CN104761544B - The selective depressant of the important mutant of clinic of EGFR tyrosine kinase - Google Patents

Abstract

The present invention relates to the selective depressants of the important mutant of clinic of EGFR tyrosine kinase.The present invention relates to the officinal salts of the compound of formula A and they, the pharmaceutical composition comprising such compound and salt, with and application thereof.

Description

The selective depressant of the important mutant of clinic of EGFR tyrosine kinase

Background of invention

1. invention field

The present invention relates to the officinal salt of the compound of Formulas I-III and they, it is related to the medicine comprising such compound and salt Compositions, and relate to and application thereof.The compound of the present invention and salt inhibit inhibiting the drug resistance of therapy treatment to send out EGFR Important kinases in exhibition, especially EGF-R ELISA EGFR, especially its mutant, and can be used for treating or improving different Normal cell proliferative disorders, such as cancer.

2. background information

The present invention relates to the biaryl amino-compounds for the inhibitor that can be used as protein kinase PKs.PKs is to communicate in the cell In very important signal transduction entity, they are by catalytic phosphatase group from serving as phosphodonor (phosphodonor) herein The hydroxyl that is transferred in the amino acid side chain on protein of ATP and modify many protein.PKs usually shifts phosphate group Serine or threonine hydroxyl on to protein, they are serine/threonine kinase (S/TKs) in this case.Under It on the phenolic hydroxyl for the tyrosine side chain that phosphoric acid is transferred on target protein by the one most common kinases type and is albumen junket ammonia Acid kinase (PTKs).In general, tyrosine kinase is incorporated in the intracellular domain of great transmembrane protein, the transmembrane protein is extracellular There is cognate ligand binding structural domain, thus ligand binding activates tyrosine kinase in structural domain.Such molecule is receptor junket ammonia Acid kinase (RTKs).Less common a kind of targeting hydroxyl associated with the various lipids pure and mild sphingol of such as phosphatidyl-4 is simultaneously claimed Make lipid kinase.Some of them are closely related with certain PKs in structure, due to this and due to lipid kinase identical as PKs Approach in the activity that often needs to, they usually consider together with PKs.Most rare classification is can be by serine/Soviet Union's ammonia The dual-specificity kinase (DSKs) of acid and tyrosine hydroxyl all phosphorylations.In general, in cell normal amount PK classification none Seeming can be by the substrate phosphorylation of other types, although certain loss of specificity can be induced in laboratory conditions, one It is same in a little tumours, wherein expressing the mutant forms of PK or in which may largely be overexpressed PK.In structure, kinases phase When being fully understood.There are kinase domain, it can be holoprotein or be only a small portion of much bigger modularization albumen Point, this structural domain have about 35 kD basic conserved structure, be made of two leaves (lobe), N-terminal structural domain mainly by Beta sheet is constituted, and biggish C-terminal structural domain is mainly made of alpha-helix.Exist between the two leaves and combines ATP and substrate Drastic crack gap.Binding Capacity domain is quite greatly and quite variable, and the spy for distinguishing different protein substrate and holding phosphorylation It is anisotropic.This species specificity may be it is very variable, some enzymes such as MEK only has a kind of known substrate, and other enzymes can be by egg Hundreds of different dis in white matter.

Phosphorylation can change the conformation of protein, and enzyme is usually converted to active form from inactive form, or vice versa , or protein is made to associate closely with binding partners, with cause cellular localization or function multiprotein complex assembling or Variation in decomposition.Many transductants that signal neutralizes in from cell surface to core to cell are PKs or are controlled by PKs.Therefore, The inhibitor of the kinase activity of PKs has the influence of highly significant to cellular signal transduction, to decay to the normal of external signal Response and the usually unsuitable excessive response as caused by the mutation of signal transduction molecule itself.The access of even now is in vivo Distribution is very extensive and participates in most of body functions in a manner of such or is such and may be caused by their dysfunction Disease, the inhibitor of PKs is particularly useful for treating cancer and immune disorders, all confirms extensively in both kinds of Diseases The overactivity of PKs and they usually play a key effect in driving lysis itself.

Many different types of kinase inhibitors, some granted and listings that succeeded are developed.Seem effectively to press down Make the molecular scaffold of many kinases first is that a series of threefold rings, two of them, usually all three rings are aromatics, are being tied U-shaped structure is likely to form when closing on kinases.Two distal loops by key or can pass through the various companies being made of 1-3 atomic link Base is connect to be directly connected on center ring.Center ring --- it is almost nitrogenous heteroaromatic system always --- forms 1-3 and arrives Just before so-called DFG ring (the not structure changes in kinases, the activity conformation for being just able to achieve the enzyme must be properly positioned) The end N- and C- leaf between kinases hinge area in backbone residue on hydrogen bond.This end of the inhibitor also takes up sharp A part of the adenine combined area (it is often very hydrophobic) of enzyme, and when two rings for constituting " dry (stems) " of the U occupy Often fill the fat pipe of a part in the space generally occupied by the remainder of ATP molecule.Although considerable swash to specific The compatibility of enzyme from be conducive to interact with the unique structural determinant of being expected in target kinase and/or be unfavorable for Be not intended to the selected substituent group of the kinase interactions inhibited to decorate these center rings, but to many compatibilities of various kinases and Various torsions and bending angle of the selectivity between these three rings, and optimize some substituent groups to the compatibility of target kinase It not direct in itself may interact with protein, but the most stable conformation of these three rings relative to each other can be can control.Therefore The effect of some substituent groups be influence the inhibition molecule whole interior energy, with stablize be conducive in conjunction with conformation and it is indirect With kinase interactions.

Kinases have shown be many lysises very important effector, especially in cancer.By kinases with Many different levels control cell Proliferation and under the normal environments for cell Proliferation, it is necessary to send signal from extracellular, herein They are integrated on receptor and activated receptor.Many important receptors in cellular signal transduction are kinases, especially RTKs, or It is directly coupled on kinases --- the kinases itself is activated receptor activation.Once these kinases have been activated, they are activated again Signal transduction cascade, this is usually directed to several other kinases in the amplification wave of phosphorylation, this eventually lead to the transcription in core because The activation of son.The activation of transcription factor, which causes to generate, to be executed the intracellular various programs and (including the cell is made to initially enter increasing Grow those of period program) protein.Once newly synthesized protein will continue to this in general, this process continues a few hours Process, without further extracellular input.If proliferative cell cycle starts, the first histone matter of synthesis includes Their activator and active cell of further transcription factor and the later phases of driving cell cycle are replicated and were divided The effector of journey.Kinases is the main controller of each step during this.When this process do not have it is appropriately controlled and thin When born of the same parents execute the cell cycle in the case where no external control appropriate, they are converted and if immune system does not disappear Them are gone out, then is likely to form tumour.

When checking transformed cells, their constant features first is that Hyperphosphorylationof, shows that too many kinases is inappropriate Ground activation.This may be caused by mutation extremely diversified in cell.Such as their own pair is inadequately generated by cell Receptor connects the ligand of one of kinases.Or multiple volumes as not having its expression of suitable control or the gene as present in cell Outer copy, one of these kinases may be seriously overexpressed.Another very common gene defect is the mutation in kinases code area, This, which causes, is in activity in composition and does not need the kinases that proper signal activates it.Sometimes the kinases is not inadequately in work Property, but lose phosphatase by mutation or deletion (this is considered limiting its signal transduction by removing phosphoric acid from target molecule) It is living.The inspection of cell culture tumour and isolate from clinical tumor is all almost always in the phosphorylation of tumour cell It was found that this kind of defect.

In the late 1980s, it was found that several small molecule kinase inhibitors.These molecules are almost always incorporated in Its binding site is competed in the catalysis crack of kinases and with ATP.Therefore they be ATP emulative, since then since find it is big Most inhibitor belong to this kind.But it finds once in a while and protein substrate competition, substrate competitive kinase inhibition Agent or the kinase inhibitor more generally all competed with ATP and substrate (double inhibitor) or neither with Receptor Competition, also not with bottom The kinase inhibitor (noncompetitive inhibitor) of object competition.After taking into account Premeabilisation of cells difference, it is found that these compounds are separating Kinase inhibition detection in effect and the kinase inhibition in cell between exist very good correlation.To many kinases Speech, there is also excellent correlations between the phosphorylation loss and the inhibition of cell Proliferation of downstream target.Due to tens kinds Different kinases is thousands of time to show this correlation, clearly confirms that abnormal kinase signal transduction will cause transformed cells not Controlled proliferation blocks the kinases of overactivity that can terminate the proliferation in many cases.In many cases, kinase inhibitor The apoptosis that transformed cells can actually be induced alone, so that tumor regression.This may be due to usually activating in tumour cell Several rush Apoptosis mechanisms and abnormal Phosphorylation may sufficiently participate in inhibiting apoptotic process and occurring.Although these energy in cell Power can prevent the good proof in nude mice as the tumour of xenograft growth late at the beginning, but as reagent changes Into, the conventional growth for confirming kinase inhibitor and can slowing down the tumour of the targeted kinases oncogene of expression, better reagent Cause tumor size usually to degenerate to immeasurablel degree, and the seldom regrowth of tumour after stopping administration, shows possibility The tumour of animal is cured.In addition, in vivo efficacy is related to cell activity and enzymatic activity after associated with tumour exposure Connection.

Clinic proves to come slower, possible partly because clinical tumor is usually more swollen than what is grown under conditions of careful control Tumor is much more complex, partly because mouse is much more strong than people in terms of biochemistry and can tolerate bigger drug relative dosage, And it is primarily due to be generally difficult to know which is the kinases for being suitble to inhibit in any given tumour.But Imatinib --- Quite potent inhibitor with the carcinogenic TK BCR-ABL of the fusion of really outstanding pharmacokinetic property, in 2000 It is approved for chronic myelocytic leukemia (CML).It is clinical that this kinase inhibitor provides very compellent learning concept It proves, because about 2/3 CML patient's (its tumour is almost by definition containing there are two types of one of the BCR-ABL of form) admirably rings It should treat and leukaemia cell usually almost disappears from circulation.Surprisingly, the mutation around this blocking is seen Get up very slowly, or even the drug is still valid in 80% patient after treatment 10 years.This not yet confirms to be ordinary circumstance, can It can be partly because most of tumours find much more late than CMLs in their biohistory and have the much longer time to become base Because of heterogeneity, and partly because few tumour depends on a kind of oncogene as CML dependent on BCR-ABL.

Epidermal growth factor RTK(EGFR, erbB-1) two kinds of inhibitor Gefitinibs and Erlotinib in about 10 years Before be approved for lung cancer.EGFR is one of the kinases most often lacked of proper care seen in solid tumor, usually 50% or more tumour Discovery is overexpressed or is mutated in type, including non-small cell lung cancer (NSCLC).Although these inhibitor are to varied overexpression The xenograft of EGFR has excellent activity, but very limited activity is observed in NSCLC, only about 10% Patient responds the drug and average response only continues 1 year or so, although the respondent of discovery persistently much once in a while.When being rung When going through of the person of answering finds that most of good response persons have one of several mutation in EGFR, contains wild type (wt) The respondent of EGFR usually not good response.As analysis these mutant, especially EGFR L858R and EGFR del746- When 750, it is found that they all have the property inherently activated, it means that they drive increasing in the case where no external signal It grows, ATP(higher K is also weaker combined than wt EGFR_m), while having similar with wt EGFR to the affine of the inhibitor Property.It means that since these inhibitor are ATP emulative, than being easier to compete ATP from enzyme in wt and cut off susceptible prominent Kinase activity in variant, with the practical inhibitor effectiveness promoted in mutant.Meanwhile the proliferation and survival ratio of these tumours are big Most tumors rely more on EGFR signal transduction, because of the signal reliably overacfivity since original mutation event.

It, may be in the initiator cell initially converted if lung cancer is to usual rather late when discovery as described above, solid tumor (founder cell) average 6-12 after occurring.It is multiple to their DNA that one of property of transformed cells is that they lose The control of quality control processed, therefore their spontaneous mutation rate is more much higher than unconverted cell.Since mutation is easiest in DNA Generation and these cellular replications obtain very fast in reproduction process, this further increases mutation rate.As a result it is, as tumour is old Change, obtain ever-increasing mutation count, this occurs in a random basis, so that passing through tumorigenic subclone at any time --- With the science of heredity slightly different and slightly different each other with primary tumor.These subclones are not only involved in and body itself Struggle for existence, also mutual struggle for existence, because competing their obtainable limited resources between themselves.If changing advantage The environment of tumor colonies, so that it becomes less shake down relatively (such as by adding its effective inhibitor), it If preceding much unsuccessful secondary clone is not influenced by the inhibitor, the ecological niche vacated can be taken over.Alternatively, if The clone is not killed thoroughly or completely cuts through proliferation, will continue to generate mutation, and if the inhibition is avoided in mutation, it is this Subclone can be freely proliferated now, and be not inhibited the obstruction of the parental clone (parental clone) of agent or inhibition.Therefore Natural selection prediction, cancer should can generate drug resistance just as infectious disease, and since the tumour in single host is sub- Competition between clone greatly drives the selection course, and overall effect is to promote more aggressive subclone, and tumour is usual Become more fatal with its evolution.

As respondent of the tracking to Gefitinib and Erlotinib, occurring and several different genes for drug resistance is found Variation is associated.Tumour seems to drive tumour using entirely different signal transducting system in the case where extremely rare, but resistance to Pharmacological property is usually directed to the adjustment (tweaking) of primal system.EGFR constitutes RTKs together with erbB-2, erbB-3 and erbB-4 ErbB subfamily member.These receptors are initiated the ligand activation of their dimerization, although EGFR-EGFR homodimer phase When being usually used in signal transduction, but the more common process in this family is that ligand is made to cause Heterodimerization, so that signal passes Leading entity is such as EGFR:erbB-2 or erb-B2:erbB-3 and ligand appropriate.Reactivate the simplest side of the system Formula is to improve the expression of one of other erbBs, this is even also often seen before the treatment, and helps that more table excessively be construed to Tumour up to wt EGFR is not responding to EGFR inhibition.Rather relevant mechanism is related to RTK HGFR, although be not erbB family at Member, but have been displayed and form the carcinogenic heterodimer of height with erbB-3 when being overexpressed, and the overexpression of HGFR is to EGFR The common mechanism of the drug resistance of inhibitor.At least in lab setup, HGFR inhibitor is added in these cells and is restored To the sensibility of EGFR inhibitor.The third and the most common drug resistance mode are the further mutation of EGFR, this reduces it To the sensibility of EGFR inhibitor." gatekeeper " mutation T 790M most commonly so-called in these, and then right Being often found to have double mutant (such as L858R T790M) in the initial response person of EGFR inhibitor generation drug resistance NSCLCs.Such subclone is to exist always or only occur being unknown after the treatment, but seem that the mutation most probable exists In short term response person it is existing and later generate drug resistance long-term respondent in as fresh mutation occur.

Initially believe, these mutation spatially hinder inhibitor to be integrated on mutant enzyme, therefore reduce the affine of them Property and effect.But it is closer studies have shown that the most common mutation has extremely low influence to inhibitor compatibility, but lead to ATP The binding affinity of wt EGFR is restored or may up to 10 times, as a result achievable inhibitor concentration no longer high enough to Treat shutoff signal conduction in effectiveness.In principle, it is only necessary to sufficiently improve the compatibility of inhibitor to overcome the ATP of raising Compatibility, but be difficult to do so in practice, because Gefitinib and Erlotinib are very potent, secondary nanomoles EGFR inhibitor with good PK property, but there was only active to the tumour of wt EGFR driving.In addition, although T790M is prominent Variant does not reduce EGFR to the compatibility of Erlotinib and Gefitinib, but its certain limitation can be in both inhibitor The approach of compatibility is improved in anilinoquinazoline chemical type.Therefore, in order to find to the bigger affine of T790M type mutant Property, new chemical template is had checked, and some, the U-shaped inhibitor of especially type discussed above seems at this It is quite promising in one field.

EGFR receptor plays a significant role in whole body, (is all especially in proliferation in entire GI epithelium and skin Very active tissue) in.Since two kinds of major dose-limiting toxicities of EGFR inhibitor are fash and serious GI disorder, These are almost affirmed largely (mechanistically based) based on mechanism.As long as tumour is driven by wt EGFR Dynamic, this is difficult to be avoided by rationally designing, and especially for oral agents, wherein gastrointestinal tract exposure is inevitable, but if Tumour then can reduce the toxicity observed to disclosed clinical candidate, the candidate is all in benzene by mutation EGFR driving In amido quinazoline template and undoubtedly to 790 wt(T) EGFR compares the mutant receptors of the family (they is in position 790 M) clearly more powerful effect.When treatment has become the tumour for depending on the driving of T790M mutant, this is undoubtedly not up to best Mode.It assume that several inhibitor modes are more suitable for treating the expected tumour that this drug resistance process occurs, thus EGFR inhibits Agent has the compatibility to T790M mutant improved, and all these examples is all in the literature, some nowadays in clinical test In.This patent application describes the compounds for meeting one of these standards.

The EGFR inhibitor for being significantly higher than wt EGFR to the compatibility of mutation EGFR should be able to inhibit under optimal dose by this Proliferation in the tumour of mutant driving, while in unconverted tissue (wherein wt EGFR is responsible for EGFR signal transduction) There is EGFR signal transduction relatively extremely low (if any) to influence.This should allow to give the mutant of significantly larger dosage Selective EGFR inhibitor, to improve the effect and therapeutic index of the tumour driven to mutant.It is noted that due to mutant To ATP combine influence, this substantially in Erlotinib and Gefitinib respondent there is a situation where, wherein mainly due to The mutant of response reduces the compatibility of competitive ligand ATP, they are actually more more sensitive to inhibitor than wt EGFR.It is existing Having been discovered that several third generation EGFR inhibitors, some of them are in clinic.These compounds are usually can not retroactive inhibition Agent, initially based on U-shaped hexichol amine pyrimidine bracket, but this has extended to several associated supports, but all with icotype knot It closes on hexichol amine pyrimidine.In general, these compounds are the very potent of the mutation EGFRs containing T790M mutation Inhibitor, and it is less potent to wt EGFR and some other mutation.Due to this mode, it is believed that wt can be significantly reduced (mechanism-based) toxicity based on mechanism that EGFR inhibits, while keeping to the tumour by appropriate EGFR mutation driving Extremely strong inhibition effect.Therefore such compound especially can be before to a line Erlotinib or Gefitinib therapy Sensitive patient is used as second-line therapy after becoming drug resistance.These inhibitor can not only inhibit mutation appropriate by force as before Receptor also itself should will not inhibit (mechanism-induced) toxicity of the significant mechanism induction of initiation by EGFR This point is realized simultaneously.Big cyclammonium inhibitor of the invention is irreversible EFGR inhibitor, have with as these reagent classes Inhibit the selective mode and excellent pharmacokinetic property inhibited better than wt EGFR to mutant EGFR, it is therefore evident that being Excellent reagent for NSCLC and the second line treatment of any other tumour of the driving of this subfamily by being mutated EGFR kinases.

The 1990s mid-term develop improve inhibitor effectiveness another method.Recognize a part of TKs benefit With the cysteine residues on ATP combination groove edge to form the hydrogen bond with the ribose of ATP, and it is most of thus using Soviet Union's ammonia Acid.This cysteine (the C in EGFR is all contained in EGFR family⁷⁹⁷).Speculate that this cysteine may be connected inhibitor On allcylating moiety alkylation, the inhibitor is incorporated in ATP-binding site and is provided about in hcy thiolactone electrophilic Body.To keep this concept effective, allcylating moiety must have low inherent reactivity, because in terms of potential PK and toxicity reason all Be not intended to its do not make any distinction between with internal a large amount of nucleophilic precursor reactant.To make alkylating agent with highly selective suitable with this certainty Weak electrophilic precursor reactant, according to display, which itself have to have high (non-covalent) compatibility and necessary to binding site Preferentially so that the weak electrophilic body is combined close to the conformation of electrophilic body.Finally, it was found that, which needs the blood relative to inhibitor Slurry half-life period quickly carries out, and otherwise it, which had not been reacted with crucial cysteine, to wash out from internal.It has found in this way Irreversible inhibiting compound, and find that they are not only the much potent internal EGFR of the reversible inhibitor more equivalent than theoretically Inhibitor, as additional benefit, they also make the difference (at least in the case where anilinoquinazoline and correlation 3- cyano quinolines) ErbB-2 and erbB-4 inhibits template as the very potent inhibitor of all erbBs, it was demonstrated that if binding pattern is really good, It may be less most important to the high non-covalent compatibility of target.It is into clinical most of second generation EGFR inhibitors The irreversible inhibitor of EGFR, uses acrylamide derivative as electrophilic body, and seems generally in clinic than reversible suppression Preparation is more effective.Clinical test, which is tended to what is paid close attention to, is considered as simplest registration policy (registration Strategy), receive Gefitinib/Erlotinib respondent and after forming drug resistance second line treatment they.Due at this It is frequently observed response in kind setting, these compounds can obviously overcome the T790M drug resistance of many patients, may not be such as This, because most of design before finding the catastrophe.But this clinical strategy also means that not yet answer is closed In irreversible inhibitor whether by wt EGFR(and possible wt erbB-2/4) it is overexpressed in the tumour driven with useful The same noticeable problem of clinical activity, but in view of between wt EGFR and double mutant ATP compatibility it is similar Property, it appears that it is possible to really such.

WO 2013/014448 is disclosed as a series of of potent and mutant selectivity irreversible EGFR inhibitor 4- (bicyclic aryl) -2- (3- acrylamido anilino-) pyrimidine, they usually have low compatibility to wt receptor.The compound Often there is the quite similar effect down to secondary nanomolar range to EGFRdel746-750 with L858RT790M double mutant Power, despite the fact that the former is susceptibility mutations body, the latter is the mutant for the inhibitor of resistance to anilinoquinazoline.To wt EGFT Activity tend to inefficient 40 to > 100 times.Early clinic data are shown in the plurality with T790M mutation in its NSCLSs Response rate (actual shrinkage) in patient, very slight fash is observed in many patients, shows DLTs no longer by wt The control that EGFR inhibits.

As discussed above, the activity of the allcylating moiety in irreversible inhibitor (such as michael acceptor) is verified very It is important.It is few to reach expected target if it is too strong to the reactivity of nucleophile, and there may be modified big of a large amount of electrophilics Molecule is to cause additional risk of toxicity.In addition, plasma half-life is extremely short, this throws into question in many tumours.By having enough to meet the need slowly Oncogene driving tumour can and under the irreversible inhibitor for being exposed to short pulse long-term Inhibit proliferaton signal transduction, Because of the subsequent permanent deactivation of the oncogene.But if tumour constantly generates new carcinogenic protein, plasma drug level is quick Drop to effective concentration hereinafter, and no longer inhibit newly synthesized receptor, the inhibitor signal transduction pathway is administered until next time Ability activity recovery.Even if tumour does not have enough to meet the need oncogene quickly, this pulse administration also provide select by force pressure using as Apparent drug resistance approach releases the control generated to oncogene.

If checking the acrylamide core of irreversible inhibitor, several factors can be used for influencing it as michael acceptor Reactivity；More it is more than and uses most of allcylating moieties, because michael reaction is inherently reversible.They usually control the electricity of alkene The double bond of sub- density, electron deficient is more advantageous to michael reaction in terms of dynamics and thermodynamics.But the polarity of olefinic double bonds Degree is also important, and the low electron density on relatively low electron density ratio α carbon on β-carbon is more important.Finally, being gathered around by space It squeezes (steric crowding) and stablizes the double bond relative to Michael-adduct, because Michael's addition makes two plane trigonometries The ligancy of shape (or being line style in the case where alkynes) atom improves 1, and becomes tetrahedron (plane triangle).

The electron density of Michael's system depends in part on the property of carbonyl.Although carbonyl is acyl always in acrylamide Amine, but the group being bonded with the amide nitrogen will affect its ability to whole system contribution electronics, and therefore shadow quite significantly Ring the whole electron density of michael acceptor π system.In WO 2013/014448, most of acrylamides are had There are two the substituent group (ether and tertiary amine) of strong electron and a benzyl rings closer to neutral substituent group (2- aminopyrimidine) Replace, but and by control acrylamide nitrogen-atoms it is achievable almost, this substitute mode may generally make the π system Michael's addition is inactivated.Extreme at another, 6- acrylamido Pyridopyrimidine has been described as irreversible EGFR suppression Preparation, but for simple acrylamide, chemical reactivity is really too strong so that can not treat effectively.By providing intramolecular hydrogen Key donor can also indirectly control carbonyl itself.This hydrogen bond has omits one of lone electron pair of ketonic oxygen compared with normal Micro- population effect for being pulled away from oxygen atom, so that entire π system slightly more electron deficient and polarization.But although this strategy is effective, But it is related to the loss that building is enough the chemical scaffold (the chemical scaffold) and conformational flexibility that allow hydrogen bond to be formed, Because the formation of cyclic annular hydrogen bond is related to serious space constraint always.If this implements to be more advantageous to goal response on molecule Conformation, this is more important more than electronic effect, but if it implements unfavorable conformation, then may totally block the hair of goal response It is raw.

The reactive most common mode for influencing michael acceptor is that functional group is arranged on olefinic carbon.In general, In alpha position, unique significant effect is electro --- electron-withdrawing group makes the π system more electron deficient and improves reactivity, And electron substituent group increases π-electron density and keeps the system lower to the reactivity of Michael's addition.Although electron-withdrawing group Property theoretically can remove bigger electron density simultaneously from β carbon from α-carbon or through mesomeric effect by inductive effect Therefore change the integral polarity and its whole electron density of the system, but seem to have no difference in practice and all lack α-electronics group tends to activate michael acceptor by force, inactivates michael acceptor to α-electronics substituent group.In β-carbon Place, for electronics and space reasons, substituting effect is often more complicated.For acrylamide, β atom must accommodate michael acceptor Volume, if it has had the substituent group greater than hydrogen, this will cause the space constraint being initially not present in adduct.Perhaps Multi-substituent, such as alkyl can improve the electron density of entire π-system and improve its thermodynamic stability, and these effects Michael reaction can be slowed down and reduce the overall favorable thermodynamics of the reaction, so that it is easier that inverse michael reaction occurs.β The electron-withdrawing group of position usually passes through their influence acceleration michael reactions to the whole electron density of π-system, but now The property of their electron attraction also becomes important.Simple inductivity electron-withdrawing group (such as halogen or CF₃) can be further The electron density of β atom is reduced, so that π-system more polarity, but intermediary electron-withdrawing group (such as carbonyl) can be drawn from alpha position More electron densities, so that olefinic double bonds more electron deficient and more hypopolarization, and compared with absolutely not substituent group, it can sufficiently improve The thermodynamic driving force of the reaction, while slowing down integral power.

When carrying out simple enzymatic determination with irreversible inhibitor, it is less likely to obtain true inhibition constant.If the chemical combination Object has a good non-covalent compatibility, and irreversible warhead is only with its target response on enzyme and the enzyme is pure and 100% It is active, and the whole thermodynamics of bonding has the Δ G greater than about 3 kcals, then IC₅₀It theoretically should be organized enzyme The half of concentration, but the IC measured in practice₅₀It is usually much higher.The dynamics that this may be attributed to alkylated reaction is very slow, But situation be not usually in this way, and the speed regardless of alkylated reaction, IC₅₀It is more much bigger than enzyme concentration.

As the case where most pharmaceutical agents, the cellular potency of irreversible EGFR inhibitor is often below their separation Enzymatic determination effect.Drug permeation cell film and must not be pumped out by resistance protein.The other potential targets of many of cell can (sequester) drug can be invalidly detained, and it can suffer from the metabolism in cell.It, can for irreversible inhibitor The quantity of the emulative good nucleophile of energy is greatly improved in raji cell assay Raji, it is meant that indiscriminate michael acceptor is existing There are many potential target for reaction, and with michael acceptor reactivity improve, merely due to this reason, cellular potency is just Always it reduces.

In the detection of entire animal, situation becomes obvious more complicated.Inhibitor is not added in static system, It is added in dynamical system, many processes carry out simultaneously.(drug is wherein mainly even ignored to the drug of parenteral administration Absorption Characteristics) for, the drug must sufficiently be distributed in target tissue and around stop long enough with useful effect. The circulatory system in conjunction with excretion and metabolic process is it is meant that the drug for being even delivered to target also tends to only of short duration presence.It is right In the inhibitor of fast reaction, short-time contact target may be enough that it is made irreversibly to inhibit target, but the suppression for slow reaction Preparation, the contact with target may be too short so that reaction can not be made to proceed to completion on most of target molecules, and slow alkylation inhibits Agent may show to be very similar to (usual unoptimizable) reversible inhibitor.Therefore, start to realize, only prepare extremely low reactivity Michael acceptor be not enough to prepare acceptable medicament.But as discussed above, most of real tumours are often quite fast Ground has enough to meet the need their oncogene, the EGFR protein for being such as mutated and/or being overexpressed, therefore the reaction of Rapid Alkylation Under target protein Property stronger michael acceptor need that there is sizable half-life period so that major part or entire period between administration, week It encloses in the presence of sufficient inhibitor so that newly synthesized receptor to be alkylated.In practice, long-time blood plasma needed for accomplishing this point Exposure can only low compound be realized by providing the inherent reactivity of michael acceptor, so that it largely keeps circulation many Hour, many nucleophiles without touching in vivo with it (including are largely recycled and glutathion inside cell, are mainly used Way first is that capturing electrophilic xenobiotics (xenochemicals)) irreversibly react.

A series of intrinsic paddy Guang of irreversible EGFR inhibitors is described in J Med Chem 56,7025 (2013) Sweet peptide reactivity.Under the conditions of one group of standard, 23- > 10, the half-life period of glutathione addition in 000 minute are described, and retouch 25-400 minutes effective ranges are stated.Since the end-stage half-life period of Afatinib in human body is 37 hours (Clin Pharmacokinet 52,1101 (2013)), Canertinib be about 3.5 hours (Clin Cancer Res, 12, 4645 (2006)), but they are respectively provided with 25 and 23 minutes half-life period in glutathione measurement, and nothing is suspected to have other metabolism Factor plays a role herein.Due to dacomitinib --- it is 4- anilino- -7- alkane as Canertinib as Afatinib Oxygroup quinazoline -6- acrylamide derivative, but there is as Afatinib amino crotonamide side chain rather than Canertinib Unsubstituted acrylamide --- in human body with about 50 hours plasma half-life and in this measurement be 49 hours, This strongly suggests that removing terminal acrylic amide can provide PK advantage in human body, and the rate of this and michael reaction there is not pass System.It should be noted, however, that being different from the aniline of the very electron rich of 2013/014448 compound of WO, these inhibitor are respectively Acrylamide nitrogen be bonded on the 7- alkoxy -4- anilinoquinazoline of appropriate electron deficient.This application is without proposing to compare propylene The long any alkylation side chain of acyl group, it is most likely that be because the intrinsic michael acceptor property of such compound is too weak so that nothing Effect, this is a bit emphasized in J Med Chem 56,7025 (2013).But as discussed immediately above, such chemical combination The estimated pharmacokinetics having the advantages that better than corresponding acrylamide of object, and the meeting when suitably adjusting their electron density Irreversible inhibitor is generated in the anilino-pyrimidine template discussed in WO 2013/014448.The purpose of the disclosure is to disclose With those of disclosing in WO 2013/014448 there is extended crotonamide (or bigger) side on similar anilino-pyrimidine The compound of chain, wherein reducing the electron density of michael acceptor systematically to keep being enough to provide T790M mutation EGFR The selectivity of inhibitor can not the treatment of retroactive inhibition and the reactivity of pharmacodynamics advantage, while keep do not have on michael acceptor There is the associated excellent pharmacokinetic property of terminal double bond.

Summary of the invention

Part of the present invention provides the optionally adjustable albumen for causing the drug resistance for inhibiting therapy to existing EGFR- base and swashs Enzyme, the especially protein kinase of I receptor tyrosine kinase (RTK) family or erbB family, most particularly certain of EGFR receptor The active new compound and its officinal salt of a little mutant forms.This inhibitory activity influences biological function, including but unlimited In cell Proliferation and cell invasion, inhibit transfer, inducing apoptosis or inhibition angiogenesis.Additionally provide pharmaceutical composition and medicine Object, described pharmaceutical composition and drug only include the compound of the present invention or salt or comprising combining with other therapeutic agents or moderator The compound of the present invention or salt.

The present invention relates to the compounds of formula A

Or its officinal salt, wherein

X¹It is CH or N；

Y isOr

R¹Selected from hydrogen, fluorine, chlorine, methyl, CF₃、CHF₂And cyano；

R²Selected from methoxyl group, ethyoxyl, isopropoxy, cyclopropyl oxygroup, methyl, ethyl, isopropyl and cyclopropyl；

R³Selected from H, halogen, C_1-6Alkyl, C_1-6Halogenated alkyl, C_2-6Halo alkynyl, optionally by R⁷And C_1-6It is alkyl-substituted C_2-6Alkynyl, R⁴N-C_2-6Alkyl-NR⁴R⁴、R⁷；

R⁴It independently is H, C_1-6Alkyl, C_2-6Hydroxyalkyl, C_1-6Alkoxyalkyl or C_2-6Alkyl NR⁸R⁹Or two R⁴Group Carbon atom connected to them forms 3-12 unit monocycle or bicyclic ring systems, wherein most two carbon atoms are by N, NR⁴、O、S (O)_xWith S (O) (NR¹⁴) substitution；

R⁵It is H, F, Cl, CF₃、CHF₂、CF₂C_1-6Alkyl, CF₂CH₂NR⁸R⁹、CH₂NR⁸R⁹Or C_1-6Alkyl；

R^6eAnd R^6zIt independently is H, F, Cl, CF₃、CHF₂、C_1-6Alkyl, aryl, heteroaryl, naphthenic base, heterocycle, (CH₂)_mCHR⁴R⁷、CF₂C_1-6Alkyl, CF₂(CH₂)_mCHR⁴R⁷Or C (R₄)₂R⁷；

R^6tIt is C_1-6Alkyl, C_3-6Naphthenic base, aryl, heteroaryl, heterocycle, (CH₂)_mCHR⁴R⁷、C(R₄)₂R⁷；

R⁷It is OH, NR⁸R⁹、OCH₂(CH₂)_mNR⁸R⁹、C_1-6Alkoxy, C_1-6Alkoxy -C_1-6Alkoxy, C_2-6Hydroxyl alkoxy, Oxetanyl, oxetanyl oxygroup, oxetanyl amino, tetrahydrofuran base, tetrahydrofuran base oxygroup, Tetrahydrofuran base amino, Oxyranyle, Oxyranyle oxygroup, Oxyranyle amino, oxepane alkyl, oxa- ring Heptan alkyl oxy, oxepane alkyl amino, azetidinyl, azetidinyl oxygroup, azetidinyl amino, pyrrole Cough up alkyl, pyrrolidinyl oxygroup, pyrrolidinyl amino, piperidyl, piperidyl oxygroup, piperidyl amino, nitrogen heterocyclic heptyl, nitrogen Trioxepane base oxygroup, nitrogen heterocyclic heptyl amino, dioxolane base, dioxane base, morpholino, thiomorpholine Generation, thiomorpholine generation-S, S- dioxide, Piperazino (piperazino), Dioxepane base, Dioxepane base Oxygroup, Dioxepane base amino, oxaza heptane base, oxaza alkyl oxy in heptan, oxaza alkyl amino in heptan, Diazesuberane base, Diazesuberane base oxygroup, Diazesuberane base amino；And as R⁷A part all NH It can be optionally by R⁴Replace；

R⁸And R⁹It independently is H, C_1-6Alkyl, C_3-6Alkenyl, C_3-6Alkynyl, C_3-7Naphthenic base, C_3-7Cycloalkenyl, C₁-C₆Acyl group, 4-12 circle heterocyclic ring base, C₆-C₁₂Aryl or 5-12 unit's heteroaryl；Or R⁸And R⁹Atom connected to them forms 4-12 unit monocycle Or bicyclic ring systems, wherein most two carbon atoms are by N, NR⁴、O、S(O)_xWith S (O) (NR¹⁴) substitution；And R⁸And R⁹It can be independent Or together by most three independently selected from hydroxyl, C_1-6Alkoxy, C_1-6Hydroxyalkyl, C_1-6Alkoxy -C_1-6Alkyl, C_1-6Alcoxyl Base-C_1-6Alkoxy, C_2-6The substituent group of hydroxyl alkoxy replaces；

R¹⁰It is H, C_1-6Alkyl；

X²It is C, CH or N；

X³It is CH or N,

X⁴It is CR⁴Or N or NR¹⁰；

X⁵And X⁶All being C or one can be N；

Two keys in a-e are double bonds and the other three is singly-bound, so that atom X²-X⁶All there are two double bond and its phases Even；

Key f is usually double bond, only works as X⁴And X⁵Or X⁴And X⁶It is nitrogen and X²-X⁶In the other three be C, CH or CR⁴ When, two key f can also be singly-bound；

M is 0-4；

Condition is as follows:

X²-X⁶At least one of and not more than three be N or NR⁴；

R⁵、R^6eAnd R^6zMiddle only one can be halogen or containing with carbon itself that be bonded directly in the acrylamide system The halogen of atom Direct Bonding.

DESCRIPTION OF THE PREFERRED

The term as used herein " halogen (halogen) " or " halogenated (halo) " refer to fluorine, chlorine, bromine or iodine (F, Cl, Br, I).It is halogenated preferably to refer to fluorine or chlorine.

Term " alkyl " refers to saturation monovalent aliphatic alkyl, including having the straight chain and branched group of specified carbon atom number. Term " C_1-6Alkyl " or " C₁-C₆Alkyl " refers to the linear or branched alkyl group containing 1 to 6 carbon atom, such as methyl, ethyl, just Propyl, isopropyl, normal-butyl, isobutyl group, sec-butyl, tert-butyl, amyl, hexyl etc..Similarly, term " C_1-4Alkyl " or “C₁-C₄Alkyl " refers to the linear or branched alkyl group containing 1 to 4 carbon atom, such as methyl, ethyl, n-propyl, isopropyl, positive fourth Base, isobutyl group, sec-butyl, tert-butyl etc..

The term as used herein " halogen (halogen) " or " halogenated (halo) " refer to fluorine, chlorine, bromine or in some cases Under, replace alkyl that can name referring in particular to substituent group.It is taken for example, " halogenated alkyl " refers to by one or more halogenic substituents In generation, has the alkyl of specified carbon atom number, and usually contains 1-6 carbon atom and 1,2 or 3 halogen atom (i.e. " C₁-C₆Halogen Substituted alkyl ").Therefore, C₁-C₆Halogenated alkyl includes trifluoromethyl (- CF₃) and difluoromethyl (- CF₂H).

Similarly, " hydroxyalkyl " refers to the alkane with specified carbon atom number replaced by one or more hydroxyl substituents Base, and usually contain 1-6 carbon atom and 1,2 or 3 hydroxyl (i.e. " C₁-C₆Hydroxyalkyl ").Therefore, C₁-C₆Hydroxyalkyl includes hydroxyl Methyl (- CH₂) and 2- ethoxy (- CH OH₂CH₂OH).

Term " C_1-6Alkoxy ", " C₁-C₆Alkoxy " or " OC_1-6Alkyl " refer to the straight chain containing 1 to 6 carbon atom or Branched alkoxy, such as methoxyl group, ethyoxyl, positive propoxy, isopropoxy, n-butoxy, isobutoxy, sec-butoxy, tertiary fourth Oxygroup, amoxy, hexyloxy etc..Term " C_1-4Alkoxy ", " C₁-C₄Alkoxy ", " OC_1-4Alkyl " refers to containing 1 to 4 carbon The straight or branched alkoxyl of atom, such as methoxyl group, ethyoxyl, positive propoxy, isopropoxy, n-butoxy, isobutoxy, secondary Butoxy, tert-butoxy etc..

Term " C_3-6Cycloalkyloxy ", " C₃-C₆Cycloalkyloxy " or " OC_3-6Naphthenic base " refers to containing 3 to 6 carbon atoms Cyclic alkoxy, such as cyclopropyl oxygroup, cyclobutoxy group, cyclopentyloxy.

" alkoxyalkyl " refers to the alkyl with specified carbon atom number replaced by one or more alkoxy substituents. Alkoxyalkyl is usually in moieties containing 1-6 carbon atom and by 1,2 or 3 C₁-C₄Alkoxy substituent replaces.This The group of sample is sometimes be described as C herein₁-C₄Alkoxy -C₁-C₆Alkyl." aminoalkyl " refers to be taken by one or more What generation or unsubstituted amino replaced has the alkyl of specified carbon atom number, further defines such group herein.

Aminoalkyl is replaced containing 1-6 carbon atom and usually in moieties by 1,2 or 3 amino-substituent.Cause This, C₁-C₆Aminoalkyl includes such as amino methyl (- CH₂NH₂), N, N- dimethylarnino-ethyl (- CH₂CH₂N(CH₃)₂、3- (N-Cyclopropylamino) propyl (- CH₂CH₂CH₂NH-^CPr) andNPyrrolidinyl ethyl (- CH₂CH₂ NPyrrolidinyl).

" alkenyl " refers to the alkyl as defined herein being made of at least two carbon atoms and at least one carbon-to-carbon double bond. Alkenyl usually have 2 to 20 carbon atoms ("C₂-C₂₀Alkenyl "), preferably 2 to 12 carbon atoms ("C₂-C₁₂Alkenyl "), more preferable 2 To 8 carbon atom (" C₂-C₈Alkenyl ") or 2 to 6 carbon atom (" C₂-C₆Alkenyl ") or 2 to 4 carbon atoms ("C₂-C₄Alkenyl "). Representative example includes vinyl, 1- acrylic, 2- acrylic, 1-, 2- or 3- cyclobutenyl etc.."C₂-C₆Alkenyl " refers to containing 2 To 6 carbon atoms at least one in two sp²The linear chain or branched chain group of double bond between hydbridized carbon atoms.If their bands Substituted base or substituent group as other groups occur, such as in O- (C₂-C₆) in alkenyl, above-mentioned definition is also suitable.Suitably C₂-C₆The example of alkenyl is positive acrylic, isopropenyl, n-butene base, isobutenyl, n-pentene base, secondary pentenyl, n-hexylene Base, secondary hexenyl etc..Alkenyl can be unsubstituted or be replaced by the identical group for being described herein as suitable for alkyl.

" alkynyl " refers to the alkyl as defined herein being made of at least two carbon atoms and at least one carbon-carbon triple bond. Alkynyl has 2 to 20 carbon atom (" C₂-C₂₀Alkynyl "), preferably 2 to 12 carbon atom (" C₂-C₁₂Alkynyl "), more preferable 2 to 8 A carbon atom (" C₂-C₈Alkynyl ") or 2 to 6 carbon atom (" C₂-C₆Alkynyl ") or 2 to 4 carbon atom (" C₂-C₄Alkynyl ").Generation Table example includes, but are not limited to acetenyl, 1- propinyl, 2-propynyl, 1-, 2- or 3- butynyl etc..Alkynyl can not by The identical group for replacing or being described herein as being suitable for alkyl replaces."C₂-C₆Alkynyl " refers to containing 2 to 6 carbon atoms and extremely The linear chain or branched chain group of few three keys between two sp hydbridized carbon atoms.If they are with substituent group or as it The substituent group of its group occurs, such as in O- (C₂-C₆) in alkynyl, above-mentioned definition is also suitable.Suitable C₂-C₆The example of alkynyl It is propinyl, butynyl, pentynyl, hexin base etc..

" alkylidene " used herein refers to that two other groups can be linked together has specified carbon atom number Bivalent hydrocarbon radical.It is sometimes referred to-(CH₂) n-, wherein n is 1-8, and n is preferably 1-4.When specified, alkylidene can also be by it Its group replaces and may include one or more degrees of unsaturation (i.e. alkenylene or alkynylene part) or ring.The open valency of alkylidene It does not need in the opposite end of chain.Therefore ,-CH (Me)-and-C (Me)₂It is also included in the range of term " alkylidene ", cyclic group Group such as cyclopropane -1,1- diyl and unsaturated group such as ethenylidene (- CH=CH-) or allylidene (- CH₂CH=CH-) it is same It is included in the range of term " alkylidene ".When alkylidene is described as optionally being substituted, substituent group includes as described herein Those of be typically found on alkyl.

" sub- miscellaneous alkyl " refers to the non-conterminous carbon atom of one or more of wherein alkylidene chain by-N- ,-O- ,-P- or-S- The alkylidene as described above of substitution, is presented as such as-N (R)-,-P (=O) (R)-,-S (O)_xOr-S (=O) (=NR)-, wherein R is H or C₁-C₄Alkyl and x are 0-2.For example, group-O- (CH₂)_1-4Be ' C₂-C₅'-sub- miscellaneous alkyl, wherein corresponding alkylidene One of carbon atom is substituted by O.

" aryl " or " aromatics " refers to the pi-electron system with total conjugated and full carbon monocycle or condensed ring with armaticity It is polycyclic.Term " C₆-C₁₂Aryl " and " C₆-₁₂Aryl " includes in this term and including having 6 to 12 carbon atoms and in ring Heteroatomic aromatic ring system is free of in system.The example of aryl is phenyl and naphthalene.Aryl can be substituted or unsubstituted. C₆-C₁₂Substituent group on the adjacent ring carbon atom of aryl may be combined to form optionally by one or more substituent groups, as oxo, C₁-C₆5- the or 6- member carbocyclic ring ring that alkyl, hydroxyl, amino and halogen replace, or optionally by one or more substituent groups, as oxo, C₁-C₆What alkyl, hydroxyl, amino and halogen replaced is selected from N, O and S (O) containing 1,2 or 3_xThe ring of (wherein x is 0,1 or 2) is miscellaneous 5- the or 6- circle heterocyclic ring ring of atom.The example of aryl includes phenyl, xenyl (biphenyl), naphthalene, anthryl, phenanthryl, dihydro Change indenyl, indenyl and tetralyl.Aryl can be unsubstituted or be substituted like that as further described herein.

" heteroaryl " or " heteroaromatic " refer to containing specified annular atom number and in aromatic ring include at least one be selected from N, O and Monocycle or condensed-bicyclic or polycyclic ring system of the hetero atom of S as ring members with known armaticity.Heteroatomic addition is real Armaticity in existing 5- member ring and 6- member ring.In general, heteroaryl contains 5 to 20 annular atoms (" 5-20 unit's heteroaryl "), it is excellent It selects 5 to 14 annular atoms (" 5-14 unit's heteroaryl "), more preferable 5 to 12 annular atoms (" 5-12 unit's heteroaryl ") or 5 to 6 rings Atom (" 5-6 unit's heteroaryl ").Heteroaryl ring is connected in base molecule via the annular atom of hetero-aromatic ring, to keep armaticity. Therefore, 6- unit's heteroaryl ring can be connected in base molecule via ring C atom, and 5- unit's heteroaryl ring can be via ring C or N atom It is connected in base molecule.Heteroaryl can be unsubstituted or be substituted like that as further described herein.Such as this paper institute With " 5-6 unit's heteroaryl " refers to the monocycle with 5 or 6 annular atoms containing 1,2 or 3 ring hetero atom selected from N, O and S Group, but including with 4 nitrogen, remaining annular atom be C and also with total conjugated π-electron system tetrazole radical.5- or Substituent group on the adjacent cyclic atom of 6- unit's heteroaryl may be combined to form optionally by one or more substituent groups, such as oxo, C₁- C₆The condensed 5- or 6- member carbocyclic ring ring that alkyl, hydroxyl, amino and halogen replace, or optionally by one or more substituent groups, such as oxygen Generation, C₁-C₆What alkyl, hydroxyl, amino and halogen replaced is selected from N, O and S (O) containing 1,2 or 3_x(wherein x is 0,1 or 2) The condensed 5- or 6- circle heterocyclic ring ring of ring hetero atom.If described fused rings itself are aromatics, it is miscellaneous to be referred to as condensed (bicyclic) Aromatic species, no matter whether second ring contains hetero atom.Pharmaceutically acceptable heteroaryl is sufficiently stable to be connected to chemical combination of the invention On object, it is formulated into pharmaceutical composition and then delivers medicine to the heteroaryl for needing its patient.

The example of heteroatomic 5- unit's heteroaryl ring containing 1,2 or 3 independently selected from O, N and S includes pyrrole radicals, thiophene Pheno base, pyrazolyl, imidazole radicals, oxazolyl, isoxazolyl, thiazolyl, isothiazolyl, triazolyl, tetrazole radical, dislikes two at furyl Oxazolyl and thiadiazolyl group.Preferred 6- unit's heteroaryl ring contains 1 or 2 nitrogen-atoms.The example of 6- unit's heteroaryl is pyridyl group, rattles away Piperazine base, pyrimidine radicals and pyrazinyl.The example of condensed heteroaryl ring includes benzofuran, benzothiophene, indoles, benzimidazole, Yin Azoles, quinolone, isoquinolin, purine, pyrrolopyrimidine, naphthyridines and carbazole.

" arlydene " used herein refers to be obtained and respectively removing hydrogen atom on two carbon atoms from core by aromatic hydrocarbons The bivalent group arrived.In common embodiment, arlydene ring is the substitution of 1,2- bis- or 1, the disubstituted arlydene of 3-.Sub- virtue The aryl rings of base portion point can be optionally suitble to the group of aryl rings to replace to this on open valency position and replace indicated journey Degree.Arlydene ring is preferably C₆-C₁₂Arlydene ring, such as 1,2- phenylene or 1,3- phenylen moiety.

Similarly, " inferior heteroaryl " used herein, which refers to, passes through two carbon atoms or a carbon original from core by hetero-aromatic ring Bivalent group obtained from hydrogen atom is respectively removed on son and a nitrogen-atoms.In common embodiment, inferior heteroaryl ring It is the substitution of 1,2- bis- or the disubstituted inferior heteroaryl of 1,3-.The heteroaryl ring of heteroarylene moieties is optionally suitble to heteroaryl ring Group replaces to this and replaces indicated degree.The inferior heteroaryl ring that inferior heteroaryl ring is preferably 5-12 member, may condense, more It is preferred that 5-6 member inferior heteroaryl ring, each can be optionally substituted.

Term " heteroalicyclic ", " heterocycle " or " heterocycle " is used interchangeably herein to indicate containing specified annular atom Number, including at least one be selected from N, O and S hetero atom as ring members it is non-aromatic, be saturated or the unsaturated ring system in part, Wherein the heterocyclic ring is connected in base molecule via annular atom (it can be C or N).Heteroalicyclic ring can be fused to one or In a number of other heteroalicyclics or carbocyclic ring ring, the condensed ring can be saturation, part is unsaturated or aromatics.Heteroalicyclic ring 1 to 4 hetero atom selected from N, O and S is preferably comprised as ring members, more preferable 1 to 2 ring hetero atom, condition is such miscellaneous Aliphatic ring does not contain two adjacent oxygen atoms.Heteroalicyclyl can it is unsubstituted or be described herein as be suitable for alkyl, The identical group of aryl or heteroaryl replaces.

Preferred heteroalicyclyl includes according to the 3-12 member heteroalicyclyl of definition herein, 5-8 circle heterocyclic ring base (or heterolipid Ring group), 4-12 member heteroalicyclic monocycle and 6-12 member heteroalicyclic it is bicyclic." 3-12 member heteroalicyclic " used herein refers to tool There are the monocycle or bicyclic radicals of 3 to 12 annular atoms, wherein 1,2,3 or 4 annular atom is selected from N, O, P (O), S (O)_x(wherein X is 0,1,2) and the hetero atom of S (=O) (=NR), remaining annular atom is C.The ring can also have one or more double bonds.But The ring does not have the pi-electron system of total conjugated.Substituent group on two ring carbon atoms may be combined to form to be selected containing 1,2 or 3 From N, O and S (O)_xThe carbocyclic ring of the ring hetero atom of (wherein x is 0,1 or 2) or 5- the or 6- member bridged ring of heteroalicyclic.The heterolipid ring Base is optionally by oxo, hydroxyl, amino, C₁-C₆Alkyl etc. replaces.

In common embodiment, heteroalicyclyl contains 3-12 ring members, including carbon and non-heteroatoms, preferably 4- 6 ring members.In certain preferred embodiments, the substituent group comprising 3-12 member heteroalicyclyl is selected from azetidinyl, pyrrole Alkyl, piperidyl, piperazinyl, morpholinyl and thiomorpholine basic ring are coughed up, each optionally there can be chemical meaning in such substitution It is substituted in the degree of justice.

It is to be understood that in addition to oxo or azepine group are connected on N, P or S to be formed and be such as, but not limited to nitro, oxygen Phosphino- (phosphinyl), phosphono amido (phosphinamido), sulfo group oximido (sulfoximino) and sulfonyl etc Group or in the case where certain hetero-aromatic rings, such as triazine, triazole, tetrazolium, oxadiazoles, thiadiazoles it is outer, typically no more than two N, O or S atom are continuously coupled.

" naphthenic base " refers to containing the non-aromatic of specified carbon atom number, saturation or the unsaturated carbocyclic ring ring system in part, It can be the monocycle being connected in base molecule via the carbon atom of cycloalkyl ring, bridging or condensed-bicyclic or polycyclic ring body System.Naphthenic base of the invention usually contains 3 to 12 carbon atom (" C₃-C₁₂Naphthenic base "), preferably 3 to 8 carbon atom (" C₃-C₈ Naphthenic base ").Other naphthenic base include part the unsaturated part (" C with 4 to 7 carbon₄-C₇Cycloalkenyl ").It is representative real Example include for example cyclopropane, cyclobutane, pentamethylene, cyclopentene, hexamethylene, cyclohexene, cyclohexadiene, cycloheptane, cycloheptatriene, Adamantane etc..Naphthenic base can be unsubstituted or be replaced by the identical group for being described herein as suitable for alkyl.It is used herein “C₃-C₆Naphthenic base " refers to full carbon monocycle or condensed ring polycyclic moiety with 3 to 6 carbon atoms.

" cycloalkyl-alkyl " can be used for describing via alkylidene linker (linker) (usually C₁-C₄Alkylidene) connection Cycloalkyl ring (usually C on to base molecule₃-C₈Naphthenic base).Cycloalkyl-alkyl is by the carbon atom in carbocyclic ring ring and linker Sum describes and usually contains 4-12 carbon atom (" C₄-C₁₂Cycloalkyl-alkyl ").Therefore, Cvclopropvlmethvl is C₄Naphthenic base Alkyl, and cyclohexyl-ethyl is C₈Cycloalkyl-alkyl.Cycloalkyl-alkyl can be unsubstituted or in naphthenic base and/or alkylidene The identical group for being described herein as being suitable for alkyl on part replaces.

" aryl alkyl " refers to the virtue as described herein being connected in base molecule via alkylidene or similar linker Base.Aryl alkyl is described by the total number of carbon atoms in the ring and linker.Therefore benzyl is C₇Aryl alkyl, phenylethyl are C₈Aryl alkyl.Aryl alkyl usually contains 7-16 carbon atom (" C₇-C₁₆Aryl alkyl "), wherein aryl moiety contains 6- 12 carbon atoms, and alkylene moiety contains 1-4 carbon atom.Such group can also be expressed as-C₁-C₄Alkylidene-C₆-C₁₂ Aryl.

" heteroaryl alkyl " refers to the heteroaryl as described above being connected in base molecule via alkylidene linker, with The difference of " aryl alkyl " is that at least one annular atom of aromatic fractions is the hetero atom selected from N, O and S.Herein sometimes It is described according to the sum of the non-hydrogen atom (i.e. C, N, S and O atom) of (not including substituent group) in the ring and the linker of combination miscellaneous Aryl alkyl.Thus, for example, pyridylmethyl can be referred to as " C₇"-heteroaryl alkyl.In general, unsubstituted heteroaryl Alkyl contains 6-20 non-hydrogen atom (including C, N, S and O atom), and wherein heteroaryl moieties usually contain 5-12 atom, sub- Moieties usually contain 1-4 carbon atom.Such group can also be expressed as-C₁-C₄Alkylidene -5-12 unit's heteroaryl.

Similarly, " alkoxy aryl " and " heteroarylalkoxy " refers to via sub- miscellaneous alkyl linker (i.e.-O- alkylene Base -) it is connected to aryl and heteroaryl in base molecule, wherein (i.e. according to the non-hydrogen atom in the ring and the linker of combination C, N, S and O atom) sum these groups are described.Therefore ,-O-CH₂Phenyl and-O-CH₂Pyridyl group is referred to as C respectively₈Virtue Base alkoxy and C₈Heteroarylalkoxy.

When aryl alkyl, alkoxy aryl, heteroaryl alkyl or heteroarylalkoxy are described as optionally being substituted, take It can be on divalent linker part or on the aryl or heteroaryl moieties of the group for base.It is optionally present in alkylidene or Asia Substituent group on miscellaneous alkyl part is identical as those of usually describing to alkyl or alkoxy above, and be optionally present in aryl or Substituent group on heteroaryl moieties is identical as those of usually describing to aryl or heteroaryl above.

" hydroxyl " refers to-OH group.

" acyl group " refers to univalent perssad-C (O) alkyl, and wherein moieties have specified carbon atom number (usual C₁-C₈, excellent Select C₁-C₆Or C₁-C₄) and can be applied to alkyl group replace.Therefore, C₁-C₄Acyl group includes-C (O) C₁-C₄Alkyl replaces Base, such as-C (O) CH₃.Similarly, " acyloxy " refers to univalent perssad-OC (O) alkyl, and wherein moieties have specified carbon Atomicity (usual C₁-C₈, preferably C₁-C₆Or C₁-C₄) and can be applied to alkyl group replace.Therefore, C₁-C₄Acyloxy Including-OC (O) C₁-C₄Alkyl substituent, such as-OC (O) CH₃。

Term " monocycle or bicyclic ring systems " refers to aromatics, the unsaturated ring of saturation or part containing specified annular atom number System and can optionally include one or more hetero atoms selected from N, O and S as ring members, wherein heterocyclic ring is via can be C Or the annular atom of N is connected in base molecule.It include term " naphthenic base ", " aryl ", " heterocycle " and " miscellaneous in this term Aryl ".Monocycle or bicyclic ring systems of the invention usually contains 4 to 12 member atoms (" 4-12 unit monocycle or bicyclic ring body System ").Bicyclic system can condense (spiral shell) via 1,1-, 1,2- condenses (condensed) or 1, and > 2- condenses (end of the bridge) connection.It is representative real Example include pentamethylene, cyclopentene, hexamethylene, norborny (norbornyl), spiral shell [2.3] hexane, phenyl, xenyl, naphthalene, Anthryl, phenanthryl, pyrrole radicals, thienyl, furyl, pyrazolyl, imidazole radicals, oxazolyl, isoxazolyl, thiazolyl, azetidin Alkyl, pyrrolidinyl, piperidyl, piperazinyl, benzothienyl, indyl etc..

All alkyl, alkenyl, alkynyl, naphthenic base, cycloalkenyl, monocycle and bicyclic heterocycle, aryl (monocycle and bicyclic), heteroaryl Base (monocycle and bicyclic), cycloalkyl-alkyl, aryl alkyl, alkoxy aryl, heteroaryl alkyl or heteroarylalkoxy (including are appointed What C_1-6Alkyl, C_2-6Alkenyl, C_2-6Alkynyl, C_3-6Naphthenic base, C_4-6Cycloalkenyl, C_6-12The saturation list of bicyclic alkyl, 4-12 atom The saturated bicyclic heterocycle of ring heterocycle or 6-12 atom, all C_6-12Aryl monocycle or bicyclic and 6-12 atom heteroaryl list Ring is bicyclic) can optionally be replaced by multiple substituent groups, the substituent group independently selected from halogen, hydroxyl, oxo, hydroxyl amino, Oximido, diazanyl, hydrazono-, cyano, nitro, azido, NR⁸R⁹、OC_1-6Alkyl, OC_3-6Alkenyl, OC_3-6Alkynyl, C_1-6Alkyl, OC_3-6Naphthenic base, OC_3-7Cycloalkenyl, C_1-6Acyl group, C_1-6Acyloxy, N (R⁸)COR⁴、CO₂R⁴、CONR⁸R⁹、NR⁸CONR⁸R⁹、 NR⁸CO₂R⁴、OCO₂R⁴、OCONR⁸R⁹、S(O)_xR⁴、S(R⁴)(=O)=NR⁸、S(=O)(=NR⁸)NR⁸R⁹、SO₂NR⁸R⁹、NR⁸SO₂R⁴、 NR⁸SO₂NR⁸R⁹、-NR⁸S(=O)(=NR⁸)R⁴、-N=S(=O)(R⁴)R⁴、-N=S(=O)(NR⁸R⁹)R⁴、ONR⁸R⁹、ON(R⁸)COR⁴、 ONR⁸CONR⁸R⁹、ONR⁸CO₂R⁴、ONR⁸SO₂R⁴、ONR⁸SO₂NR⁸R⁹。

As used in preparation and embodiment, following term has shown meaning: " 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 nanomole；" mmol " refers to mmoles You；" mol " refers to mole；" M " refers to molar concentration, and " mM " refers to millimolar concentration, and " μM " refers to micro-molar concentration, and " nM " is Refer to nanomolar concentration, " L " refers to liter, and " mL " refers to milliliter, and " μ L " refers to microlitre.

The officinal salt of the compound of the present invention includes its acid-addition salts and alkali salt (including disalt).

Suitable acid-addition salts are formed by the acid of formation nontoxic salts.Example includes acetate, aspartate, benzoic acid Salt, benzene sulfonate, bicarbonate/carbonate, disulfate/sulfate, borate, camsilate, citrate, two sulphur of second Hydrochlorate, esilate, formates, fumarate, gluceptate, gluconate, glucuronate, hexafluorophosphate, oxybenzene Acyl benzoic acid salt (hibenzate), hydrochloride/chloride, hydrobromate/bromide, hydriodate/iodide, isethionic acid Salt, lactate, malate, maleate, malonate, mesylate, Methylsulfate, naphthoate, 2- naphthalene sulfonate, cigarette Hydrochlorate, nitrate, Orotate, oxalates, palmitate, embonate, phosphate/phosphor acid hydrogen salt/dihydric phosphate, sugarcane Sugar lime, stearate, succinate, tartrate, toluene fulfonate and trifluoroacetate.

Suitable alkali salt is formed by the alkali of formation nontoxic salts.Example includes aluminium salt, arginine 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, referring to " the Handbook of Pharmaceutical of Stahl and Wermuth Salts:Properties, Selection, and Use " (Wiley-VCH, Weinheim, Germany, 2002).

By optionally mixing the solution of the compound with required acid or alkali, can easily prepare of the invention The officinal salt of compound.Salt can be precipitated from solution and be collected by filtration or can by evaporate solvent recovery.The salt In degree of ionization can change from complete ionization to almost unionization.

The compound of the present invention containing one or more asymmetric carbon atoms can be with two or more alloisomerisms The form of body exists.If the compound of the present invention contains alkenyl or alkenylene, it is understood that there may be geometry cis/trans (or Z/E) Isomers.If the compound contains such as ketone group or oximido or aromatic fractions, it may occur that tautomerism (" is mutually cashed As ").The result is that single compound may show more than one isomerism.

Including all stereoisomers, several of the compound of the present invention in the range of claimed compound of the invention What isomers and tautomeric form, including showing the compound of more than one isomerism and mixing for one or more Close object.It further include acid-addition salts or alkali salt, wherein counter ion is optical activity, such as D-lactate or L-lysine salt, or anti- Ion is racemic, such as DL- tartrate or DL- arginine salt.

Cis/trans isomers can be by the way that well known to a person skilled in the art traditional technologies, such as chromatography and substep to tie Crystallization separation.

The compound of the present invention is also possible to show atropisomerism, wherein limited rotation (joins especially about connection The key of two aryl rings in virtue) cause different rotational isomers can not mutual inversion of phases and whole under Normal Environmental Temperature A molecule is kept also very likely can not mutual inversion of phases at heat-staple temperature.In this case, claimed to be turned by resistance The different stereoisomers that isomerism generates.

The traditional technology for being used to prepare/separating single enantiomter include synthesized by suitable optical voidness precursor chirality or Using such as chiral high pressure liquid chromatography (HPLC), especially Simulation moving bed (SMB) construction in resolution of racemic object (or The racemate of salt or derivative).

Alternatively, racemate (or racemic precursor) can be reacted with suitable optically-active compound, such as alcohol, or in formula (I) in the case that compound contains acid or alkaline part, with acid or alkali, such as tartaric acid or the reaction of 1- phenylethylamine.Institute Obtaining diastereomeric mixtures can be separated by chromatography and/or Steppecd crystallization and pass through method well known to technical staff for one Kind or two kinds of diastereoisomers are converted to corresponding pure enantiomter.

Chipal compounds (and its chiral precursor) of the invention can use chromatography (usual HPLC) in asymmetric resin For the mobile phase that upper use is made of hydrocarbon with the acquisition of enantiomter enriched form, the hydrocarbon is usually heptane or hexane, containing 0 to 50% isopropanol (usual 2 to 20%) and 0 to 5% alkylamine (usual 0.1% diethylamine).It is mixed that the concentration of eluate provides enrichment Close object.

The mixture of stereoisomer can be separated by traditional technology well known by persons skilled in the art.[see, for example, E " Stereochemistry of Organic Compounds " (Wiley, the New York, 1994) of L Eliel].

The present invention includes the compound of the present invention of all pharmaceutical isotope labellings, wherein one or more atom quilts With same atoms ordinal number but atomic mass or mass number are different from the atomic mass being generally found in nature or mass number Atom substitution.

The example for the isotope being suitable for inclusion in the compound of the present invention includes the isotope of hydrogen, such as²H and³H, carbon it is same Position element, such as¹¹C、¹³C and¹⁴C, the isotope of chlorine, such as³⁶Cl, the isotope of fluorine, such as¹⁸F, the isotope of iodine, such as¹²³I and¹²⁵I, nitrogen Isotope, such as¹³N and¹⁵N, the isotope of oxygen, such as¹⁵O、¹⁷O and¹⁸O, the isotope of phosphorus, such as³²The isotope of P and sulphur, such as³⁵S。

The compound of the present invention of certain isotope labellings, for example, comprising it is radioisotopic those, can be used for drug And/or substrate tissue distribution research.In view of their easy being incorporated to property and ready-made detection means, radioactive isotope tritium is (i.e.³H) and carbon-14 (i.e.¹⁴C) it is particularly useful for this purposes.

With higher isotope, such as deuterium, i.e.,²H substitution can provide by the certain treatment benefits of higher metabolic stability bring Place, such as increased Half-life in vivo or reduced dose requirements, therefore be preferred in some cases.

With Positron emitting isotopes, such as¹¹C、¹⁸F、¹⁵O and¹³N substitution can be used for positron emission computerized tomography (PET) Research is with detection substrate receptor share.

Usually by traditional technology well known by persons skilled in the art or by with appended embodiment and preparation described in Those similar methods replace the unlabelled reagent used before to prepare same position using the reagent of isotope labelling appropriate The compound of the present invention of element label.

The compound of the present invention can be administered in the form of prodrug.Therefore, possible little or no pharmacology itself is living Property the compound of the present invention certain derivatives when delivering medicine in body or on body can for example pass through hydrolytic rupture turn Be melted into have the required active other formulas of formula 1(or 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)。

" Design of Prodrugs " (Elsevier, 1985) with such as such as H. Bundgaard can for example be passed through Described in certain parts known to those skilled in the art for " precursor portions (pro-moieties) " substitute change of the invention Functional group appropriate present in object is closed to prepare prodrug.

Some examples of such prodrug include:

When the compound contains carboxylic acid functional (-- COOH), prodrug includes its ester, such as uses C₁-C₆Alkyl substitutes hydrogen；

When the compound contains alcohol functional group (-- OH), prodrug includes its ether, such as uses C₁-C₆Alkanoyloxymethyl (- C₁-C₆Pivaloyloxymethyl) substitution hydrogen；With

When the compound contains primary amino group or secondary amino functionalities (- NH₂Or-NHR, wherein R is not H) when, prodrug includes Its amide, such as with (C₁-C₁₀) alkanoyl (- C₁-C₁₀Acyl group) substitute one or two hydrogen.

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

Finally, certain compounds of formula A itself may act as the prodrug of other compounds of formula A.

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

Wherein

R¹Selected from hydrogen, fluorine, chlorine, methyl, CF₃、CHF₂And cyano；

R²Selected from methoxyl group, ethyoxyl, isopropoxy, cyclopropyl oxygroup, methyl, ethyl, isopropyl and cyclopropyl；

R³Selected from H, halogen, C_1-6Alkyl, C_1-6Halogenated alkyl, C_2-6Halo alkynyl, optionally by R⁷And C_1-6It is alkyl-substituted C_2-6Alkynyl, R⁴N-C_2-6Alkyl-NR⁴R⁴、R⁷；

R⁴It independently is H, C_1-6Alkyl, C_2-6Hydroxyalkyl, C_1-6Alkoxyalkyl or C_2-6Alkyl NR⁸R⁹Or two R⁴Group Carbon atom connected to them forms 3-12 unit monocycle or bicyclic ring systems, wherein most two carbon atoms are by N, NR⁴、O、S (O)_xWith S (O) (NR¹⁴) substitution；

R⁵It is H, F, Cl, CF₃、CHF₂、CF₂C_1-6Alkyl, CF₂CH₂NR⁸R⁹、CH₂NR⁸R⁹Or C_1-6Alkyl；

R^6eIt is C_1-6Alkyl, aryl, heteroaryl, naphthenic base, heterocycle, (CH₂)_mCHR⁴R⁷、CF₂(CH₂)_mCHR⁴R⁷Or C (R₄)₂R⁷；

R^6zIt is H, F, Cl, CF₃、CHF₂、CF₂C_1-6Alkyl or C_1-6Alkyl；

R⁷It is OH, NR⁸R⁹、OCH₂(CH₂)_mNR⁸R⁹、C_1-6Alkoxy, C_1-6Alkoxy -C_1-6Alkoxy, C_2-6Hydroxyl alkoxy, Oxetanyl, oxetanyl oxygroup, oxetanyl amino, tetrahydrofuran base, tetrahydrofuran base oxygroup, Tetrahydrofuran base amino, Oxyranyle, Oxyranyle oxygroup, Oxyranyle amino, oxepane alkyl, oxa- ring Heptan alkyl oxy, oxepane alkyl amino, azetidinyl, azetidinyl oxygroup, azetidinyl amino, pyrrole Cough up alkyl, pyrrolidinyl oxygroup, pyrrolidinyl amino, piperidyl, piperidyl oxygroup, piperidyl amino, nitrogen heterocyclic heptyl, nitrogen Trioxepane base oxygroup, nitrogen heterocyclic heptyl amino, dioxolane base, dioxane base, morpholino, thiomorpholine Generation, thiomorpholine generation-S, S- dioxide, Piperazino, Dioxepane base, Dioxepane base oxygroup, dioxa Cycloheptyl alkyl amino, oxaza heptane base, oxaza alkyl oxy in heptan, oxaza alkyl amino in heptan, diaza cycloheptyl Alkyl, Diazesuberane base oxygroup, Diazesuberane base amino；And as R⁷All NH of a part can be optionally by R⁴ Replace；

R⁸And R⁹It independently is H, C_1-6Alkyl, C_3-6Alkenyl, C_3-6Alkynyl, C_3-7Naphthenic base, C_3-7Cycloalkenyl, C_1-C₆Acyl group, 4-12 circle heterocyclic ring base, C_6-C₁₂Aryl or 5-12 unit's heteroaryl；Or R⁸And R⁹Atom connected to them forms 4-12 unit monocycle Or bicyclic ring systems, wherein most two carbon atoms are by N, NR⁴、O、S(O)_xWith S (O) (NR¹⁴) substitution；And R⁸And R⁹It can be independent Or together by most three independently selected from hydroxyl, C_1-6Alkoxy, C_1-6Hydroxyalkyl, C_1-6Alkoxy -C_1-6Alkyl, C_1-6Alcoxyl Base-C_1-6Alkoxy, C_2-6The substituent group of hydroxyl alkoxy replaces；

R¹⁰It is H, C_1-6Alkyl；

X²It is C, CH or N；

X³It is CH or N,

X⁴It is CR⁴Or N or NR¹⁰；

X⁵And X⁶All being C or one can be N；

Two keys in a-e are double bonds and the other three is singly-bound, so that atom X²-X⁶All there are two double bond and its phases Even；

Key f is usually double bond, only works as X⁴And X⁵Or X⁴And X⁶It is nitrogen and X²-X⁶In the other three be C, CH or CR⁴ When, two key f can also be singly-bound；

M is 0-4；

Condition is as follows:

X²-X⁶At least one of and not more than three be N or NR⁴；

If R^6eIt is CF₂(CH₂)_mCHR⁴R⁷, then R⁵And R^6zIt is not F, Cl, CF₃、CHF₂Or CF₂C_1-6Alkyl, but if R^6eIt is not CF₂(CH₂)_mCHR⁴R⁷, then R⁵And R^6zFirst is that F, Cl, CF₃、CHF₂Or CF₂C_1-6Alkyl；

Or the compound of structure (II)

Or its officinal salt, wherein

R¹Selected from hydrogen, fluorine, chlorine, methyl, CF₃、CHF₂And cyano；

R²Selected from methoxyl group, ethyoxyl, isopropoxy, cyclopropyl oxygroup, methyl, ethyl, isopropyl and cyclopropyl；

R³Selected from H, halogen, C_1-6Alkyl, C_1-6Halogenated alkyl, C_2-6Halo alkynyl, optionally by R⁷And C_1-6It is alkyl-substituted C_2-6Alkynyl, R⁴N-C_2-6Alkyl-NR⁴R⁴、R⁷；

R⁴It independently is H, C_1-6Alkyl, C_2-6Hydroxyalkyl, C_1-6Alkoxyalkyl or C_2-6Alkyl NR⁸R⁹Or two R⁴Group Carbon atom connected to them forms 3-12 unit monocycle or bicyclic ring systems, wherein most two carbon atoms are by N, NR⁴、O、S (O)_xWith S (O) (NR¹⁴) substitution；

R⁵It is H, CH₂NR⁸R⁹Or C_1-6Alkyl；

R^6eIt is C_1-6Alkyl, aryl, heteroaryl, naphthenic base, heterocycle, (CH₂)_mCHR⁴R⁷、CF₂(CH₂)_mCHR⁴R⁷Or C (R₄)₂R⁷；

R^6zIt is H or C_1-6Alkyl；

R⁷It is OH, NR⁸R⁹、OCH₂(CH₂)_mNR⁸R⁹、C_1-6Alkoxy, C_1-6Alkoxy -C_1-6Alkoxy, C_2-6Hydroxyl alkoxy, Oxetanyl, oxetanyl oxygroup, oxetanyl amino, tetrahydrofuran base, tetrahydrofuran base oxygroup, Tetrahydrofuran base amino, Oxyranyle, Oxyranyle oxygroup, Oxyranyle amino, oxepane alkyl, oxa- ring Heptan alkyl oxy, oxepane alkyl amino, azetidinyl, azetidinyl oxygroup, azetidinyl amino, pyrrole Cough up alkyl, pyrrolidinyl oxygroup, pyrrolidinyl amino, piperidyl, piperidyl oxygroup, piperidyl amino, nitrogen heterocyclic heptyl, nitrogen Trioxepane base oxygroup, nitrogen heterocyclic heptyl amino, dioxolane base, dioxane base, morpholino, thiomorpholine Generation, thiomorpholine generation-S, S- dioxide, Piperazino, Dioxepane base, Dioxepane base oxygroup, dioxa Cycloheptyl alkyl amino, oxaza heptane base, oxaza alkyl oxy in heptan, oxaza alkyl amino in heptan, diaza cycloheptyl Alkyl, Diazesuberane base oxygroup, Diazesuberane base amino；And as R⁷All NH of a part can be optionally by R⁴ Replace；

R⁸And R⁹It independently is H, C_1-6Alkyl, C_3-6Alkenyl, C_3-6Alkynyl, C_3-7Naphthenic base, C_3-7Cycloalkenyl, C_1-C₆Acyl group, 4-12 circle heterocyclic ring base, C_6-C₁₂Aryl or 5-12 unit's heteroaryl；Or R⁸And R⁹Atom connected to them forms 4-12 unit monocycle Or bicyclic ring systems, wherein most two carbon atoms are by N, NR⁴、O、S(O)_xWith S (O) (NR¹⁴) substitution；And R⁸And R⁹It can be independent Or together by most three independently selected from hydroxyl, C_1-6Alkoxy, C_1-6Hydroxyalkyl, C_1-6Alkoxy -C_1-6Alkyl, C_1-6Alcoxyl Base-C_1-6Alkoxy, C_2-6The substituent group of hydroxyl alkoxy replaces；

R¹⁰It is H, C_1-6Alkyl；

X²It is C, CH or N；

X³It is CH or N,

X⁴It is CR⁴Or N or NR¹⁰；

X⁵And X⁶All being C or one can be N；

Two keys in a-e are double bonds and the other three is singly-bound, so that atom X²-X⁶All there are two double bond and its phases Even；

Key f is usually double bond, only works as X⁴And X⁵Or X⁴And X⁶It is nitrogen and X²-X⁶In the other three be C, CH or CR⁴ When, two key f can also be singly-bound；

M is 0-4；

Condition is as follows:

X²-X⁶At least one of and not more than three be N or NR⁴；

If R^6eIt is CF₂(CH₂)_mCHR⁴R⁷, then R⁵It cannot be CF₂C_1-6Alkyl；

Or the compound of structure (III)

Or its officinal salt, wherein

R¹Selected from hydrogen, fluorine, chlorine, methyl, CF₃、CHF₂And cyano；

R²Selected from methoxyl group, ethyoxyl, isopropoxy, cyclopropyl oxygroup, methyl, ethyl, isopropyl and cyclopropyl；

R³Selected from H, halogen, C_1-6Alkyl, C_1-6Halogenated alkyl, C_2-6Halo alkynyl, optionally by R⁷And C_1-6It is alkyl-substituted C_2-6Alkynyl, R⁴N-C_2-6Alkyl-NR⁴R⁴、R⁷；

R⁴It independently is H, C_1-6Alkyl, C_2-6Hydroxyalkyl, C_1-6Alkoxyalkyl or C_2-6Alkyl NR⁸R⁹Or two R⁴Group Carbon atom connected to them forms 3-12 unit monocycle or bicyclic ring systems, wherein most two carbon atoms are by N, NR⁴、O、S (O)_xWith S (O) (NR¹⁴) substitution；

R^6tIt is C_1-6Alkyl, C_3-6Naphthenic base, aryl, heteroaryl, heterocycle, (CH₂)_mCHR⁴R⁷、C(R₄)₂R⁷；

R⁷It is OH, NR⁸R⁹、OCH₂(CH₂)_mNR⁸R⁹、C_1-6Alkoxy, C_1-6Alkoxy -C_1-6Alkoxy, C_2-6Hydroxyl alkoxy, Oxetanyl, oxetanyl oxygroup, oxetanyl amino, tetrahydrofuran base, tetrahydrofuran base oxygroup, Tetrahydrofuran base amino, Oxyranyle, Oxyranyle oxygroup, Oxyranyle amino, oxepane alkyl, oxa- ring Heptan alkyl oxy, oxepane alkyl amino, azetidinyl, azetidinyl oxygroup, azetidinyl amino, pyrrole Cough up alkyl, pyrrolidinyl oxygroup, pyrrolidinyl amino, piperidyl, piperidyl oxygroup, piperidyl amino, nitrogen heterocyclic heptyl, nitrogen Trioxepane base oxygroup, nitrogen heterocyclic heptyl amino, dioxolane base, dioxane base, morpholino, thiomorpholine Generation, thiomorpholine generation-S, S- dioxide, Piperazino, Dioxepane base, Dioxepane base oxygroup, dioxa Cycloheptyl alkyl amino, oxaza heptane base, oxaza alkyl oxy in heptan, oxaza alkyl amino in heptan, diaza cycloheptyl Alkyl, Diazesuberane base oxygroup, Diazesuberane base amino；And as R⁷All NH of a part can be optionally by R⁴ Replace；

R⁸And R⁹It independently is H, C_1-6Alkyl, C_3-6Alkenyl, C_3-6Alkynyl, C_3-7Naphthenic base, C_3-7Cycloalkenyl, C_1-C₆Acyl group, 4-12 circle heterocyclic ring base, C_6-C₁₂Aryl or 5-12 unit's heteroaryl；Or R⁸And R⁹Atom connected to them forms 4-12 unit monocycle Or bicyclic ring systems, wherein most two carbon atoms are by N, NR⁴、O、S(O)_xWith S (O) (NR¹⁴) substitution；And R⁸And R⁹It can be independent Or together by most three independently selected from hydroxyl, C_1-6Alkoxy, C_1-6Hydroxyalkyl, C_1-6Alkoxy -C_1-6Alkyl, C_1-6Alcoxyl Base-C_1-6Alkoxy, C_2-6The substituent group of hydroxyl alkoxy replaces；

R¹⁰It is H, C_1-6Alkyl；

X²It is C, CH or N；

X³It is CH or N,

X⁴It is CR⁴Or N or NR¹⁰；

X⁵And X⁶All being C or one can be N；

Two keys in a-e are double bonds and the other three is singly-bound, so that atom X²-X⁶All there are two double bond and its phases Even；

Key f is usually double bond, only works as X⁴And X⁵Or X⁴And X⁶It is nitrogen and X²-X⁶In the other three be C, CH or CR⁴ When, two key f can also be singly-bound；

M is 0-4；

Condition is as follows:

X²-X⁶At least one of and not more than three be N or NR⁴。

The compound of the present invention includes:

。

Synthetic method

The compound of the present invention and its intermediate can be with many sides known to ordinary technicians in the field of organic synthesis Formula preparation.Reagent and raw material are that those of ordinary skill in the art are easy to get.

The particularly useful reference that can be used conveniently to prepare the synthetic method of the compound of the present invention is found in Larock, R. C. Comprehensive Organic Transformations, VCH:New York, 1989, simultaneously through this reference Enter herein.About the another of the suitable protecting base selected for protecting reactive functional groups present in compound of the present invention One useful reference is Greene and Wuts, Protective Groups In Organic Synthesis, Wiley& Sons, 1991, it is incorporated herein by this reference also like as abundant illustrate.

The compound of the present invention can be prepared by various technologies, and some of them are described below.Those skilled in the art can manage Solution, these methods be it is representative and not restrictive.In WO 2013/014448, WO 2013/169401 and J Med Chem The many that can be used for constructing the compound of the present invention is described in 56,7025 (2013) (all the full text is incorporated herein by reference) Reaction.

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

。

Scheme 1. segment I, II and III are totally assembled into final product

The overall synthesis of these compounds includes the steps that building segment I, II and III appropriate, followed by usually abides by Follow the final assembly program of scheme 1.2- chlorine on required 4- (bicyclic aryl) -2- chlorine pyrimidine (I) is in those skilled in the art It is coupled under the conditions of well known with 3- nitroaniline appropriate (or 3- amino -5- nitro-pyrimidine) (II).This can be acid catalyzed It is the coupling of Buchwald type shown in process or process as above, but it is also possible to coupling that alkali causes, to there is several differences Approach form the key of this key.3- nitro is reduced into corresponding amine, this can be carried out in a manner of diversified Reaction.It is certain to restore group, as the presence of alkynyl may be such that this reaction complicates, but can be used for the reagent of this conversion Diversity, which allows, firmly believes that it is completed.If R⁴It is not H, R⁴It can be introduced in this step.Standard reductive alkylation is enumerated herein, but Other technologies can be used, for example, if group is not interfered in the molecule, with alkyl halide direct alkylation.But big In most cases, R⁴It is H.Pass through aromatic amine is acylated, the completion point with the acid derivative of acid chloride or other suitable (III) The assembling of son, this may require using peptide coupling agent, and many is known.In some cases, which may be herein It is not completed in step, and further chemical process may be needed.For example, it may be possible to protecting group is removed from (III), or (III) amine that may be fitted without in this stage, this then can be installed, such as the displacement for passing through allyl halide --- in this field In have the governed technology of precedent greatly.

Although not illustrating herein, it is to be understood that, there are many possible variant in this approach, they it is verified It is advantageous in some cases, it will not materially affect whole strategy and be that those of ordinary skill in the art are obvious.It discusses below State several possible variants, but these are intended to illustrate possible variant, and be not intended to cover those of ordinary skill in the art it is aobvious and All possibilities being clear to.

The example discussed is if 5- substituent group makes 4- inactivations, by aniline idol before installing biaryl key It is linked on the position 2- of 2,4- dichloro pyrimidine, or using the 5- substituent group of ortho positioned, then converts it into required substituent group.

Second example is nitro need not be used as the protection form of the final 3- amine on aniline in coupling.With 2- chlorine pyrimidine radicals biaryl for example can convert it into amine using 3- halogenated aniline, and using known method during being coupled. Or 3- amido functional group can be introduced before coupling, as long as it can suitably and selectively be protected.This is even related to It is coupled with before segment (I) coupling with segment (III), although the applicability of this method is likely to limited.

In some cases, it can be advantageous to be coupled segment (II) aniline and segment (I) biaryl, thus R²And/or R³Base Group is not last group, but its useful synthon, then can next described after this coupling or even The step of after (if those skilled in the art think most appropriate) carry out chemistry manipulation to prepare final R²And/or R³Substituent group.

The alternative assembling of 2. diaryl-amine core of scheme

Due to the broad applicability of Buchwald coupling, aryl halide (preferred aryl groups chlorine or aryl bromide) can be used 2- ammonia Base -4- bicyclic heteroaryl pyrimidine arylation.2- amino -4- chlorine pyrimidine is all easy to get, and 2- amino -4- chlorine pyrimidine itself have been displayed with Suzuki reaction occurs for diversified heteroaromatic boronic acid to form the biaryl of corresponding 4- pyrimidine radicals connection, without protecting 2- amino, such as (Org Biomol Chem 9,3139 (2011), Bioorg Med Chem 19,5756 (2011), Eur J Org Chem 4532 (2011)).Alternatively, can be by simple by intermediate chloride (II) with ammonia treatment under high pressure Singly it is converted to corresponding 2- amino-compound.Biggish difference can occur in the potential chemistry of aryl, because of preparation now Aryl halide rather than aniline are discussed as the alternative synthetic method of (II) segment by some different changes of this change bring below It learns.

Above-mentioned variation does not all change the elementary tactics of the synthesis, but simply changes the time peace of certain basic steps It arranges and may provide certain specific changes in raw material.It is least compatible by avoiding which kind of the selection of final approach depends on Conversion and the chemistry being easiest to is provided.

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

。

The synthesis of scheme 3. biaryl segment (I) a-e

The synthesis of most of 4- bicyclic aryl -2- chlorine pyrimidines described in present patent application has greatly precedent that can follow.In scheme 2 In, the known priority that all these approach all utilize 2,4- dihalopyrimidines to react at 4- prior to 2-, so as to It is monosubstituted that 4- occurs for high yield.Various technologies can be used, using indoles and imidazoles A, B, C, in classical alkali (base) condition The lower direct nucleophilic displacement for realizing chlorine.But, if it is desired, such reaction can also as to Buchwald that G is shown as or The widely commercially available property of Suzuki coupling operation, heteroaromatic boronic acid and ester allows Suzuki reaction to be optionally used for C, and to the greatest extent Pipe D utilizes Sonagashira to react as shown, and then on the acetylene of nucleophilic addition to electron deficient, but this reaction can also regard It needs to react as Suzuki and carry out.E and F is using Suzuki reaction assembling, and required borate is commercially available.All these In situation, replace if necessary to further, corresponding heteroaryl bromide, which is converted to required boric acid, to be direct and have precedent can greatly It follows.

Above scheme 2 does not include f(R¹ = CHF₂) or g(R¹ = CF₃) subbreed.Corresponding dichloro pyrimidine is not in f series Well known, and inactivate adjacent halogen known to trifluoromethyl, so that 2,4- bis- chloro- 5- trifluoromethyl pyrimidines are first at 2- and big Most nucleophilic precursor reactants --- difluoromethyl may also show this effect.

It is preferentially changed in the position 4- known to 2,4- dichloro pyrimidine -5- formaldehyde.Therefore Suzuki or Buchwald coupling can be at this Corresponding aldehyde is generated in a little series, and these aldehyde are easy by DAST, SF₄Etc. the difluoromethyl derivative for being converted to corresponding (I), The relatively low degree of functionality of these molecules causes to be unlikely to occur serious side reaction.Similar program can be used for (I) g, thus (I) e cyano compound or another acid derivative appropriate are converted to 5- carboxylic acid, can then pass through HF/SF₄Or other technologies It is converted to trifluoromethyl derivative (I) g.The acid can be from the oxidation of corresponding alcohol.But in many cases, simpler Single solution may be the first two steps simply reversed in final assembling --- and keep the chloro- 5- trifluoromethyl of 2,4- bis- phonetic The biaryl coupling needed for then carrying out at 4- to realize required 2- displacement is reacted in pyridine with aniline/aminopyridine (II) first.

If aryl halide is coupled on aminopyrimidine, similar reverse may be implemented.The chloro- 5- trifluoromethyl of 2,4- bis- is phonetic Pyridine will not selectively be reacted with ammonia very much and product is the 1:1 mixture of 2 and 4- displacement, shows CF₃Ortho position inactivation has big Space components (steric component).If necessary to improve the 2- selectivity of this method, it is possible to lower 2- choosing It selects and is more difficult to obtain 5- difluoromethyl analog, then using large volume (bulky) amine such as dibenzylamine, then in 4- Suzuki idols Connection and amine deprotection can make the yield of this approach higher and avoid the separation of possible difficulty.

In some cases, it can be advantageous at pyrimidine ring together by the bracket assembled of anilino- biaryl pyrimidine, especially It is when 6,5- bicyclic heteroaryl is connected on pyrimidine via carbon.As shown in following scheme 3b, if heteroaryl is in its 3- Position is replaced by acetyl group, and the entire bracket group of the compound of the present invention can be made with DMF aldolisation, then with the condensation of N- aryl guanidine It is fitted together.Since aniline amidification (amidinate) used in the present invention, this approach can be the present invention by many modes Described in several classes of compounds feasible alternative is provided.Due to 3- acetyl compounds indoles, N- methyl indol, It is known that this alternative synthetic method connected all 4- carbon in pyrazolo [1,5-a] pyridine and imidazo [1,2-a] pyridine series Pyrimidine serial is feasible.

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

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

1. checking anilino compounds of the invention, they require to prepare 2,4,5- substituted anilines of several different series. At 2-, R²It can be one of several low alkyl groups or lower alkoxy.The group may be existing, such as in 3- benzyl halide or In the halogenated methyl phenyl ethers anisole of 3- or it can be in or be not in setting for the transition metal-catalyzed halide group for passing through suitably activate down later Change addition.4- bit substituent R³It is more various.It can be H, F, alkoxy, alkyl, alkynyl or tertiary amine, several in these groups It is considerably complicated in structure, there is additional polar functional group thereon, to help the physical chemistry and medicine generation for improving the inhibitor Kinetic property.As described in above-mentioned overall plan 1,1- are fixed as amino, and 3- are fixed as nitro.But it if utilizes The Buchwald of aminopyrimidine is coupled, then 1- must be halogen, and Cl or Br is preferred most of the time.3- advantageously For nitro, but it can be amino, and most probable has protecting group, such as Boc, but if electronics or three-dimensional effect (sterics) make It is inactivated relative to 1- amine, may be not protected.It is also possible to halogen, then the amine in transition metal-catalyzed reaction Change or it can be carbonyl substituent group, is then reset in Curtius or Beckman reaction.Therefore, work as R³When not being hydrogen, There is the quaternary benzene of a large amount of 1,2,4,5- to can be used as the synthon of amino aniline ring.Work as R³When being hydrogen, commercially available 1,2,4- replace Benzene synthon can be used for claimed all corresponding compounds in the application, and convert them to segment (II) Chemistry needed for any example is completely in the limit of power of those of ordinary skill in the art.

Some document systems of the quaternary benzene derivative of 1,2,4,5- of the synthon suitable for this patent are given below It is standby.As long as in the ortho position of nitro or contraposition, there are halogens, can introduce amino or alkoxy via the nucleophilic displacement of halogen.Even if These displacements, the especially displacement of fluorine (fluoride) can also occur for existing electron donating group in the ring.In Buchwald class Chloride or bromide can be used in the reaction of type even to introduce amino or alkoxy side after having removed all nitros Chain, it can also be used to aromatic ring is coupled on aminopyrimidine as discussed above.Several synthons have 1,3- dinitro substituent group simultaneously And there are some documents to point out that a nitro is better than another selective reduction.Although being unfavorable for yield, non-selective nitro Reduction is not big problem, as long as isomers can be separated.Since the two nitros finally require to restore, it is different not have " wrong " Structure body is reduced, as long as another required chemistry for constructing segment (II) can be carried out, because of " unacceptable " " 3- nitre The first reduction of base " can simply be solved and protecting it, then reduction " 1- nitro " with Boc and being coupled on chlorine pyrimidine Certainly.Work as R³It, can be anti-using Suzuki or Sonagashira on 4- halogen (preferably bromine or iodine) when being carbon-based substituent group It answers.If there is ortho position or contraposition nitrogen, it can use Finkelstein reaction for chloride and be converted to corresponding iodide.

Some documents preparation of the scheme 4. for the quaternary benzene synthon of 1,2,4,5- of the compound of the present invention

By correct R²Group is inserted into above-mentioned synthon --- doing so if necessary --- and can use in each case It realizes and does not need to synthesize any such R in commercial reagent²Part.Most of R proposed³Group can also be with can be by them The form being incorporated in aniline ring is bought.Commercially available very diversified amine, including diamines and triamine, this is equally applicable to Alcohol, alkoxyl alcohol and amino alcohol, many with cyclic structure therein is building up to, SAR is less likely to require any of these Partial novel synthesis.But this is not suitable for the R of more complicated carbon connection³Part, especially propargylamine.Such compound Two kinds it is general preparation be shown in scheme 5.

The preparation of 5. 3- aminobutyne derivative of scheme

Propargyl chloride (or being acetic acid esters in the presence of copper catalyst) is relatively easy to replace with amine, even if leaving group is tertiary amine (tertiary).Alternatively, under conditions of forming imonium ion with secondary amine processing ketone can directly with various metallic acetylides Reaction as shown in the second reaction equation to generate required compound.In this case, acetylene is then successfully used to Gao Xuan Selecting property Sonagashira reaction, to form the 2- alkynyl arylamine of type needed for the 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 may be at 4- or may be farther With solubilized amine.Such side chain has greatly precedent that can follow and does not need to be discussed further in the literature.Many of which tool There is the too low reactivity as michael acceptor so that can not play a role on the aniline for be rich in electronics, to these side chains Speech, it is necessary to make N- aryl substituent slightly less than electron rich and therefore lower inactivation.Other michael acceptor side chains contain Alkynes, this makes them be inherently michael acceptor more active than corresponding alkene.The many alkynes that can be used are big in the literature There is precedent that can follow, but describes the synthesis of some specific alkynes in this section.In the electronic property for not changing N- aryl moiety (electronics) main policies that michael acceptor is further activated in the case where are the electrophilic halogen atoms of addition.By In the relatively high molecular weight and lipophilicity of the compound based on this bracket, electrophilic strategy is confined to for fluorine atom being incorporated to In michael acceptor, and various mono-, two- and borontrifluoride α is discussed below, beta-unsaturated acid is as segment (III) mikey The synthesis of your receptor.The problem done so is that fluorine is often quite good at activation Michael systems, and in many situations In, which must further be substituted to improve reactivity and the necessary phase of N- aryl by electronics or Space design Work as electron rich.Another problem is that these compounds can be E or Z stereoisomer mostly, some preparations can be generated than less Desired isomers, and most of can generate E/Z mixture.But reaction condition can be usually manipulated to improve required isomery The percentage of body, and isomers needed for various isolation technics separate can be used, these manipulations are all that those skilled in the art are ripe It knows.

The document of 6. 2- and 3- fluoroolefins acid of scheme synthesizes

The most straightforward approach for activating the olefin(e) acid ester system for Michael's addition is that fluorine atom is placed directly within to alkene carbon On.A fluorine atom on 2- or 3- carbon atom is provided with sufficient activation, therefore alkyl and double bond is added slightly to drop Low whole activation is wise.Scheme 6 shows some documents preparation of the fluorination alkene of compound for use in the present invention.

Shown in first four kinds prepare all quite general, and may be allowed many different alkyl, because they all rely on α- Fluoroester precursor is reacted with carbonyl.Therefore one or two alkyl can be introduced to the position 3- of final acrylamide system, with Significantly adjust its reactivity.In the reaction of top, the allylic bromination tendency of these compounds is also illustrated.In acid/ester stage or It is had good grounds in this field after being coupled on aniline with amine displacement allyl bromide, bromoallylene.In existing literature synthesis, do not go out The special preparation of existing 2- alkyl -3- fluoro cinnamate or crotonates, but as shown in three reactions on the lower, 3,3- bis- replace Acrylic acid derivative has several general synthesis, and a large amount of precedents show that these ester products can fully (cleanly) be hydrolyzed into Required carboxylic acid.Triphen phosphino- fluoromethane (triphenylphosphanylfluoromethylidene) (J Org Chem 40, 2796 (1975), seemingly member the only known in its family) it can may be methylated with Methyl triflate C-, It is subsequently used for Wittig reaction, although even root (parent) looks quite problematic.It is reported that with SF₄ Obtain the E/Z- of 2,3- dialkyl group -3- fluoro acrylic ester when handling 2- alkyl 'beta '-ketoester with appropriate yield in complex mixture Mixture (Zhurnal Organicheskoi Khimii, 18,782 (1982)), it is contemplated that raw material are cheap, even if complete It is not attempt to improve the reaction entirely, also can get the required quaternary alkene of sufficient quantity, this may be very versatile.2- alkyl- 3- fluoro cinnamate and crotonates can be prepared as shown in scheme 7.Separate the iodo- 2- fluoro alkene intermediate of 1- phenyl simultaneously Methyl is transferred to α-carbon when being handled with LDA and boron alkyl acid esters such as catecholborane derivative, to form vinyl borine. These can generate a kind of isomers of required compound under Pd catalysis with carbon monoxide carboxy methylation again.

The synthesis of scheme 7. 2- alkyl -3- fluoro cinnamate and beta-unsaturated esters

。

Some preparations of scheme 8.2- trifluoromethyl chain acid ester

。

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

There is several methods that for synthesizing 2- trifluoromethyl crotonates and more advanced analog.The some of them side of being illustrated in In case 8.First reaction is very significant, and because introducing 2,3- double bond at the end of the synthesis, is carrying out sulfoxide oxidation- It can be by addition or hydrogenation modification 4,5- double bond, to obtain diversified 3,3- dialkyl group -2- trifluoromethyl propylene before elimination Amide.

Horner-Wadsworth-Emmons reaction on trifluoromethyl ketone confirms to be the quite general of this kind of compound Preparation, which is mainly or entirely E- stereoselectivity.Across acetylenic acid ester (ynoate) addition HI can be complete Z- choosing Selecting property and independent of trifluoromethyl (Tet Letters 36,2469 (1995)), therefore by as third reaction Shown in HI is added in 3- alkyl propiolic acid, then with trifluoromethyl cuprate (trifluoromethyl cuprate) Reagent coupling, can simply reverse the stereoselectivity of second reaction.By using Pd catalysis Stille coupling rather than The copper coupling of author's description, can make the 4th reaction much general.

Although document appears not to the method for preparing 2- difluoromethyl crotonamide and more advanced homologue well, Since possible tautomerism is melted into very reactive 3,3- difluoropropenes amide isomers, such compound may not used. But the recent synthesis (J Fluorine Chem 130,682 (2009)) of 3,3- difluoro pyruvates allows with based on phosphine The approach of acid esters obtains these compounds (J Fluorine Chem 113,177 (2002)).The difluoromethyl of vinyl halide Change (J Amer Chem Soc 134,5524 (2012), Angew Chem Int Edn 51,12090 (2012), with this Class reagent combines the ability (J Fluorine Chem 111,85 (2001)) of 1,1- dibromoalkene list difluoromethyl Them are allowed to prepare as follows.

The preparation of 10. 2- difluoromethyl chain -2- olefin(e) acid ester derivant of scheme

If tautomerization is a problem, crystal structure shows have space to leave 1,1- difluoro second in this position Base, although this functional group seems that setting not in the alpha-position of chain -2- olefin(e) acid ester is known, 3,3- bis- fluoro- 2- oxo fourths Acid esters is known (J Org Chem 60,5174 (1999)) and can be with square with as corresponding trifluoroacetone esters of gallic acid Formula (J Fluorine Chem 113,177 (2002)) reacts to form these compounds with Wittig reagent.

The synthesis of scheme 11 4,4- difluoro crotonates and climbing groundsel acid esters (senecoate)

Scheme 11 shows that 4,4- difluoro crotonates and 4,4- difluoro climbing groundsel acid esters are (any all to have as side chain Sufficient activity) document synthesis.But at least for crotonates, this is too active so that be not available, can be by prolonging Long alkyl chain improves this activity, this can as shown in scheme 12 using 2, the 2- difluoro alkanoate being easy to get and Claisen condensation, then multistep restores, or 2,2-, bis- fluoro aldehyde, then Horner-Wadsworth-Emmons is anti-by being formed It should realize.Since Bromodifluoroacetic acid ethyl ester and diethy-aceto oxalate are all easy to be converted to 2,2-, bis- fluoroester of more long-chain and aldehyde, hold Easy-to-use alkyl halide C- is alkylated, and the slight modifications of program can get diversified 4,4- difluoro chain acid ester in Utilization plan 11 Side chain.

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

Scheme 13 shows that being used to form the big of bis- fluorine chain -2- olefin(e) acid of E-3- methyl -4,4- has the governed approach of precedent, non- Often similar to some chemistry in scheme 9.Such compound is also valuable, because being all fluorine disclosed in the application Change minimum activation in michael acceptor.

The preparation of 13. E-3- methyl -4,4- of scheme, two fluorine chain -2- olefin(e) acid

Scheme 14 (a) shows a kind of noticeable reaction, thus two-or three-fluorine crotonates can at low temperature with To form 2- methyl analogue, this has in crotonates and difluoro crotonic acid 2- methylmalonic acid transesterification 1- and 2- carbon atom Intermediate electrophilicity between ester, but steric hindrance is lower than difluoro climbing groundsel acid esters.Although in the literature and indirect having precedent can It follows, but by the way that in molecule, as carried out this conversion on those of preparation molecule in scheme 12, extended alkyl side chain is expected to stand Body selectively generates bis- fluorine chain -2- olefin(e) acid ester of E-2- methyl -4,4-, this can generate remaining trisubstituted isomery in the series Body, this method are illustrated in scheme 14 (b).

The preparation of scheme 14. 4,4- difluoro tiglate and more advanced homologue

The synthesis for the acetylenic acid side chain that many amino replace is discussed in J Med Chem 49,1475 (2006), these It is incorporated herein by this reference.The system of many 3- amino -3,3- dialkyl group propyne derivatives has been described in this application before It is standby.These compounds can carboxylation be used on acetylene 1- carbon with preparing as described in J Med Chem 49,1475 (2006) The more space of the compound of the present invention is obstructed but the aminoalkynyl side chain of solubilising.

In scheme 15, the synthesis of the fluoro- 3- nitropyridine of bromo- 2, the 6- bis- of 5- is described in two steps.It is this novel Compound is the very versatile synthon of the diamino-pyridine of formula (II), and instantiates the formula of being used to prepare (II) in this scenario Compound pyridine ring several different conversions.Since nitro and bromine can pass through reduction or the amination conversion of copper catalysis respectively At amine linking group (J Org Chem 77,6908,7471 (2012), Eur J the Org Chem 1854 with biaryl part (2010)) protection step, is then optionally carried out, the displacement that can use any fluorine is implanted into required substituent group.First nucleophile 2- fluorine is replaced to the property of can choose, but then replaces 6- fluorine under the condition (forcing conditions) of slightly more strength, Therefore by selection displacement series, the molecule can be constructed so that bromine or nitro become the linker with biaryl core.Pass through utilization Nickel phosphine and phosphine oxide chemistry of complex Selective activation C-F key with various alkyl/aryl metal derivative cross-couplings, into one Versatility (the J Org Chem 67,8991 (2002), Org of the step enhancing fluoro- 3- nitropyridine of the bromo- 2,6- bis- of 5- Letters, 14,3316 (2012)).

The synthesis of 15. 3,5- diamino-pyridine linker of scheme

In addition to the known compound having been described above, many other compounds as known in the art can be used as in synthesis Raw material, and be given in Table 1 below constitute unintentionally a series of such compounds that exclusiveness is enumerated and about they Bibliography.In many cases, take passages the compound bibliography show it is also clear suitable in addition to those of clearly covering For the chemical conversion of other compounds of the invention, and it will be appreciated by those skilled in the art that they are broadly suitable for this The other aspects of invention.

Other known compounds of table 1. for synthesis。

Treatment method

The invention further relates to treatment method and purposes, combine including giving individually or with other therapeutic agents or moderator The compound of the present invention or its officinal salt.

In one embodiment, the present invention relates to treat or inhibit the cell Proliferation of mammal, cell invasion, turn Move, the method for apoptosis or angiogenesis, including given to the mammal therapeutically effective amount the compound of the present invention or its Officinal salt.

In another embodiment, the present invention relates to treat or inhibit the cell Proliferation of mammal, cell invasion, turn It moves, the method for apoptosis or angiogenesis, including giving the therapeutically effective amount combined with second therapeutic agent to the mammal The compound of the present invention or its officinal salt, wherein the total amount of the compound of the present invention and the second therapeutic agent effectively treat or Inhibit the cell Proliferation, cell invasion, transfer, apoptosis or angiogenesis.

In one embodiment, the second therapeutic agent is selected from mitotic inhibitor, alkylating agent, antimetabolic Object, insertion antibiotic, growth factor receptor inhibitors, radiation, cell cycle inhibitor, enzyme, topoisomerase enzyme inhibitor, biological response Modifying agent, antibody, cytotoxin, antihormone and antiandrogen antitumor agent.

In other embodiments, the cell Proliferation, cell invasion, transfer, apoptosis or angiogenesis are by RTKs's The member of erbB family, mainly EGFR, most likely the T790M mutant form of EGFR mediates.

In another embodiment, the cell Proliferation, cell invasion, transfer, apoptosis or angiogenesis with selected from following Cancer it is associated: spongioblastoma, lung cancer (such as squamous cell carcinoma, non-small cell lung cancer, gland cancer, bronchioloalveolar Cancer (BAC), the BAC with focal infiltration, the gland cancer with BAC feature and large cell carcinoma), cancer of pancreas, head and neck cancer (such as squama Shape cell cancer), breast cancer, colorectal cancer, epithelioma (such as squamous cell carcinoma), oophoroma and prostate cancer and overexpression ErbB family member or carcinogenic Activating mutations body (no matter these protein whether being overexpressed in tumour) containing erbB family Any other cancer.

Another embodiment of the present invention is related to the compound of the present invention, is used as drug, it is especially useful in treatment is wherein EGFR and/or mutation EGFR albumen (such as L858R/T790M EGFR) active inhibition can cause the disease of benefit, such as cancer. A further embodiment of the invention is related to the compound of the present invention or its officinal salt is used to prepare for treating EGFR mediation The purposes of the drug with EGFR inhibitory activity of disease and/or the patient's condition, especially above-listed disease and/or the patient's condition.

Term " therapeutically effective amount " refers to the change for alleviating one or more symptoms of treated illness to a certain extent Close the dosage of object.About treatment of cancer, therapeutically effective amount, which refers to have, to be reduced tumor size, inhibits (being slowed or stopped) swollen Tumor metastasis, inhibition (are slowed or stopped) tumour growth or tumor invasion and/or alleviate to a certain extent relevant to cancer The amount of the effect of one or more symptom or symptom.

Diagnostician (as those skilled in the art) is cured mainly to obtain in a similar situation using routine techniques and by observation As a result, can readily determine that therapeutically effective amount.When determining therapeutically effective amount (dosage), cures mainly diagnostician and consider to be permitted It is multifactor, including but not limited to: the species of mammal；Its figure, age and general health；The disease specific being related to； The infringement degree or seriousness of disease；The response of individual patient；The specific compound of administration；Mode of administration；Drug-delivery preparation is peculiar Bioavilability；Selected dosage regimen；Concomitant medication；With other correlation circumstances.

Unless otherwise specified, the term as used herein " treatment " refers to the applicable illness of the term or the patient's condition or such illness Or reverse, mitigation, process inhibition or the prevention of one or more symptoms of the patient's condition.Term " treatment " also refers to as defined immediately above " treatment " treatment behavior.Term " treatment " further includes the adjuvant treatment of mammal.

" cancer " used herein refers to any pernicious and/or invasive growth as caused by abnormal cell growth or swollen Tumor, including solid tumor, leukemia, bone marrow cancer or the lymphatic system cancer to form their cell type name.The example of solid tumor Including but not limited to sarcoma and cancer.The example of leukemia includes but is not limited to leukaemia, lymthoma and myeloma.Term " cancer " packet Include but be not limited to derived from internal privileged site primary carcinoma, from its initial position be diffused into other body parts metastatic carcinoma, Recurrence and Second primary tumors of the original primary carcinoma after alleviation --- there is the people's of different types of cancer history newly to send out primary before Cancer.

In another embodiment, the present invention provides the method for inhibiting cell Proliferation, including makes cell and effectively inhibit thin The compound of the present invention or the contact of its officinal salt of the amount of born of the same parents' proliferation.In another embodiment, the present invention, which provides, induces carefully The method of born of the same parents' apoptosis, including contacting cell and the compound as described herein of effective amount for inducing Apoptosis.

" contact " is to instigate the compound of the present invention or officinal salt and expression mutation EGFR or in particular cell types The cell for playing one of other target kinases of transformation is put together so that the compound can directly or indirectly influence The activity of EGFR or other kinases.Contact can be external (i.e. in artificial environment, such as, but not limited in test tube or culture medium In) or realization (i.e. in living body, such as, but not limited in mouse, rat or rabbit) in vivo.

In some embodiments, the cell is in cell line, in cancerous cell line.In other embodiments, institute Cell is stated in tissue or tumour, and the tissue or tumour can be internal in mammal (including people).

Can be realized by the way that the compound can be delivered to any method of action site the compound of the present invention to Medicine.These methods include oral route, intraduodenal route, parenteral injection (including vein, it is subcutaneous, intramuscular, intravascular or Infusion), part and rectally.

Adjustable dosage regimen is to provide optimal required response.For example, single bolus (bolus) can be given, it can At any time by giving several divided doses or proportional reduction or enhancer as indicated by the emergency for the treatment of condition Amount.It is consistent with dosage for ease of being administered, it is especially advantageous that parenteral compositions is prepared with unit dosage forms.

Unit dosage forms used herein refer to that the physics for being suitable as the single dose of mammal to be treated is discrete Unit；Constituent parts contain the active ingredient of predetermined amount in conjunction with required pharmaceutical carrier, being computed therapeutic effect needed for generating Object.The specification of unit dosage forms of the invention depends on and depends directly on the unique property of (a) chemotherapeutics and to be realized specific Treatment or prevention effect, and (b) for the intrinsic limit in the compounding art for the such reactive compound for treating individual sensitivity System.

Dosage appropriate becomes with the type and severity of the patient's condition for the treatment of, and may include single dose or multi-agent.It cures mainly Diagnostician's understanding, should be according to the personnel of individual need and execution or supervision composition administration for any specific mammal Professional judgement and adjust specific dosage regimen at any time, dosage range listed in this article be merely exemplary and be not intended to limit Make the range or practice of composition claimed.For example, (can be may include according to pharmacokinetics or pharmacodynamic parameter Clinical effect, such as toxic effect and/or laboratory evaluation) adjust dosage.Therefore, the present invention includes in the patient determined such as technical staff Dosage escalation.The determination of the suitable dosage and dosage regimen of chemotherapeutics is well known in related fields and is understood to learning this In the limit of power of the technical staff of teaching content disclosed in text.

Can by comparing they external activity and determine chemical combination of the invention in vivo activity in an animal model The effective dose of object.Need for the amount of the compound for the treatment of or its active salt or derivative not only with selected specific salts and Become, also become with administration route, the age of the property of the patient's condition for the treatment of and patient and physical condition, finally by attending physician or Clinician's discretion.

The compound of the present invention can deliver medicine to patient with daily about 0.1 to about 2,000 milligram of dosage level.It is right For the normal adult that about 70 kilograms of weight, the dosage that about 0.01 to about 10 milligram of daily per kilogram of body weight is preferred 's.But specific dosage used can change.For example, dosage may depend on many factors, requirement, treatment including patient The patient's condition severity and compound used therefor pharmacological activity.Determination to the optimal dose of particular patient is art technology Well known to personnel.In some cases, may be more than sufficient lower than the dosage level of above range lower limit, and in other situations In, larger dose can be used without will cause harmful side effect, precondition be such larger dose is first divided into it is several Smaller dose is for whole day administration.

Pharmaceutical composition of the invention can be prepared in the form of in bulk, single unit dose or multiple unit doses, be wrapped Dress is sold." unit dose " used herein is the discrete amount of the pharmaceutical composition of the active constituent comprising predetermined amount.Activity The amount of ingredient is generally equal to the convenient score of the dosage or this dosage of giving the active constituent of individual, such as this dosage Half or 1/3.

The relative quantity of active constituent, pharmaceutical acceptable carrier and any supplementary element in pharmaceutical composition of the invention can become Change, identity, figure and the physical condition of the individual depending on treatment and the administration route for further depending on the composition.Example Such as, the composition may include the active constituent of 0.1% to 100% (w/w).

Pharmaceutical composition and preparation method thereof suitable for delivering the compound of the present invention be those skilled in the art it is aobvious and It is clear to.Such composition and preparation method thereof be found in for example ' Remington's Pharmaceutical Sciences', the 19th edition (Mack Publishing Company, 1995), the disclosure of which full text is incorporated by this through this Text.

The compound of the present invention can be taken orally.Oral administration may relate to swallow, so that the compound enters stomach and intestine Road, or oral cavity or sublingual administration can be used, thus the compound directly enters blood flow from oral cavity.It is suitble to the preparation of oral administration Including solid pharmaceutical preparation, as tablet, the capsule containing particle, liquid or powder, pastille (being filled including liquid), masticatory, it is more-and Nanometer-fine granule, gelling agent, solid solution, liposome, film (including mucous membrane patch), ovum pill (ovules), spray and liquid Body preparation.

Liquid preparation includes suspension, solution, syrup and elixir.Such preparation can be used as filling out for soft capsule or hard capsule Material, and generally includes carrier, such as water, ethyl alcohol, polyethylene glycol, propylene glycol, methylcellulose or suitable oil, and it is a kind of or Numerous emulsifiers and/or suspending agent.Liquid preparation can also be prepared by the reconstruct of solid.

The compound of the present invention can be also used in instant, the fast dosage form collapsed, such as the Expert of Liang and Chen Opinion in Therapeutic Patents, 11 (6), described in 981986 (2001) those, the disclosure of which is complete Text is incorporated herein by this reference.

For Tabules, according to dosage, drug may make up the 1 weight % to 80 weight % of dosage form, more generally composition dosage form 5 weight % to 60 weight %.In addition to drug, tablet usually also contains disintegrating agent.The example of disintegrating agent include primojel, Sodium carboxymethylcellulose, calcium carboxymethylcellulose, cross-linked carboxymethyl cellulose receive, Crospovidone, polyvinylpyrrolidone, methyl Hydroxypropyl cellulose, starch, pregelatinized starch and the sodium alginate that cellulose, microcrystalline cellulose, low alkyl group replace.In general, Disintegrating agent accounts for the 1 weight % to 25 weight % of dosage form, preferably 5 weight % to 20 weight %.

Usually using adhesive to assign tablet formulation cohesion.Suitable adhesive include microcrystalline cellulose, gelatin, Sugar, polyethylene glycol, natural and paragutta, polyvinylpyrrolidone, pregelatinized starch, hydroxypropyl cellulose and hydroxypropyl methyl Cellulose.Tablet can also contain diluent, such as the lactose monohydrate, anhydrous of spray drying (monohydrate), sweet dew Alcohol, xylitol, dextrose, sucrose, D-sorbite, microcrystalline cellulose, starch and calcium phosphate dibasic dihydrate.

Tablet can include optionally also surfactant, such as lauryl sodium sulfate and Tween 80 and glidant, such as two Silica and talcum.When it is present, surfactant can account for 0.2 weight % of tablet to 5 weight %, and glidant can account for tablet 0.2 weight % to 1 weight %.

Tablet usually also contains lubricant, as magnesium stearate, calcium stearate, zinc stearate, sodium stearyl fumarate with And the mixture of magnesium stearate and lauryl sodium sulfate.Lubricant usually accounts for the 0.25 weight % to 10 weight % of tablet, preferably Account for the 0.5 weight % to 3 weight % of tablet.

Other possible ingredients include antioxidant, colorant, flavoring agent, preservative and odor mask.

Tablet mixture can be with direct pressing or with roll pressing to form tablet.Alternatively, tablet mixture or mixture A part wet granulation, non-slurry pelletizing or melt pelletization, melt can be solidified or be squeezed out before tabletting.Final preparation may include one A or multiple layers, and can be coating or uncoated；It even can be packed into capsule.

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

The above-mentioned preparation of various administration fashions discussed above can be configured to quick-release and/or adjust release (modified Release).Adjusting release formulation includes that sustained release, sustained release, pulse release, controlled release, Targeting delivery and sequencing are released It puts.Suitable tune release formulation for the purpose of the present invention is described in United States Patent (USP) No. 6,106,864.In Verma et al., Pharmaceutical Technology On-line, 25 (2), visible other suitable release tech in 1-14 (2001) Details, such as high energy dispersions and infiltration and coated bead.It is described in WO 00/35298 using chewing gum (chewing Gum controlled release) is realized.

The compound of the present invention can also be directly administered in blood flow, in muscle or in internal organs.Suitable parenterally Administration mode includes in intravenous, intra-arterial, peritonaeum, in intrathecal, intra-ventricle, urethra, in breastbone, encephalic, intramuscular and subcutaneous. Suitable Parenteral administration device includes needle (including micropin) syringe, needleless injector and infusion techniques.

Parenteral formulation is usually aqueous solution, can contain excipient, (preferably such as salt, carbohydrate and buffer PH value to 3 to 9), still, for some purposes, they are more suitable for being configured to sterile non-aqueous solution or will be with suitable media Object, the dried forms being used in combination such as aseptic apirogen water.

Using well known to a person skilled in the art standard pharmaceutical techniques, parenteral formulation is easily implemented in aseptic condition Under preparation (such as passing through freeze-drying).

Preparation technique appropriate can be used, such as solubilizer is added to improve formula used in parenteral solutions preparation (I) solubility of compound.

Parenteral administration preparation can be configured to quick-release and/or adjust release.Therefore, the compound of the present invention can be configured to Implanted storage cavern agent (depot) administration of solid, semisolid or thixotropic fluid to be released as the tune for providing reactive compound.Such system The example of agent includes the bracket and poly- (glycolide-dl- lactide) or PGLA microballoon for being coated with drug.

The compound of the present invention can with solvable macromolecular body, such as cyclodextrin and its suitable derivative or contain polyethylene glycol Polymer combine to improve solubility, dissolution rate, the taste masking effect, biology when they are used with any of above mode of administration Availability and/or stability.For example, it is found that Drug-cyclodextrin complexes are generally used for most of dosage forms and administration route. Inclusion and non-inclusion complex can be used.As the alternative solution with drug direct combination, cyclodextrin may be used as auxiliary and add Add agent, that is, is used as carrier, diluent or solubilizer.Be most commonly used to these purposes is α-, β-and gamma-cyclodextrin, and the example can See international patent application Nos. WO 91/11172, WO 94/02518 and WO 98/55148.

Term " combination therapy " refers to that the compound of the present invention is in succession or same together at least one medication or medicament When be administered.Combination therapy includes using the compound of the present invention and other controlling in separated dosage form or in identical pharmaceutical preparation Treat agent.The compound of the present invention can be used with one or more therapeutic agents and (simultaneously, is administered in succession or respectively).

In one embodiment of the invention, combining with the compound of the present invention and pharmaceutical composition as described herein makes Anticancer agent is anti-angiogenic agent (such as tumour is prevented to form the reagent of new blood vessel).The example packet of anti-angiogenic agent Include such as VEGF inhibitor, VEGFR inhibitor, TIE-2 inhibitor, PDGFR inhibitor, angiogenin inhibitor, PKCI3 suppression Preparation, CQX-2(cyclo-oxygenase II) inhibitor, integrin (α-v/ β -3), MMP-2(matrix metalloproteinase 2) inhibitor and MMP-9(matrix metalloproteinase 9) inhibitor.Preferred anti-angiogenic agent includes Sutent (SutenFM), bevacizumab (Avastin^TM) and Axitinib (AG 13736).

Additional anti-angiogenic agent includes vatarani (CGP 79787), Sorafenib (Nexavar^TM), piperazine Jia Tani Sodium (Macugen^TM), Vande Thani (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).

The anti-angiogenic agent that can be used in combination with the compound of the present invention and pharmaceutical composition as described herein its Its example includes celecoxib (Celebrex^TM), SC 69124 (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-blood Pipe generating agent includes exisulind (Aptosyn TM), sasapyrin (AmigesicTM), Diflunisal (Dolobid^TM), cloth Ibuprofen (Motrin^TM), Ketoprofen (Orudis^TM), Nabumetone (Relafen^TM), piroxicam (FeldeneTM), naproxen (AIeveTM, Naprosyn TM), Diclofenac (Voltaren TM), indocin (Indocin^TM), sulindac (ClinoriI^TM), tolmetin (tolmetin) (Tolectin^TM), Etodolac (Lodine^TM), ketorolac (ToradoI^TM) and Olsapozine (Daypro^TM).Other anti-angiogenic agents include ABT 510(Abbott), A Leisita (apratastat) (TMI 005), AZD 8955(AstraZeneca), incyclinide(Metastat^TM) and PCK 3145(Procyon).Its Its anti-angiogenic agent includes A Quting (Neotigason TM), plitidepsin(aplidine TM), cilengtide (EMD 121974), Combretastatin A-4 4(CA4P), Suwei A amine (fenretinide) (4HPR),Halofuginone hydrobromide (Tempostatin^TM), Panzem^TM(methoxyestradiol), PF-03446962(Pfizer), Rui Masita (BMS 275291), catumaxomab (Removab^TM), lenalidomide (Revlimid^TM), squalamine (EVIZONTM), Thalidomide (Thalomid^TM), Ukrain^TM(NSC 631570), Vitaxin^TM(MEDI 522) and zoledronic acid (Zometa TM).

In another embodiment, the anticancer agent be so-called signal transduction inhibitor (such as inhibit control cell growth, Break up the mode exchanged in the cell with the adjusting molecule of the basic process of survival).Signal transduction inhibitor includes small molecule, resists Body and antisense molecule.Signal transduction inhibitor includes such as kinase inhibitor (such as tyrosine kinase inhibitor or serine/Soviet Union Histidine kinase inhibitor) and cell cycle inhibitor.Signal transduction inhibitor more specifically includes 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 more targeting kinase inhibitors.It is preferred that Signal transduction inhibitor include Gefitinib (Iressa^TM), Cetuximab (Erbitux^TM), Erlotinib (Tarceva^TM), Herceptin (Herceptin^TM), Sutent (Sutent^TM) and Imatinib (Gleevec^TM).

The signal transduction inhibitor that can be used in combination with the compound of the present invention and pharmaceutical composition as described herein Other examples include BMS 214662(Bristol-Myers Squibb), Luo Nafani (Sarasar^TM), pelitrexol (AG 2037), matuzumab (EMO 7200), Buddhist nun's trastuzumab (TheraCIM h-R3^TM), Victibix (Vectibix^TM), Vande Thani (Zactima^TM), pazopanib (SB 786034), ALT 110(Alteris Therapeutics), BIBW 2992(Boehringer Ingelheim) and Cervene^TM(TP 38).Signal transduction inhibitor Other examples include PF-2341 066(Pfizer), PF-299804(Pfizer), Canertinib, handkerchief trastuzumab (Omnitarg^TM), Lapatinib (Tycerb^TM), pelitinib (EKB 569), Miltefosine (Miltefosin^TM), BMS 599626(Bristol-Myers Squibb), Lapuleucel-T(Neuvenge^TM), NeuVax^TM(E75 cancer vaccine), Osidem^TM, wood benefit replace Buddhist nun (TAK-165), Victibix (Vectibix^TM), Lapatinib (Tycerb^TM), pelitinib (EKB And handkerchief trastuzumab (Omnitarg 569)^TM).Other examples of signal transduction inhibitor include ARRY 142886(Array Biopharm), everolimus (Certican^TM), Zuo Tamosi (Endeavor^TM), tamiros (temsirolimus) (ToriseI^TM) and AP 23573(ARIAO).In addition, other signals transduction inhibitor includes XL 647(Exelixis), Suo La Non- Buddhist nun (Nexavar^TM), LE-AON(Georgetown University) and GI-4000(Globelmmune).Other signals turn Leading inhibitor includes ABT 751(Abbott), alvocidib(Flavopiridol), BMS 387032(Bristol Myers), EM 1421(Erimos), N- (the chloro- 1H- indoles -7- base of 3-) -1,4- 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 be used in combination with reagent of the invention, with Erlotinib, Gefitinib, drawing Pa replaces other erbB races for Buddhist nun, Conmana, Afatinib, linatinib, peletinib and dacomitinib to inhibit Agent is considered especially significant.All there is all these compounds enough wild type erbB kinase inhibiting activities to be based on having (mechanism-based) dose-limiting toxicity of mechanism, but can be administered with tolerance and show good clinic Activity.One of their main weakness is that the tumour for responding these medicaments very well is tended to erbB mutation, the mutation Make the tumour to the inhibitor quite sensitive, but when being combined with second of mutation, it is intended to the tumour be made to be resistant to very much these Reagent.The selection pressure for accelerating this process has been discussed above.The compound of the present invention targets main resistant mutants, And since they have extremely low activity to wild-type enzyme, (mechanism based) poison based on mechanism will not be dramatically increased Property.But they can be such that the double mutant in evolution is under selection disadvantage identical with original sensitive mutant, therefore pole Slow down or may entirely prevent the appearance of persister greatly.Therefore, this combination is verified clinically highly useful.

The present invention considers that the compound of the present invention is used together with classical antitumor agent.Classical antitumor agent includes hormone tune Save agent such as hormone, antihormones, Androgen receptor agonists, androgen antagonist and anti-estrogen therapy agent, histon deacetylase (HDAC) (HOAC) inhibitor, gene silencing agent or gene activator, ribalgilase, proteosomics, topoisomerase I inhibit Agent, camptothecin derivative, Topoisomerase II inhibitors, alkylating agent, antimetabolite, poly- (AOP- ribose) polymerase -1 (PARP-1) inhibitor, Antitubulin, antibiotic, the spindle poison of plant origin, iridium-platinum complex, gene Therapeutic agent, antisense oligonucleotides, blood-vessels target agent (VTAs) and Statins.

The example for the antitumor agent being used in combination with the compound of the present invention includes Velcade(bortezomib), 9- amino Camptothecine, Belotecan, camptothecine, Diflomotecan, edotecarin, Exatecan (Daiichi), gefitinib, 10- hydroxyl Camptothecine, Irinotecan HCI(Camptosar), Lurtotecan, Orathecin(Rubitecan, Supergen), topotecan, Camptothecine, 10-hydroxycamptothecine, 9-aminocamptothecin, Irinotecan, edotecarin, topotecan, Aclarubicin, Ah mould Element, Amonafide, Amrubicin, anthracycline (annamycin), daunorubicin, Doxorubicin, Elsamitrucin, epirubicin, Etoposide, idarubicin, galarubicin, hydroxycarbamide, Nemorubicin, mitoxantrone (mitoxantrone), pirarubicin, Pixantrone (pixantrone), procarbazine, butterfly mycin, Sobuzoxane, his fluorine pool glycosides (tafluposide), valrubicin, Zinecard(dexrazoxane), mustargen N- oxide, cyclophosphamide, hemel, AP-5280, apaziquone, Brostallicin, bendamustine, busulfan, carboquone, Carmustine, Chlorambucil, Dacarbazine, Estramustine, Fotemustine, glufosfamide, ifosfamide, lomustine, Mafosfamide, mustargen (mechlorethamine), melphalan, Dibromannitol, mitolactol, mitomycin C, mitoxantrone, Nimustine, Ranimustine, Temozolomide, thio-tepa and Platinum coordination alkylated compound, such as cis-platinum, Paraplatin(carboplatin), eptalatin, lobaplatin, Nedaplatin, Eloxatin(Ao Shali Platinum, Sanofi), streptozotocin, satraplatin (satrplatin) and combinations thereof.

Present invention further contemplates that the compound of the present invention and dihydrofolate reductase inhibitor (such as methotrexate (MTX) and Trimetrexate Glucuronate), purine antagonist (such as Ismipur riboside, mercaptopurine, 6- thioguanine, Cladribine, clofarabine (Clolar), fludarabine, nelarabine and Raltitrexed), Pyrimidine antagonists (such as 5 FU 5 fluorouracil), Alimta(pemetrexed Disodium (premetrexed disodium)), capecitabine (Xeloda TM), cytarabine, Gemzar^TM(gemcitabine) replaces Add fluorine, doxifluridine, Carmofur, cytarabine (including octadecyl phosphate (ocfosfate), phosphate stearate, Sustained release and liposomal form), enocitabine, 5-azacitidine (Vidaza), Decitabine and ethynylcytidine) and other anti-generations Thank object such as Eflornithine, hydroxycarbamide, formyl tetrahydrofolic acid, nolatrexed (Thymitaq), 3- aminopyridine-2-formaldehyde-sulphur For semicarbazones (triapine), Trimetrexate and N- (5- [N- (3,4- dihydro -2- methyl -4- oxoquinazolin -6- Ji Jia Base)-N- methylamino] -2- thenoyl)-Pidolidone and combinations thereof is used together.

With the compound of the present invention, it is optionally anti-swollen with classics used in the conjoint therapy of one or more other medicaments Other examples of oncocyte toxin agent include taxol albumin combination particle injection suspension (Abraxane) (Abraxis BioScience, Inc.), Ba Tabulin (Batabulin) (Amgen), vinflunine (Bristol- Myers Squibb Company), actinomycin D, bleomycin, mitomycin C, neoearcinostain (Zinostatin), vincaleukoblastinum, vincristine, Eldisine, vinorelbine (Navelbine), docetaxel (Taxotere), Ao Tasai, taxol (including Taxoprexin, A kind of DHA/ taxol-conjugate), cis-platinum, carboplatin, Nedaplatin, oxaliplatin (Eloxatin), satraplatin, Irinotecan (Camptosar), capecitabine (Xeloda), oxaliplatin (Eloxatin), docetaxel alitretinoin (Taxotere Alitretinoin), Canfosfamide(Telcyta^TM), DMXAA(Antisoma), ibandronic acid, L-ASP, training Door winter enzyme (Oncaspar^TM), Efaproxiral (Efaproxyn^TMRadiotherapy)), bexarotene (Targretin^TM), replace rice Li Fen, Theratope^TM(Biomira), vitamin A acid (Vesanoid^TM), Tirapazamine (Trizaone^TM), motexafin gadolinium (Xcytrin^TM) Cotara^TM(mAb) and NBI-3001(Protoxb Therapeutics), polyglutamic acid-paclitaxel (Xyotax^TM) and combinations thereof.

Embodiment

Abbreviation

DMSO dimethyl sulfoxide

DTT dithiothreitol (DTT)

ATP atriphos

EDTA ethylenediamine tetra-acetic acid

Ki enzyme inhibition constant

DMEM Dulbecco's improves Eagle culture medium

NCS newborn bovine serum

PBS phosphate buffered saline (PBS)

PMSF phenylmethanesulfonyl fluoride

ELISA enzyme linked immunosorbent assay (ELISA)

IgG immunoglobulin G

FBS fetal calf serum

BDNF brain-derived neurotrophic factor.

The synthesis of the compound of the present invention

。

16. 2- of scheme (3- but-2-enamides base/amyl- 2- acrylamide base -4- fluoroanilino) -4- heteroaryl pyrimidineA-E Synthesis。

1. compound of embodimentASynthesis

As described in WO 2008/137027, containing anhydrous K₂CO₃DMF in 1 equivalents of methanol processing 2 ,-two fluoro- 1, 5- dinitrobenzene, then with sodium dithionite by 2- anisyl nitro selective reduction at amine, and to provide 4- fluoro- for chromatographic isolation 2- methoxyl group -5- nitroaniline.As described in WO 2013/014448, in the hot amylalcohol containing p-methyl benzenesulfonic acid with 3- (2- Chlorine pyrimidine-4-yl) pyrazolo [1,5-a] pyridine condensation generation triaryl bracket.Nitro is restored with Raney's nickel or Fe/AcOH, newly Manufactured amine is acylated with 4- bromine crotonyl chloride in THF at 0 DEG C as described in US 6297258.Such as institute in US 6297258 It states and replaces 4- bromine completion compound in situ with N methyl piperazineASynthesis.

2. compound of embodimentBSynthesis

Use the KRuIO of Polymer-supported₄Or other oxidations well known by persons skilled in the art are by known 3- (1- azepine Cyclobutane base) propyl alcohol is oxidized to corresponding aldehyde.It is reacted with carboxymethyl Wittig reagent, then with LiOH saponification generation 5- (1- nitrogen Azetidinyl) amyl- 2- olefin(e) acid, corresponding acid chloride, and the triaryl amine amine described in embodiment 1 are converted to oxalyl chloride Change, to generate compoundB。

3. compound of embodimentCSynthesis

Keep the fluoro- 2- methoxyl group -5- nitroaniline of 4- described in embodiment 1 and the chloro- 4- of 2- (N- methyl indol -3- base) phonetic Pyridine, which is condensed, is simultaneously reduced into corresponding amine for nitro, and it is described in embodiment 1 under conditions of be coupled and generate with 4- bromine crotonyl chloride Required 4- bromine crotonocyl amination triaryl amine bracket.With the displacement of bis- (2- ethoxy) amine under conditions of described in the US 6297258 Bromine completes compoundCSynthesis.

4. compound of embodimentDSynthesis

The synthesis is identical as the synthesis of compound C, only in the last one step, with 4- (piperidin-1-yl) piperidines into Row bromine is replaced to generate compoundD。

5. compound of embodimentESynthesis

The synthesis is identical as the synthesis of compound C, only in the last one step, carries out bromine displacement with piperidines to generate CompoundE。

17. 2- of scheme (3- but-2-enamides base anilino-) -4- heteroaryl pyrimidineF-ISynthesis。

6. compound of embodimentFSynthesis

Make commercially available 4- nitro-o-toluidine (R=H) and 2- ethyl -5- nitroaniline (R=Me) and the chloro- 4- of 2- (N- first Base indol-3-yl) pyrimidine condensation, nitro is reduced into corresponding amine in this case, and the condition described in embodiment 1 The coupling of lower and 4- bromine crotonyl chloride generates required 4- bromine crotonocyl amination triaryl amine bracket.As R=H, in US 6297258 Described under conditions of with piperidines displacement bromine complete compoundFSynthesis.

7. compound of embodimentGSynthesis

Bromine crotonamide (wherein R=CH as described in example 6 above₃) with 3- hydroxy azetidine in US Compound is completed in reaction under conditions of described in 6297258GSynthesis.

8. compound of embodimentHSynthesis

As described in WO 2013/014448,2- methoxyl group -5- nitroaniline and 3- (2- chlorine pyrimidine-4-yl) pyrazolo Condensation of [1,5-a] pyridine in the hot amylalcohol containing p-methyl benzenesulfonic acid generates triaryl bracket appropriate.Nitro is reduced into Corresponding amine, and it is described in embodiment 1 under conditions of generated with the coupling of 4- bromine crotonyl chloride needed for 4- bromine crotonocyl amination Triaryl amine bracket.1- methyl octahydro pyrrolo- [3,4- is used under conditions of described in the US 6297258b] pyrroles's displacement bromine completion CompoundHSynthesis.

9. compound of embodimentISynthesis

The synthesis and compoundHSynthesis it is identical, only in the last one step, with 2- methyl -2,7- diaza spiro Ring [4.4] nonane carries out bromine displacement to generate compoundI。

18. 2- of scheme (3- but-2-enamides base -4- aminoalkynyl anilino-) -4- heteroaryl pyrimidineJ-MSynthesis。

10. compound of embodimentJSynthesis

By di-nitrated fluoro- 1, the 5- dinitrobenzene of the bromo- 2- of preparation 4- of 3- bromofluorobenzene, fluorine is selectively set by hydroxide ion It changes, then such as (J Org Chem 56,5958 (1991) and ChemMedChem 6,1411 (2011) is described uses Lian Erya Sodium sulphate selective reduction ortho position nitro.The synthesis of the crucial aniline for 18 compound of scheme is completed in neighbour's methylation, such as WO It is even with the chloro- 4- of 2- (N- methyl indol -3- base) pyrimidine in the hot amylalcohol containing p-methyl benzenesulfonic acid described in 2013/014448 Then connection is protected with Boc acid anhydride to generate triaryl bracket appropriate.It is reacted using Sonagashira by 4- methyl-1-(3- first Base butyl- 3- alkynyl) piperazine is coupled on bromobenzene amine moiety, nitro is reduced into amine with iron in acetic acid, is optionally added in addition Acid to remove Boc protecting group.Compound is completed and with crotonyl chloride acylated aniline aminoJSynthesis.

11. compound of embodimentThe synthesis of K

Then the crucial biaryl bromaniline of synthesis as described in example 10 above is reacted using Sonagashira by N- Propargyl pyrroles is coupled to thereon.Nitro reduction as described in example 10 above also removes Boc group, will with 4- bromine crotonyl chloride The amine is acylated, and as described in US 6297258 with dimethylamine displacement bromine to complete compoundKPreparation.

12. compound of embodimentLSynthesis

The preparation bromo- 2- methoxyl group -5- nitroaniline of 4- as described in example 10 above, then such as institute in WO 2013/014448 It states with 3- (2- chlorine pyrimidine-4-yl) pyrazolo [1,5-a] pyridine coupler in the hot amylalcohol containing p-methyl benzenesulfonic acid, it is appropriate to generate Triaryl bracket.Then it is reacted using Sonagashira and is coupled to N- propargyl morpholine thereon.Nitre is restored with iron and acetic acid Base is acylated with 4- bromine crotonyl chloride to generate corresponding amine, and as described in US 6297258 with piperazine displacement bromine to complete CompoundLPreparation.

13. compound of embodimentMSynthesis

Then the crucial biaryl bromaniline of synthesis as described in example 12 above is reacted using Sonagashira by 4- Methyl-1-(3- methyl butyl- 3- alkynyl) piperazine is coupled to thereon.With iron and acetic acid reduction nitro to generate corresponding amine, make With classical peptide coupling condition with 4- (methoxy ethoxy) crotonic acid (by 4- bromocrotonic acid and methyl cellosolve acid sodium (sodium methoxyetholate) is made) it is acylated to complete compoundMSynthesis.

18. 2- of scheme (3- but-2-enamides base -4- fluoroalkyl/alkynyl anilino-) -4- heteroaryl pyrimidineN-PConjunction At。

14. compound of embodimentNSynthesis

Preparation 2- (the bromo- 2- methoxyl group -5- nitrobenzene amido of 4-) -4- (1- (tert-butoxy carbonyl as described in example 10 above Base) indol-3-yl) pyrimidine.Then commercially available 3,3,3- trifluoropropyne utilizes Sonagashira reaction to be coupled to form three through bromide Propynylated (trifluoroptpopynated) aniline of fluorine.Then iron and acetic acid or familiar to those skilled in the art other are used Nitro is reduced into corresponding amine by reducing agent, is simultaneously replaced as described in US 6297258 with piperazine with 4- bromine crotonyl chloride is acylated Bromine is to complete compoundNPreparation.

15. compound of embodimentOSynthesis

Second from the bottom kind of compound of embodiment 14 uses N, N, the processing of N'- trimethyl ethylenediamine as described in US 6297258 To complete compoundOPreparation.

16. compound of embodimentPSynthesis

Preparation 2- (the bromo- 2- methoxyl group -5- nitrobenzene amido of 4-) -4- (indol-3-yl) pyrimidine as described in example 10 above And indole nitrogen is methylated with NaH/ methyl iodide.Then the bromide is carbonylated using classics Heck carbonylation conditions, and use The aldehyde is converted to difluoromethyl by DAST.Nitro is reduced into corresponding amine with iron and acetic acid or Raney's nickel, and by such as US The conjunction of compound P is completed described in 6297258 with the acylation of 4- bromine crotonyl chloride and with bis- (2- ethoxy) amine displacement bromides At.

19. 2- of scheme (3- (2- (fluorine/trifluoromethyl) but-2-enamides base) anilino-) -4- heteroaryl pyrimidineQ-T's Synthesis。

17. compound of embodimentQSynthesis

2- (the fluoro- 2- methoxyl group -5- nitrobenzene amido of 4-) -4- (1- methyl indol -3- base) pyrimidine (WO is handled with dimethylamine 2013/014448), to replace fluorine atom.Then nitro is reduced into corresponding amine, and with E, the fluoro- 3- methyl butene of the bromo- 2- of 4- Acyl chlorides (acid preparation, Eur J Chem 2603 (1998), and pass through oxalyl chloride or other sides well known by persons skilled in the art Method is converted to acid chloride) it is acylated using condition described in US 6297258.Then the allyl bromide, bromoallylene is replaced to produce with pyrrolidines Raw compoundsQ。

18. compound of embodimentRSynthesis

3- (2- (the fluoro- 2- methoxyl group -5- nitrobenzene amido of 4-) pyrimidine-4-yl) pyrazolo [1,5-a] is handled with dimethylamine Pyridine (WO 2013/014448), to replace fluorine atom.Then nitro is reduced into corresponding amine, and using in US 6297258 The fluoro- 3- methyl butene acylated with acid chloride of the condition E described in embodiment 17, the bromo- 2- of 4-.Then 3- methoxypyrrole is used Alkane replaces the allyl bromide, bromoallylene to generate compoundR。

19. compound of embodimentSSynthesis

The 2- fluoro- 3- methyl but-2-ene acyl chlorides (processing of respective acids is used under conditions of described in the US 6297258 (synthesis 122 (1975)), uses oxalyl chloride) by 3- (2- (5- amino -4- (N- methyl-N- (2, N, N- dimethylamino Base ethyl) -2- methoxy-pllenylamine base) pyrimidine-4-yl) -4,5,6,7- tetrahydro-pyrazole simultaneously [1,5-a] pyridine (WO 2013/ 014448) acylated to generate compoundS。

20. compound of embodimentTSynthesis

3- (2- (the fluoro- 2- methoxyl group -5- nitrobenzene amido of 4-) pyrimidine-4-yl)-is handled with 3- dimethylamino pyrrolidines 4,5,6,7- tetrahydro-pyrazoles simultaneously [1,5-a] pyridine pyrimidine (WO 2013/014448), to replace fluorine atom.Then nitro is restored At corresponding amine, and with the fluoro- 3- methyl but-2-ene acylated with acid chloride (referring to embodiment 19) of 2- to generate compound T.

20. 2- of scheme (3- (2- (fluorine/trifluoromethyl) but-2-enamides base) anilino-) -4- heteroaryl pyrimidineU-X's Synthesis。

21. compound of embodimentUSynthesis

As described in example 17 above by 2- (the fluoro- 2- methoxyl group -5- nitrobenzene amido of 4-) -4- (1- methyl indol -3- base) Pyrimidine (WO 2013/014448) is converted to corresponding dimethylaminoaniline.This then in coupling agent, such as EDAC/HOBT or (the amyl- 2- olefin(e) acid condensation of the fluoro- 4- methyl of diethylamino -2- is to generate compound with Z-2- in the presence of HATU or pyBOPU。Z- 2- (the amyl- 2- olefin(e) acid of the fluoro- 4- methyl of diethylamino -2- is prepared in three steps by 2- bromine isobutylaldehyde --- with two in ether Ethamine replace (Dalton Trans 5945 (2008)), then with 2- fluorine phosphinylidyne yl acetate (2- Fluorophosphoinoacetate Horner-Wadsworth-Emmons reaction (Synthesis 427 (2002))) occurs And saponification.

22. compound of embodimentVSynthesis

Make 2- (the fluoro- 2- methoxyl group -5- nitrobenzene amido of 4-) -4- (1- methyl indol -3- base) pyrimidine (WO 2013/ 014448) it is reacted with N methyl piperazine, restores nitro then to generate free 3- amino.This then with known Z- α-fluorine cortex cinnamomi Acid and peptide coupling agent condensation generate compoundV。

23. compound of embodimentWSynthesis

With N methyl piperazine processing 3- (2- (the fluoro- 2- methoxyl group -5- nitrobenzene amido of 4-) pyrimidine-4-yl) pyrazolo [1, 5-a] pyridine (WO 2013/014448), to replace fluorine atom.Then nitro is reduced into corresponding amine, and with the fluoro- 3- of Z-2- (1- methyl piperidine -3- base) acrylic acid is (by known 1- methyl piperidine-as described in Tet Letters 32,339 (1991) 3- formaldehyde and 2,2- bis- chloro- 3,3,3- trifluoroacetic acid methyl esters are prepared in two steps, are then saponified) it is acylated to generate compoundW。

24. compound of embodimentXSynthesis

3- (2- (the fluoro- 2- methoxyl group -5- nitrobenzene amido of 4-) pyrimidine-4-yl) -4,5,6,7- is handled with N methyl piperazine Tetrahydro-pyrazole simultaneously [1,5-a] pyridine (WO 2013/014448), to replace fluorine atom.Then nitro is reduced into corresponding amine, And as described in example 23 above with the fluoro- 3- of Z-2- (1- methyl piperidine -3- base) propylene acylating acid to generate compound X.

21. 2- of scheme (3- (3 (- fluorine/(two/tri-) methyl fluoride) but-2-enamides base) anilino-) -4- heteroaryl pyrimidine Y-ACSynthesis。

25. compound of embodimentYSynthesis

In the chloro- 4- of 2- (imidazo [1,2-a] pyridin-3-yl) pyrimidine (WO 2010/097335) such as WO 2013/014448 It is described to be reacted with the fluoro- 2- methoxyl group -5- nitroaniline of 4-.Then M- methyl-N- (2- dimethyl aminoethyl) amine displacement fluorine is used, Nitro is reduced into corresponding amine with iron and acetic acid.This then generates compound with the fluoro- 2- methyl crotonic acyl chlorides condensation of 3-Y。

26. compound of embodimentZSynthesis

The chloro- 4- of 2- (1- methyl indol -3- base) pyrimidine as described in WO 2013/014448 with the fluoro- 2- methoxyl group -5- of 4- Nitroaniline reaction.Then M- methyl-N- (2- dimethyl aminoethyl) amine displacement fluorine is used, is reduced into nitro with iron and acetic acid Corresponding amine.This, which is then reacted with 3- difluoromethyl crotonyl chloride, generates compoundZ。

27. compound of embodimentAASynthesis

With N methyl piperazine processing 3- (2- (the fluoro- 2- methoxyl group -5- nitrobenzene amido of 4-) pyrimidine-4-yl) pyrazolo [1, 5-a] pyridine (WO 2013/014448), to replace fluorine atom.Then nitro is reduced into corresponding amine with iron in acetic acid, and It is acylated to generate compound with 3- trifluoromethyl crotonyl chlorideAA。

28. compound of embodimentABSynthesis

3- (2- (5- amino -4- (4- methylpiperazino) -2- methoxyl group -5- anilino-) pyrimidine-4-yl) -4,5,6,7- Simultaneously [1,5-a] pyridine (it is the intermediate in embodiment 4) 2,3- dimethyl -4,4- difluoro crotonic acid and peptide are even for tetrahydro-pyrazole Join agent, as PyBOP is acylated to generate compoundAB。

29. compound of embodimentACSynthesis

2- (5- amino -4- dimethyl is handled with the bromo- 2- methyl -3- trifluoromethyl crotonic acid of 4- and coupling agent, such as PyBOP Amino -2- methoxy-pllenylamine base) -4- (1- methyl indol -3- base) pyrimidine (intermediate in embodiment 21), then such as US With dimethylamine displacement bromine to generate compound described in 6297258AC。

22. 2- of scheme (3- (bis- fluorine chain -2- acrylamide base of 4,4-) anilino-) -4- heteroaryl pyrimidineAD-AGSynthesis。

30. compound of embodimentADSynthesis

Acetaldehyde is as described in J Org Chem 45,28 (1980) and J Fluorine Chem 36,293 (1987) The amyl- 2- acetylenic acid of 4,4- difluoro is converted in 4 steps.Such as New J Chem 27 is carried out after the stereotaxis addition of HI, The methylation of the catalysis of Pd described in 432 (2003) and Synthesis 543 (2002) substitutes iodate with methyl bromide zinc Object.The acid can use peptide coupling reagent such as PyBOP and the condensation of aforementioned triaryl amine now to generate compoundAD。

31. compound of embodimentAESynthesis

The bromine of N- methyl -3- hydroxy azetidine sodium salt displacement bromoacetaldehyde acetal is smoothly used in DMF.In the acetal It after hydrolysis, reacts the aldehyde with methyl propiolic acid lithium at low temperature, is oxidized to gained secondary alcohol accordingly with catalytic perruthenate Ketone.The ketone is converted to gem-dimethyl compound by DAST, is used across three key addition HI and as described in example 30 above after saponification Methyl bromide zinc substitution.The acid is then condensed with triaryl amine, is only that fluoride displacement step with the difference in embodiment 30 It is carried out with morpholine, to generate compoundAE。

32. compound of embodimentAFSynthesis

The Grignard Reagent derived from Chloromethyl methyl ether is set to react the corresponding α -one of generation at low temperature with diethy-aceto oxalate Ester.This is then converted to corresponding 2,2- difluoro ester with DAST, and is reduced into corresponding aldehyde with DIBAL at low temperature.With The aldehyde is converted to corresponding beta-unsaturated esters by Horner-Wadsworth-Emmons reaction, this ester by with 2- methyl-prop two The exchange reaction of sour sodium " methylation " (J Org Chem 63,7525 (1998)) on the position 2-.Use BCl₃Remove methyl ether simultaneously The ester is removed by saponification.Gained carboxylic acid is using PyBOP or similar reagents with triaryl amine coupling appropriate as described above to produce Raw compoundsAF。

33. compound of embodimentAGSynthesis

2 methyl propanal is converted to the fluoro- 3- of corresponding 4,4- bis- using with identical chemical process described in embodiment 30 Methyl alkenoic acid.Its followed by PyBOp and triaryl amine intermediate condensation appropriate or its can be equally converted to oxalyl chloride For corresponding acid chloride to implement to be coupled, this generates compoundAG。

23. 2- of scheme (3- (alkynes -2- amide groups) anilino-) -4- heteroaryl pyrimidineAH-AKSynthesis。

34. compound of embodimentAHSynthesis

Chlorine is caused to be replaced by amine with the amine processing chloro- 3- methyl butyne of 3- under various conditions (usual copper catalysis), this In situation, the use of bis- (methoxy ethyl) amine generates required amino acetylene.Such as document (US 6297258, J Med Chem 49,1475 (2006)) described in via grignard (or lithium) acetylide formed and CO₂By this compound carboxylation to generate 4- (bis- (methoxy ethyl) amino of N, N-) amyl- 2- acetylenic acid of -4- methyl.This then in the presence of condensing agent such as PyBOP with triaryl amine (by 3- (2- (the fluoro- 2- methoxyl group -5- nitrobenzene amido of 4-) pyrimidine-4-yl) -4,5,6,7- tetrahydro-pyrazole simultaneously [1,5-a] pyridine (WO 2013/014448) is made and being reduced into amine with methyl cellosolve displacement fluorine and then by nitro under alkaline condition) Coupling is to generate final product compoundAH。

35. compound of embodimentAISynthesis

4- methyl-4- (4- methyl piperazine is prepared by 3- acetoxy-3-methyl butyne and N methyl piperazine in two steps Piperazine -1- base) amyl- 2- olefin(e) acid, this is related to amine displacement of copper catalysis, then the carboxylation of acetylene series Grignard Reagent.It is then in dehydrating agent As in the presence of PyBOP with triaryl amine (by 2- (the fluoro- 2- methoxyl group -5- nitrobenzene amido of 4-) -4- (imidazo [1,2-a] pyridine - 3- yl) pyrimidine (it is the intermediate in embodiment 25) by with 2- methyl amino ethanol handle, then use Fe/AcOH or other Nitro is reduced into amine and is made by method such as catalytic hydrogenation) it is coupled to generate compoundAI。

36. compound of embodimentAJSynthesis

3- (2- (the fluoro- 2- methoxyl group -5- nitrobenzene amido of 4-) pyrimidine-4-yl) pyrazolo [1,5-a] is handled with pyrrolidines Pyridine (WO 2013/014448), to replace fluorine atom.Then nitro is reduced into corresponding amine with iron in acetic acid, and contracted Mixture, as used 4- methyl -4- (4- methylpiperazine-1-yl) amyl- 2- olefin(e) acid (embodiment in the presence of EDAC/OBT, PyBOP or HATU 35) it is acylated to generate compoundAJ。

37. compound of embodimentAKSynthesis

Tertiary propargyl ethanol is generated with front three ethyl-acetylene lithium processing N-Boc piperidin-4-one, is post-processed with KF aqueous solution, so Acetic anhydride acetylation is used in the presence of DMAP afterwards.Then the acetate is replaced in the presence of copper (I) catalyst with methylamine, new to introduce Propargylamine in the presence of DMAP with Boc acid anhydride protect.The formation that acetylene series Grignard Reagent is realized with MeMgBr, such as document (US 6297258, J Med Chem 49,1475 (2006)) described in use CO₂Carboxylation generates 3- (1- tert-butoxycarbonyl -4- (N- Methyl-N- tertbutyloxycarbonylamino) piperidin-4-yl) propiolic acid.2- (the fluoro- 2- methoxyl group -5- nitrobenzene amido of 4-) -4- Yin Diindyl -3- base) pyrimidine (WO 2013/014448) Boc acid anhydride N-protected will with Fe/AcOH then with azetidine displacement fluorine Nitro is reduced into amine.Then with above-mentioned N ,-two Boc propiolic acid derivative of N ' and peptide coupling agent such as PyBOP by triaryl amine it is acylated with Generate compoundAK。

24. 2- of scheme (5- (chain -2- acrylamide base) pyridin-3-yl amino) -4- heteroaryl pyrimidineAL-ANSynthesis。

38. compound of embodimentALSynthesis

The fluoro- 3- nitropyridine of the bromo- 2,6- bis- of 5- is prepared as described in scheme 15.With N, N, N '-trimethyl ethylenediamine and ring Sodium propoxide replaces 2- fluorine and 6- fluorine in succession, and is using the water solubility as described in Eur J Org Chem 1854 (2010) Cu^IIWith ammonia displacement bromine to generate 5- amino -6- cyclopropyl oxygroup -2- (N- methyl (2- diformazan in the ammonia-water systems of-salen complex compound Base aminoethylamino) -3- nitropyridine.This under common acid catalysed conditions with the chloro- 4- of 2- (1- methyl indol -3- base) pyrimidine (WO 2013/014448) coupling, although can conveniently implement under alkaline condition in dipolar aprotic solvent such as DMF This displacement.Then nitro is restored with Fe/AcOH, gained amino is with amyl- 2- alkene acylated with acid chloride to generate compoundAL。

39. compound of embodimentAMSynthesis

The fluoro- 3- nitropyridine of the bromo- 2,6- bis- of 5- is prepared as described in scheme 15.With methanol under mild alkaline conditions and use 2-methyl cellosolve sodium replaces 2- fluorine and 6- fluorine in succession under more strong alkaline condition.With Fe/AcOH reduction nitro to generate 3- ammonia Bromo- 2- methoxyl group -6- (methoxy ethoxy) pyridine of base -5-, under alkaline condition with the chloro- 4- of 2- (pyrazolo [1,5-a] pyrrole Pyridine -3- base) pyrimidine (WO 2013/014448) coupling.Then using system ammonia displacement bromine described in embodiment 38, gained Amine then uses 4- (methoxy ethoxy) crotonic acid (it prepares description in embodiment 13) and suitable coupling agent such as PyBOP acyl Change to generate compoundAM。

40. compound of embodimentANSynthesis

The fluoro- 3- nitropyridine of the bromo- 2,6- bis- of 5- is prepared as described in scheme 15.The sodium salt of 4-Boc amino oxetanes Replace 2- fluorine and 6- fluorine in succession at room temperature or at higher temperatures at low temperature and with azetidine.Then hydrosulfurous acid is used Sodium or nickel boride reduction nitro (Tet Letters 34,3083 (1993)) are to generate 3- amino -6- azetidine -1- The bromo- 2- of base -5- (N-Boc- oxetanes -3- base amino) pyridine.This under alkaline condition with 3- (2- chlorine pyrimidine-4-yl)- Simultaneously [1,5-a] pyridine (WO 2013/014448) is coupled 4,5,6,7- tetrahydro-pyrazoles, then with ammonia as described in example 38 above For water by bromo-amine, gained amine is acylated with 4- bromine crotonyl chloride, and is completed by being replaced as described in US 6297258 with pyrrolidines The synthesis is to generate compoundAN。

41. N- of embodiment [5- [[the chloro- 4- of 5- (1H-3- indoles) -2- pyrimidine radicals] amino] -2- [3- (diethylin) Propyl- 1- alkynes] -4- methoxyphenyl] acrylamide preparation route and step

。

Experimental procedure:

The preparation of compound 2:

18.47 g (150.00 mmol) 2- aminoanisole and 120 mL second are added in one 500 mL there-necked flasks Nitrile, stirring and dissolving.At 0 DEG C, 26.70 g NBS (150.00 mmol) slowly are added portionwise to it and stir at this temperature It mixes.After 1 hour, reaction terminates.100 mL saturation Na is added into this reaction system₂S₂O₃Aqueous solution, and be extracted with ethyl acetate (300 mL*3).Merge organic phase, uses anhydrous Na₂SO₄It dries, filters, is concentrated.Crude product is through silica gel column chromatography (PE/EA=10/ 1) the bromo- 21.20 g(104.92 mmol of 2- aminoanisole of 4- is isolated and purified to obtain), yield 69.95%.

The preparation of compound 3:

At 0-5 DEG C, 10.10 g (50.00 mmol) is added in the sulfuric acid solution for being 85% to 80 mL mass fractions The bromo- 2- aminoanisole of 4- and stirring and dissolving.At this temperature, 6.10 g (50.00 mmol) nitric acid is added portionwise to it Guanidine.It finishes, control 0-5 DEG C of temperature and stirs 1 hour.Then, reaction system is slowly poured into 200 mL, 50% NaOH solution In, there is solid precipitation.Sediment is filtered, is washed, it is dry, through column chromatography (PE/EA=10/1,6/1) isolate and purify 7.10 G(28.70 mmol) the bromo- 5- nitro 2- aminoanisole of 4-, yield 57.48%.

The preparation of compound 5:

In 120 mL sec-butyl alcohols, 3.31 g (12.55 mmol) compound, 4,3.24 g (18.82 mmol) is added P-methyl benzenesulfonic acid and the bromo- 5- nitro 2- aminoanisole of 3.10 g (12.55 mmol) 4-.This mixture stirs at 100 DEG C Reaction 20 hours, TLC detection, end of reaction.It is cooled to room temperature, is concentrated to give black solid, successively through acetonitrile and methyl- tert fourth Base ether washs to obtain 2.85 g of solid (6.00 mmol) compound, 5 crude product, yield 47.84%.

The preparation of compound 6:

In the in the mixed solvent of 80 mL ethyl alcohol and 20 mL water, 2.80 g (5.90 mmol) compound, 5,2.00 g is added (35.80 mmol) Fe powder and 2.00 g NH₄Cl (37.40 mmol).In N₂Under protection, the mixture is anti-in 60 DEG C of stirrings It answers 6 hours, TLC detection, end of reaction.Reaction solution is filtered while hot, filtrate is concentrated to give yellow solid, and the solid crude product is through column layer Analysis (PE/EA=5/1) isolates and purifies to obtain 1.10 g (2.48 mmol) compound 6, yield 42.03%.

The preparation of compound 8:

In the anhydrous THF of 30 mL, 1.10 g (2.48 mmol) compound 6 and 383.61 mg (2.97 is added Mmol) DIEA, stirring and dissolving.At 0 DEG C, 268.65 mg (2.97 mmol) acryloyl chloride slowly is added dropwise to it, drop finishes, TLC is detected after stirring 0.5 hour at this temperature, and reaction terminates.5 mL ice water are added into the system, stir 20 minutes.Water Mutually extracted with ethyl acetate (20 mL*3).Merge organic phase, through anhydrous Na₂SO₄After drying, precipitation is depressurized in filtering.It is thick to produce The inverted preparation chromatography preparative separation of product obtains 0.48 g (0.96 mmol) yellow solid compound 8, yield 38.87%.

Compound N-[5- [[the chloro- 4- of 5- (1H-3- indoles) -2- pyrimidine radicals] amino] -2- [3- (diethylin) propyl- 1- Alkynes] -4- methoxyphenyl] acrylamide preparation:

In the mixed solution equipped with 8 mL triethylamines and 6 mL DMF, it is added 400.00 mg (861.49 μm of ol) 8,120.94 mg of compound (172.30 μm of ol) Pd (PPh₃)₂Cl₂, 199.10 mg (172.30 μm of ol) Pd (PPh₃)₄ With 16.41 mg (86.15 μm of ol) CuI, stirring and dissolving.In N₂Under protection, 1.62 g (14.57 mmol) chemical combination is added to it Object 9.The system is heated to 110 °C of microwave reactions 2 hours under the conditions of anhydrous and oxygen-free.TLC detection reaction terminates.It is cooled to 25 DEG C, 15 mL H are added into system₂O, water phase are extracted with ethyl acetate (15 mL*3).Merge organic phase, through anhydrous Na₂SO₄ After drying, filtering, decompression precipitation obtains dark oil object.The inverted preparation chromatography preparative separation of crude 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- pyrimidine radicals] amino] -2- [3- (diethyl Amido) propyl- 1- alkynes] -4- methoxyphenyl l] acrylamide, yield 9.87%.

Chromatography target compound content 98.05%, ESI-MS(m/z): 528 (M+H)⁺。

42. N- of embodiment [5- [[4- (1H-3- indoles) -2- pyrimidine radicals] amino] -2- [3- (diethylin) propyl- 1- Alkynes] -4- methoxyphenyl] acrylamide preparation route and step

。

The preparation of compound 3:

In 100 mL sec-butyl alcohols, it is right that 2.30 g (10.00 mmol) compound, 2,2.58 g (15.00mmol) are added Toluenesulfonic acid and the bromo- 5- nitro 2- aminoanisole of 2.47 g (10.00 mmol) 4-.Mixture is reacted 20 at 100 DEG C Hour, TLC detection, reaction is finished.It is cooled to room temperature, is concentrated to give black solid, successively washed through acetonitrile and methyl tertiary butyl ether(MTBE) Obtain 2.55 g (5.79 mmol) compound, 3 crude product, yield 57.95%.

The preparation of compound 4:

In 80 mL ethyl alcohol, be added 2.00 g (4.54 mmol) compound, 3,2.03 g Fe (36.34 mmol) and 2.00 g NH₄Cl (37.40 mmol)。N₂Under protection, mixture reacts 6 hours when 60 DEG C, and TLC detection, reaction is finished.Reaction Liquid filtering, is concentrated to give yellow solid, and solids crude capo chromatography (PE/EA=5/1) isolates and purifies to obtain 1.25 g (3.05 Mmol) compound 4, yield 67.18%.

The preparation of compound 5:

In the anhydrous THF of 40 mL, 1.22 g (2.97 mmol) compound 4 and 422.23 mg (3.27 is added Mmol) DIEA, stirring and dissolving.At 0 DEG C, 295.70 mg (3.27 mmol) acryloyl chloride slowly is added dropwise to it, drop finishes, It stirs at this temperature.TLC is detected after 0.5 hour, and reaction terminates.5 mL ice water are added into system, stir 20 minutes.Water Mutually extracted with ethyl acetate (20 mL*3).Merge organic phase, through anhydrous Na₂SO₄After drying, precipitation is depressurized in filtering.Crude product 0.65 g (1.4 mmol) yellow solid compound 5, yield 47.14% are obtained through reverse chromatograms preparative separation.

Compound N-[5- [[4- (1H-3- indoles) -2- pyrimidine radicals] amino] -2- [3- (diethylin) propyl- 1- alkynes] -4- Methoxyphenyl] acrylamide preparation:

In the mixing for filling 15 mL triethylamines and 10 mL DMF not solution, 600.00 mg (1.29 are added Mmol) compound 5,72.56 mg (103.38 μm of ol) Pd (PPh₃)₂Cl₂, 119.46 mg (103.38 μm of ol) Pd (PPh₃)₄With 12.30 mg (84.62 μm of ol) CuI, stirring and dissolving.In N₂Under protection, 2.43 g (21.85 are added to it Mmol) compound 6.System is under the conditions of anhydrous and oxygen-free, and microwave reaction 3 hours at 90 DEG C, TLC detection reaction terminates.It is cooled to 25 DEG C, 30 mL H are added into system₂O, water phase are extracted with ethyl acetate (30 mL*3).Merge organic phase, through anhydrous Na₂SO₄After drying, filtering, decompression precipitation obtains dark oil object.Crude product obtains 50.00 mg through reverse chromatograms preparative separation (101.10 μm of ol) compound as white solid N- [5- [[4- (1H-3- indoles) -2- pyrimidine radicals] amino] -2- [3- (diethylamine Base) propyl- 1- alkynes] -4- methoxyphenyl] acrylamide, yield 7.84%.

Chromatography target compound content 93.58%, ESI-MS(m/z): 495 (M+H)⁺。

43. N- of embodiment [5- [[4- (1- methyl -3- indoles) -2- pyrimidine radicals] amino] -2- [3- (diethylin) propyl- 1- alkynes] -4- methoxyphenyl] acrylamide preparation route and step

。

The preparation of compound 3:

In 80 mL sec-butyl alcohols, 2.44 g (10.04 mmol) compound, 2,2.60 g (15.06mmol) is added to first Benzene sulfonic acid and the bromo- 5- nitro 2- aminoanisole of 2.48 g (10.04 mmol) 4-.It is small that mixture is reacted to 20 at 90 DEG C When, TLC detection, reaction is finished.It is cooled to room temperature, is concentrated to give black solid, is successively washed through acetonitrile and methyl tertiary butyl ether(MTBE) 2.58 g (5.68 mmol) compound, 3 crude product, yield 56.56%.

The preparation of compound 4:

In 80 mL ethyl alcohol, be added 2.20 g (4.84 mmol) compound, 3,2.17 g Fe (38.72 mmol) and 2.07 g NH₄Cl (38.74 mmol)。N₂Under protection, mixture reacts 6 hours when 60 DEG C, and TLC detection, reaction is finished.Reaction Liquid filtering, is concentrated to give yellow solid, and solids crude capo chromatography (PE/EA=3/1) isolates and purifies to obtain 1.40 g (3.30 Mmol) compound 4, yield 68.17%.

The preparation of compound 5:

In the anhydrous THF of 40 mL, 1.40 g (3.30 mmol) compound 4 and 469.09 mg (3.36 is added Mmol) DIEA, stirring and dissolving.At 0 DEG C, 328.52 mg (3.36 mmol) acryloyl chloride slowly is added dropwise to it, drop finishes, It stirs at this temperature.TLC is detected after 0.5 hour, and reaction terminates.5 mL ice water are added into system, stir 20 minutes.Water Mutually extracted with ethyl acetate (20 mL*3).Merge organic phase, through anhydrous Na₂SO₄After drying, precipitation is depressurized in filtering.Crude product 640.50 mg (1.34 mmol) yellow solid compound 5, yield 40.60% are obtained through reverse chromatograms preparative separation.

Compound N-[5- [[4- (1- methyl -3- indoles) -2- pyrimidine radicals] amino] -2- [3- (diethylin) propyl- 1- Alkynes] -4- methoxyphenyl] acrylamide preparation:

In the mixing for filling 15 mL triethylamines and 10 mL DMF not solution, 600.00 mg (1.25 are added Mmol) compound 5,70.44 mg (100.34 μm of ol) Pd (PPh₃)₂Cl₂, 115.96 mg (100.34 μm of ol) Pd (PPh₃)₄With 11.94 mg (62.72 μm of ol) CuI, stirring and dissolving.In N₂Under protection, 2.43 g (21.85 are added to it Mmol) compound 6.System is under the conditions of anhydrous and oxygen-free, and microwave reaction 3 hours at 90 DEG C, TLC detection reaction terminates.It is cooled to 25 DEG C, 30 mL H are added into system₂O, water phase are extracted with ethyl acetate (30 mL*3).Merge organic phase, through anhydrous Na₂SO₄After drying, filtering, decompression precipitation obtains dark oil object.Crude product obtains 30.00 mg through reverse chromatograms preparative separation (58.99 μm of ol) compound as white solid N- [5- [[4- (1- methyl -3- indoles) -2- pyrimidine radicals] amino] -2- [3- (diethyl Amido) propyl- 1- alkynes] -4- methoxyphenyl] acrylamide, yield 4.72%.

Chromatography target compound content 96.12%, ESI-MS(m/z): 509 (M+H)⁺。

44. N- of embodiment [2- [3- (diethylin) propyl- 1- alkynyl] -4- methoxyl group -5- [(4- pyrazoles [1,5-a] -3- Pyridine -2- pyrimidine) amino] phenyl] and acrylamide preparation route and step

。

The preparation of compound 3:

In 100 mL sec-butyl alcohols, it is right that 2.31 g (10.00 mmol) compound, 2,2.58 g (15.00mmol) are added Toluenesulfonic acid and the bromo- 5- nitro 2- aminoanisole of 2.47 g (10.00 mmol) 4-.Mixture is reacted 20 at 100 DEG C Hour, TLC detection, reaction is finished.It is cooled to room temperature, is concentrated to give black solid, successively washed through acetonitrile and methyl tertiary butyl ether(MTBE) Obtain 2.20 g (4.99 mmol) compound, 3 crude product, yield 49.86%.

The preparation of compound 4:

In 50 mL acetone and 2 mL H₂In the mixed solution of O, 2.08 g (4.71 mmol) compound 3,4.31 is added G Zn (65.99 mmol) and 2.02 g NH₄Cl (37.71 mmol).It at 25 DEG C, stirs 1 hour, TLC detection, reaction Finish.Reaction solution filtering, is concentrated to give solid crude product, and solids crude capo chromatography (PE/EA=3/1) isolates and purifies to obtain 1.41 g (3.43 mmol) compound 4, yield 72.78%.

The preparation of compound 5:

In the anhydrous THF of 40 mL, 1.00 g (2.43 mmol) compound 4 and 314.06 mg (2.43 is added Mmol) DIEA, stirring and dissolving.At 0 DEG C, 241.93 mg (2.67 mmol) acryloyl chloride slowly is added dropwise to it, drop finishes, It stirs at this temperature.TLC is detected after 0.5 hour, and reaction terminates.5 mL ice water are added into system, stir 20 minutes.Water Mutually extracted with ethyl acetate (20 mL*3).Merge organic phase, through anhydrous Na₂SO₄After drying, precipitation is depressurized in filtering.Crude product 0.68 g (1.37 mmol) yellow solid compound 5, yield 56.46% are obtained through reverse chromatograms preparative separation.

Compound N-[2- [3- (diethylin) propyl- 1- alkynyl] -4- methoxyl group -5- [(4- pyrazoles [1,5-a] -3- pyridine - 2- pyrimidine) amino] phenyl] and acrylamide preparation:

In the mixing for filling 15 mL triethylamines and 10 mL DMF not solution, 620.00 mg (1.33 are added Mmol) compound 5,4.83 mg (6.88 μm of ol) Pd (PPh₃)₂Cl₂, 7.95 mg (6.88 μm of ol) Pd (PPh₃)₄With 0.82 Mg (4.30 μm of ol) CuI, stirring and dissolving.In N₂Under protection, 2.51 g (22.61 mmol) compound 6 is added to it.System Under the conditions of anhydrous and oxygen-free, microwave reaction 3 hours at 90 DEG C, TLC detection reaction terminates.25 DEG C are cooled to, is added into system 30 mL H₂O, water phase are extracted with ethyl acetate (30 mL*3).Merge organic phase, through anhydrous Na₂SO₄After drying, filtering subtracts Pressure-off is molten to obtain dark oil object.Crude product obtains 45.00 mg (90.80 μm of ol) compound N-[2- through reverse chromatograms preparative separation [3- (diethylin) propyl- 1- alkynyl] -4- methoxyl group -5- [(4- pyrazoles [1,5-a] -3- pyridine -2- pyrimidine radicals) amino] phenyl] Acrylamide, yield 6.83%.

Chromatography target compound content 91.17%, ESI-MS(m/z): 496 (M+H)⁺。

Kinase inhibition measurement

Using commercially available detection kit known to a person of ordinary skill in the art and service measurement formula (I) compound swash Enzyme inhibits.These kits and service for measuring various kinases, including but not limited to ALK, ABL, AXL, Aur B C, BLK, erbB-2, erbB-4. EGFR, mutation 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、 The inhibition of InsR and Atk.These detection kits and the commercial supplier of service include Promega Corporation and Reaction Biology Corporation, EMD Millipore and CEREP.In addition to commercially available detection kit and service, Pass through the kinase inhibiting activity of the compound of following measuring methods measurement formula (I-III).

The purification of epidermal growth factor recipient tyrosine kinase

People's EGF receptor tyrosine is separated from the A431 people's epidermoid carcinoma cell for being overexpressed EGF receptor by following method Kinases.Cell is in roller bottle in 50% Delbuco ' s Modified Eagle and 50% HAM F- containing 10% fetal calf serum Growth in 12 nutrient mediums (Gibco).About 10⁹A cell is containing 20 mM 2- (4N- [2- ethoxy] piperazine -1- base) Ethanesulfonic acid (hepes), pH 7.4,5 mM ethylene glycol bis- (2- amino-ethyl ether) N, N, N', N'- tetraacethyl, 1% Triton X- LOO, 10% glycerol, 0.1 mM sodium orthovanadate, 5 mM sodium fluorides, 4 mM pyrophosphates, 4 mM benzamides, 1 mM, bis- sulphur threose Alcohol, 80 μ g/mL Aprotinins, 40 μ g/mL leupeptins and 1mM phenylmethylsulfonyl fluoride 2 volume buffers in crack.? Be centrifuged under 25,000xg after ten minutes, supernatant at 40 DEG C with before 10 milliliters with 50 mM Hepes, 10% glycerol, 0.1% Triton X-100 and 150mM NaCI, pH 7.5(equilibration buffer) balance wheat germ lectin avidin agarose balance 2 hours. Wash off the protein of pollution from resin with the 1M NaCl in equilibration buffer, and with the 0.5M N- acetyl in equilibration buffer Base -1-D- aminoglucose, then 1mM urea elutes enzyme.The enzyme is eluted with 0.1 mg/ml EGF.Such as pass through the poly- of Coomassie blue stain The assessment of acryl amide electrophoresis gel, receptor seem uniform.

The detecting step of Caliper Mobility Shift Kinase Assay method:

Measurement 1. determines the IC of the test anti-single mutation EGFR (EGFR_d746-750) of compound₅₀Value

Test compound with 100 % dimethyl sulfoxides (DMSO) is configured to 500 μM, then with 100 % DMSO hole-specifically into The concentration gradient dilution that 4 times of row, dilutes 10 gradients." feminine gender " and " positive " control wells are with 100 % DMSO generations of 100 μ L It replaces.Wherein, " feminine gender " control wells are no compound group, and " positive " control wells are no kinases group.Then, 10 μ are added in 96 orifice plates L compound and 90 μ 1 × kinase buffer liquids of L.384 orifice plates are added in the above-mentioned compound containing 10%DMSO of 5 μ L, then by 10 384 orifice plates are added in 2.5 × kinase solution of μ L (containing 12.5 nM EGFR_d746-750,5 mM DTT, 1 × kinase buffer liquid) In.Incubation at room temperature after ten minutes, be added 10 μ L 2.5 × substrate solution (contain 7.5 μM of peptide substrates, 35 μM of ATP, 25 mM MgCl2,12.5 mM MnCl2,1 × kinase buffer liquid) starting reaction.25 μ L are added in 28C after being incubated for 1 hour whole Only liquid terminates reaction.Oscillation carries out read plate to EZ Reader II after centrifugation, finally according to conversion value and " feminine gender " and " sun Property " readings of control wells calculates the inhibiting rate under each concentration of compound, the mapping of binding compounds concentration calculates IC₅₀Value.

Measurement 2. determines the IC of the test anti-double mutations EGFR (EGFR_T790M/L858R) of compound₅₀Value

Test compound with 100 % dimethyl sulfoxides (DMSO) is configured to 500 μM, then with 100 % DMSO hole-specifically into The concentration gradient dilution that 4 times of row, dilutes 10 gradients." feminine gender " and " positive " control wells are with 100 % DMSO generations of 100 μ L It replaces.Wherein, " feminine gender " control wells are no compound group, and " positive " control wells are no kinases group.Then, 10 μ are added in 96 orifice plates L compound and 90 μ 1 × kinase buffer liquids of L.384 orifice plates are added in the above-mentioned compound containing 10%DMSO of 5 μ L, then by 10 384 holes are added in 2.5 × kinase solution of μ L (containing 25 nM EGFR_T790M/L858R, 5 mM DTT, 1 × kinase buffer liquid) In plate.Incubation at room temperature after ten minutes, be added 10 μ L 2.5 × substrate solution (contain 7.5 μM of peptide substrates, 47.5 μM ATP, 25 mM MgCl2,1 × kinase buffer liquid) starting reaction.28C is added 25 μ L terminate liquids and terminates instead after being incubated for 1 hour It answers.Oscillation carries out read plate to EZ Reader II after centrifugation, finally according to conversion value and " feminine gender " and " positive " control wells Reading calculates the inhibiting rate under each concentration of compound, and the mapping of binding compounds concentration calculates IC₅₀Value.

Measurement 3. determines the IC of the test anti-Wild type EGFR of compound₅₀Value

Test compound with 100 % dimethyl sulfoxides (DMSO) is configured to 500 μM, then with 100 % DMSO hole-specifically into The concentration gradient dilution that 4 times of row, dilutes 10 gradients." feminine gender " and " positive " control wells are with 100 % DMSO generations of 100 μ L It replaces.Wherein, " feminine gender " control wells are no compound group, and " positive " control wells are no kinases group.Then, 10 μ are added in 96 orifice plates L compound and 90 μ 1 × kinase buffer liquids of L.384 orifice plates are added in the above-mentioned compound containing 10%DMSO of 5 μ L, then by 10 2.5 × kinase solution of μ L (containing 20 nM EGFR, 5 mM DTT, 1 × kinase buffer liquid) is added in 384 orifice plates.Incubation at room temperature After ten minutes, be added 10 μ L 2.5 × substrate solution (contain 7.5 μM of peptide substrates, 5.75 μM of ATP, 25 mM MgCl2,25 mM MnCl2,1 × kinase buffer liquid) starting reaction.The termination of 25 μ L terminate liquids is added in 28C after being incubated for 1 hour Reaction.Oscillation carries out read plate to EZ Reader II after centrifugation, finally according to conversion value and " feminine gender " and " positive " control wells Reading calculate the inhibiting rate under each concentration of compound, the mapping of binding compounds concentration calculates IC₅₀Value.

Test result column of the part of compounds in measurement 1-3 are in table 1 below.

Table 1

No	Structure	Measure 1 (single mutant) (nM)	Measure 2 (double mutants) (nM)	Measure 3 (wild types) (nM)
					Embodiment 41	N- [5- [[the chloro- 4- of 5- (1H-3- indoles) -2- pyrimidine radicals] amino] -2- [3- (diethylin) propyl- 1- alkynes] -4- methoxyphenyl] Acrylamide	2.6	2.7	5.4
Embodiment 42	N- [5- [[4- (1H-3- indoles) -2- pyrimidine radicals] amino] -2- [3- (diethylin) propyl- 1- alkynes] -4- methoxyphenyl] propylene Amide	9.9	9.0	22.5
					Embodiment 43	N- [5- [[4- (1- methyl -3- indoles) -2- pyrimidine radicals] amino] -2- [3- (diethylin) propyl- 1- alkynes] -4- methoxyphenyl] Acrylamide	51	12.6	750.5
Embodiment 44	N- [2- [3- (diethylin) propyl- 1- alkynyl] -4- methoxyl group -5- [(4- pyrazoles [1,5-a] -3- pyridine -2- pyrimidine) amino] benzene Base] acrylamide	29.2	7.1	237

Other kinase inhibition measurements

The compound of formula (I-III) is measured to other kinases according to program known to persons of ordinary skill in the art The measuring method of inhibition.These measuring methods include, but are not limited to be related to the measuring method of the inhibition of following kinases:

● wild type c-Met kinases measures wild type c-Met as described in International Publication No. WO 2011/069761 The inhibition of kinases, entire content are incorporated herein by this reference.

● LCK and BLK kinases such as United States Patent (USP) No. 7 measures the inhibition of LCK and BLK kinases described in 125,875, Its entire content is incorporated herein by this reference.

Compound as described herein screens in the following manner.Suitable for measuring the kinase activity of compound as described herein The kinases of following procedure includes, but are not limited to: Lck, Lyn, Src, Fyn, Syk, Zap-70, Itk, Tec, Btk, EGFR, mutation EGFR, ErbB2, ErbB-4, Kdr, Flt-1, Flt-3, Tek, c-Met, InsR and Atk.Kinases is in Escherichia coli or rod-shaped disease The kinase domain being fused on glutathione S-transferase (GST) or overall length structure are expressed as in poison-High Five expression system Build the fusion protein of object or polyhistidine label.By basic foregoing affinity chromatography (Lehr et al., 1996；Gish Et al., 1995) they are purified to close to homogeneous.In some cases, kinases mentions before measurement activity with purification or part Pure regulatory polypeptide coexpression or mixing.It is living essentially by blas (Braunwalder et al., 1996) measurement kinases Property and inhibition.In short,³²PO₄It is poly- that the synthesis substrate on the bioactivity surface for being attached to titer plate is transferred to from ATP (Glu-Tyr) basis of assessment enzymatic activity is served as on 4:1 or poly- (Arg-Ser) 3:1.After incubation period, by using first The 0.5% phosphoric acid washing plate is added liquid scintillator, then counts in liquid scintillation detector, measures the phosphoric acid of transfer Amount.By making to be merged on the substrate being incorporated on plate³²The compound concentration that P amount reduces by 50% measures IC₅₀.It is other similar Method also can be used --- and thus phosphoric acid is transferred in the solution or fixed (i.e. solid phase) contains tyrosine, serine, Soviet Union In the peptide or peptide substrate of propylhomoserin or histidine (they are independent or combine, or in conjunction with other amino acid).For example, it is also possible to make With scintillation proximity method (Wu et al., 2000), ELISA(Cleaveland et al., 1990), fluorescence polarization (Seethala and Menzel, 1998) it detects phosphoric acid with homogenizing time resolved fluorometric method (HTRF, Kolb et al., 1998) and turns to peptide or polypeptide It moves.Alternatively, measuring kinase activity using antibody based method, thus use antibody or polypeptide more as reagent detection phosphorylation target Peptide.

Reference

Braunwalder et al. (1996) Anal. Biochem. 234 (1): 23-26.

Cleaveland et al. (1990) Anal Biochem. 190 (2): 249-53.

Gish et al. (1995) Protein Eng. 8 (6): 609-614.

Kolb et al. (1998) Drug Discov. Today. 3:333-342.

Lehr et al. (1996) Gene 169 (2): 27527-9.

Seethala et al. (1998) Anal Biochem. 255 (2): 257-62.

Wu et al. (2000) Comb Chem High Throughput Screen. 3 (1): 27-36.

ErbB cell detection program summary

1. EGF Rat-1 DNA is synthesized

Rat fibroblast system (Rat-I) is layered in (plated out) flat hole plate in complete medium and makes it Attachment is whole night.Then make cell in the culture medium containing 0.1% bovine serum albumin(BSA) (BSA) it is hungry whole night, with or without chemical combination Object dilution preculture 1 hour, then with 1 ng/ml epidermal growth factor (EGF), 50 ng/ml platelet derived growth factors (PDGF), 3 ng/ml fibroblast growth factors (FGF) or the activation of 10 ng/ml insulin-like growth factor-is (IGF-1) Whole night.Pass through³H- thymidine is incorporated to the horizontal measurement proliferation in DNA.It is issued by comparing existing compared with reference material in compound Existing thymidine is incorporated to level to measure IC₅₀'s。

2. the EGF-R autophosphorylation in A431

By people's epidermoid carcinoma cell (A431；ATCC, Manassas, Va.) (plated is layered in complete medium Out) in flat hole plate and make its attachment whole night.Then make cell hungry in the culture medium containing 0.5% fetal calf serum (FCS), use Or do not have to chemical compound diluted liquid preculture, then activated 3 minutes with 50 ng/ml EGF.By cell cracking and pass through SDS-PAGE Protein isolate matter.By using the phosphoric acid junket on the western blot determination EGF-R of anti-phospho-EGF-R- specific antibody Propylhomoserin is horizontal.IC is measured by comparing the phosphotyrosine levels found in the presence of compound compared with reference material₅₀'s。

3. HRG β l T47D DNA is synthesized

By human breast tumor cell line (T47D；ATCC, Manassas, Va.) (plated is layered in complete medium Out) in flat hole plate and make its attachment whole night.Then make cell hungry in the culture medium containing 0.1% bovine serum albumin(BSA) (BSA) It starves whole night, with or without chemical compound diluted liquid preculture 1 hour, is then activated with 150 ng/ml Heregulin(HRG β l) whole Night.Pass through³H- thymidine is incorporated to the horizontal measurement proliferation in DNA.It is found in the presence of compound compared with reference material by comparing Thymidine be incorporated to level to measure IC₅₀'s。

4. the ErbB2 autophosphorylation in T47D

By human breast cancer cell (T47D；ATCC, Manassas, Va.) (plated is layered in complete medium Out) in flat hole plate and make its attachment whole night.Then make cell hungry in the culture medium containing 0.1% bovine serum albumin(BSA) (BSA) It starves, with or without chemical compound diluted liquid preculture, is then activated 10 minutes with 900 ng/ml HRG β 1.By cell cracking and lead to Cross SDS-PAGE protein isolate matter.By using the western blot determination ErbB2 of anti-phospho-ErbB2- specific antibody On phosphotyrosine levels.It is surveyed by comparing the phosphotyrosine levels found in the presence of compound compared with reference material Determine IC₅₀'s。

5. HRG β l 3T3-Her2/3 DNA is synthesized

Apoptosis (3T3) overall length people's ErbB-2 and ErbB-3 stable transfection (Carraway et al., J Biol Chem (1995) 270,7111-6)).This cell line is layered on (plated out) flat hole in complete medium In plate and make its attachment whole night.Use cell starvation in the culture medium containing 0.1% bovine serum albumin(BSA) (BSA) whole night, Or do not have to chemical compound diluted liquid preculture 1 hour, then activated whole night with 25 ng/ml Heregulin(HRG β l).Pass through³H- Thymidine is incorporated to the horizontal measurement proliferation in DNA.It is incorporated to by comparing the thymidine found in the presence of compound compared with reference material Level measures IC₅₀'s。

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

Human pneumonocyte system PC9(exons 19 deletes EGFR) it is obtained from American type Culture Collection. PC9 cell is stored in the RPMI 1640 containing 10% fetal calf serum and 2 mM glutamine.Cell is containing 5% CO₂Plus It is cultivated at 37 DEG C in wet incubator.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 endogenous p-EGFR in cell pyrolysis liquid The measuring method of cells phosphorylation.It is inoculated with 40 (10000, μ L cells in the medium in 384 orifice plate of Corning black transparent bottom Cells/well) and at 37 DEG C, 5% CO₂Lower culture is whole night.Using Echo 555, given to cell in 100% DMSO by sound wave The compound of middle serial dilution.Plate is further cultured for 2 hours, and then after culture medium is sucked out, it is slow that 40 μ L x cracking is added into each hole Fliud flushing.Greiner black height combines the captured antibody coating of 384 orifice plates, is then closed with 3% BSA.Removing confining liquid (block) after, 15 μ L lysates are transferred to Greiner black height in conjunction in 384 orifice plates and cultivating 2 hours.Suction and After PBS board-washing, 20 μ L detection antibody is added and cultivates 2 hours.In suction and with after PBS board-washing, 20 μ L are added QuantaBlu fluorescence peroxidase substrate (Thermo Fisher Scientific catalog number (Cat.No.) 15169) is simultaneously cultivated 1 hour. 20 μ L QuantaBlu terminate liquids are added in plate and utilize 352 nanometers of excitation wavelengths and 460 on Envision plate reader Nanometer emission wavelength reads fluorescence.It is output in suitable software package (such as Origin) with the data that each compound obtains to implement Curve fitting analysis.IC is measured by compound concentration needed for calculating 50% effect of offer as this data₅₀Value.

L858R/T790M EGFR(double mutant) cells phosphorylation measurement

Human pneumonocyte system NCI-HI975 is obtained from American type Culture Collection.NCI-HI975 is thin Born of the same parents are stored in the RPMI 1640 containing 10% fetal calf serum and 2 mM glutamine.Cell is containing 5% CO₂Humidification incubator In cultivated at 37 DEG C.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 it is thin in cell pyrolysis liquid for measuring endogenous p-EGFR The measuring method of born of the same parents' phosphorylation.It is (10000 thin to be inoculated with 40 μ L cells in the medium in 384 orifice plate of Corning black transparent bottom Born of the same parents/hole) and at 37 DEG C, 5% CO₂Lower culture is whole night.Using Echo 555, given in 100% DMSO by sound wave to cell The compound of serial dilution.Plate is further cultured for 2 hours, and after culture medium is sucked out, 40 μ L 1x lysis buffers are added into each hole. Greiner black height combines the captured antibody coating of 384 orifice plates, is then closed with 3% BSA.Removing confining liquid (block) Afterwards, 15 μ L lysates are transferred to Greiner black height in conjunction in 384 orifice plates and cultivating 2 hours.It is washed in suction and with PBS After plate, 20 μ L detection antibody is added and cultivates 2 hours.In suction and with after PBS board-washing, 20 μ L QuantaBlu fluorescence mistakes are added Oxide zymolyte (Thermo Fisher Scientific catalog number (Cat.No.) 15169) is simultaneously cultivated 1 hour.By 20 μ L QuantaBlu Terminate liquid is added in plate and is read on Envision plate reader using 352 nanometers of excitation wavelengths and 460 nanometer emission wavelength Fluorescence.It is output in suitable software package (such as Origin) with the data that each compound obtains to implement curve fitting analysis.By This data measure IC by compound concentration needed for calculating 50% effect of offer₅₀Value.

BaF3 transfection system

BaF3 is to immortalize mouse pro-B cell line, relies on IL-3 growth and survives and do not express ErbB family member. Those of ordinary skill in the art are with erbB albumen (including but not limited to wt EGFR, wt erbB-2, wt erbB-4, L858R- EGFR, del746-750-EGFR and T790M-EGFR) appropriate structuring cDNAs transfect BaF3 cell (Blood 97,1050 (2001)) it, (is not still deposited through the independent growths in the case where IL-3 is not present or in the presence of appropriate erbB ligand then In IL-3) growth come select transfection clone, so as to select and separate with required erbB family member transfection BaF3 cell System.These cell lines then can be used to measure the cell IC of each member of erbB family as described in the literature₅₀s.(Cancer Res 71,7587 (2011)、Cancer Res 67, 11924 (2006) Proc Natnl Acad Sci USA 103, 7817 (2006)).

Various other kinases.Such as United States Patent (USP) No. 6, the inhibition of various other kinases is measured described in 881,737, including But be 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, entire content are incorporated herein by this reference.

Animal xenograft tumor model

General procedure.It will be from the known ATCC cell line with related oncogene or from appropriate turn transfected intentionally The cell of change is suspended in culture medium appropriate, and by 5 x 10⁶Or 1 x 10⁷Flank of a cell infusion to nu/nu mouse In.Alternatively, can be with the segment (generally about 1 cubic centimetre) of passage of tumor in trochar placement body to cause tumour.When swollen When tumor reaches size (usually in 100-300 nanogram range) for being suitble to experiment, animal is randomly divided by 6-10 mouse structure At match group, and be fed for by mouth Zhou Qiang give medium or test product once or twice daily.Tumour is measured using slide calliper rule Volume.With the calculating of (gross tumor volume of n-th day 0 day gross tumor volume/0 day of gross tumor volume-the) × 100 n-th day Increase percentage of the volume of xenograft tumours relative to the 0th day (starting that day that test-compound is administered).With (1-medicine In object treatment group in average increase %/medium treatment group of gross tumor volume gross tumor volume average increase %) × 100 calculate it is each Median tumor growth suppression percentage of the medication therapy groups relative to medium treatment group.Count aobvious using single tail t test evaluation Work property.

Wild type EGFR heterograft measures to measure the effect to the tumour for being overexpressed wt EGFR, can be used By A431 epidermoid or the xenograft of LoVo colon cancer cell culture.

EGFR del746-750 heteroplastic transplantation model is in order to measure to the tumour for being overexpressed EGFR-del746-750 Effect can be used by the xenograft of PC9 NSCLC cell culture.

EGFR L858R heteroplastic transplantation model can make to measure the effect to the tumour for being overexpressed EGFR-L858R With the xenograft by H3255 NSCLC cell culture.

EGFR L858R/T790M double mutations heteroplastic transplantation model in order to measure to be overexpressed EGFR-L858R/ The effect of the tumour of T790M double mutant can be used by the xenograft of H1975 NSCLC cell culture.

ErBB-2 heterograft measure in order to measure to be overexpressed wt erbB-2 tumour effect, can be used by The xenograft of N87 gastric cancer or BT474 breast cancer cell culture.

Pharmacodynamics measurement is made with appropriate time interval with any of above tumour (preferably 200-300 after oral administration Milligram size) mouse euthanasia.Tumor resection, it is quick-frozen and be dispersed in using Qiagen Tissue-Lyser containing protease In the non denatured lysis buffer of inhibitors of phosphatases.The homogenate is cracked 1 hour at 4 DEG C, by centrifugal clarification, then Phosphor EGFR/erbB-2/3/4 and total receptor are analyzed by quantitative immuning engram method.The phospho-RTK of each RTK band Its total RTK signal normalization of signal.Alternatively, can use eERK and phosphor-ERK antibody appropriate passes through similar techniques Measure the ratio of the total ERK and phosphor-ERK in tumour.

Claims (7)

1. Formula 1. (A) compound

   Or its officinal salt, wherein

   X¹It is CH；

   Y is

   R¹Selected from hydrogen, fluorine, chlorine；

   R²Selected from methoxyl group, ethyoxyl, isopropoxy；

   R³Selected from by NR⁸R⁹Substituted C_2-6Alkynyl；

   R⁴For H；

   R⁵It is H；

   R^6eAnd R^6zIt independently is H；

   R⁸And R⁹It independently is C_1-6Alkyl；

   X²It is C；

   X³It is CH；

   X⁴It is N or NR¹⁰；

   R¹⁰It is H, C_1-6Alkyl；

   X⁵It is C or N；

   X⁶It is C；

   Two keys in a-e are double bonds and the other three is singly-bound, so that atom X²-X⁶All there are two double bond is coupled；

   Key f is double bond.
2. 2. the compound of claim 1, so that R¹It is hydrogen.
3. 3. the compound of claim 1, so that X⁴It is NH or NCH₃, X¹And X³It is CH and X²、X⁵And X⁶It is C.
4. 4. the compound of claim 1, so that R¹And R⁵It is hydrogen, X⁴It is NH or NCH₃, X¹And X³It is CH, X²、X⁵And X⁶It is C, and R^6z It is H.
5. 5. compound is selected from:

   Or their officinal salt.
6. 6. pharmaceutical composition, it includes the compound or pharmaceutically acceptable salt thereof of any one of claim 1-5 and pharmaceutical acceptable carrier or Excipient.
7. 7. the compound or pharmaceutically acceptable salt thereof of any one of claim 1-5 is in preparation for treating or inhibiting the thin of mammal Born of the same parents' proliferation, cell invasion, transfer, apoptosis or angiogenesis drug in purposes.