careram ^tM ),iP 751(lnvedus), SC-58125(Pharmacia) and Etoricoxib (Arcoxia TM).Other anti-angiogenic agent comprises exisulind (Aptosyn TM), sasapyrin (AmigesicTM), diflunisal (Dolobid ^tM), Ibuprofen BP/EP (Motrin ^tM), Ketoprofen (Orudis ^tM), nabumetone (Relafen ^tM), piroxicam (FeldeneTM), Naproxen Base (AIeveTM, Naprosyn TM), diclofenac (Voltaren TM), INDOMETHACIN (Indocin ^tM), sulindac (ClinoriI ^tM), tolmetin (tolmetin) (Tolectin ^tM), R-ETODOLAC (Lodine ^tM), ketorolac (ToradoI ^tM) and Taisho) (Daypro ^tM).Other anti-angiogenic agent comprises ABT 510(Abbott), A Leisita (apratastat) (TMI 005), AZD 8955(AstraZeneca), incyclinide(Metastat ^tM) and PCK 3145(Procyon).Other anti-angiogenic agent comprises A Quting (Neotigason TM), plitidepsin(aplidine TM), cilengtide(EMD 121974), Combretastatin A-4 4(CA4P), fenretinide (fenretinide) (4 hPR),halofuginone hydrobromide (Tempostatin ^tM), Panzem ^tM(methoxyestradiol), PF-03446962(Pfizer), Rui Masita ( bMS275291), appropriate rope monoclonal antibody (Removab is blocked ^tM), Revlimid (Revlimid ^tM), squalamine (EVIZONTM), Thalidomide (Thalomid ^tM), Ukrain ^tM(NSC 631570), Vitaxin ^tM(MEDI 522) and Zoledronic acid (Zometa TM).

In another embodiment, this carcinostatic agent is so-called signal transduction inhibitor (Molecular regulator of the primary process of such as inhibitory control Growth of Cells, differentiation and survival is in the mode of intracellular communication).Signal transduction inhibitor comprises small molecules, antibody and antisense molecule.Signal transduction inhibitor comprises such as kinase inhibitor (such as tyrosine kinase inhibitor or serine/threonine kinase inhibitor) and cell cycle inhibitor.Signal transduction inhibitor more specifically comprises such as farnesyl protein transferase inhibitor, EGF inhibitor, ErbB-1(EGFR), ErbB-2, pan erb, IGF1 R inhibitor, MEK, c-Kit inhibitor, FLT-3 inhibitor, K-Ras inhibitor, Pl3 kinase inhibitor, JAK inhibitor, STAT inhibitor, Raf kinase, Akt inhibitor, mTOR inhibitors, P70S6 kinase inhibitor, WNT pathway inhibitor and so-called many targets kinase inhibitor.Preferred signal transduction inhibitor comprises Gefitinib (Iressa ^tM), Cetuximab (Erbitux ^tM), erlotinib (Tarceva ^tM), Herceptin (Herceptin ^tM), Sutent (Sutent ^tM) and imatinib (Gleevec ^tM).

BMS 214662(Bristol-Myers Squibb can be comprised with other examples of the signal transduction inhibitor of compound of the present invention and pharmaceutical composition conbined usage as herein described), Luo Nafani (Sarasar ^tM), pelitrexol(AG 2037), horse trastuzumab (EMO 7200), Buddhist nun's trastuzumab (TheraCIM h-R3 ^tM), Victibix (Vectibix ^tM), ZD6474 (Zactima ^tM), pazopanib (SB 786034), ALT 110(Alteris Therapeutics), BIBW 2992(Boehringer Ingelheim) and Cervene ^tM(TP 38).Other example of signal transduction inhibitor comprises PF-2341 066(Pfizer), PF-299804(Pfizer), card is how for Buddhist nun, handkerchief trastuzumab (Omnitarg ^tM), lapatinibditosylate (Tycerb ^tM), training profit for Buddhist nun (EKB 569), miltefosine (Miltefosin ^tM), BMS 599626(Bristol-Myers Squibb), Lapuleucel-T(Neuvenge ^tM), NeuVax ^tM(E75 cancer vaccine), Osidem ^tM, wood profit for Buddhist nun (TAK-165), Victibix (Vectibix ^tM), lapatinibditosylate (Tycerb ^tM), training profit is for Buddhist nun (EKB 569) and handkerchief trastuzumab (Omnitarg ^tM).Other example of signal transduction inhibitor comprises ARRY 142886(Array Biopharm), everolimus (Certican ^tM), Zuo Tamosi (Endeavor ^tM), CCI-779 (temsirolimus) (ToriseI ^tM) and AP 23573(ARIAO).In addition, other signal transduction inhibitor comprises XL 647(Exelixis), Xarelto (Nexavar ^tM), LE-AON(Georgetown University) and GI-4000(Globelmmune).Other signal transduction inhibitor comprises ABT 751(Abbott), alvocidib(Flavopiridol), BMS 387032(Bristol Myers), EM 1421(Erimos), N-(the chloro-1H-indoles of 3--7-base)-Isosorbide-5-Nitrae-benzene disulfonic acid amide (indisulam) (E 7070), seliciclib(CYC 200), BIO 112(Onc Bio), BMS 387032(Bristol-Myers Squibb), PO 0332991(Pfizer) and AG 024322(Pfizer).

In the signal transduction inhibitor that can use with agents of the present invention, be considered to especially meaningful for erlotinib, Gefitinib, lapatinibditosylate, Conmana, Ah method for other erbB group inhibitor of Buddhist nun, HKI-272, peletinib and dacomitinib.All these compounds all have enough wild-type erbB kinase inhibiting activities to have (mechanism-based) dose-limiting toxicity based on mechanism, but can show good clinical activity with tolerance administration.The tumour that one of their main weakness is to respond very well these medicaments tends to have erbB sudden change, and described sudden change makes this tumour to this inhibitor quite sensitive, but with the second suddenly change in conjunction with time, tend to make this tumour tolerate very much these reagent.Discuss the selective pressure accelerating this process above.The main resistant mutants of targeting compounds of the present invention, and because they have pole low activity to wild-type enzyme, (mechanism based) toxicity based on mechanism significantly can not be increased.But, under they can make the double mutant in evolution be in the selection inferior position identical with original sensitive mutant, therefore greatly slow down or the appearance of persister may be prevented completely.Therefore, this combination is verified very useful clinically.

The present invention considers that compound of the present invention uses together with classical antineoplastic agent.Classical antineoplastic agent comprises hormone regulator as hormone, hormone antagonist, Androgen receptor agonists, androgen antagonist and anti-estrogen therapy agent, histon deacetylase (HDAC) (HOAC) inhibitor, gene silencing agent or gene activator, rnase, proteosomics, topoisomerase I inhibitor, camptothecin derivative, Topoisomerase II inhibitors, alkylating agent, metabolic antagonist, poly-(AOP-ribose) polysaccharase-1(PARP-1) inhibitor, Antitubulin, microbiotic, the spindle poison of plant origin, iridium-platinum complex, gene therapeutic agents, antisense oligonucleotide, blood-vessels target agent (VTAs) and Statins.

Velcade(Velcade is comprised) with the example of the antineoplastic agent of compound conbined usage of the present invention, 9-aminocamptothecin, Belotecan, camptothecine, Diflomotecan, edotecarin, Exatecan (Daiichi), gefitinib, 10-hydroxycamptothecine, irinotecan HCI(Camptosar), lurtotecan, Orathecin(Rubitecan, Supergen), topotecan, camptothecine, 10-hydroxycamptothecine, 9-aminocamptothecin, irinotecan, edotecarin, topotecan, aclarubicin, Zorubicin, amonafide, amrubicin, anthracycline (annamycin), daunorubicin, Dx, elsamitrucin, epirubicin, Etoposide, idarubicin, galarubicin, hydroxyurea, Nemorubicin, mitoxantrone (mitoxantrone), pirarubicin, China fir fine jade (pixantrone), Procarbazine, butterfly mycin, sobuzoxane, he moors glycosides (tafluposide) by fluorine, valrubicin, Zinecard(dexrazoxane), mustargen N-oxide compound, endoxan, altretamine, AP-5280, apaziquone, brostallicin, bendamustine, busulfan, carboquone, carmustine, Chlorambucil, Dacarbazine, estramustine, fotemustine, glufosfamide, ifosfamide, lomustine, Mafosfamide, mustargen (mechlorethamine), melphalan, mitobronitol, mitolactol, ametycin, mitoxantrone, nimustine, ranomustine, Temozolomide, the alkylated compound of thio-tepa and platinum coordination, as cis-platinum, Paraplatin(carboplatin), eptalatin, lobaplatin, S 254, Eloxatin(oxaliplatin, Sanofi), U-9889, Satraplatin (satrplatin) and combination thereof.

The present invention also considers compound of the present invention and dihydrofolate reductase inhibitor (as methotrexate and trimetrexate glucuronate), purine antagonist is (as Ismipur riboside, mercaptopurine, 6-Tioguanine, CldAdo, Clofarex (Clolar), fludarabine, Nelzarabine and Raltitrexed), Pyrimidine antagonists (as 5 FU 5 fluorouracil), Alimta(pemetrexed disodium (premetrexed disodium)), capecitabine (Xeloda TM), cytosine arabinoside, Gemzar ^tM(gemcitabine), Tegafur, doxifluridine, carmofur, cytosine arabinoside (comprises octadecyl phosphoric acid salt (ocfosfate), phosphoric acid salt stearate, slowly-releasing and liposomal form), enocitabine, 5-azacitidine (Vidaza), Decitabine and ethynylcytidine) and other metabolic antagonist as eflornithine, hydroxyurea, formyl tetrahydrofolic acid, Nolatrexed (Thymitaq), 3-aminopyridine-2-formaldehyde-thiosemicarbazone (triapine), trimetrexate and N-(5-[N-(3, 4-dihydro-2-methyl-4-oxoquinazolin-6-ylmethyl)-N-methylamino]-2-thenoyl)-Pidolidone and combination use together.

With compound of the present invention, other example of the classical antitumor cell toxin agent used in the conjoint therapy of optional and one or more other medicaments comprises taxol albumin bound injecting particles suspension (Abraxane) (Abraxis BioScience, Inc.), Ba Tabulin (Batabulin) (Amgen), Vinflunine (Bristol-Myers Squibb Company), dactinomycin, bleomycin, ametycin, neocarzinostatin (Zinostatin), vinealeucoblastine(VLB), vincristine(VCR), vindesine, vinorelbine (Navelbine), docetaxel (Taxotere), Ao Tasai, taxol (comprising Taxoprexin, a kind of DHA/ taxol-conjugate), cis-platinum, carboplatin, S 254, oxaliplatin (Eloxatin), Satraplatin, irinotecan (Camptosar), capecitabine (Xeloda), oxaliplatin (Eloxatin), docetaxel alitretinoin (Taxotere alitretinoin), Canfosfamide(Telcyta ^tM), DMXAA(Antisoma), Ibandronic acid, L-ASP, pegaspargase (Oncaspar ^tM), Efaproxiral (Efaproxyn ^tM-radiotherapy)), bexarotene (Targretin ^tM), Tesmilifene, Theratope ^tM(Biomira), vitamin A acid (Vesanoid ^tM), Win-59075 (Trizaone ^tM), motexafin gadolinium (Xcytrin ^tM) Cotara ^tM(mAb) and NBI-3001(Protoxb Therapeutics), polyglutamic acid-paclitaxel (Xyotax ^tM) and combination.

Embodiment

abbreviation

DMSO methyl-sulphoxide

DTT dithiothreitol (DTT)

ATP Triphosaden

EDTA ethylenediamine tetraacetic acid (EDTA)

Ki enzyme level constant

DMEM Dulbecco's improves Eagle substratum

NCS new-born calf serum

PBS phosphate buffered saline (PBS)

PMSF phenylmethanesulfonyl fluoride

ELISA enzyme-linked immunosorbent assay

IgG immunoglobulin G

FBS foetal calf serum

BDNF Brain Derived Neurotrophic Factor.

the synthesis of compound of the present invention

。

scheme 16. 2-(3-but-2-enamides base/penta-2-alkene amide group-4-fluoroanilino)-4-heteroaryl pyrimidine a-E synthesis.

Embodiment 1. compound asynthesis

As described in WO 2008/137027, containing anhydrous K ₂cO ₃dMF in fluoro-1, the 5-dinitrobenzene of 1 equivalents of methanol process 2 ,-two, then with V-Brite B, the selective reduction of 2-anisyl nitro is become amine, and chromatographic separation provides the fluoro-2-methoxyl group of 4--5-N-methyl-p-nitroaniline.As described in WO 2013/014448, in containing the hot amylalcohol of tosic acid, produce triaryl support with 3-(2-chloropyrimide-4-base) pyrazolo [1,5-a] pyridine condensation.With Raney's nickel or Fe/AcOH reduction nitro, newly formed amine uses 4-bromine crotonyl chloride acidylate as described in US 6297258 at 0 DEG C in THF.Compound is completed with N methyl piperazine original position displacement 4-bromine as described in US 6297258 asynthesis.

Embodiment 2. compound bsynthesis

Use the KRuIO of Polymer-supported ₄or known 3-(1-azetidinyl) propyl alcohol is oxidized to corresponding aldehyde by other oxidation well known by persons skilled in the art.With carboxymethyl Wittig reagent react, then produce 5-(1-azetidinyl) penta-2-olefin(e) acid with LiOH saponification, it changes into corresponding chloride of acid with oxalyl chloride, and with the triaryl amine amination described in embodiment 1, to produce compound b.

Embodiment 3. compound csynthesis

Make the fluoro-2-of the 4-described in embodiment 1 methoxyl group-5-N-methyl-p-nitroaniline with the chloro-4-of 2-(N-skatole-3-base) pyrimidine condensation and nitroreduction is become corresponding amine, and the 4-bromine crotonoyl amination triaryl amine support needed for producing with the coupling of 4-bromine crotonyl chloride under condition described in embodiment 1.Compound is completed with two (2-hydroxyethyl) amine displacement bromine under the condition described in US 6297258 csynthesis.

Embodiment 4. compound dsynthesis

This synthesis is identical with the synthesis of Compound C, just in the end in a step, carries out bromine displacement to produce compound with 4-(piperidin-1-yl) piperidines d.

Embodiment 5. compound esynthesis

This synthesis is identical with the synthesis of Compound C, just in the end in a step, carries out bromine displacement to produce compound with piperidines e.

scheme 17. 2-(3-but-2-enamides base anilino)-4-heteroaryl pyrimidine f-I synthesis.

Embodiment 6. compound fsynthesis

Make commercially available 4-nitro-o-toluidine (R=H) and 2-ethyl-5-N-methyl-p-nitroaniline (R=Me) and the chloro-4-of 2-(N-skatole-3-base) pyrimidine condensation, in this case nitroreduction is become corresponding amine, and the 4-bromine crotonoyl amination triaryl amine support needed for producing with the coupling of 4-bromine crotonyl chloride under condition described in embodiment 1.As R=H, under the condition described in US 6297258, complete compound with piperidines displacement bromine fsynthesis.

Embodiment 7. compound gsynthesis

Bromine crotonamide (wherein R=CH as described in example 6 above ₃) complete compound with the reaction of 3-hydroxy azetidine under the condition described in US 6297258 gsynthesis.

Embodiment 8. compound hsynthesis

As described in WO 2013/014448,2-methoxyl group-5-N-methyl-p-nitroaniline and 3-(2-chloropyrimide-4-base) pyrazolo [1,5-a] pyridine are producing suitable triaryl support containing the condensation in the hot amylalcohol of tosic acid.Nitroreduction is become corresponding amine, and the 4-bromine crotonoyl amination triaryl amine support needed for producing with the coupling of 4-bromine crotonyl chloride under condition described in embodiment 1.With 1-methyl octahydro pyrrolo-[3,4-under the condition described in US 6297258 b] pyrroles replaces bromine and complete compound hsynthesis.

Embodiment 9. compound isynthesis

This synthesis and compound hsynthesis identical, just in the end in a step, carry out bromine displacement to produce compound with 2-methyl-2,7-diazaspiracyclic [4.4] nonane i.

scheme 18. 2-(3-but-2-enamides base-4-aminoalkynyl anilino)-4-heteroaryl pyrimidine j-M synthesis.

Embodiment 10. compound jsynthesis

By the bromo-2-of di-nitrated preparation 4-fluoro-1 of 3-bromofluorobenzene, 5-dinitrobenzene, fluorine is replaced by hydroxide ion selectivity, then as (J Org Chem 56,5958 (1991) and ChemMedChem 6,1411 (2011) described V-Brite B selective reduction ortho position nitros.Neighbour has methylated for the synthesis of the crucial aniline of scheme 18 compound; its as described in WO 2013/014448 in containing the hot amylalcohol of tosic acid with the chloro-4-of 2-(N-skatole-3-base) pyrimidine coupling, then with the protection of Boc acid anhydride to produce suitable triaryl support.Utilize Sonagashira to react 4-methyl isophthalic acid-(3-methyl fourth-3-alkynyl) piperazine is coupled in bromaniline part, in acetic acid, with iron, nitroreduction is become amine, optionally add other acid to remove Boc protecting group.By completing compound with crotonyl chloride acylated aniline amino jsynthesis.

Embodiment 11. compound the synthesis of K

Synthesize crucial biaryl bromaniline as described in example 10 above, then utilize Sonagashira to react and N-propargyl pyrroles is coupled on it.Nitroreduction as described in example 10 above also removes Boc group, with 4-bromine crotonyl chloride by this amine acidylate, and replaces bromine to complete compound with dimethylamine as described in US 6297258 kpreparation.

Embodiment 12. compound lsynthesis

Prepare the bromo-2-methoxyl group of 4--5-N-methyl-p-nitroaniline as described in example 10 above, then as described in WO 2013/014448 in containing the hot amylalcohol of tosic acid with 3-(2-chloropyrimide-4-base) pyrazolo [1,5-a] pyridine coupler, produce suitable triaryl support.Then utilize Sonagashira to react N-propargyl morpholine is coupled on it.Reduce nitro to produce corresponding amine with iron and acetic acid, and it uses 4-bromine crotonyl chloride acidylate, and replaces bromine to complete compound with piperazine as described in US 6297258 lpreparation.

Embodiment 13. compound msynthesis

Synthesize crucial biaryl bromaniline as described in example 12 above, then utilize Sonagashira to react and 4-methyl isophthalic acid-(3-methyl fourth-3-alkynyl) piperazine is coupled on it.Reduce nitro to produce corresponding amine with iron and acetic acid, and it uses classical peptide coupling condition 4-(methoxy ethoxy) β-crotonic acid (being made up of 4-bromocrotonic acid and methyl cellosolve acid sodium (sodium methoxyetholate)) acidylate to complete compound msynthesis.

scheme 18. 2-(3-but-2-enamides base-4-fluoroalkyl/alkynyl anilino)-4-heteroaryl pyrimidine n-P synthesis.

Embodiment 14. compound nsynthesis

Prepare 2-(the bromo-2-methoxyl group of 4--5-oil of mirbane amido)-4-(1-(tert-butoxycarbonyl) indol-3-yl) pyrimidine as described in example 10 above.Then commercially available 3,3,3-trifluoropropyne utilize Sonagashira to react and form trifluoropropyl ethynylation (trifluoroptpopynated) aniline through bromide coupling.Then use iron, with other reductive agent that acetic acid or those skilled in the art are familiar with, nitroreduction is become corresponding amine, it also replaces bromine to complete compound with piperazine by 4-bromine crotonyl chloride acidylate as described in US 6297258 npreparation.

Embodiment 15. compound osynthesis

Second from the bottom kind of compound of embodiment 14 uses the process of N, N, N'-trimethylammonium quadrol to complete compound as described in US 6297258 opreparation.

Embodiment 16. compound psynthesis

Prepare 2-(the bromo-2-methoxyl group of 4--5-oil of mirbane amido)-4-(indol-3-yl) Kui Linpyrimido quinoline NaH/ methyl-iodide as described in example 10 above indole nitrogen is methylated.Then this bromide uses classical Heck carbonylation conditions carbonylation, and uses DAST that this aldehyde is changed into difluoromethyl.With acetic acid or Raney's nickel, nitroreduction is become corresponding amine with iron, and pass through as described in US 6297258 with 4-bromine crotonyl chloride acidylate and the synthesis completing compound P with two (2-hydroxyethyl) amine displacement bromide.

scheme 19. 2-(3-(2-(fluorine/trifluoromethyl) but-2-enamides base) anilino)-4-heteroaryl pyrimidine q-T synthesis.

Embodiment 17. compound qsynthesis

With dimethylamine process 2-(the fluoro-2-methoxyl group of 4--5-oil of mirbane amido)-4-(1-skatole-3-base) pyrimidine (WO 2013/014448), to replace fluorine atom.Then nitroreduction is become corresponding amine; and with E; (the acid preparation of the bromo-2-of 4-fluoro-3-methyl butene acyl chlorides; Eur J Chem 2603 (1998), and change into chloride of acid by oxalyl chloride or other method well known by persons skilled in the art) use the condition acidylate described in US 6297258.Then this allyl bromide 98 is replaced to produce compound with tetramethyleneimine q.

Embodiment 18. compound rsynthesis

With dimethylamine process 3-(2-(the fluoro-2-methoxyl group of 4--5-oil of mirbane amido) pyrimidine-4-yl) pyrazolo [1,5-a] pyridine (WO 2013/014448), to replace fluorine atom.Then nitroreduction is become corresponding amine, and use the E described in condition embodiment 17 described in US 6297258, the bromo-2-of 4-fluoro-3-methyl butene acylated with acid chloride.Then this allyl bromide 98 is replaced to produce compound with 3-methoxypyrrolidin r.

Embodiment 19. compound ssynthesis

With (the process (synthesis 122 (1975)) of respective acids of 2-fluoro-3-methyl but-2-ene acyl chlorides under the condition described in US 6297258; use oxalyl chloride) by 3-(2-(5-amino-4-(N-methyl-N-(2; N; N-dimethyl aminoethyl)-2-methoxy-pllenylamine base) pyrimidine-4-yl)-4; 5; 6,7-tetrahydro-pyrazole also [1,5-a] pyridine (WO 2013/014448) acidylate produces compound s.

Embodiment 20. compound tsynthesis

With 3-dimethylamino tetramethyleneimine process 3-(2-(the fluoro-2-methoxyl group of 4--5-oil of mirbane amido) pyrimidine-4-yl)-4,5,6,7-tetrahydro-pyrazoles also [1,5-a] pyridine pyrimidine (WO 2013/014448), to replace fluorine atom.Then nitroreduction is become corresponding amine, and with 2-fluoro-3-methyl but-2-ene acylated with acid chloride (see embodiment 19) to produce compound T.

scheme 20. 2-(3-(2-(fluorine/trifluoromethyl) but-2-enamides base) anilino)-4-heteroaryl pyrimidine u-X synthesis.

Embodiment 21. compound usynthesis

As described in example 17 above 2-(4-fluoro-2-methoxyl group-5-oil of mirbane amido)-4-(1-skatole-3-base) pyrimidine (WO 2013/014448) is changed into corresponding dimethylaminoaniline.This is subsequently at coupling agent, and as EDAC/HOBT or HATU or pyBOP exist lower and Z-2-, (condensation of the fluoro-4-methylpent of diethylamino-2--2-olefin(e) acid is to produce compound u.Z-2-(the fluoro-4-methylpent of diethylamino-2--2-olefin(e) acid is prepared in three steps by 2-bromine isobutyric aldehyde---and with diethylamine displacement (Dalton Trans 5945 (2008)) in ether, then there is Horner-Wadsworth-Emmons with 2-fluorine phosphoryl acetic ester (2-fluorophosphoinoacetate) and react (Synthesis 427 (2002)) and saponification.

Embodiment 22. compound vsynthesis

2-(the fluoro-2-methoxyl group of 4--5-oil of mirbane amido)-4-(1-skatole-3-base) pyrimidine (WO 2013/014448) and N methyl piperazine are reacted, and the nitro that then reduces is amino to produce free 3-.This produces compound with known Z-α-fluoro cinnamic acid and peptide coupling agent condensation subsequently v.

Embodiment 23. compound wsynthesis

With N methyl piperazine process 3-(2-(the fluoro-2-methoxyl group of 4--5-oil of mirbane amido) pyrimidine-4-yl) pyrazolo [1,5-a] pyridine (WO 2013/014448), to replace fluorine atom.Then nitroreduction is become corresponding amine; and with the fluoro-3-of Z-2-(1-methyl piperidine-3-base) vinylformic acid (as Tet Letters 32; by known 1-methyl piperidine-3-formaldehyde and 2 described in 339 (1991); 2-bis-chloro-3; 3; 3-trifluoroacetic acid methyl esters is prepared in two steps, then saponification) acidylate to be to produce compound w.

Embodiment 24. compound xsynthesis

With N methyl piperazine process 3-(2-(the fluoro-2-methoxyl group of 4--5-oil of mirbane amido) pyrimidine-4-yl)-4,5,6,7-tetrahydro-pyrazoles also [1,5-a] pyridine (WO 2013/014448), to replace fluorine atom.Then nitroreduction is become corresponding amine, and use the fluoro-3-of Z-2-(1-methyl piperidine-3-base) vinylformic acid acidylate to produce compounds X as described in example 23 above.

scheme 21. 2-(3-(3 (-fluorine/(two/tri-) methyl fluoride) but-2-enamides base) anilino)-4-heteroaryl pyrimidine y-AC synthesis.

Embodiment 25. compound ysynthesis

The chloro-4-of 2-(imidazo [1,2-a] pyridin-3-yl) pyrimidine (WO 2010/097335) reacts with the fluoro-2-methoxyl group of 4--5-N-methyl-p-nitroaniline as described in WO 2013/014448.Then use M-methyl-N-(2-dimethyl aminoethyl) amine to replace fluorine, with acetic acid, nitroreduction is become corresponding amine with iron.Condensation of fluoro-2-methyl crotonic acyl chlorides produces compound with 3-subsequently for this y.

Embodiment 26. compound zsynthesis

The chloro-4-of 2-(1-skatole-3-base) pyrimidine reacts with the fluoro-2-methoxyl group of 4--5-N-methyl-p-nitroaniline as described in WO 2013/014448.Then use M-methyl-N-(2-dimethyl aminoethyl) amine to replace fluorine, with acetic acid, nitroreduction is become corresponding amine with iron.This reacts with 3-difluoromethyl crotonyl chloride subsequently and produces compound z.

Embodiment 27. compound aAsynthesis

With N methyl piperazine process 3-(2-(the fluoro-2-methoxyl group of 4--5-oil of mirbane amido) pyrimidine-4-yl) pyrazolo [1,5-a] pyridine (WO 2013/014448), to replace fluorine atom.Then in acetic acid, with iron, nitroreduction is become corresponding amine, and by 3-trifluoromethyl crotonyl chloride acidylate to produce compound aA.

Embodiment 28. compound aBsynthesis

3-(2-(5-amino-4-(4-methylpiperazino)-2-methoxyl group-5-anilino) pyrimidine-4-yl)-4; 5; 6; 7-tetrahydro-pyrazole also [1; 5-a] pyridine (it is the intermediate in embodiment 4) is with 2; 3-dimethyl-4,4-difluoro β-crotonic acid and peptide coupling agent, if PyBOP acidylate is to produce compound aB.

Embodiment 29. compound aCsynthesis

With the bromo-2-methyl of 4--3-trifluoromethyl β-crotonic acid and coupling agent, as PyBOP process 2-(5-amino-4-dimethylamino-2-methoxy-pllenylamine base)-4-(1-skatole-3-base) pyrimidine (intermediate in embodiment 21), then as described in US 6297258, replace bromine to produce compound with dimethylamine aC.

scheme 22. 2-(3-(4,4-difluoro chain-2-alkene amide group) anilino)-4-heteroaryl pyrimidine aD-AG synthesis.

Embodiment 30. compound aDsynthesis

Acetaldehyde, as J Org Chem 45,28 (1980) and J Fluorine Chem 36, changes into 4,4-difluoro penta-2-acetylenic acid in 4 steps described in 293 (1987).After the stereotaxis addition of HI, carry out methylating of the Pd catalysis as described in New J Chem 27,432 (2003) and Synthesis 543 (2002), it substitutes iodide with methyl bromide zinc.This acid can utilize peptide coupling reagent if PyBOP and aforementioned triaryl amine condensation are to produce compound now aD.

Embodiment 31. compound aEsynthesis

Reposefully with the bromine of N-methyl-3-hydroxy azetidine sodium salt displacement bromoacetaldehyde acetal in DMF.After this acetal hydro, this aldehyde is reacted with methyl-prop acetylenic acid lithium at low temperatures, with catalytic perruthenate, gained secondary alcohol is oxidized to corresponding ketone.This ketone is changed into gem-dimethyl compound by DAST, also substitutes with methyl bromide zinc as described in example 30 above after saponification across triple bond addition HI.This acid subsequently with triaryl amine condensation, the difference in itself and embodiment 30 is only that fluorochemical displacement step morpholine carries out, to produce compound aE.

Embodiment 32. compound aFsynthesis

Grignard reagent derived from Chloromethyl methyl ether is reacted at low temperatures to oxalic acid diethyl ester and produces corresponding α-one ester.This changes into corresponding 2,2-difluoro esters with DAST subsequently, and is reduced into corresponding aldehyde with DIBAL at low temperatures.With Horner-Wadsworth-Emmons reaction, this aldehyde is changed into corresponding unsaturated ester, this ester is by " methylate " on 2-position with the permutoid reaction of 2-Methylpropanedioic acid sodium (J Org Chem 63,7525 (1998)).Use BCl ₃removing methyl ether also removes this ester by saponification.Gained carboxylic acid utilizes PyBOP or similar reagents and triaryl amine coupling suitable as above to produce compound aF.

Embodiment 33. compound aGsynthesis

The chemical process identical with described in embodiment 30 is used 2 methyl propanal to be changed into 4,4-bis-corresponding fluoro-3-methyl alkenoic acids.It utilizes PyBOp and suitable triaryl amine intermediate condensation subsequently, or it can change into corresponding chloride of acid to implement coupling with oxalyl chloride equally, and this produces compound aG.

scheme 23. 2-(3-(alkynes-2-amide group) anilino)-4-heteroaryl pyrimidine aH-AK synthesis.

Embodiment 34. compound aHsynthesis

Under various condition (usual copper catalysis), cause chlorine to be replaced by amine with the chloro-3-methyl butyne of amine process 3-, in this case, the amino acetylene needed for use generation of two (methoxy ethyl) amine.Formed and CO via grignard (or lithium) acetylide as described in document (US 6297258, J Med Chem 49,1475 (2006)) ₂by this compound carboxylation to produce 4-(N, N-two (methoxy ethyl) are amino)-4-methylpent-2-acetylenic acid.This subsequently condensing agent as PyBOP exist under with triaryl amine (by 3-(2-(the fluoro-2-methoxyl group of 4--5-oil of mirbane amido) pyrimidine-4-yl)-4,5,6,7-tetrahydro-pyrazole also [1,5-a] pyridine (WO 2013/014448) by making with subsequently nitroreduction being become amine with methyl cellosolve displacement fluorine in the basic conditions) coupling to be to produce final product compound aH.

Embodiment 35. compound aIsynthesis

Prepare 4-methyl-4-(4-methylpiperazine-1-yl) penta-2-olefin(e) acid in two steps by 3-acetoxy-3-methyl butyne and N methyl piperazine, this relates to the amine displacement of copper catalysis, the then carboxylation of acetylene series Grignard reagent.Its then dewatering agent as PyBOP exist under with triaryl amine (by 2-(the fluoro-2-methoxyl group of 4--5-oil of mirbane amido)-4-(imidazo [1,2-a] pyridin-3-yl) pyrimidine (it is the intermediate in embodiment 25) by with the process of 2-methyl amino ethanol, then nitroreduction is become amine by Fe/AcOH or other method such as catalytic hydrogenation and make) coupling to produce compound aI.

Embodiment 36. compound aJsynthesis

With tetramethyleneimine process 3-(2-(the fluoro-2-methoxyl group of 4--5-oil of mirbane amido) pyrimidine-4-yl) pyrazolo [1,5-a] pyridine (WO 2013/014448), to replace fluorine atom.Then in acetic acid, with iron, nitroreduction is become corresponding amine, and at condensing agent, as EDAC/OBT, PyBOP or HATU exist lower 4-methyl-4-(4-methylpiperazine-1-yl) penta-2-olefin(e) acid (embodiment 35) acidylate to produce compound aJ.

Embodiment 37. compound aKsynthesis

Produce tertiary propargyl ethanol with trimethylammonium acetylene lithium process N-Boc piperidin-4-one-, it, with the aftertreatment of the KF aqueous solution, then uses diacetyl oxide acetylize under DMAP exists.Then this acetate replaces with methylamine under copper (I) catalyzer exists, and the new propargylamine introduced is protected with Boc acid anhydride under DMAP exists.Realize the formation of acetylene series Grignard reagent with MeMgBr, as described in document (US 6297258, J Med Chem 49,1475 (2006)), use CO ₂carboxylation produces 3-(1-tert-butoxycarbonyl-4-(N-methyl-N-tertbutyloxycarbonylamino) piperidin-4-yl) propynoic acid.2-(the fluoro-2-methoxyl group of 4--5-oil of mirbane amido)-4-indol-3-yl) pyrimidine (WO 2013/014448) Boc acid anhydride N-protected, then replace fluorine with azetidine, with Fe/AcOH, nitroreduction is become amine.Then use above-mentioned N, N '-two Boc propiolic acid derivative and peptide coupling agent as PyBOP by triaryl amine acidylate to produce compound aK.

scheme 24. 2-(5-(chain-2-alkene amide group) pyridin-3-yl is amino)-4-heteroaryl pyrimidine aL-AN synthesis.

Embodiment 38. compound aLsynthesis

Bromo-2, the 6-bis-fluoro-3-nitropyridines of 5-are prepared as described in scheme 15.With N, N, N '-trimethylammonium quadrol and ring sodium propylate in succession replace 2-fluorine and 6-fluorine, and using the water-soluble Cu as described in Eur J Org Chem 1854 (2010) ^iIbromine is replaced to produce 5-amino-6-ring propoxy--2-(N-methyl (2-Dimethylaminoethylamino)-3-nitropyridine with ammonia in the ammonia-water systems of-salen complex compound.This is chloro-4-(1-skatole-3-base) pyrimidine (WO 2013/014448) coupling with 2-under common acid catalytic condition, although conveniently can implement this displacement in the basic conditions in dipolar aprotic solvent is as DMF.Then reduce nitro with Fe/AcOH, gained amino with penta-2-alkene acylated with acid chloride to produce compound aL.

Embodiment 39. compound aMsynthesis

Bromo-2, the 6-bis-fluoro-3-nitropyridines of 5-are prepared as described in scheme 15.Under gentle alkaline condition and with 2-methyl cellosolve sodium, under more strong alkaline condition, 2-fluorine and 6-fluorine is in succession replaced with methyl alcohol.Reduce nitro to produce the bromo-2-methoxyl group of 3-amino-5--6-(methoxy ethoxy) pyridine with Fe/AcOH, its chloro-4-(pyrazolo [1,5-a] pyridin-3-yl) pyrimidine (WO 2013/014448) coupling with 2-in the basic conditions.Then use the system ammonia described in embodiment 38 to replace bromine, gained amine uses 4-(methoxy ethoxy) β-crotonic acid (its preparation is described in embodiment 13) and suitable coupling agent if PyBOP acidylate is to produce compound subsequently aM.

Embodiment 40. compound aNsynthesis

Bromo-2, the 6-bis-fluoro-3-nitropyridines of 5-are prepared as described in scheme 15.The sodium salt of 4-Boc amino oxygen azetidine replaces 2-fluorine and 6-fluorine at low temperatures and with azetidine at room temperature or at higher temperatures in succession.Then with V-Brite B or nickel boride reduction nitro (Tet Letters 34,3083 (1993)) to produce the bromo-2-of 3-amino-6-azetidine-1-base-5-(N-Boc-trimethylene oxide-3-base is amino) pyridine.This in the basic conditions with 3-(2-chloropyrimide-4-base)-4; 5; 6; 7-tetrahydro-pyrazole also [1; 5-a] pyridine (WO 2013/014448) coupling; then use ammoniacal liquor as described in example 38 above by bromo-amine, gained amine 4-bromine crotonyl chloride acidylate, and by having replaced this synthesis to produce compound with tetramethyleneimine as described in US 6297258 aN.

The syntheti c route of embodiment 41. N-[5-[[the chloro-4-of 5-(1H-3-indoles)-2-pyrimidyl] is amino]-2-[3-(diethylin) third-1-alkynes]-4-p-methoxy-phenyl] acrylamide and step

。

experimental procedure:

the preparation of compound 2:

18.47 g (150.00 mmol) 2-anisidine and 120 mL acetonitriles are added, stirring and dissolving in one 500 mL there-necked flasks.At 0 DEG C, slowly add 26.70 g NBS (150.00 mmol) to it in batches and stir at this temperature.After 1 hour, reaction terminates.The saturated Na of 100 mL is added in this reaction system ₂s ₂o ₃the aqueous solution, and be extracted with ethyl acetate (300 mL*3).Merge organic phase, use anhydrous Na ₂sO ₄drying, filters, concentrated.Thick product obtains 4-bromo-2-anisidine 21.20 g(104.92 mmol through silica gel column chromatography (PE/EA=10/1) separation and purification), yield 69.95%.

the preparation of compound 3:

At 0-5 DEG C, be add the bromo-2-anisidine of 10.10 g (50.00 mmol) 4-in the sulphuric acid soln of 85% and stirring and dissolving to 80 mL massfractions.At this temperature, add 6.10 g (50.00 mmol) Guanidinium nitrate to it in batches.Finish, control temperature 0-5 DEG C and stir 1 hour.Then, reaction system is slowly poured in 200 mL 50% NaOH solution, have solid to separate out.Throw out is filtered, washing, dry, obtain 7.10 g(28.70 mmol through column chromatography (PE/EA=10/1,6/1) separation and purification) and 4-bromo-5-nitro 2-anisidine, yield 57.48%.

the preparation of compound 5:

In 120 mL sec-butyl alcohols, add 3.31 g (12.55 mmol) compound 4,3.24 g (18.82 mmol) tosic acid and 3.10 g (12.55 mmol) 4-bromo-5-nitro 2-anisidine.This mixture stirring reaction 20 hours at 100 DEG C, TLC detects, and reacts complete.Be cooled to room temperature, concentrate to obtain black solid, it washs to obtain solid 2.85 g (6.00 mmol) compound 5 crude product through acetonitrile and methyl tertiary butyl ether, yield 47.84% first.

the preparation of compound 6:

In the mixed solvent of 80 mL ethanol and 20 mL water, add 2.80 g (5.90 mmol) compound 5,2.00 g (35.80 mmol) Fe powder and 2.00 g NH ₄cl (37.40 mmol).At N ₂under protection, this mixture was 60 DEG C of stirring reactions 6 hours, and TLC detects, and reacts complete.While hot by reacting liquid filtering, filtrate concentrates to obtain yellow solid, and this solid crude product obtains 1.10 g (2.48 mmol) compound 6 through column chromatography (PE/EA=5/1) separation and purification, yield 42.03%.

the preparation of compound 8:

In the anhydrous THF of 30 mL, add 1.10 g (2.48 mmol) compound 6 and 383.61 mg (2.97 mmol) DIEA, stirring and dissolving.At 0 DEG C, slowly drip 268.65 mg (2.97 mmol) acrylate chloride to it, drip and finish, stir TLC after 0.5 hour at this temperature and detect, reaction terminates.In this system, add 5 mL frozen water, stir 20 minutes.Aqueous phase ethyl acetate (20 mL*3) extracts.Merge organic phase, through anhydrous Na ₂sO ₄after drying, filter, decompression precipitation.Thick product obtains 0.48 g (0.96 mmol) yellow solid compound 8 through reversed-phase preparative chromatography preparative separation, yield 38.87%.

the preparation of compound N-[5-[[the chloro-4-of 5-(1H-3-indoles)-2-pyrimidyl] is amino]-2-[3-(diethylin) third-1-alkynes]-4-p-methoxy-phenyl] acrylamide:

In the mixing solutions that 8 mL triethylamines and 6 mL DMF are housed, add 400.00 mg (861.49 μm of ol) compound 8,120.94 mg (172.30 μm of ol) Pd (PPh ₃) ₂cl ₂, 199.10 mg (172.30 μm of ol) Pd (PPh ₃) ₄with 16.41 mg (86.15 μm of ol) CuI, stirring and dissolving.At N ₂under protection, add 1.62 g (14.57 mmol) compound 9 to it.This system is heated to 110 ° of C microwave reactions 2 hours under anhydrous and oxygen-free condition.TLC detection reaction terminates.Be cooled to 25 DEG C, in system, add 15 mL H ₂o, aqueous phase ethyl acetate (15 mL*3) extracts.Merge organic phase, through anhydrous Na ₂sO ₄after drying, filter, decompression precipitation obtains dark oil thing.Thick product obtains 45.00 mg (85.07 μm of ol) compound as white solid N-[5-[[the chloro-4-of 5-(1H-3-indoles)-2-pyrimidyl] is amino]-2-[3-(diethylin) third-1-alkynes]-4-p-methoxy-phenyl l] acrylamide through reversed-phase preparative chromatography preparative separation, yield 9.87%.　

Stratographic analysis target compound content 98.05%, ESI-MS(m/z): 528 (M+H) ⁺.

The syntheti c route of embodiment 42. N-[5-[[4-(1H-3-indoles)-2-pyrimidyl] is amino]-2-[3-(diethylin) third-1-alkynes]-4-p-methoxy-phenyl] acrylamide and step

。

the preparation of compound 3:

In 100 mL sec-butyl alcohols, add 2.30 g (10.00 mmol) compound 2,2.58 g (15.00mmol) tosic acid and 2.47 g (10.00 mmol) 4-bromo-5-nitro 2-anisidine.Reacted 20 hours at 100 DEG C by mixture, TLC detects, and reaction is finished.Be cooled to room temperature, concentrate to obtain black solid, it washs to obtain 2.55 g (5.79 mmol) compound 3 crude product through acetonitrile and methyl tertiary butyl ether, yield 57.95% first.

the preparation of compound 4:

In 80 mL ethanol, add 2.00 g (4.54 mmol) compound 3,2.03 g Fe (36.34 mmol) and 2.00 g NH ₄cl (37.40 mmol).N ₂under protection, mixture reacts 6 hours in 60 DEG C time, and TLC detects, and reaction is finished.Reacting liquid filtering, concentrate to obtain yellow solid, this solid crude product column chromatography (PE/EA=5/1) separation and purification obtains 1.25 g (3.05 mmol) compound 4, yield 67.18%.

the preparation of compound 5:

In the anhydrous THF of 40 mL, add 1.22 g (2.97 mmol) compound 4 and 422.23 mg (3.27 mmol) DIEA, stirring and dissolving.At 0 DEG C, slowly drip 295.70 mg (3.27 mmol) acrylate chloride to it, drip and finish, stir at this temperature.After 0.5 hour, TLC detects, and reaction terminates.In system, add 5 mL frozen water, stir 20 minutes.Aqueous phase ethyl acetate (20 mL*3) extracts.Merge organic phase, through anhydrous Na ₂sO ₄after drying, filter, decompression precipitation.Thick product obtains 0.65 g (1.4 mmol) yellow solid compound 5 through reverse chromatograms preparative separation, yield 47.14%.

the preparation of compound N-[5-[[4-(1H-3-indoles)-2-pyrimidyl] is amino]-2-[3-(diethylin) third-1-alkynes]-4-p-methoxy-phenyl] acrylamide:

In the mixing filling 15 mL triethylamines and 10 mL DMF not solution, add 600.00 mg (1.29 mmol) compound 5,72.56 mg (103.38 μm of ol) Pd (PPh ₃) ₂cl ₂, 119.46 mg (103.38 μm of ol) Pd (PPh ₃) ₄with 12.30 mg (84.62 μm of ol) CuI, stirring and dissolving.At N ₂under protection, add 2.43 g (21.85 mmol) compound 6 to it.System is under anhydrous and oxygen-free condition, and microwave reaction 3 hours when 90 DEG C, TLC detection reaction terminates.Be cooled to 25 DEG C, in system, add 30 mL H ₂o, aqueous phase ethyl acetate (30 mL*3) extracts.Merge organic phase, through anhydrous Na ₂sO ₄after drying, filter, decompression precipitation obtains dark oil thing.Thick product obtains 50.00 mg (101.10 μm of ol) compound as white solid N-[5-[[4-(1H-3-indoles)-2-pyrimidyl] is amino]-2-[3-(diethylin) third-1-alkynes]-4-p-methoxy-phenyl] acrylamide through reverse chromatograms preparative separation, yield 7.84%.　

Stratographic analysis target compound content 93.58%, ESI-MS(m/z): 495 (M+H) ⁺.

The syntheti c route of embodiment 43. N-[5-[[4-(1-methyl-3-indoles)-2-pyrimidyl] is amino]-2-[3-(diethylin) third-1-alkynes]-4-p-methoxy-phenyl] acrylamide and step

。

the preparation of compound 3:

In 80 mL sec-butyl alcohols, add 2.44 g (10.04 mmol) compound 2,2.60 g (15.06mmol) tosic acid and 2.48 g (10.04 mmol) 4-bromo-5-nitro 2-anisidine.Reacted 20 hours at 90 DEG C by mixture, TLC detects, and reaction is finished.Be cooled to room temperature, concentrate to obtain black solid, it washs to obtain 2.58 g (5.68 mmol) compound 3 crude product through acetonitrile and methyl tertiary butyl ether, yield 56.56% first.

the preparation of compound 4:

In 80 mL ethanol, add 2.20 g (4.84 mmol) compound 3,2.17 g Fe (38.72 mmol) and 2.07 g NH ₄cl (38.74 mmol).N ₂under protection, mixture reacts 6 hours in 60 DEG C time, and TLC detects, and reaction is finished.Reacting liquid filtering, concentrate to obtain yellow solid, this solid crude product column chromatography (PE/EA=3/1) separation and purification obtains 1.40 g (3.30 mmol) compound 4, yield 68.17%.

the preparation of compound 5:

In the anhydrous THF of 40 mL, add 1.40 g (3.30 mmol) compound 4 and 469.09 mg (3.36 mmol) DIEA, stirring and dissolving.At 0 DEG C, slowly drip 328.52 mg (3.36 mmol) acrylate chloride to it, drip and finish, stir at this temperature.After 0.5 hour, TLC detects, and reaction terminates.In system, add 5 mL frozen water, stir 20 minutes.Aqueous phase ethyl acetate (20 mL*3) extracts.Merge organic phase, through anhydrous Na ₂sO ₄after drying, filter, decompression precipitation.Thick product obtains 640.50 mg (1.34 mmol) yellow solid compound 5 through reverse chromatograms preparative separation, yield 40.60%.

the preparation of compound N-[5-[[4-(1-methyl-3-indoles)-2-pyrimidyl] is amino]-2-[3-(diethylin) third-1-alkynes]-4-p-methoxy-phenyl] acrylamide:

In the mixing filling 15 mL triethylamines and 10 mL DMF not solution, add 600.00 mg (1.25 mmol) compound 5,70.44 mg (100.34 μm of ol) Pd (PPh ₃) ₂cl ₂, 115.96 mg (100.34 μm of ol) Pd (PPh ₃) ₄with 11.94 mg (62.72 μm of ol) CuI, stirring and dissolving.At N ₂under protection, add 2.43 g (21.85 mmol) compound 6 to it.System is under anhydrous and oxygen-free condition, and microwave reaction 3 hours when 90 DEG C, TLC detection reaction terminates.Be cooled to 25 DEG C, in system, add 30 mL H ₂o, aqueous phase ethyl acetate (30 mL*3) extracts.Merge organic phase, through anhydrous Na ₂sO ₄after drying, filter, decompression precipitation obtains dark oil thing.Thick product obtains 30.00 mg (58.99 μm of ol) compound as white solid N-[5-[[4-(1-methyl-3-indoles)-2-pyrimidyl] is amino]-2-[3-(diethylin) third-1-alkynes]-4-p-methoxy-phenyl] acrylamide through reverse chromatograms preparative separation, yield 4.72%.　

Stratographic analysis target compound content 96.12%, ESI-MS(m/z): 509 (M+H) ⁺.

The syntheti c route of embodiment 44. N-[2-[3-(diethylin) third-1-alkynyl]-4-methoxyl group-5-[(4-pyrazoles [1,5-a]-3-pyridine-2-pyrimidine) is amino] phenyl] acrylamide and step

。

the preparation of compound 3:

In 100 mL sec-butyl alcohols, add 2.31 g (10.00 mmol) compound 2,2.58 g (15.00mmol) tosic acid and 2.47 g (10.00 mmol) 4-bromo-5-nitro 2-anisidine.Reacted 20 hours at 100 DEG C by mixture, TLC detects, and reaction is finished.Be cooled to room temperature, concentrate to obtain black solid, it washs to obtain 2.20 g (4.99 mmol) compound 3 crude product through acetonitrile and methyl tertiary butyl ether, yield 49.86% first.

the preparation of compound 4:

At 50 mL acetone and 2 mL H ₂in the mixing solutions of O, add 2.08 g (4.71 mmol) compound 3,4.31 g Zn (65.99 mmol) and 2.02 g NH ₄cl (37.71 mmol).At 25 DEG C, stir 1 hour, TLC detects, and reaction is finished.Reacting liquid filtering, concentrate to obtain solid crude product, this solid crude product column chromatography (PE/EA=3/1) separation and purification obtains 1.41 g (3.43 mmol) compound 4, yield 72.78%.

the preparation of compound 5:

In the anhydrous THF of 40 mL, add 1.00 g (2.43 mmol) compound 4 and 314.06 mg (2.43 mmol) DIEA, stirring and dissolving.At 0 DEG C, slowly drip 241.93 mg (2.67 mmol) acrylate chloride to it, drip and finish, stir at this temperature.After 0.5 hour, TLC detects, and reaction terminates.In system, add 5 mL frozen water, stir 20 minutes.Aqueous phase ethyl acetate (20 mL*3) extracts.Merge organic phase, through anhydrous Na ₂sO ₄after drying, filter, decompression precipitation.Thick product obtains 0.68 g (1.37 mmol) yellow solid compound 5 through reverse chromatograms preparative separation, yield 56.46%.

the preparation of compound N-[2-[3-(diethylin) third-1-alkynyl]-4-methoxyl group-5-[(4-pyrazoles [1,5-a]-3-pyridine-2-pyrimidine) is amino] phenyl] acrylamide:

In the mixing filling 15 mL triethylamines and 10 mL DMF not solution, add 620.00 mg (1.33 mmol) compound 5,4.83 mg (6.88 μm of ol) Pd (PPh ₃) ₂cl ₂, 7.95 mg (6.88 μm of ol) Pd (PPh ₃) ₄with 0.82 mg (4.30 μm of ol) CuI, stirring and dissolving.At N ₂under protection, add 2.51 g (22.61 mmol) compound 6 to it.System is under anhydrous and oxygen-free condition, and microwave reaction 3 hours when 90 DEG C, TLC detection reaction terminates.Be cooled to 25 DEG C, in system, add 30 mL H ₂o, aqueous phase ethyl acetate (30 mL*3) extracts.Merge organic phase, through anhydrous Na ₂sO ₄after drying, filter, decompression precipitation obtains dark oil thing.Thick product obtains 45.00 mg (90.80 μm of ol) compound N-[2-[3-(diethylin) third-1-alkynyl]-4-methoxyl group-5-[(4-pyrazoles [1 through reverse chromatograms preparative separation, 5-a]-3-pyridine-2-pyrimidyl) amino] phenyl] acrylamide, yield 6.83%.　

Stratographic analysis target compound content 91.17%, ESI-MS(m/z): 496 (M+H) ⁺.

Kinase inhibition measures

Utilize the kinase inhibition of the compound of commercially available detection kit known to a person of ordinary skill in the art and service mensuration formula (I).These test kits and service, for measuring various kinases, include but not limited to the suppression of ALK, ABL, AXL, Aur B & C, BLK, erbB-2, erbB-4. EGFR, sudden change EGFR, HPK, IRAK1, RON, ROS1, SLK, STK10, TIE2, TRK, c-Met, Lck, Lyn, Src, Fyn, Syk, Zap-70, Itk, Tec, Btk, EGFR, ErbB2, Kdr, Flt-1, Flt-3, Tek, c-Met, InsR and Atk.The commercial supplier of these detection kit and service comprises Promega Corporation and Reaction Biology Corporation, EMD Millipore and CEREP.Except commercially available detection kit and service, measured the kinase inhibiting activity of the compound of formula (I-III) by following assay method.

The purification of epidermal growth factor recipient tyrosine kinase

From A431 people's epidermoid carcinoma cell of process LAN EGF acceptor, people EGF receptor tyrosine kinase is separated by following method.Cell is growth in 50% Delbuco ' the s Modified Eagle containing 10% foetal calf serum and 50% HAM F-12 nutritional medium (Gibco) in roller bottle.About 10 ⁹individual cell is containing 20 mM 2-(4N-[2-hydroxyethyl] piperazine-1-base) ethyl sulfonic acid (hepes), pH 7.4,5 mM ethylene glycol bis (2-amino-ethyl ether) N, N, cracking in 2 volume buffer of N', N'-tetraacethyl, 1% Triton X-lOO, 10% glycerine, 0.1 mM sodium orthovanadate, 5 mM Sodium Fluorides, 4 mM pyrophosphate salts, 4 mM benzamide, 1 mM dithiothreitol (DTT), 80 μ g/mL Trypsin inhibitor,Trasylols, 40 μ g/mL leupeptins and 1mM phenylmethylsulfonyl fluoride.Under 25,000xg after centrifugal 10 minutes, supernatant liquor at 40 DEG C with before 10 milliliters with 50 mM Hepes, 10% glycerine, 0.1% Triton X-100 and 150mM NaCI, pH 7.5(level pad) the wheat germ lectin avidin agarose that balances balances 2 hours.From resin, the protein of pollution is washed off with the 1M NaCl in level pad, and with the 0.5M N-ethanoyl-1-D-glycosamine in level pad, then 1mM urea wash-out enzyme.This enzyme 0.1 mg/ml EGF wash-out.As the polyacrylamide gel electrophoresis gel by Coomassie blue stain is assessed, acceptor seems even.

The detecting step of Caliper Mobility Shift Kinase Assay method:

Measure the IC that 1. determine the anti-single mutation EGFR of test compounds (EGFR_d746-750) ₅₀value

Test compounds 100 % methyl-sulphoxides (DMSO) are mixed with 500 μMs, then hole-specifically carry out the concentration gradient dilution of 4 times with 100 % DMSO, dilute 10 gradients." feminine gender " and " positive " control wells 100 % DMSO of 100 μ L replace.Wherein, " feminine gender " control wells is without compound group, and " positive " control wells is without kinases group.Subsequently, 10 μ L compounds and 90 μ L 1 × kinase buffer liquid are added in 96 orifice plates.Above-mentioned for the 5 μ L compound containing 10%DMSO is added 384 orifice plates, then 10 μ L 2.5 × kinase solution (containing 12.5 nM EGFR_d746-750,5 mM DTT, 1 × kinase buffer liquid) is added in 384 orifice plates.Incubated at room is after 10 minutes, add 10 μ L 2.5 × substrate solution (containing 7.5 μMs of peptide substrates, 35 μMs of ATP, 25 mM MgCl2,12.5 mM MnCl2,1 × kinase buffer liquid) initial action.28C adds 25 μ L stop buffer termination reactions after hatching 1 hour.Vibration, carries out reading plate to EZ Reader II after centrifugal, finally calculates the inhibiting rate under each concentration of compound according to the reader of conversion value and " feminine gender " and " positive " control wells, and binding compounds concentration is mapped calculating IC ₅₀value.

Measure the IC that 2. determine the anti-double mutations EGFR (EGFR_T790M/L858R) of test compounds ₅₀value

Test compounds 100 % methyl-sulphoxides (DMSO) are mixed with 500 μMs, then hole-specifically carry out the concentration gradient dilution of 4 times with 100 % DMSO, dilute 10 gradients." feminine gender " and " positive " control wells 100 % DMSO of 100 μ L replace.Wherein, " feminine gender " control wells is without compound group, and " positive " control wells is without kinases group.Subsequently, 10 μ L compounds and 90 μ L 1 × kinase buffer liquid are added in 96 orifice plates.Above-mentioned for the 5 μ L compound containing 10%DMSO is added 384 orifice plates, then 10 μ L 2.5 × kinase solution (containing 25 nM EGFR_T790M/L858R, 5 mM DTT, 1 × kinase buffer liquid) is added in 384 orifice plates.Incubated at room is after 10 minutes, add 10 μ L 2.5 × substrate solution (containing 7.5 μMs of peptide substrates, 47.5 μMs of ATP, 25 mM MgCl2,1 × kinase buffer liquid) initial action.28C adds 25 μ L stop buffer termination reactions after hatching 1 hour.Vibration, carries out reading plate to EZ Reader II after centrifugal, finally calculates the inhibiting rate under each concentration of compound according to the reader of conversion value and " feminine gender " and " positive " control wells, and binding compounds concentration is mapped calculating IC ₅₀value.

Measure the IC that 3. determine test compounds against wild type EGFR ₅₀value

Test compounds 100 % methyl-sulphoxides (DMSO) are mixed with 500 μMs, then hole-specifically carry out the concentration gradient dilution of 4 times with 100 % DMSO, dilute 10 gradients." feminine gender " and " positive " control wells 100 % DMSO of 100 μ L replace.Wherein, " feminine gender " control wells is without compound group, and " positive " control wells is without kinases group.Subsequently, 10 μ L compounds and 90 μ L 1 × kinase buffer liquid are added in 96 orifice plates.Above-mentioned for the 5 μ L compound containing 10%DMSO is added 384 orifice plates, then 10 μ L 2.5 × kinase solution (containing 20 nM EGFR, 5 mM DTT, 1 × kinase buffer liquid) is added in 384 orifice plates.Incubated at room is after 10 minutes, add 10 μ L 2.5 × substrate solution (containing 7.5 μMs of peptide substrates, 5.75 μMs of ATP, 25 mM MgCl2,25 mM MnCl2,1 × kinase buffer liquid) initial action.28C adds 25 μ L stop buffer termination reactions after hatching 1 hour.Vibration, carries out reading plate to EZ Reader II after centrifugal, finally calculates the inhibiting rate under each concentration of compound according to the reader of conversion value and " feminine gender " and " positive " control wells, and binding compounds concentration is mapped calculating IC ₅₀value.

Part of compounds is measuring the row of the test result in 1-3 in table 1 below.

Table 1

No	Structure	Measure 1 (single mutant) (nM)	Measure 2 (double mutant) (nM)	Measure 3 (wild-type) (nM)
					Embodiment 41	N-[5-[[the chloro-4-of 5-(1H-3-indoles)-2-pyrimidyl] is amino]-2-[3-(diethylin) third-1-alkynes]-4-p-methoxy-phenyl] acrylamide	2.6	2.7	5.4
Embodiment 42	N-[5-[[4-(1H-3-indoles)-2-pyrimidyl] is amino]-2-[3-(diethylin) third-1-alkynes]-4-p-methoxy-phenyl] acrylamide	9.9	9.0	22.5
					Embodiment 43	N-[5-[[4-(1-methyl-3-indoles)-2-pyrimidyl] is amino]-2-[3-(diethylin) third-1-alkynes]-4-p-methoxy-phenyl] acrylamide	51	12.6	750.5
Embodiment 44	N-[2-[3-(diethylin) third-1-alkynyl]-4-methoxyl group-5-[(4-pyrazoles [1,5-a]-3-pyridine-2-pyrimidine) is amino] phenyl] acrylamide	29.2	7.1	237

Other kinase inhibition measures

The compound of mensuration formula (I-III) is carried out to the assay method of other kinase whose suppression according to program known to persons of ordinary skill in the art.These assay methods include, but not limited to the assay method relating to following kinase whose suppression:

● wild-type c-Met kinases.as described in International Publication No. WO 2011/069761, measure the kinase whose suppression of wild-type c-Met, its whole content is incorporated herein by this reference.　　

● lCK and BLK kinases.as U.S. Patent No. 7,125, measure the kinase whose suppression of LCK and BLK described in 875, its whole content is incorporated herein by this reference.

Compound as herein described screens in the following manner.The kinases being applicable to the follow procedure of the kinase activity measuring compound as herein described includes, but are not limited to: Lck, Lyn, Src, Fyn, Syk, Zap-70, Itk, Tec, Btk, EGFR, sudden change EGFR, ErbB2, ErbB-4, Kdr, Flt-1, Flt-3, Tek, c-Met, InsR and Atk.Kinases is expressed as the fusion rotein being fused to kinase domain on glutathione S-transferase (GST) or total length construction or polyhistidine mark in intestinal bacteria or baculovirus-High Five expression system.By basic foregoing affinity chromatography (people such as Lehr, 1996; The people such as Gish, 1995) they are purified to close to homogeneous.In some cases, kinases measure before activity with purify or Partial purification regulatory polypeptide coexpression or mix.Kinase activity and suppression is measured essentially by blas people such as (, 1996) Braunwalder.In brief, ³²pO ₄transfer to the synthesis substrate be attached to the bioactivity surface of titer plate poly-(Glu-Tyr) 4:1 or poly-(Arg-Ser) 3:1 from ATP and serve as the basis assessing enzymic activity.After incubation period, by first with this plate of 0.5% phosphoric acid washing, add liquid scintillator, then count in liquid scintillator detector, measure the amount of the phosphoric acid of transfer.By making to be merged on the substrate that is incorporated on plate ³²the compound concentration that P measures reduction by 50% measures IC ₅₀.Other similar method also can be used---thus phosphoric acid to be transferred in the solution or in fixing (i.e. solid phase) peptide containing tyrosine, Serine, Threonine or Histidine (their separately or combination, or are combined with other amino acid) or peptide substrate.Such as, the people such as scintillation proximity method (people such as Wu, 2000), ELISA(Cleaveland can also be used, 1990), fluorescence polarization (Seethala and Menzel, 1998) and homogenizing time resolved fluorometric method people such as (, 1998) HTRF, Kolb detect the transfer of phosphoric acid to peptide or polypeptide.Or, antibody based method can be utilized to measure kinase activity, use antibody or polypeptide to detect phosphorylation target polypeptide as reagent thus.

bibliography

The people such as Braunwalder (1996). Anal. Biochem. 234 (1): 23-26.　

The people such as Cleaveland (1990). Anal Biochem. 190 (2): 249-53.　

The people such as Gish (1995). Protein Eng. 8 (6): 609-614.　

The people such as Kolb (1998). Drug Discov. Today. 3:333-342.　

The people such as Lehr (1996). Gene 169 (2): 27527-9.　

The people such as Seethala (1998). Anal Biochem. 255 (2): 257-62.　

The people such as Wu (2000). Comb Chem High Throughput Screen. 3 (1): 27-36.

erbB cell detection program summary

1. EGF Rat-1 DNA synthesizes

Rat fibroblast system (Rat-I) to be layered in perfect medium in (plated out) flat hole plate and to make it adhere to whole night.Then make cell containing 0.1% bovine serum albumin (BSA) substratum in hunger whole night, with or without diluted chemical compound liquid preculture 1 hour, then use 1 ng/ml Urogastron (EGF), 50 ng/ml Thr6 PDGF BBs (PDGF), 3 ng/ml fibroblast growth factors (FGF) or 10 ng/ml insulin-like growth factor-is (IGF-1) to activate whole night.Pass through ³h-thymidine is incorporated to the level determination propagation in DNA.Be incorporated to level by the thymidine comparing discovery under compound exists compared with contrast and measure IC ₅₀' s.

2. the EGF-R autophosphorylation in A431

By people's epidermoid carcinoma cell (A431; ATCC, Manassas, Va.) to be layered in perfect medium in (plated out) flat hole plate and to make it adhere to whole night.Then make cell hungry in the substratum containing 0.5% foetal calf serum (FCS), use or without diluted chemical compound liquid preculture, then activate 3 minutes with 50 ng/ml EGF.By lysis and by SDS-PAGE isolated protein.By using the phosphotyrosine levels on the western blot determination EGF-R of anti-phospho-EGF-R-specific antibody.IC is measured by comparing the phosphotyrosine levels found under compound exists compared with contrast ₅₀' s.

3. HRG β l T47D DNA synthesizes

By human breast tumor cell line (T47D; ATCC, Manassas, Va.) to be layered in perfect medium in (plated out) flat hole plate and to make it adhere to whole night.Then make cell in containing the substratum of 0.1% bovine serum albumin (BSA) hunger whole night, with or without diluted chemical compound liquid preculture 1 hour, then use 150 ng/ml Heregulin(HRG β l) activation whole night.Pass through ³h-thymidine is incorporated to the level determination propagation in DNA.Be incorporated to level by the thymidine comparing discovery under compound exists compared with contrast and measure IC ₅₀' s.

4. the ErbB2 autophosphorylation in T47D

By HBT's cell (T47D; ATCC, Manassas, Va.) to be layered in perfect medium in (plated out) flat hole plate and to make it adhere to whole night.Then make cell hungry in the substratum containing 0.1% bovine serum albumin (BSA), use or without diluted chemical compound liquid preculture, then activate 10 minutes with 900 ng/ml HRG β 1.By lysis and by SDS-PAGE isolated protein.By using the phosphotyrosine levels on the western blot determination ErbB2 of anti-phospho-ErbB2-specific antibody.IC is measured by comparing the phosphotyrosine levels found under compound exists compared with contrast ₅₀' s.

5. HRG β l 3T3-Her2/3 DNA synthesizes

Apoptosis (3T3) has used total length people ErbB-2 and ErbB-3 stable transfection (people such as Carraway, J Biol Chem (1995) 270,7111-6)).This clone to be layered in perfect medium in (plated out) flat hole plate and to make it adhere to whole night.Then make cell in containing the substratum of 0.1% bovine serum albumin (BSA) hunger whole night, with or without diluted chemical compound liquid preculture 1 hour, then use 25 ng/ml Heregulin(HRG β l) activation whole night.Pass through ³h-thymidine is incorporated to the level determination propagation in DNA.Be incorporated to level by the thymidine comparing discovery under compound exists compared with contrast and measure IC ₅₀' s.

Exon l9 deletes EGFR(and activates single mutant) cells phosphorylation mensuration

Human pneumonocyte system PC9(exons 19 deletes EGFR) available from American type Culture Collection.PC9 cell is kept in the RPMI 1640 containing 10% foetal calf serum and 2 mM glutamine.Cell is containing 5% CO ₂humidification incubator at 37 DEG C cultivate.According to R & D Systems DuoSet IC Human Phospho-EGF R ELISA(R & D Systems catalog number (Cat.No.) #DYCI095) described in program carry out for measuring the cells phosphorylation of endogenous p-EGFR in cell pyrolysis liquid assay method.40 μ L cells (10000 cells/well) are inoculated in the medium and at 37 DEG C, 5% CO in 384 orifice plates at the bottom of Corning black transparent ₂lower cultivation whole night.Utilize Echo 555, give the compound of serial dilution in 100% DMSO by sound wave to cell.Plate cultivates 2 hours again, then after sucking-off substratum, adds 40 μ L x lysis buffers in each hole.Greiner black height to be captured antibody bag quilt in conjunction with 384 orifice plates, then closes with 3% BSA.After removing confining liquid (block), 15 μ L lysates are transferred to Greiner black height in conjunction with cultivating 2 hours in 384 orifice plates.In suction with after washing plate with PBS, add 20 μ L and detect antibody and cultivate 2 hours.In suction with after washing plate with PBS, add 20 μ L QuantaBlu fluorescence peroxidase substrate (Thermo Fisher Scientific catalog number (Cat.No.) 15169) and cultivate 1 hour.20 μ L QuantaBlu stop buffers to be added in plate and to read plate instrument to utilize 352 nanometer excitation wavelengths and 460 nanometer emission wavelength read fluorescence at Envision.Output in suitable software package (as Origin) by the data that each compound obtains to implement curve fitting analysis.There is provided the compound concentration needed for 50% effect by this data by calculating and measure IC ₅₀value.

L858R/T790M EGFR(double mutant) cells phosphorylation mensuration

Human pneumonocyte system NCI-HI975 is available from American type Culture Collection.NCI-HI975 cell is kept in the RPMI 1640 containing 10% foetal calf serum and 2 mM glutamine.Cell is containing 5% CO ₂humidification incubator at 37 DEG C cultivate.According to R & D Systems DuoSet IC Human Phospho-EGF R ELISA(R & D Systems catalog number (Cat.No.) #DYCI095) described in program carry out for measuring the cells phosphorylation of endogenous p-EGFR in cell pyrolysis liquid assay method.40 μ L cells (10000 cells/well) are inoculated in the medium and at 37 DEG C, 5% CO in 384 orifice plates at the bottom of Corning black transparent ₂lower cultivation whole night.Utilize Echo 555, give the compound of serial dilution in 100% DMSO by sound wave to cell.Plate cultivates 2 hours again, after sucking-off substratum, adds 40 μ L 1x lysis buffers in each hole.Greiner black height to be captured antibody bag quilt in conjunction with 384 orifice plates, then closes with 3% BSA.After removing confining liquid (block), 15 μ L lysates are transferred to Greiner black height in conjunction with cultivating 2 hours in 384 orifice plates.In suction with after washing plate with PBS, add 20 μ L and detect antibody and cultivate 2 hours.In suction with after washing plate with PBS, add 20 μ L QuantaBlu fluorescence peroxidase substrate (Thermo Fisher Scientific catalog number (Cat.No.) 15169) and cultivate 1 hour.20 μ L QuantaBlu stop buffers to be added in plate and to read plate instrument to utilize 352 nanometer excitation wavelengths and 460 nanometer emission wavelength read fluorescence at Envision.Output in suitable software package (as Origin) by the data that each compound obtains to implement curve fitting analysis.There is provided the compound concentration needed for 50% effect by this data by calculating and measure IC ₅₀value.

BaF3 transfection system

BaF3 is immortalization mouse pro-B clone, and it relies on IL-3 growth and survives and do not express ErbB family member.Those of ordinary skill in the art are with the cDNAs transfection BaF3 cell (Blood 97 of the appropriate structuring of erbB albumen (including but not limited to wt EGFR, wt erbB-2, wt erbB-4, L858R-EGFR, del746-750-EGFR and T790M-EGFR), 1050 (2001)), then by independent growths when there is not IL-3 or under suitable erbB part exists the growth of (still there is not IL-3) select the clone of transfection, to select and the BaF3 clone be separated with required erbB family member transfection.These clones can subsequently for such as measuring the cell IC of each member of erbB family described in document ₅₀s.（Cancer Res 71,7587 (2011)、Cancer Res 67, 11924 (2006) Proc Natnl Acad Sci USA 103, 7817 (2006)）。

other kinases various.as U.S. Patent No. 6,881, other kinase whose suppression various is measured described in 737, include but not limited to Lck, Lyn, Src, Fyn, Syk, Zap-70, Itk, Tec, Btk, EGFR, ErbB2, Kdr, Flt-1, Flt-3, Tek, c-Met, InsR and Atk, its whole content is incorporated herein by this reference.

animal xenograft tumor model

general procedure.By from the known ATCC clone with relevant oncogene or from having a mind to the cell suspension of suitable conversion of transfection in suitable substratum, and by 5 x 10 ⁶or 1 x 10 ⁷individual cell infusion is in the flank of nu/nu mouse.Or, the fragment (usually about 1 cubic centimetre) of interior generation tumour can be placed to cause tumour with trochar.When tumour reaches size (usually in 100-300 nanogram range) being applicable to experiment, animal is divided at random the coupling group be made up of 6-10 mouse, and once a day or pass twice through mouth week strong feeding and give vehicle or test product.Slide calliper rule are used to measure gross tumor volume.The increase per-cent that the volume of the xenograft tumours of n-th day (started that day of administration test-compound) relative to the 0th day is calculated with (gross tumor volume-gross tumor volume of 0 day/0th of n-th day day gross tumor volume) × 100.With (in 1-medication therapy groups gross tumor volume average increase %/vehicle treatment group in gross tumor volume average increase %) × 100 calculate each medication therapy groups relative to vehicle treatment group median tumor growth suppress per-cent.Use single tail t test evaluation statistical significance.

wild type EGFR xenotransplantation measures.in order to measure the effect of the tumour to process LAN wt EGFR, the heterograft cultivated by A431 epidermoid or LoVo colon cancer cell can be used.

eGFR del746-750 heteroplastic transplantation model.in order to measure the effect of the tumour to process LAN EGFR-del746-750, can use by the heterograft of PC9 NSCLC cell cultures.

eGFR L858R heteroplastic transplantation model.in order to measure the effect of the tumour to process LAN EGFR-L858R, can use by the heterograft of H3255 NSCLC cell cultures.

eGFR L858R/T790M double mutations heteroplastic transplantation model.in order to measure the effect of the tumour to process LAN EGFR-L858R/T790M double mutant, can use by the heterograft of H1975 NSCLC cell cultures.

erBB-2 xenotransplantation measures.in order to measure the effect of the tumour to process LAN wt erbB-2, the heterograft cultivated by N87 cancer of the stomach or BT474 breast cancer cell can be used.

pharmacodynamics measures.the mouse euthanasia with any above-mentioned tumour (preferred 200-300 milligram size) is made after oral administration with appropriate time interval.Tumor resection, quick-frozen in the non denatured lysis buffer utilizing Qiagen Tissue-Lyser to be dispersed in containing proteolytic enzyme and inhibitors of phosphatases.By this homogenate cracking 1 hour at 4 DEG C, by centrifugal clarification, then analyze phosphor EGFR/erbB-2/3/4 and total acceptor by quantitative immuning engram method.Its total RTK signal standardization of phospho-RTK signal of each RTK band.Or, suitable eERK and phosphor-ERK antibody can be utilized to measure the ratio of total ERK and the phosphor-ERK in tumour by similar techniques.

Claims

1. the compound of formula (A)

Or its pharmacologically acceptable salt, wherein

X ¹cH or N;

Y is or

R ¹⁰h, C _1-6alkyl;

X ²c, CH or N;

X ³cH or N,

X ⁴cR ⁴or N or NR ¹⁰;

X ⁵and X ⁶be all C or can be N;

M is 0-4;

Condition is as follows:

X ²-X ⁶in at least one and no more than three be N or NR ⁴;

2. the compound of the claim 1 of formula (I)

Or its pharmacologically acceptable salt, wherein

R ^6eand R ^6zh, F, Cl, CF independently ₃, CHF ₂, C _1-6alkyl, aryl, heteroaryl, cycloalkyl, heterocyclic radical, (CH ₂) _mcHR ⁴r ⁷, CF ₂c _1-6alkyl, CF ₂(CH ₂) _mcHR ⁴r ⁷or C (R ₄) ₂r ⁷;

R ¹⁰h, C _1-6alkyl;

X ²c, CH or N;

X ³cH or N,

X ⁴cR ⁴or N or NR ¹⁰;

X ⁵and X ⁶be all C or can be N;

M is 0-4;

Condition is as follows:

X ²-X ⁶in at least one and no more than three be N or NR ⁴;

R ⁵, R ^6eand R ^6zin one and only have one must be halogen or containing with the halogen of the carbon atom Direct Bonding be bonded directly in described alkene acid amides system itself.

3. the compound of the claim 1 of formula (II)

Or its pharmacologically acceptable salt, wherein

R ⁵h, CH ₂nR ⁸r ⁹or C _1-6alkyl;

R ^6eand R ^6zh, C independently _1-6alkyl, aryl, heteroaryl, cycloalkyl, heterocyclic radical, (CH ₂) _mcHR ⁴r ⁷, CF ₂c _1-6alkyl, CF ₂(CH ₂) _mcHR ⁴r ⁷or C (R ₄) ₂r ⁷;

R ⁸and R ⁹be C independently _1-6alkyl, C _3-6thiazolinyl, C _3-6alkynyl, C _3-7cycloalkyl, C _3-7cycloalkenyl group, C _1-c ₆acyl group, 4-12 unit heterocyclic radical, C _6-c ₁₂aryl or 5-12 unit heteroaryl; Or R ⁸and R ⁹the atom be connected with them forms 4-12 unit's monocycle or bicyclic ring systems, and wherein maximum two carbon atoms are by N, NR ⁴, O, S (O) _xwith S (O) (NR ¹⁴) substitute; And R ⁸and R ⁹can separately or together by maximum three independently selected from hydroxyl, C _1-6alkoxyl group, C _1-6hydroxyalkyl, C _1-6alkoxy-C _1-6alkyl, C _1-6alkoxy-C _1-6alkoxyl group, C _2-6the substituting group of hydroxyl alkoxyl group replaces;

R ¹⁰h, C _1-6alkyl;

X ²c, CH or N;

X ³cH or N,

X ⁴cR ⁴or N or NR ¹⁰;

X ⁵and X ⁶be all C or can be N;

M is 0-4;

Condition is as follows:

X ²-X ⁶in at least one and no more than three be N or NR ⁴;

R ⁵, R ^6eand R ^6zonly one is CF ₂(CH ₂) _mcHR ⁴r ⁷or CF ₂c _1-6alkyl.

4. the compound of the claim 1 of formula (III)

Or its pharmacologically acceptable salt, wherein

R ³be selected from H, F, C _1-6alkyl, C _1-6haloalkyl and optionally by C _1-6alkyl and R ⁷the C replaced _2-6alkynyl and R ⁷;

R ¹⁰h, C _1-6alkyl;

X ²c, CH or N;

X ³cH or N,

X ⁴cR ⁴or N or NR ¹⁰;

X ⁵and X ⁶be all C or can be N;

M is 0-4;

Condition is as follows:

X ²-X ⁶in at least one and no more than three be N or NR ⁴.

5. the compound of claim 1, makes R ¹hydrogen.

6. the compound of claim 5, makes X ⁴nH or NCH ₃, X ¹and X ³be CH and X ², X ⁵and X ⁶c.

7. the compound of claim 2, makes R ¹and R ⁵hydrogen, X ⁴nH or NCH ₃, X ¹and X ³cH, X ², X ⁵and X ⁶c, R ^6ecF ₂(CH ₂) _mcHR ⁴r ⁷or CF ₂c _1-6alkyl, and R ^6zh or CH ₃.

8. the compound of claim 3, makes R ¹hydrogen, X ⁴nH or NCH ₃, X ³cH, X ², X ⁵and X ⁶c, R ^6ecF ₂(CH ₂) _mcHR ⁴r ⁷or CF ₂c _1-6alkyl, and R ^6zand R ⁵h or CH ₃.

9. the compound of claim 4, makes R ¹hydrogen, X ⁴nH or NCH ₃, X ³cH, X ², X ⁵and X ⁶c.

10. the compound of the claim 2 of formula (IV), wherein:

R ¹⁰h, C _1-6alkyl.

The compound of the claim 2 of 11. formulas (V), wherein:

R ¹⁰h, C _1-6alkyl.

The compound of the claim 2 of 12. formulas (VI), wherein:

R ¹⁰h, C _1-6alkyl.

The compound of the claim 2 of 13. formulas (VII), wherein:

R ¹⁰h, C _1-6alkyl.

The compound of the claim 3 of 14. formulas (VIII), wherein:

R ⁵h, CH ₂nR ⁸r ⁹or C _1-6alkyl;

R ^6eand R ^6zbe H, C independently _1-6alkyl, aryl, heteroaryl, cycloalkyl, heterocyclic radical, (CH ₂) _mcHR ⁴r ⁷, CF ₂c _1-6alkyl, CF ₂(CH ₂) _mcHR ⁴r ⁷or C (R ₄) ₂r ⁷;

R ¹⁰h, C _1-6alkyl.

The compound of the claim 3 of 15. formulas (IX), wherein:

R ⁵h, CH ₂nR ⁸r ⁹or C _1-6alkyl;

R ¹⁰h, C _1-6alkyl.

The compound of the claim 3 of 16. formulas (X), wherein:

R ⁵h, CH ₂nR ⁸r ⁹or C _1-6alkyl;

R ^6eand R ^6zbe H, C independently _1-6alkyl, aryl, heteroaryl, cycloalkyl, heterocyclic radical, (CH ₂) _mcHR ⁴r ⁷, CF ₂c _1-6alkyl, CF ₂(CH ₂) _mcHR ⁴r ⁷or C (R ₄) ₂r ⁷ _;

R ¹⁰h, C _1-6alkyl.

The compound of the claim 3 of 17. formulas (XI), wherein:

R ⁵h, CH ₂nR ⁸r ⁹or C _1-6alkyl;

R ¹⁰h, C _1-6alkyl.

The compound of the claim 4 of 18. formulas (XII), wherein:

R ¹⁰h, C _1-6alkyl.

The compound of the claim 4 of 19. formulas (XIII), wherein:

R ¹⁰h, C _1-6alkyl.

The compound of the claim 4 of 20. formulas (XIV), wherein:

R ¹⁰h, C _1-6alkyl.

The compound of the claim 4 of 21. formulas (XI), wherein:

R ¹⁰h, C _1-6alkyl.

22. compounds, it is selected from:

Or their pharmacologically acceptable salt.

23. pharmaceutical compositions, it comprises compound or pharmaceutically acceptable salt thereof any one of claim 1-22 and pharmaceutically acceptable carrier or vehicle.

Compound or pharmaceutically acceptable salt thereof any one of 24. claim 1-22 is for the preparation for the treatment of or suppress in the medicine of mammiferous cell proliferation, cell invasion, transfer, apoptosis or vasculogenesis purposes.