CN101370944A

CN101370944A - Compounds and methods of identifying, synthesizing, optimizing and profiling protein modulators

Info

Publication number: CN101370944A
Application number: CNA2006800514988A
Authority: CN
Inventors: 杰勒德·M·豪斯
Original assignee: Individual
Current assignee: Individual
Priority date: 2005-11-23
Filing date: 2006-11-24
Publication date: 2009-02-18

Abstract

This invention relates to methods of identifying, synthesizing, optimizing and profiling compounds that are inhibitors or activators of proteins, both naturally occurring endogenous proteins as well as certain variant forms of endogenous proteins, and novel methods of identifying such variants. The method accelerates the identification and development of compounds as potential therapeutically effective drugs by simplifying the pharmaceutical discovery and creation process through improvements in hit identification, lead optimization, biological profiling, and rapid elimination of toxic compounds. Implementation results in overall cost reductions in the drug discovery process resulting from the corresponding increases in efficiency.

Description

Identify, the Compounds and methods for of synthetic, optimization and profiling protein modulators

The cross reference of related application

The application is the part continuation application of the PCT International Application PCT/US06/33890 of submission on August 29th, 2006, PCT/US06/33890 is the part continuation application of the PCT International Application PCT/US2005/18412 of submission on May 23rd, 2005, it requires the U.S.Ser.No.60/739 of submission on November 23rd, 2005,477, the U.S.Ser.No.60/739 that on November 23rd, 2005 submitted to, 476, the U.S.Ser.No.60/741 that on December 2nd, 2005 submitted to, 767, the U.S.Ser.No.60/751 that on December 16th, 2005 submitted to, 030, the U.S.Ser.No.60/783 that on March 13rd, 2006 submitted to, 106, the U.S.Ser.No.60/785 that on March 23rd, 2006 submitted to, 904, the U.S.Ser.No.60/785 that on March 23rd, 2006 submitted to, the U.S.Ser.No.60/789 that on April 4th, 817 and 2006 submitted to, 379 right of priority.

Technical field

The present invention relates to identify, synthesize, optimize and be characterized by the compound of protein inhibitor or activator, described protein is the variant form of naturally occurring endogenous protein and some endogenous protein, and relates to the novel method of identifying described variant.The quick eliminating of improvement, guide's optimization, bio-identification and toxic chemical by hitting evaluation is simplified drug discovery and production method, and described method has been quickened as the evaluation of the compound of potential treatment active drug and research and development.Because the corresponding increase of efficient, this enforcement cause the total cost in the drug discovery process to reduce.

Background of invention

Important composition at the discovery/production method of the modern novel drugs that has the selectivity protein target in people's cell comprises:

1. the ＂ that identify to suppress or activate selected target protein hits the ＂ compound.(based on these purposes, definition is hit to being divided into positive compound and can having some investigator's desirable effect and pharmaceutical properties in given test.Yet in modern pharmacy research, only in fact hitting just can become final clinical candidate after substance is further modified);

2. the initial compound that hits is further studied and improved to the selection lead compound on its basis;

3. be mainly used in by a series of designs and improve lead compound the inhibition of target protein or the chemically modified of activation character, optimize lead compound (its chemical structure with original hit compound relevant or consistent), but described chemically modified can also improve bioavailability, plasma half-life or reduction toxicity;

4. characterizing the biological activity spectrum (guide who comprises optimization) of specifying lead compound compares with other non-target protein with definite, it is to the relative specificity and the selectivity of selected target protein, some non-target protein and target protein itself closely related (for example other member of protein families);

5. design the outer and interior zooscopy of body of clinical precursor with evaluation agent weight range, carinogenicity, absorption, distribution, metabolism, drainage, pharmacokinetics, oral administration biaavailability (if necessary), pharmacodynamics, toxicity and correlation parameter;

6. the healthy volunteer with suffer from the clinical trial that carries out among the patient of disease, its effective therapeutic is disposed be considered to useful.

The present invention relates to the novel method of substantially improved above-mentioned steps 1-4.Described method also can be used for producing and optimize compound, and described compound is than identifying, optimize with present accepted standard and simple method or the model experiment medicine of sign being much effective and toxicity is much lower.

As a part of making great efforts the advanced new pharmaceutical technology of research and development, researched and developed method described herein, measurable by inventing, reliable methodology, described pharmaceutical technology is become " drug discovery " process into one be described as " drug manufacture " process more accurately, described methodology reduces time and the great number cost relevant with drug discovery/research and development method simultaneously for those skilled in the art provide essential instrument to produce the novel drugs of specific protein important in the target human diseases.

The chemical sproof sexual development that carries out is for using the sign of many types of drug long-term treatments, especially in the treatment field of cancer and infectious diseases among the patient.Identified the molecular mechanism of the resistance phenomenon that mediates some type, and in other situation, the mechanism of tolerance acquisition and that form again is still unknown at present.

Think that at first the relevant chemical sproof a kind of mechanism of inductive (acquisition) comprises that the protein expression that is called P-glycoprotein (P-gp) increases in the cancer therapy field.P-gp is arranged in cytolemma and plays the medicine efflux pump.This protein can pump the cytotoxic chemical promoting agent from cell, comprise the anticarcinogen of many types.Therefore, the incremental adjustments of P-glycoprotein produces the resistance to multiple medicine usually.P-glycoprotein incremental adjustments in tumour cell can be represented a kind of defense mechanism, and this mechanism develops in mammalian cell so that prevent to be subjected to the infringement of cytotoxic chemical promoting agent.At present identified other the relevant resistance protein that has with the similar function of P-gp, comprised multiple medicines thing-resistance-member of associated protein family, such as MRP1 and ABCG2.Under any circumstance, when research and development had the lower compound of specificity and toxicity to specified target albumen, P-glycoprotein descended in conjunction with the importance of cartridge clip (ABC) translocator in clinical significance resistance with relevant ATP-.

The molecular mechanism of the acquired resistance that another kind is possible is that optional signal pathway causes cell survival and the metabolism that continues, even original medicine is still effective to its target.In addition, the change of medicine born of the same parents intracellular metabolite also can cause the therapeutic efficiency forfeiture.In addition, can producer express and gene amplification result's change, thereby cause the proteic expression of specified target to increase or reduce, and need the increase drug dose usually so that keep same function (Adcock and Lane, 2003).

Sudden change inductive resistance is generally situation about occurring in the infectious diseases field.For example, several viral reverse transcriptase of encoding in human immune deficiency (HIV) viral genome or medicines of virus protease of being suppressed at been have have been researched and developed.Fully established in the literature and used reverse transcriptase inhibitors for example to treat AIDS patient that HIV-infects repeatedly finally to have produced the virus mutant form that the susceptibility to medicine lowers.The mutant forms that described enzyme is given in the sudden change that occurs in the gene of coding reversed transcriptive enzyme is reduced by the influence of medicine.

Consider that mistake is introduced into the genomic ratio of HIV, chemical sproof appearance is not unexpected in the HIV therapeutic process.Known hiv reverse transcriptase has wrong taxis especially, and wherein forward mutation rate is about 3.4 x 10 ^-5Plant sudden change/base pair/replication cycle (Mansky etc., J.Virol.69:5087-94 (1995)).Yet the similar mutation rate of the endogenous gene of encoding in mammalian cell is hanged down one more than the order of magnitude.

New evidence shows that resistance also may be because of sudden change result generation (Gorre etc., Science, 2001 of the gene that relates to the drug targets of encoding; PCT/US02/18729).In this case, make the patient contact concrete therapeutant, behind the appointment cancer drug of the protein of paying close attention to such as targeting specific (POI or ＂ target ＂ albumen), lie in the hypertrophy of one group of cell of the sudden change that occurs in the proteinic gene that is encoded to the therapeutant target.Whether the hypertrophy of also not understanding at present this cell mass because of due to the cell that is pre-existing in of the patient's of the sudden change of the implicit POI that produces drug resistance little per-cent, or whether this class sudden change can activate or suppress in the therapeutical agent process of described POI or generation again afterwards in animal or human's contact.In arbitrary situation, this class sudden change result all can produce the albumen (hereinafter being defined as theramutein) of sudden change, and it is subjected to the influence degree of described therapeutant lower or may be unaffected fully.

Chronic granulocytic leukemia (CML) is characterised in that marrow my late grandfather (progenitor) hyper-proliferative that keeps differentiation capability in this disease stable or chronic phase process.Multi-thread evidence has been established the imbalance as the Abl Tyrosylprotein kinase of the pathogenic oncogene among the CML of some form.This imbalance is usually relevant with the chromosome translocation that is called Philadelphia chromosome (Ph), causes by the expressing fusion protein of forming with the BCR gene product of Abelson Tyrosylprotein kinase fusion, and formation has the p210 of tyrosine kinase activity thus ^Bcr-AblRelevant fusion rotein is called p190 ^Bcr-Abl, its produce by the different breakpoints in the BCR gene and the verified patient who appears at acute lymphoblastic leukemia (ALL) with the Philadelphia chromosome positive (Ph+) in (Melo, 1994; Ravandi etc., 1999).Conversion seems because of many signal pathways, comprises due to those pathway activations that relate to RAS, MYC and JUN.Imatinib mesylate (" STI-571 " or

) be that (Druker etc., NEJM 2001, p.1038) for the 2-phenylamino pyrimidine of ATP-binding site of kinase domain of target Abl.Also had been found that the inhibitor and the Kit Tyrosylprotein kinase of platelet-derived somatomedin (PDGF) beta receptor subsequently by other method, wherein the latter relates to the generation (vide infra) of gastrointestinal stromal tumor.

Till in the recent period, do not observe as yet in using the process of specifying the proteic specific inhibitor treatment of endogenous cell, sudden change in its corresponding endogenous gene can cause the different expression of protein, and the cell function of described protein variant tolerates described inhibitor.Work (Gorre etc., Science 293:876-80 (2001) that CharlesSawyers and colleague are done; PCT/US02/18729) confirmed that first use can suppress p210 ^Bcr-AblBehind the medicine of Tyrosylprotein kinase (being STI-571) the treatment patient, produce p210 at coding ^Bcr-AblThe cell mass of clinical meaning may appear having among the described patient of implicit sudden change in the gene of the target protein that contains the Abelson tyrosine kinase domain of cancer.Various these class sudden changes produce p210 ^Bcr-AblMutant forms, they are lower than the form that produces initial cancer to the reactivity of Gleevec treatment.Sudden change that it should be noted that appearance has been given the pharmaceutically-active relevant antagonism of kinases inhibitor mutain, has kept the kinase whose original substrate specificity of mutain to a certain degree simultaneously.Before the work of Gorre etc., those skilled in the art it is generally acknowledged can be at the compound of contact inhibition Abelson protein kinase, among the patient such as STI-571 observed resistance type may because of in other mechanism of said medicine resistance one or more or by some other still uncomprehending mechanism causes, but under any circumstance, described resistance all can relate to the target (protein or other) that is different from drug targets POI.

Therefore, treat clinical relevant also may be extremely useful for the protein mutant form of existing therapy target.This class mutain (as the theramuteins of definition hereinafter) generally acknowledged and is being interpreted as important target in the relapsed cancer, and becomes in other disease and have importance.Existence is to the demand of therapeutical agent, this class therapeutical agent to may be before general active drug therapy, in the process or this class drug resistance variant form of the cell protein that produces afterwards have activity.A crucial purpose of the present invention is to provide a kind of general method, the biological activity spectrum that those skilled in the art can utilize described method to identify from high flux screening (HTS) system to hit, produce and optimize lead compound and characterize described compound, and not needing to depend on older method, for example acellular radioligand is in conjunction with test etc.Another crucial purpose of the present invention is to provide can be with acting on the compound that overcomes the chemical sproof potential therapeutical agent of sudden change-inductive in the protein that endogenous occurs.

Summary of the invention

Method described herein relates to based on the drug discovery of cell response and the generation of production system, and the utilization of described system defines, the adjusting (being called phenotypic response) of predetermined cell characteristic is measured the ability that appointed compound (chemical reagent, conditioning agent) activated or suppressed selected target protein as instrument.By this method of repeated application, can utilize method described herein to come identification of protein conditioning agent (as herein defined), on described conditioning agent, carry out the guide and optimize, and biological target protein specificity and the selectivity that characterizes described conditioning agent.

Can use invention described herein to any target protein and any eukaryotic cell type, yet condition is at first to identify and instruction utilizes the present invention to be called a fundamental element of phenotypic response according to this paper.An embodiment of described method provides the inhibitor of the selected target protein of discriminating or the ability of promoting agent to those skilled in the art.Another embodiment permission those skilled in the art carry out quick guide and optimize research to obtain the clinical candidate compound of potential.Another embodiment to those skilled in the art provide design have to specified target protein have the desirability specificity and to described protein with respect to different but closely related target protein family member has the optionally ability of compound, described target protein family member may exist with some target.

The improvement of compound (comprising the medicine of having ratified) therapeutic efficiency is important problem that repeats in the study of pharmacy.Usually a method that adopts is from known chemical structure, then described structure is carried out other chemically modifieds to improve its effectiveness, specificity (at target protein) or other parameter relevant with the patient treatment effect.In some cases, initial structure can be a known drug.Can be to use acellular in other cases simply or hit based on the initial screening that the primary cell shaker test is identified.In other cases, described compound can be based on that screening is hit or the initial chemical structure with bottom line definition of other model structure, and is commonly referred to " skeleton ".With regard to the object of the invention, skeletal definition is for to remove one or more side chains or cyclosubstituted chemical structure with respect to representative compounds, and it has identical skeleton to described representative compounds in other respects.For example, the 3rd compound can be thought skeleton in the table 4.

The use that important contribution of the present invention is a phenotypic response, and determine first compound with respect to the cell-specific of second compound so that determine whether first compound shows improved cell-specific with respect to second compound.The described method representative of report first is to a basic progress of prior art in the invention described herein.Prior art depends on acellular test macro, its protein that utilizes purifying or reorganization to produce comes the test compounds activity, and relatively appointed compound to target protein with to other proteinic effects, described other protein common with target protein (closely or not close) are relevant.Can find the example of many these class art methods in the literature, comprise people such as Hanke, 1996, people such as Warmuth, US2003/0162222 A1, Knight and Shokat, 2005 and reference wherein.Aspect identifying and optimizing the cell-specific and therapeutic efficiency of specifying skeleton, the described acellular method of old type is compared with the present invention, and it is much lower or invalid fully that efficient is wanted.Substantially improved of the present invention is from least three key elements.

First, the notion of phenotypic response, when using, provide that allow to identify may be with the system of more effective mode on improved, the function and the interactional compound of target protein with the cell-specific of measuring appointed compound (for example by determining that its CSG measures).

Second, the invention provides the method for authenticating compound, described compound can also interact with other cellular components that is different from target protein, and (it includes but not limited to the upstream or the downstream component of signal transduction path, described signal transduction path relates to target protein for example list or oligomeric protein, protein complex, protein/nucleic acid complexes etc.), it works in the specific signals pathway or the periphery of the cell pathway that works in cell of target protein therein, with the morbid state that promotes to pay close attention to, for example selected human cancer form.Because the complicacy of the signal transduction cascade that exists in more senior orderly organism (for example people) cell, the prior art of current state can not be understood all mechanism that work about specified target protein wherein fully in cell.

The 3rd, the present invention has got rid of the compound with other non-target protein cross reactions, and described non-target protein does not participate in the based signal pathway of the morbid state that works for target protein wherein.The present invention gets rid of the direct compare test of the ability of described compound (it will produce disadvantageous side effect to the patient) from the cell-specific of the compound that uses phenotypic response, and it has fundamentally got rid of the effect of control cells.Cause the cell-specific that reduces if compare the effect of nominative testing compound with reference compound, can get rid of this compound immediately so.Whether test compounds is lower to the effect of target protein, or whether test compounds does not participate in non-target protein interaction (described signal transduction path is regulated the phenotypic response relevant with target protein) of the signal transduction path of target protein with other, or whether test compounds is just Cytotoxic, and these all are incoherent and are for academic interest.Test compounds treatment validity that crucial a bit is is lower and as further considering.This has saved the time and efforts of estimating various chemical structures for the art technology researchist.For the reader importantly, identification may at the target in the acellular test macro very effective force and height compounds effective may show that low relatively CSG measures and therefore may be by very fast eliminating, thereby saved time and previous resource.

The aforesaid key advantage of the present invention is not found in the prior art, and the improvement in essence of the relative prior art of the present invention is provided.These advantages are applied to all potential treatment target protein, but difficult, highly drug-fast target protein (being known as theramuteins) is (WO 2005/115992) that is even more important.

As using result of the present invention, simplified and improved the problem of improving and optimize appointed compound (with respect to the lower compound of other efficient) greatly.Those skilled in the art are simply since first compound, and no matter whether it is that medicine, the screening of approval hit or paid close attention to proteinic basic framework for known inhibition or activation, and uses the starting point of first compound as the reference purpose.Then, use synthetic other compounds of essential drugs chemical synthesis process of standard in present this area, (this paper are also referred to as " initial compounds " or " reference compound ") such as the analogue that described other compounds are first compound, homologue, isomer.At other partial references of this paper these chemical synthesis process of part, and the reader can also be with reference to people such as Burbaum, 1995 and people such as Goodnow, 2003 general references as described method.In case synthesized other compounds, those skilled in the art bring into use method of the present invention rather than are often referred to the result's's (with trial described compound and other proteinic cross reactions being minimized target protein and a collection of other non-target protein by the test new compound) who is obtained by acellular test art methods.On the contrary, the application of the invention, those skilled in the art can be instructed the improvement of initial compounds chemical structure by direct reference based on the result of the CGS acquisition of each testing compound of cell tests systems measurement of phenotypic response of the present invention by use.Most important ground, integral body has been got rid of and has been continued to depend on result acellular, the protein purification test, comprise above-mentioned in people such as Hanke (1996) and Knight and Shokat (2005) " kinasepanel " of reference, and be better than by compound than the out-of-date methods acquisition from the compound of implementing present method, shown in the activity of this paper compounds identified, it is effective (as shown in table 4) to highly drug-fast theramuteinp210 Bcr-Abl T315I.Any compound that does not limit those skilled in the art's independent test gained in cell free system is in order to independent affirmation (if necessary), but this never needs for implementing the present invention.

Before the present invention, the drug discovery system based on cell response of not confirming as yet can identify and the inhibitor or the promoting agent of selected target protein sorted, and need not combine with target protein really to establish the compound of studying in conjunction with test or enzyme test (when target protein is enzyme) with reference to acellular, purifying protein part earlier.

These results confirm first, use the test macro based on cell response to be evaluation proteinic inhibitor of specified target or promoting agent in the positive compound as main tool from scoring high flux screening (HTS).In case these results confirm that also evaluation is hit or lead compound can activate or suppress specified target protein (by any method, comprise embodiment disclosed herein or pass through classical acellular HTS method), described compound also can be that (that is, can carry out the guide to described compound optimizes) that chemistry is optimized adopts fully based on the cell tests system of phenotypic response and need not depend on inhibition or the activation capacity of acellular protein purification test macro with individual authentication/affirmation each serialization compound of synthetic in guide's optimizing process subsequently.Only this embodiment to those skilled in the art saved a large amount of time, energy and a large amount of lab resources, these are adopting classical acellular protein purification test, radioligand in conjunction with generations such as test with independently confirm to suppress or activation character is to spend usually.

The method that this paper confirms is used development and the ongoing proteinic specific mutant form that causes cancer that relates to chronic lymphocytic leukemia (CML).Described albumen is called the Abelson protein kinase, causes that with it form of cancer is a for example known target of imatinib mesylate (imatinib) of some tyrosine kinase inhibitor.Yet as discussed in more detail below, described target protein can have the mutant form of resistance to produce with the restraining effect to imatinib in the patient.The kinase whose described form of Abelson is called theramutein.In one embodiment of the invention, identified the suitable lead compound that can suppress or activate appointment theramutein.In another embodiment of the invention, optimize lead compound.Described method is effective to the bio-identification that evaluation is hit, the described guide that hits optimizes (no matter being how to have identified described hitting at first) and relates to the compound of non-theramutein endogenous target protein.The theramutein that use is made up of the implicit kinase whose mutant form of Abelson of giving highly drug-fast T315I sudden change has confirmed the general use of described method.

The invention further relates to and be the inhibitor of proteinic variant form or the promoting agent of activator.The invention still further relates to and be the inhibitor of some variant form of endogenous protein or the promoting agent of activator.Special concern be inhibitor and activator by the endogenous protein variant of the genes encoding that has suddenlyd change, described variant produces after contact is known as the chemical active agent of the inhibitor of the corresponding endogenous protein that do not suddenly change or activator usually or at first obtains at least identifying after producing.This proteinoid variant (mutain) is called ＂ theramuteins ＂ in this article, and they can be spontaneous occur (and being pre-existing in sudden change in some cases) or described mutant can be used as the selective pressure that use produces when the appointment chemical active agent of the not mutant form that can suppress described theramutein (this paper is called " prototheramutein ") is treated organism result's generation in vivo.Be appreciated that in some cases prototheramutein can be " the wild-type ＂ form (for example producing the protein of disease because of imbalance) of POI.In other situation, prototheramutein is " wild-type " the proteinic variant that causes disease, and it has suddenlyd change and has impelled the disease state as the described result that formerly suddenlys change to take place thus.The back one type example of Prototheramutein is P210 ^BCR-ABLCancer protein, and implicit Threonine (T) is called P210 to this proteinic mutant forms that Isoleucine (I) suddenlys change on 315 ^{BCR-ABL-T315I}, and be the example of theramutein.Name " P210 used herein ^BCR-ABL" with term " p210 ^Bcr-Abl", " wild-type Bcr-Abl protein " etc. be synonym.

Theramuteins is the rare endogenous protein of a class, and they have implied and have given the sudden change of described protein to the resistance of medicine, and known described medicine suppresses or activate their the corresponding body of not sudden change with the treatment effective means.The endogenous gene of a few this proteinoid of known coded shows this class sudden change in some cases at present.In one embodiment, the present invention relates to suppress the composition of proteinic some the resistance mutant (theramuteins) of Abelson Tyrosylprotein kinase, described Abelson tyrosine-kinase zymoprotein is called P210-Bcr-Abl in the literature at first, and it relates to the generation of chronic granulocytic leukemia.

Method of the present invention is particularly related to the specific inhibitor of identification of protein or specific, activated dose method.Term " specificity " application (definition in vide infra) in term " inhibitor " or " activator " in context refers to the cell function of described inhibitor or activator conjugated protein and inhibition or activation of protein, but debond and activation or suppress various other protein or non--protein target in the cell.Those skilled in the art fully understand, and the time spent of doing of protein inhibitor or activator is discussed in medical literature, in the mutability that exists aspect the related notion of the notion of specific inhibitor or specific, activated dose and target protein " specificity " to a certain degree.Therefore, with regard to purpose of the present invention, material is the specific inhibitor of specifying theramutein or specific, activated dose, condition is that described material can or activate described protein with the prescribed concentration inhibition, make that corresponding phenotypic response (phenoresponse) is adjusted in a suitable manner, and the phenotypic response (if any) to corresponding control cells does not have appreciable effect under identical prescribed concentration, and described corresponding control cells is marking protein not basically.

In certain embodiments, material can be the conditioning agent of two closely-related protein such as one of prototheramutein and its corresponding theramutein.In other embodiments, except that being the conditioning agent of prototheramutein and theramutein, material can also be regulated the activity of proteins with identity function.As mentioned above, remove inhibition p210 ^{Bcr Abl}Outside the Tyrosylprotein kinase, the PDGF beta receptor (also being Tyrosylprotein kinase) that imatinib mesylate can also be suppressed at the c-kit oncoprotein (also being Tyrosylprotein kinase) of overexpression in some gastrointestinal stromal tumor and express in some chronic myelomonocytic leukemia (CMML).This compounds is called " appropriate specificity " inhibitor sometimes.

The present invention also provides the general method that can be used to identify activation or suppress the material of theramutein, described material with theramutein activation or be suppressed to same degree and preferably in addition greater than the degree (yet those skilled in the art fully understand the described ＂ wild-type ＂ form of this proteinoid and suddenly change in the process of the corresponding disease that produces described protein participation) of the known drug material that can suppress this proteinic corresponding ＂ wild-type ＂ form.

In a preferred embodiment, the invention provides P210 with general formula I ^{BCR-ABL-T315I}The inhibitor of theramutein:

Wherein:

Ring A is 5-, 6-or 7-unit ring or 7-to 12-unit condensed-bicyclic;

X ¹Be selected from N, N-R ⁰Or C-R ¹

X ²Be selected from N, N-R ⁰Or C-R ¹

Dotted line is represented two keys of choosing wantonly;

Each R ¹Be independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF ₃, NO ₂, OR ¹¹,-(CH ₂) _pC (O) (CH ₂) _qR ¹¹,-(CH ₂) _pC (O) N (R ¹²) (R ¹³) ,-(CH ₂) _pC (O) O (CH ₂) _qR ¹¹,-(CH ₂) _pN (R ¹¹) (CH ₂) _qC (O) R ¹¹,-(CH ₂) _pN (R ¹²) (R ¹³) ,-(CH ₂) _pN (R ¹¹) (CH ₂) _qR ¹¹,-N (R ¹¹) SO ₂R ¹¹,-OC (O) N (R ¹²) (R ¹³) ,-SO ₂N (R ¹²) (R ¹³), the group formed of halogen, aryl and heterocycle, and in addition or alternatively, two R on the adjacent ring atom ¹Group forms and contains 0-3 heteroatomic 5-or 6-unit fused rings;

N is 0-6;

Each R ¹¹Be independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl and heterocycle;

Each R ¹²And R ¹³Be independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl and heterocycle; Or R ¹²And R ¹³Can form with the nitrogen that they connected and randomly to contain a first ring of extra heteroatomic 5-to 7-; Wherein 5-to 7-unit ring can be chosen wantonly by 1 to 3 and independently be selected from alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF ₃, NO ₂, OR ⁰, CO ₂R ⁰, C (O) R ⁰, halogen, aryl and heterocyclic substituting group replace;

P is 0-4;

Q is 0-4;

R ²Be selected from-CR ²¹ _a-,-NR ²² _b-and-(C=R ²³)-;

Each R ²¹Be independently selected from H, halogen ,-NH ₂,-N (H) (C _1-3Alkyl) ,-N (C _1-3Alkyl) ₂,-O-(C _1-3Alkyl), OH and C _1-3Alkyl;

Each R ²²Be independently selected from H and C _1-3Alkyl;

R ²³Be selected from O, S, N-R ⁰And N-OR ⁰

R ³Be selected from-CR ³¹ _c-,-NR ³² _d-,-SO ₂-and-(C=R ³³)-;

Each R ³¹Group be selected from H, halogen ,-NH ₂,-N (H) (R ⁰) ,-N (R ⁰) ₂,-O-R ⁰, OH and C _1-3Alkyl;

Each R ³²Group is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CO ₂R ⁰, C (O) R ⁰, aryl and heterocycle;

R ³³Be selected from O, S, N-R ³⁴And N-OR ⁰

R ³⁴Be selected from H, NO ₂, CN, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl and heterocycle;

R ⁴Be selected from-CR ⁴¹ _e-,-NR ⁴² _f-,-(C=R ⁴³)-,-SO ₂-and-O-;

Each R ⁴¹Be selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, CO ₂R ⁰, C (O) R ⁰, aralkyl, aryl and heterocycle;

Each R ⁴²Group is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CO ₂R ⁰, C (O) R ⁰, aryl and heterocycle;

Each R ⁴³Be selected from O, S, N-R separately ⁰And N-OR ⁰

Condition is to work as R ²For-NR ²² _b-and R ⁴For-NR ⁴² _f-time, R ³Be not-NR ³² _d-; R ³And R ⁴Be selected from respectively-(C=R when different ³³)-and-(C=R ⁴³)-; And R ³And R ⁴Be not selected from simultaneously-SO ₂-;

R ⁵Be selected from-Y-R ⁶With-Z-R ⁷

Y is selected from chemical bond, O, NR ⁰

R ⁶Be selected from alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl and heterocycle;

Z is for having 1-4 carbon atom and randomly by halogen, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CO ₂R ⁰, C (O) R ⁰, C (O) N (R ⁰) ₂, CN, CF ₃, N (R ⁰) ₂, NO ₂And OR ⁰In the hydrocarbon chain of one or more replacements;

R ⁷For H or be selected from aryl and heterocycle;

Each R ⁰Be independently selected from H, alkyl, cycloalkyl, aralkyl, aryl and heterocycle;

A is 1 or 2;

B is 0 or 1;

C is 1 or 2;

D is 0 or 1;

E is 1 or 2; And

F is 0 or 1.

The invention provides the new mode in basis of treatment cancer and other disease, wherein no matter by which kind of mechanism, all follow the drug resistance that appraisable (clinical significant) theramutein-mediates after using existing medical compounds treatment, by being provided at that theramuteins produces and (Wakai etc. when identifying like this, 2004 have reported an example, and wherein theramutein can produce in continued treatment scheme process) or before the clinical significance colony hypertrophy of the cell of expressing theramutein, give drug candidate in advance and carry out.In addition, if the pharmacological agent validity in the individual subgroup of proteinic certain theramutein that expresses drug targeting to specified disease is lower, the present invention can be by providing the treatment that effectively adapts to those experimenters at the drug candidate material of described theramutein so.

The invention provides measure chemical active agent as the conditioning agent of theramutein in the cell whether at least with as the same effective means of the material of known corresponding prototheramutein conditioning agent.An embodiment of this method comprises makes the control cells of expressing prototheramutein and can showing reactive phenotypic characteristic (relevant with the function of prototheramutein in the cell) contact with known prototheramutein conditioning agent, the test cell of expressing theramutein and also can showing reactive phenotypic characteristic (relevant with the function of theramutein in the cell) is contacted with chemical active agent, and the reaction of the control cells of the reaction of compare test cell and processing; So that determine that this chemical active agent is effective equally with the material as known prototheramutein conditioning agent at least as the conditioning agent of theramutein.In some other embodiment, one type control cells may not expressed prototheramutein fully.In other embodiments, the amount of the theramutein that expresses of the amount of the control cells prototheramutein that can express and test cell is identical substantially.In other embodiments, under certain conditions, the degree of the reactive phenotypic characteristic that control cells can show is identical substantially with test cell.In other embodiments, test cell can be expressed appointment protein, and described protein seldom or is not basically expressed in the control cells expression.

The protein of special concern of the present invention relates to the Theramuteins of regulatory function for those, such as enzyme; Protein kinase; Tyrosylprotein kinase; Receptor tyrosine kinase; Serine threonine protein kinase; Two-way specificity protein kinase; Proteolytic enzyme; Matrix metalloproteinase; Phosphoric acid esterase; Cell cycle control albumen; Docking protein is such as the IRS family member; Cell surface receptor; G-albumen; Ionic channel; DNA-and RNA-are conjugated protein; Polysaccharase etc.Do not plan to place restrictions on and to be used for theramutein of the present invention or other proteinic types.Simultaneously, known three kinds of theramuteins:BCR-ABL, c-Kit and EGFR.

May (comprise with the protein that exists in the cell, for example theramutein or prototheramutein) relevant any reactive phenotypic characteristic can be used for present method, comprise: the temporary transient feature of the cell of phosphorylation state of growth or cultural characters, theramutein substrate (or other modify) and any type for example, as define and go through.

Accompanying drawing is described

Accompanying drawing 1 has shown that the compound 2 (C2) of different concns is to unconverted vehicle Control Ba/F3 cell (for the IL-3 dependency) and expression ＂ wild-type ＂ p210 ^Bcr-AblBa/F3 cell (called after p210 ^Bcr-Abl-wt) and express p210 ^{Bcr-Abl-T315I}The growth of the Ba/F3 cell of drug resistance mutant strain and the effect of survival rate.Measure cell counting and survival rate as what describe in detail in this specification sheets with the automatic cytological counter.Cell counting is represented by solid color bar; Cell survival rate is represented by dashed bars.Notice that STI-571 effectively suppresses P210 cell line growth (grey bar), and even under 10 μ M concentration, can not suppress T315I cell line growth (white bars).500nM C2 shows maximum specificity breach in this dose response series scope.The C2 of the STI-571 of 10 μ M and 500nM is compared (white bars) to the effect of T315I clone.Abbreviation: DMSO: methyl-sulphoxide (solvent that is used for medicine dissolution).

Accompanying drawing 2 has shown that the compound 6 (C6) of different concns is to unconverted vehicle Control Ba/F3 cell and expression p210 ^{Bcr-Abl-T315I}The growth of the Ba/F3 cell of drug resistance mutant strain and the effect of survival rate.All are described in detail all as institute in the accompanying drawing 1.

Accompanying drawing 3 has shown by the different compounds identified in the screening relatively with regard to its different measurement results to the specificity breach that obtains with regard to the effect in the clone of expressing prototheramutein and theramutein.Compound 3 (C3) has shown the preferred example of the ability that is used for authenticating compound, and this compound is tackled the effect of prototheramutein mutually to effect that theramutein applied even greater than it.(E group) .A group: control group DMSO treatment; B: negative allos specificity breach; C: slight male allos specificity breach; D: remarkable male homology specificity breach; E: positive allos specificity breach.Referring to the text that is used to explain.A group: control group DMSO treatment; B: negative allos specificity breach; C: slight male allos specificity breach; D: remarkable male homology specificity breach; Referring to explaining part.

Accompanying drawing 4 has shown and has been used for the active reorganization P210 Bcr-Abl wild-type that detects of autophosphorylation and the radioautogram of T315I mutant kinase domain.200ng protein in advance with test substances incubation 10 minutes under standard autophosphorylation reaction conditions, is added radiolabeled ATP then and makes to be reflected at and carried out under 30 ℃ 30 minutes, after this by the SDS-PAGE sample separation.Gel is carried out silver dyeing, dry in a vacuum and contact X-mating plate.Attention is in the 10 μ MSTI effective while of 571 couples of wild-type P210 Bcr-Abl, and it is in fact in concentration even invalid to the T315I kinase domain when reaching 100 μ M." P210 clone " refers to and expresses p210 ^BCR-ABL-wtCell." T315I clone " refers to and expresses p210 ^{BCR-ABL-T315I}Cell.

The chemical structure of the representational compound of accompanying drawing 5 expression the present invention.

The chemical structure of the representational compound of accompanying drawing 6 expression the present invention.

The chemical structure of the representational compound of accompanying drawing 7 expression the present invention.

The chemical structure of the representational compound of accompanying drawing 8 expression the present invention.

The chemical structure of the representational compound of accompanying drawing 9 expression the present invention.

The chemical structure of the representational compound of accompanying drawing 10 expression the present invention.

The chemical structure of the representational compound of accompanying drawing 11 expression the present invention.

The chemical structure of the representational compound of accompanying drawing 12 expression the present invention.

The chemical structure of the representational compound of accompanying drawing 13 expression the present invention.

Accompanying drawing 14 expression has the restraining effect of the supposition compound of 1 cell-specific breach to growth velocity for test cell and control cells.

Accompanying drawing 15 expression has the restraining effect of the supposition compound of 40 cell-specific breach to growth velocity for test cell and control cells.

Accompanying drawing 16 is illustrated in and significantly is lower than Cytotoxic apparent IC ₅₀Concentration under the growth-inhibiting effect of imatinib mesylate.

The C2 of accompanying drawing 17 expression different concns and various C2 analogue are to expressing p210 ^{Bcr-Abl-T315I}The effect of the growth of the Ba/F3 cell of resistance mutant.

There are down the standard acellular albumen kinases autophosphorylation measurement result of T3151 mutant kinase structural domain in C2 and various C2 analogue that accompanying drawing 18 is illustrated in 20 μ M concentration.

Detailed Description Of The Invention

Term used herein " halogen (halo) " or " halogen (halogen) " comprise Fluorine, chlorine, bromine and iodine.

Term used herein " alkyl " is paid close attention to is not with the replacement of 1-6 carbon atom and not Substituted straight chain and branched alkyl. Preferred alkyl comprises methyl, ethyl, propyl group, isopropyl Base, butyl, isobutyl group, the tert-butyl group etc. In addition, alkyl can be randomly by one or more Be selected from halogen, CN, CO₂R、C(O)R、C(O)NR ₂、NR ₂, ring-amino, NO₂With getting of OR Generation base replacement.

What term used herein " cycloalkyl " was paid close attention to is to replace and unsubstituted cycloalkyl. Preferred cycloalkyl contains the monocycle of 3-7 carbon atom for those, and comprises cyclopropyl, ring penta Base, cyclohexyl etc. Other cycloalkyl can be selected from C₇-C ₁₀Bicyclic ring system or C₉-C ₁₄Three ring systems. In addition Outward, cycloalkyl can be randomly by one or more halogen, CN, CO of being selected from₂R、C(O)R、 C(O)NR ₂、NR ₂, ring-amino, NO₂Replace with the substituting group of OR.

Term used herein " alkenyl " is paid close attention to is to replace and unsubstituted straight chain and propping up Alkenyl group. Preferred alkenyl contains the alkenyl of 2-6 carbon atom for those. Other alkene Base can be randomly by one or more halogen, CN, CO of being selected from₂R、C(O)R、C(O)NR ₂、NR ₂, ring-amino, NO₂Replace with the substituting group of OR.

What term used herein " alkynyl " was paid close attention to is to replace and unsubstituted straight chain and side chain Alkynyl. Preferred alkynyl contains the alkynyl of 2-6 carbon atom for those. In addition, alkynyl can be appointed Selection of land is by one or more halogen, CN, CO of being selected from₂R、C(O)R、C(O)NR ₂、NR ₂, ring-amino, NO₂Replace with the substituting group of OR.

What term used herein " aralkyl " was paid close attention to is as substituent with aromatic group Alkyl, described aromatic group can be substituted and not be substituted. Aralkyl can randomly exist Replaced by one or more substituting groups on the aryl, described substituting group is selected from halogen, CN, CF₃、 NR ₂, ring-amino, NO₂、OR、CF ₃、-(CH ₂) _xR、-(CH ₂) _xC(O)(CH ₂)yR、 -(CH ₂) _xC(O)N(R′)(R＂)、-(CH ₂) _xC(O)O(CH ₂) _yR、-(CH ₂) _xN(R′)(R＂)、 -N(R)SO ₂R、-O(CH ₂) _xC(O)N(R′)(R＂)、-SO ₂N(R′)(R＂)、 -(CH ₂) _xN(R)-(CH ₂) _y-R、-(CH ₂) _xN(R)-C(O)-(CH ₂) _y-R、 -(CH ₂) _xN(R)-C(O)-O-(CH ₂) _y-R、-(CH ₂) _x-C(O)-N(R)-(CH ₂) _y-R、 -(CH ₂) _xC(O)N(R)-(CH ₂) _y-R、-O-(CH ₂) _x-C(O)-N(R)-(CH ₂) _y-R, replacement and unsubstituted alkyl, replacement and unsubstituted cycloalkyl, replacement and unsubstituted aralkyl, replacement and unsubstituted alkenyl, replacement and unsubstituted alkynyl, replacement and unsubstituted aryl and replacement and unsubstituted heterocycle, the heterocycle of the alkynyl of the aralkyl of the alkyl of wherein said replacement, the cycloalkyl of replacement, replacement, the alkenyl of replacement, replacement, the aryl of replacement and replacement can be by halogen, CN, CF₃、CO ₂R、C(O)R、C(O)NR ₂、NR ₂, ring-amino, NO₂With the one or more replacements among the OR.

What term used herein " heterocyclic radical " or ＂ heterocycle ＂ paid close attention to is assorted former with at least one Son is as aromatics and the non--aromatics cyclic group of ring members. Preferred heterocyclic radical contains 5 for those Or 6 annular atomses comprise at least one heteroatomic heterocyclic radical, and comprise: cyclic amine, Such as morpholino, piperidino, pyrrolidines and etc.; With the cyclic ethers class, such as oxolane, four Hydrogen pyrans etc. Aromatic heterocyclic radical, what be also referred to as " heteroaryl " concern is to comprise 1-3 Heteroatomic list-ring is assorted-aromatic group, for example pyrroles, furans, thiophene, imidazoles, oxazole, Thiazole, triazole, pyrazoles, pyridine, pyrazine, pyridazine, pyrimidine etc. Term used herein is assorted Aryl also comprises with many rings of two or more rings mixes-aromatic systems, and two atoms wherein are Two adjacent rings share (these rings are ＂ that ＂ condenses), and wherein at least one in the ring is assorted virtue Base, for example other ring can be cycloalkyl, cycloalkenyl group, aryl, heterocycle and/or heteroaryl. Many The example of ring heteroaromatic system comprise quinoline, isoquinolin, tetrahydroisoquinoline, quinoxaline, Quinaxoline, benzimidazole, benzofuran, purine, imidazopyridine, BTA etc. In addition, heterocyclic radical can randomly be replaced by following group: halogen, CN, CF₃、NR ₂, ring-amino, NO₂、OR、CF ₃、-(CH ₂) _xC(O)(CH ₂) _yR、-(CH ₂) _xC(O)N(R′)(R＂)、 -(CH ₂) _xC(O)O(CH ₂) _yR、-(CH ₂) _xN(R′)(R＂)、-N(R)SO ₂R、-O(CH ₂) _xC(O)N(R′) (R＂)、-SO ₂N(R′)(R＂)、-(CH ₂) _xN(R)-(CH ₂) _y-R、-(CH ₂) _xN(R)-C(O)-(CH ₂) _y-R、-(CH ₂) _xN(R)-C(O)-O-(CH ₂) _y-R、-(CH ₂) _x-C(O)-N(R)-(CH ₂) _y-R、 -(CH ₂) _xC(O)N(R)-(CH ₂) _y-R、-O-(CH ₂) _x-C(O)-N(R)-(CH ₂) _y-R, replace and unsubstituted alkyl, replacement and unsubstituted cycloalkyl, replacement and unsubstituted aralkyl, replacement and unsubstituted alkenyl, replacement and unsubstituted alkynyl, replacement and unsubstituted aryl and replacement and unsubstituted heterocycle, the heterocycle of the alkynyl of the aralkyl of the alkyl of wherein said replacement, the cycloalkyl of replacement, replacement, the alkenyl of replacement, replacement, the aryl of replacement and replacement can be by halogen, CN, CF₃、CO ₂R、C(O)R、C(O)NR ₂、NR ₂, ring-amino, NO₂With the one or more replacements among the OR.

What term used herein " ring-amino " was paid close attention to is with at least one nitrogen-atoms conduct The aromatics of ring members and non--aromatics cyclic group. Preferred ring is amino to contain 5 or 6 rings for those The ring amino that comprises at least one nitrogen-atoms of atom, and comprising: morpholino, piperidino, Pyrrolidines also, piperazine also, imidazoles, oxazole, thiazole, triazole, pyrazoles, pyridine, pyrazine, Pyridazine, pyrimidine etc. In addition, ring-amino can be chosen wantonly by halogen, CN, CF₃、NR ₂、NO ₂、 OR、CF ₃, replacement and unsubstituted alkyl, replacement and unsubstituted cycloalkyl, replacement and unsubstituted aralkyl, replacement and unsubstituted alkenyl, replacement and unsubstituted alkynyl, replacement and unsubstituted aryl and replacement and unsubstituted heterocyclic substituted, the heterocycle of the alkynyl of the aralkyl of the alkyl that wherein replaces, the cycloalkyl of replacement, replacement, the alkenyl of replacement, replacement, the aryl of replacement and replacement can be by one or more halogens, CN, CF₃、CO ₂R、 C(O)R、C(O)NR ₂、NR ₂、NO ₂Replace with OR.

What term used herein " aryl " or " aromatic group " were paid close attention to is single-ring aromatic group Group's (such as phenyl, pyridine radicals, pyrazolyl etc.) and many ring ring systems (naphthyl, quinoline etc.). Many rings Can be with two or more rings, two atoms wherein are that two adjacent rings share (these rings The ＂ that ＂ condenses), wherein at least one in the ring is aromatic ring, for example other ring can be cycloalkanes Base, cycloalkenyl group, aryl, heterocycle and/or heteroaryl. In addition, aryl can be randomly by one Or a plurality of substituting groups replacements, described substituting group is selected from halogen, CN, CF₃、NR ₂, ring-amino, NO₂、OR、CF ₃、-(CH ₂) _xC(O)(CH ₂) _yR、-(CH ₂) _xC(O)N(R′)(R＂)、 -(CH ₂) _xC(O)O(CH ₂) _yR、-(CH ₂) _xN(R′)(R＂)、-N(R)SO ₂R、-O(CH ₂) _xC(O) N(R′)(R＂)、-SO ₂N(R′)(R＂)、-(CH ₂) _xN(R)-(CH ₂) _y-R、-(CH ₂) _xN(R)-C (O)-(CH ₂) _y-R、-(CH ₂) _xN(R)-C(O)-O-(CH ₂) _y-R、-(CH ₂) _x-C(O)-N(R)- (CH ₂) _y-R、-(CH ₂) _xC(O)N(R)-(CH ₂) _y-R、-O-(CH ₂) _x-C(O)-N(R)-(CH ₂) _y-R, replacement and unsubstituted alkyl, replacement and unsubstituted cycloalkyl, replacement and unsubstituted aralkyl, replacement and unsubstituted alkenyl, replacement and unsubstituted alkynyl, replacement and unsubstituted aryl and replacement and unsubstituted heterocycle, the heterocycle of the alkynyl of the aralkyl of the alkyl of wherein said replacement, the cycloalkyl of replacement, replacement, the alkenyl of replacement, replacement, the aryl of replacement and replacement can be by halogen, CN, CF₃、CO ₂R、C(O)R、C(O)NR ₂、NR ₂, ring-amino, NO₂With the one or more replacements among the OR.

Term ＂ hetero atom ＂ used herein particularly refers to N, O and S as ring hetero atom.

Each R is independently selected from H, replacement and unsubstituted alkyl, replacement and unsubstituted cycloalkyl, replacement and unsubstituted aralkyl, replacement and unsubstituted aryl and replacement and unsubstituted heterocycle, and the heterocycle of the aralkyl of the alkyl of wherein said replacement, the cycloalkyl of replacement, replacement, the aryl of replacement and replacement can be by one or more halogens, CN, CF₃、OH、 CO ₂H、NO ₂、C _1-6Alkyl ,-O-(C_1-6Alkyl) ,-NH₂、-NH(C _1-6Alkyl) and-N (C_1-6Alkyl)₂Replace. Each R ' and R ＂ are independently selected from H or replacement and unsubstituted alkyl, replacement and not Substituted cycloalkyl, replacement and unsubstituted aralkyl, replacement and unsubstituted aryl With replacement and unsubstituted heterocycle, the alkyl of wherein said replacement, the cycloalkyl of replacement, get The heterocycle of the aralkyl in generation, the aryl of replacement and replacement can by one or more halogens, CN, CF₃、OH、CO ₂H、NO ₂、C _1-6Alkyl ,-O-(C_1-6Alkyl) ,-NH₂、-NH(C _1-6Alkyl) and-N (C_1-6Alkyl)₂Replace; Or R ' is connected the nitrogen that connects with them and is formed and can choose wantonly with R ＂ Ground contains at the most three first rings of extra heteroatomic 5-to 7-, and described hetero atom can be by C_1-6Alkyl Replace. Each x and each y are independently selected from 0-4.

In a preferred embodiment, the invention provides the P210 with general formula I^BCR-ABL-T315The inhibitor of Itheramutein:

Wherein:

Ring A is 5-, 6-or 7-unit ring or 7-to 12-unit condensed-bicyclic;

X ¹Be selected from N, N-R⁰Or C-R¹；

X ²Be selected from N, N-R⁰Or C-R¹；

Dotted line represents two keys of choosing wantonly;

Each R¹Be independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃、NO ₂、OR ¹¹、-(CH ₂) _pC(O)(CH ₂) _qR ¹¹、-(CH ₂) _pC(O)N(R ¹²)(R ¹³)、 -(CH ₂) _pC(O)O(CH ₂) _qR ¹¹、-(CH ₂) _pN(R ¹¹)(CH ₂) _qC(O)R ¹¹、-(CH ₂) _pN(R ¹²)(R ¹³)、 -N(R ¹¹)SO ₂R ¹¹、-OC(O)N(R ¹²)(R ¹³)、-SO ₂N(R ¹²)(R ¹³), the group that forms of halogen, aryl and heterocycle, and in addition or alternatively, two R on the adjacent ring atom¹Group forms and contains The individual heteroatomic 5-of 0-3 or 6-unit fused rings;

N is 0-6;

Each R¹¹Be independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, Aryl and heterocycle;

Each R¹²And R¹³Be independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, virtue Alkyl, aryl and heterocycle; Or R¹²And R¹³Can form with the nitrogen that they connect passable Randomly contain a first ring of extra heteroatomic 5-to 7-; Wherein 5-to 7-unit ring can be appointed Choosing by 1 to 3 independently be selected from alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF₃、NO ₂、OR ⁰、CO ₂R ⁰、C(O)R ⁰, halogen, aryl and heterocycle substituting group replace;

P is 0-4;

Q is 0-4;

R ²Be selected from-CR²¹ _a-、-NR ²² _b-and-(C=R²³)-；

Each R²¹Be independently selected from H, halogen ,-NH₂、-N(H)(C _1-3Alkyl) ,-N (C_1-3Alkyl)₂、-O-(C _1-3Alkyl), OH and C_1-3Alkyl;

Each R²²Be independently selected from H and C_1-3Alkyl;

R ²³Be selected from-O, S, N-R⁰And N-OR⁰；

R ³Be selected from-CR³¹ _c-、-NR ³² _d-、-SO ₂-and-(C=R³³)-；

R ³¹Group be selected from separately H, halogen ,-NH₂、-N(H)(R ⁰)、-N(R ⁰) ₂、-O-R ⁰, OH and C_1-3Alkyl;

R ³²Group is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CO separately₂R ⁰、 C(O)R ⁰, aryl and heterocycle;

R ³³Be selected from O, S, N-R³⁴And N-OR⁰；

R ³⁴Be selected from H, NO₂, CN, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, virtue Base and heterocycle;

R ⁴Be selected from-CR⁴¹ _e-、-NR ⁴² _f-、-(C＝R ⁴³)-、-SO ₂-and-O-;

Each R⁴¹Be selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, CO₂R ⁰、C(O)R ⁰, Aralkyl, aryl and heterocycle;

Each R⁴²Group is selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CO₂R ⁰、 C(O)R ⁰, aryl and heterocycle;

Each R⁴³Be selected from O, S, N-R⁰And N-OR⁰；

Condition is to work as R²For-NR²² _b-and R⁴For-NR⁴² _f-time, R³Be not-NR³² _d-；R ³And R⁴Be selected from respectively-(C=R when different³³)-and-(C=R⁴³)-; And R³And R⁴Be not selected from simultaneously-SO₂-；

R ⁵Be selected from-Y-R⁶With-Z-R⁷；

Y is selected from chemical bond, O, NR⁰；

Z is with 1-4 carbon atom and randomly by halogen, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CO₂R ⁰、C(O)R ⁰、C(O)N(R ⁰) ₂、CN、CF ₃、N(R ⁰) ²、NO ²And OR⁰In the hydrocarbon chain of one or more replacements;

R ⁷For H or be selected from aryl and heterocycle;

R ⁰Be selected from independently of one another H, alkyl, cycloalkyl, aralkyl, aryl and heterocycle;

A is 1 or 2;

B is 0 or 1;

C is 1 or 2;

D is 0 or 1;

E is 1 or 2; And

F is 0 or 1.

Important component part among the present invention as herein described and concept instruction are the R of The compounds of this invention²And R³The position is the member of non-any aromatics or non--aromatic ring structure all. We find band R is arranged²And/or R³The position is as the member's of aromatics or non--aromatic ring structure compound can't arbitrarily Effectively suppress T315I theramutein, and except having other preferred active group, The The compounds of this invention that lacks such ring composition on these positions is T315I theramutein The establishment agent.

In a preferred embodiment of the invention, ring A is aromatic ring.

In a preferred embodiment of the invention, X¹Or X²Be N. In another preferred side of enforcement In the case, X¹And X²Be N. In particularly preferred embodiment of the present invention, the ring A be pyridine ring or Pyrimidine ring. In a further preferred embodiment, the following structure that provides is provided ring A:

In a preferred embodiment of the invention, R⁵The group with following general formula:

Wherein:

X ³Be N or CH;

R ⁶¹Be selected from aryl and heterocycle;

Q be selected from chemical bond or have formula-O-,-(CH₂) _i-、-(CH ₂) _iC(O) (CH ₂) _j-、-(CH ₂) _i-N(R ⁶²)-(CH ₂) _j-、-(CH ₂) _iC(O)-N(R ⁶²)-(CH ₂) _j-、-(CH ₂) _iC(O)O(CH ₂) _j-、-(CH ₂) _iN(R ⁶²)C(O)-(CH ₂) _j-、-(CH ₂) _iOC(O)N(R ⁶²)-(CH ₂) _j-and-O-(CH₂) _i-C(O)N(R ⁶²)-(CH ₂) _j-group;

R ⁶²Be selected from H, alkyl, aryl and heterocycle;

H is 0 to 4;

I is 0 to 4; And

J is 0 to 4.

In further preferred embodiment of the present invention, R ⁵Be group with following general formula:

Wherein:

X ³Be N or CH;

Q ¹Be selected from chemical bond or have formula-O-,-CH ₂-,-NH-,-C (O)-NH-,-C (O) O-,-NH-C (O)-,-OC (O) NH-and-group of O-C (O) NH-;

Each R ⁷⁰Be selected from halogen, alkyl, CN, N (R ⁷¹) ₂, ring-amino, NO ₂, OR ⁷¹And CF ₃

Each R ⁷¹Be selected from H, alkyl, aryl, aralkyl and heterocycle; And k is 0 to 4.

Wherein:

X ³Be N or CH;

R ⁷⁰Be selected from halogen, alkyl, CN, N (R ⁷¹) ₂, ring-amino, NO ₂, OR ⁷¹And CF ₃And each R ⁷¹Be selected from H, alkyl, aryl, aralkyl and heterocycle.

In particularly preferred embodiments, made one or more following selections: Q ¹For-NH-; X ³Be N; Each R ⁷¹Be independently selected from H, methyl and ethyl, and each R ⁷¹Be preferably methyl; And/or R ⁷⁰Be selected from OH, OCH ₃, halogen and CF ₃

In a preferred embodiment, if R ²Or R ⁴Elected as respectively-NR ²² _b-or-NR ⁴²-, R so ³¹Be not selected from halogen ,-NH ₂,-N (H) (R ⁰) ,-N (R ⁰) ₂,-O-R ⁰Or OH.

In another preferred embodiment, the invention provides and have general formula I _aP210 ^{BCR-ABL-T315I}The inhibitor of theramutein:

Wherein:

Ring A is 5-, 6-or 7-unit ring or 7-to 12-unit condensed-bicyclic;

X ¹Be selected from N, N-R ⁰Or C-R ¹

X ²Be selected from N, N-R ⁰Or C-R ¹

Dotted line is represented two keys of choosing wantonly;

Each R ¹Be independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF ₃, NO ₂, OR ¹¹,-(CH ₂) _pC (O) (CH ₂) _qR ¹¹,-(CH ₂) _pC (O) N (R ¹²) (R ¹³) ,-(CH ₂) _pC (O) O (CH ₂) _qR ¹¹,-(CH ₂) _pN (R ¹¹) (CH ₂) _qC (O) R ¹¹,-(CH ₂) _pN (R ¹²) (R ¹³) ,-N (R ¹¹) SO ₂R ¹¹,-OC (O) N (R ¹²) (R ¹³) ,-SO ₂N (R ¹²) (R ¹³), the group formed of halogen, aryl and heterocycle, and in addition or alternatively, two R on the adjacent ring atom ¹Group forms and contains 0-3 heteroatomic 5-or 6-unit fused rings;

N is 0-6;

P is 0-4;

Q is 0-4;

Each R ²²Be independently selected from H and C _1-3Alkyl;

R ³Be selected from-CR ³¹ _c-,-NR ³² _d-,-SO ₂-and-(C=R ³³)-;

R ³³Be selected from O, S, N-R ³⁴And N-OR ⁰

R ⁴Be selected from-CR ⁴¹E-,-NR ⁴²F-,-(C=R ⁴³)-,-SO ₂-and-O-;

Each R ⁴³Be selected from O, S, N-R ⁰And N-OR ⁰

Condition is to work as R ⁴For-NR ⁴² _f-time, R ³Be not-NR ³² _d-; R ³And R ⁴Be selected from respectively-(C=R when different ³³)-and-(C=R ⁴³)-; And R ³And R ⁴Be not selected from simultaneously-SO ₂-;

R ⁵Be selected from-Y-R ⁶With-Z-R ⁷

Y is selected from chemical bond, O, N-R ⁰

R ⁷For H or be selected from aryl and heterocycle;

R ⁰Be selected from H, alkyl, cycloalkyl, aralkyl, aryl and heterocycle independently of one another;

A is 1 or 2;

B is 0 or 1;

C is 1 or 2;

D is 0 or 1;

E is 1 or 2; And

F is 0 or 1.

In another preferred embodiment, the invention provides and have general formula I _bP210 ^{BCR-ABL-T315I}The inhibitor of theramutein:

Wherein:

Ring A is 5-, 6-or 7-unit ring or 7-to 12-unit condensed-bicyclic;

X ¹Be selected from N, N-R ⁰Or C-R ¹

X ²Be selected from N, N-R ⁰Or C-R ¹

Dotted line is represented two keys of choosing wantonly;

R ¹Be selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF independently of one another ₃, NO ₂, OR ¹¹,-(CH ₂) _pC (O) (CH ₂) _qR ¹¹,-(CH ₂) _pC (O) N (R ¹²) (R ¹³) ,-(CH ₂) _pC (O) O (CH ₂) _qR ¹¹,-(CH ₂) _pN (R ¹¹) (CH ₂) _qC (O) R ¹¹,-(CH ₂) _pN (R ¹²) (R ¹³) ,-N (R ¹¹) SO ₂R ¹¹,-OC (O) N (R ¹²) (R ¹³) ,-SO ₂N (R ¹²) (R ¹³), the group formed of halogen, aryl and heterocycle, and in addition or alternatively, two R on the adjacent ring atom ¹Group forms and contains 0-3 heteroatomic 5-or 6-unit fused rings;

N is 0-6;

P is 0-4;

Q is 0-4;

Each R ²²Be selected from H and C independently of one another _1-3Alkyl;

R ⁴Be selected from-CR ⁴¹ _e-,-(C=R ⁴³)-,-SO ₂-and-O-;

R ⁴¹Be selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, CO separately ₂R ⁰, C (O) R ⁰, aralkyl, aryl and heterocycle;

Each R ⁴³Be selected from O, S, N-R ⁰And N-OR ⁰

R ⁵Be selected from-Y-R ⁶With-Z-R ⁷

Y is selected from chemical bond, O, N-R ⁰

R ⁷For H or be selected from aryl and heterocycle;

A is 1 or 2;

B is 0 or 1;

C is 1 or 2;

D is 0 or 1;

E is 1 or 2; And

F is 0 or 1.

In another preferred embodiment, the invention provides and have general formula I _cP210 ^{BCR-ABL-T315I}The inhibitor of theramutein:

Wherein:

Ring A is 5-, 6-or 7-unit ring or 7-to 12-unit condensed-bicyclic;

X ¹Be selected from N, N-R ⁰Or C-R ¹

X ²Be selected from N, N-R ⁰Or C-R ¹

Dotted line is represented two keys of choosing wantonly;

N is 0-6;

P is 0-4;

Q is 0-4;

X ³Be N, CH or C-R ²

Each R ²Be independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF ₃, NO ₂, OR ²¹,-(CH ₂) _rC (O) (CH ₂) _sR ²¹,-(CH ₂) _rC (O) N (R ²²) (R ²³) ,-(CH ₂) _rC (O) O (CH ₂) _sR ²¹,-(CH ₂) _rN (R ²¹) C (O) R ²¹,-(CH ₂) _rN (R ²²) (R ²³) ,-N (R ²¹) SO ₂R ²¹,-OC (O) N (R ²²) (R ²³) ,-SO ₂N (R ²²) (R ²³), the group formed of halogen, aryl and heterocycle, and in addition or alternatively, two R on the adjacent ring atom ²Group forms and contains 0-3 heteroatomic 5-or 6-unit fused rings;

R ²¹Be independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl and heterocycle;

R ²²And R ²¹Be independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl and heterocycle; Or R ²²And R ²¹Can form with the nitrogen that they connected and randomly to contain a first ring of extra heteroatomic 5-to 7-; Wherein 5-to 7-unit ring can be chosen wantonly by 1 to 3 and independently be selected from alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF ₃, NO ₂, OR ⁰, CO ₂R ⁰, C (O) R ⁰, halogen, aryl and heterocyclic substituting group replace;

R is 0 to 4;

S is 0 to 4;

M is 0 to 4;

R ⁴Be selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CO ₂R ⁰, C (O) R ⁰, aryl and heterocycle;

A is 0 or 1;

X ⁴Be selected from:

With

Each R ³Be independently selected from H, N (R ⁰) ₂, alkyl, cycloalkyl, alkenyl, alkynyl, CO ₂R ⁰, C (O) R ⁰, the group formed of aralkyl, aryl and heterocycle;

R ³' be selected from H, N (R ⁰) ₂, alkyl, cycloalkyl, aralkyl, aryl and heterocycle; And each R ⁰Be independently selected from H, alkyl, cycloalkyl, aralkyl, aryl and heterocycle.

In a preferred embodiment of the invention, select the R of general formula I ², R ³And R ⁴So that obtain following chemical group:

-N(R ²²)-N＝C(R ⁴¹)-

-N(R ²²)-N(R ³²)-C(＝O)-

-N(R ²²)-N(R ³²)-C(R ⁴¹)(R ⁴¹)-

-N(R ²²)-C(R ³¹)(R ³¹)-C(R ⁴¹)(R ⁴¹)-

-N(R ²²)-C(R ³¹)(R ³¹)-C(＝O)-

-N＝N-C(R ⁴¹)(R ⁴¹)-

-C(R ²¹)＝C＝C(R ⁴¹)-

-C(R ²¹)＝C(R ³¹)-C(＝O)-

-C(R ²¹)＝C(R ³¹)-C(R ⁴¹)(R ⁴¹)-

-C(R ²¹)(R ²¹)-C(R ³¹)＝C(R ⁴¹)-

-C(R ²¹)(R ²¹)-C(R ³¹)(R ³¹)-C(＝O)-

-C(R ²¹)(R ²¹)-C(R ³¹)(R ³¹)-C(R ⁴¹)(R ⁴¹)-

-C(R ²¹)(R ²¹)-N(R ³²)-C(＝O)-

-C(R ²¹)(R ²¹)-N(R ³²)-C(R ⁴¹)(R ⁴¹)-

-N(R ²²)-C(＝O)-C(R ⁴¹)(R ⁴¹)-

-N(R ²²)-C(＝O)-N(R ⁴¹)-

-N(R ²²)-C(＝O)-O-

-C(R ²¹)(R ²¹)-C(＝O)-C(R ⁴¹)(R ⁴¹)-

-C(R ²¹)(R ²¹)-C(＝O)-N(R ⁴²)-

-N(R ²²)-C(＝NR ³⁴)-N(R ⁴²)-

-C(＝O)-N(R ³²)-N(R ⁴²).

Be used for R ², R ³And R ⁴Particularly preferred chemical group comprise:

-N(R ²²)-N＝C(R ⁴¹)-

-N(R ²²)-N(R ³²)-C(＝O)-

-N(R ²²)-C(R ³¹)(R ³¹)-C(R ⁴¹)(R ⁴¹)-

-N(R ²²)-C(R ³¹)(R ³¹)-C(＝O)-

-C(R ²¹)(R ²¹)-C(＝O)-C(R ⁴¹)(R ⁴¹)-

-C(R ²¹)(R ²¹)-C(＝O)-N(R ⁴²)-

-N(R ²²)-C(＝NR ³⁴)-N(R ⁴²)-

-C(＝O)-N(R ³²)-N(R ⁴²)。

In another preferred embodiment, R ⁶Or R ⁷For choosing substituted aryl wantonly.Particularly preferred aryl comprises and replacing or unsubstituted phenyl and pyridyl.In extra or alternate embodiment, preferred substituents R ²¹And R ²²Be independently selected from group and preferably from H and CH with little spatial volume ₃, and more preferably H.

In another preferred embodiment, the invention provides P210 with general formula I I ^{BCR-ABL-T315I}The inhibitor of theramutein:

Wherein

Ring A is 5-, 6-or 7-unit ring or 7-to 12-unit condensed-bicyclic;

X ¹Be selected from N, N-R ⁰Or C-R ¹

X ²Be selected from N, N-R ⁰Or C-R ¹

Dotted line is represented two keys of choosing wantonly;

N is 0-6;

P is 0-4;

Q is 0-4;

R ⁸Be selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, CO ₂R ⁰, C (O) R ⁰, the group formed of aralkyl, aryl and heterocycle;

R ⁹Be selected from-Y-R ⁶With-Z-R ⁷

Y is selected from chemical bond, O, N-R ⁰

R ⁷For H or be selected from aryl and heterocycle; And

Each R ⁰Be independently selected from H, alkyl, cycloalkyl, aralkyl, aryl and heterocycle.

In another preferred embodiment, the invention provides and have general formula I I _aP210 ^{BCR-ABL-T315I}The inhibitor of theramutein:

Wherein

Ring A is 5-, 6-or 7-unit ring or 7-to 12-unit condensed-bicyclic;

X ¹Be selected from N, N-R ⁰Or C-R ¹

X ²Be selected from N, N-R ⁰Or C-R ¹

Dotted line is represented two keys of choosing wantonly;

R ¹Be selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF independently of one another ₃, NO ₂, OR ¹¹,-(CH ₂) _pC (O) (CH ₂) _qR ¹¹,-(CH ₂) _pC (O) N (R ¹²) (R ¹³) ,-(CH ₂) _pC (O) O (CH ₂) _qR ¹¹,-(CH ₂) _pN (R ¹¹) C (O) R ¹¹,-(CH ₂) _pN (R ¹²) (R ¹³) ,-N (R ¹¹) SO ₂R ¹¹,-OC (O) N (R ¹²) (R ¹³) ,-SO ₂N (R ¹²) (R ¹³), the group formed of halogen, aryl and heterocycle, and in addition or alternatively, two R on the adjacent ring atom ¹Group forms and contains 0-3 heteroatomic 5-or 6-unit fused rings;

N is 0-6;

P is 0-4;

Q is 0-4;

X ³Be N, CH or C-R ⁵⁰

Each R ⁵⁰Be independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF ₃, NO ₂, OR ⁵¹,-(CH ₂) _rC (O) (CH ₂) _sR ⁵¹,-(CH ₂) _rC (O) N (R ⁵²) (R ⁵³) ,-(CH ₂) _rC (O) O (CH ₂) _sR ⁵¹,-(CH ₂) _rN (R ⁵¹) C (O) R ⁵¹,-(CH ₂) _rN (R ⁵²) (R ⁵³) ,-N (R ⁵¹) SO ₂R ⁵¹,-OC (O) N (R ⁵²) (R ⁵³) ,-SO ₂N (R ⁵²) (R ⁵³), the group formed of halogen, aryl and heterocycle, and in addition or alternatively, two R on the adjacent ring atom ⁵⁰Group forms and contains 0-3 heteroatomic 5-or 6-unit fused rings;

R ⁵¹Be selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl and heterocycle;

R ⁵²And R ⁵³Be independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl and heterocycle; Or R ⁵²And R ⁵³Can form with the nitrogen that they connected and randomly to contain a first ring of extra heteroatomic 5-to 7-; Wherein 5-to 7-unit ring can be chosen wantonly by 1 to 3 and independently be selected from alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF ₃, NO ₂, OR ⁰, CO ₂R ⁰, C (O) R ⁰, halogen, aryl and heterocyclic substituting group replace;

R is 0-4;

S is 0-4;

M is 0-4; And

In another preferred embodiment, the invention provides and have general formula I I _bP210 ^{BCR-ABL-T315I}The inhibitor of theramutein:

Wherein:

R ¹⁴Be selected from H and F;

X ³Be N, CH or C-R ⁶⁰

Each R ⁶⁰Be independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF ₃, NO ₂, OR ⁰, the group formed of halogen, aryl and heterocycle;

R ⁶¹Be selected from aryl and heterocycle;

Q be selected from chemical bond or have formula-O-,-(CH ₂) _i-,-(CH ₂) _iC (O) (CH ₂) _j-,-(CH ₂) _i-N (R ⁶²)-(CH ₂) _j-,-(CH ₂) _iC (O)-N (R ⁶²)-(CH ₂) _j-,-(CH ₂) _iC (O) O (CH ₂) _j-,-(CH ₂) _iN (R ⁶²) C (O)-(CH ₂) _j-,-(CH ₂) _iOC (O) N (R ⁶²)-(CH ₂) _j-and-O-(CH ₂) _i-C (O) N (R ⁶²)-(CH ₂) _j-group;

R ⁶²Be selected from H, alkyl, aryl and heterocycle;

H is 0-4;

I is 0-4; And

J is 0-4.

At general formula I I _bIn the preferred embodiment of compound, R ⁶⁰Be selected from halogen, CF ₃And OH.Other preferred embodiment in, R ⁸Be selected from H and CH ₃

At general formula I I _bIn the preferred embodiment of compound, X ³Be N.In a further preferred embodiment, Q is selected from-(CH ₂) _i-N (R ⁶²)-(CH ₂) _j-, especially in preferred embodiments, Q is-N (R ⁶²)-.

In another preferred embodiment, the invention provides and have general formula I I _cP210 ^{BCR-ABL-T315I}The inhibitor of theramutein:

Wherein

Ring A is 5-, 6-or 7-unit ring or 7-to 12-unit condensed-bicyclic;

X ¹Be selected from N, N-R ⁰Or C-R ¹

X ²Be selected from N, N-R ⁰Or C-R ¹

Dotted line is represented two keys of choosing wantonly;

Each R ¹Be independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF ₃, NO ₂, OR ¹¹,-(CH ₂) _pC (O) (CH ₂) _qR ¹¹,-(CH ₂) _pC (O) N (R ¹²) (R ¹³) ,-(CH ₂) _pC (O) O (CH ₂) _qR ¹¹,-(CH ₂) _pN (R ¹¹) C (O) R ¹¹,-(CH ₂) _pN (R ¹²) (R ¹³) ,-N (R ¹¹) SO ₂R ¹¹,-OC (O) N (R ¹²) (R ¹³) ,-SO ₂N (R ¹²) (R ¹³), the group formed of halogen, aryl and heterocycle, and in addition or alternatively, two R on the adjacent ring atom ¹Group forms and contains 0-3 heteroatomic 5-or 6-unit fused rings;

N is 0-6;

P is 0-4;

Q is 0-4;

R ⁸Be selected from H and methyl;

X ³Be N or CH;

R ⁶¹Be selected from aryl and heterocycle;

R ⁶²Be selected from H, alkyl, aryl and heterocycle;

H is 0-4;

I is 0-4; And

J is 0-4.

In another preferred embodiment, the invention provides and have general formula I I _dP210 ^{BCR-ABL-T315I}The inhibitor of theramutein:

Wherein:

Ring A is 5-, 6-or 7-unit ring or 7-to 12-unit condensed-bicyclic;

X ¹Be selected from N, N-R ⁰Or C-R ¹

X ²Be selected from N, N-R ⁰Or C-R ¹

Dotted line is represented two keys of choosing wantonly;

N is 0-6;

P is 0-4;

Q is 0-4;

R ⁸Be selected from H and methyl;

X ³Be N or CH;

Each R ⁷¹Be selected from H, alkyl, aryl, aralkyl and heterocycle; And k is 0-4.

In another preferred embodiment, the invention provides and have general formula I I _eP210 ^{BCR-ABL-T315I}The inhibitor of theramutein:

Wherein:

Ring A is 5-, 6-or 7-unit ring or 7-to 12-unit condensed-bicyclic;

X ¹Be selected from N, N-R ⁰Or C-R ¹

X ²Be selected from N, N-R ⁰Or C-R ¹

Dotted line is represented two keys of choosing wantonly;

N is 0-6;

P is 0-4;

Q is 0-4;

R ⁸Be selected from H and methyl;

X ³Be N or CH;

Each R ⁷¹Be selected from H and alkyl.

In another preferred embodiment, the invention provides and have general formula I I _fP210 ^{BCR-ABL-T315I}The inhibitor of theramutein:

Wherein:

R ¹⁴Be selected from H and F;

R ⁸Be selected from H and methyl;

General formula I I, II _a, II _b, II _c, II _d, II _eOr II _fTypical compound comprise down array structure:

In another preferred embodiment, the invention provides P210 with general formula III ^{BCR-ABL-T315I}The inhibitor of theramutein:

Wherein

Ring A is 5-, 6-or 7-unit ring or 7-to 12-unit condensed-bicyclic;

X ¹Be selected from N, N-R ⁰Or C-R ¹

X ²Be selected from N, N-R ⁰Or C-R ¹

Dotted line is represented two keys of choosing wantonly;

N is 0-6;

P is 0-4;

Q is 0-4;

R ¹⁰Be selected from-Y '-R ¹⁸

Y ' is selected from chemical bond, O, NR ⁰-and have 1-4 carbon atom and randomly by halogen, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CO ₂R ⁰, C (O) R ⁰, C (O) N (R ⁰) ₂, CN, CF ₃, N (R ⁰) ₂, NO ₂And OR ⁰In the hydrocarbon chain of one or more replacements;

R ¹⁸Be selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CF ₃, the group formed of aryl and heterocycle; And

In another preferred embodiment, the invention provides P210 with general formula III a ^{BCR-ABL-T315I}The inhibitor of theramutein:

Wherein:

Ring A is 5-, 6-or 7-unit ring or 7-to 12-unit condensed-bicyclic;

X ¹Be selected from N, N-R ⁰Or C-R ¹

X ²Be selected from N, N-R ⁰Or C-R ¹

Dotted line is represented two keys of choosing wantonly;

N is 0-6;

P is 0-4;

Q is 0-4;

X ³Be N, CH or C-R ⁵⁰

Each R ⁵⁰Be independently selected from alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CN, CF ₃, NO ₂, OR ⁵¹,-(CH ₂) _rC (O) (CH ₂) _sR ⁵¹,-(CH ₂) _rC (O) N (R ⁵²) (R ⁵³) ,-(CH ₂) _rC (O) O (CH ₂) _sR ⁵¹, (CH ₂) _rN (R ⁵¹) C (O) R ⁵¹,-(CH ₂) _rN (R ⁵²) (R ⁵³) ,-N (R ⁵¹) SO ₂R ⁵¹,-OC (O) N (R ⁵²) (R ⁵³) ,-SO ₂N (R ⁵²) (R ⁵³), the group formed of halogen, aryl and heterocycle, and in addition or alternatively, two R on the adjacent ring atom ⁵⁰Group forms and contains 0-3 heteroatomic 5-or 6-unit fused rings;

R is 0-4;

S is 0-4;

M is 0-4; And

Each R ⁰Be selected from H, alkyl, cycloalkyl, aralkyl, aryl and heterocycle independently of one another.

In another preferred embodiment, the invention provides P210 with general formula III b ^{BCR-ABL-T315I}The inhibitor of theramutein:

Wherein:

Ring A is 5-, 6-or 7-unit ring or 7-to 12-unit condensed-bicyclic;

X ¹Be selected from N, N-R ⁰Or C-R ¹

X ²Be selected from N, N-R ⁰Or C-R ¹

Dotted line is represented two keys of choosing wantonly;

N is 0-6;

P is 0-4;

Q is 0-4;

X ³Be N or CH;

R ⁶¹Be selected from aryl and heterocycle;

R ⁶²Be selected from H, alkyl, aryl and heterocycle;

H is 0-4;

I is 0-4; And

J is 0-4.

In another preferred embodiment, the invention provides and have general formula III _cP210 ^{BCR-ABL-T315I}The inhibitor of theramutein:

Wherein:

Ring A is 5-, 6-or 7-unit ring or 7-to 12-unit condensed-bicyclic;

X ¹Be selected from N, N-R ⁰Or C-R ¹

X ²Be selected from N, N-R ⁰Or C-R ¹

Dotted line is represented two keys of choosing wantonly;

N is 0-6;

P is 0-4;

Q is 0-4;

X ³Be N or CH;

R ⁷⁰Be selected from halogen, alkyl, CN, N (R separately ⁷¹) ₂, ring-amino, NO ₂, OR ⁷¹And CF ₃

R ⁷¹Be selected from H, alkyl, aryl, aralkyl and heterocycle separately; And k is 0 to 4.

In another preferred embodiment, the invention provides and have general formula III _dP210 ^{BCR-ABL-T315I}The inhibitor of theramutein:

Wherein:

Ring A is 5-, 6-or 7-unit ring or 7-to 12-unit condensed-bicyclic;

X ¹Be selected from N, N-R ⁰Or C-R ¹

X ²Be selected from N, N-R ⁰Or C-R ¹

Dotted line is represented two keys of choosing wantonly;

N is 0-6;

P is 0-4;

Q is 0-4;

R ⁸Be selected from H and methyl;

X ³Be N or CH;

And R ⁷¹Be selected from H and alkyl separately.

In another preferred embodiment, the invention provides and have general formula III _eP210 ^{BCR-ABL-T315I}The inhibitor of theramutein:

Wherein:

R ¹⁴Be selected from H and F;

General formula III, III _a, III _b, III _c, III _dOr III _eTypical compound comprise down array structure:

In another embodiment, the invention provides P210 with general formula I V ^{BCR-ABL-T315I}The inhibitor of theramutein:

Wherein:

Ring A is 5-, 6-or 7-unit ring or 7-to 12-unit condensed-bicyclic;

X ¹Be selected from N, N-R ⁰Or C-R ¹

X ²Be selected from N, N-R ⁰Or C-R ¹

Dotted line is represented two keys of choosing wantonly;

N is 0-6;

Each R ¹²And R ¹³Be independently selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl and heterocycle; Or R ¹²And R ¹³Can form with the nitrogen that they connected and randomly to contain a first ring of extra heteroatomic 5-to 7-;

P is 0-4;

Q is 0-4;

R ²²Be selected from H and C _1-3Alkyl;

R ⁴⁴Be selected from H, alkyl, cycloalkyl ,-(C=O) R ⁰, alkenyl, alkynyl, aralkyl, aryl and heterocycle;

R ⁴⁵Be selected from-Y ＂-R ¹⁹

Y ＂ is selected from chemical bond, O, NR ⁰-and have 1-4 carbon atom and optional by halogen, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CO ₂R ⁰, C (O) R ⁰, C (O) N (R ⁰) ₂, CN, CF ₃, N (R ⁰) ₂, NO ₂And OR ⁰In the hydrocarbon chain of one or more replacements;

R ¹⁹Be selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, CF ₃, the group formed of aryl and heterocycle; And

The typical compound of general formula I V comprises array structure down:

In another embodiment, the invention provides P210 with general formula V ^{BCR-ABL-T315I}The inhibitor of theramutein:

Wherein:

Ring A is 5-, 6-or 7-unit ring or 7-to 12-unit condensed-bicyclic;

X ¹Be selected from N, N-R ⁰Or C-R ¹

X ²Be selected from N, N-R ⁰Or C-R ¹

Dotted line is represented two keys of choosing wantonly;

N is 0-6;

P is 0-4;

Q is 0-4;

R ²²Be selected from H and C _1-3Alkyl;

R ⁵⁵Be selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl and heterocycle;

R ⁵⁶Be selected from-Y ＂-R ¹⁹

In another embodiment, the invention provides and have general formula V _aP210 ^{BCR-ABL-T315I}The inhibitor of theramutein:

Wherein:

Ring A is 5-, 6-or 7-unit ring or 7-to 12-unit condensed-bicyclic;

X ¹Be selected from N, N-R ⁰Or C-R ¹

X ²Be selected from N, N-R ⁰Or C-R ¹

Dotted line is represented two keys of choosing wantonly;

N is 0-6;

P is 0-4;

Q is 0-4;

X ³Be N or C-R ⁵⁰

R ⁵²And R ⁵³Be selected from H, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl and heterocycle independently of one another; Or R ⁵²And R ⁵³Can form with the nitrogen that they connected and randomly to contain a first ring of extra heteroatomic 5-to 7-;

R is 0-4;

S is 0-4;

M is 0-4; And

General formula V or V _aTypical compound comprise down array structure:

In another embodiment, the invention provides P210 with general formula VI ^{BCR-ABL-T315I}The inhibitor of theramutein:

Wherein:

Ring A is 5-, 6-or 7-unit ring or 7-to 12-unit condensed-bicyclic;

X ¹Be selected from N, N-R ⁰Or C-R ¹

X ²Be selected from N, N-R ⁰Or C-R ¹

Dotted line is represented two keys of choosing wantonly;

N is 0-6;

P is 0-4;

Q is 0-4;

R ⁵⁶Be selected from-Y ＂-R ¹⁹

In another embodiment, the invention provides and have general formula VI _aP210 ^{BCR-ABL-T315I}The inhibitor of theramutein:

Wherein:

Ring A is 5-, 6-or 7-unit ring or 7-to 12-unit condensed-bicyclic;

X ¹Be selected from N, N-R ⁰Or C-R ¹

X ²Be selected from N, N-R ⁰Or C-R ¹

Dotted line is represented two keys of choosing wantonly;

N is 0-6;

P is 0-4;

Q is 0-4;

X ³Be N or C-R ⁵⁰

R is 0-4;

S is 0-4;

M is 0-4; And

General formula VI or VI _aTypical compound comprise down array structure:

In another preferred embodiment, the invention provides P210 with general formula VII ^{BCR-ABL-T315I}The inhibitor of theramutein:

Wherein:

Ring A is 5-, 6-or 7-unit ring or 7-to 12-unit condensed-bicyclic;

X ¹Be selected from N, N-R ⁰Or C-R ¹

X ²Be selected from N, N-R ⁰Or C-R ¹

Dotted line is represented two keys of choosing wantonly;

N is 0-6;

P is 0-4;

Q is 0-4;

Ring B is selected from the cycloalkyl that has 5 or 6 annular atomses and comprises 1 to 3 heteroatomic heterocycle that contains 5 or 6 annular atomses;

R is 0-4;

S is 0-4;

M is 0-4; And

The typical compound of general formula VII comprises array structure down:

The definition of every kind of phraseology used herein, for example alkyl, m, n, R, R ' etc. occur in any structure when once above, and are irrelevant with the definition at its other place in same structure.

Just to structure I, I _a, I _b, II and II _aIn the foregoing description of compound each, each narration is independently selected from the definition that these terms of providing in the part are provided this section to term halogen, alkyl, cycloalkyl, alkenyl, alkynyl, aralkyl, aryl, heterocyclic radical or heterocyclic.

Be appreciated that chemical structure provided herein comprises following implicit condition, promptly replace according to replacing atom and substituent permission valence link and carry out and replace producing stable compound, for example, this compound can be by such as spontaneous conversions such as rearrangement, cyclisation, eliminations.

When having one or more chiral centre in the compound of the present invention, general formula as herein described comprises each isomer and composition thereof (for example racemoid etc.).

When having one or more pairs of keys in the compound of the present invention, general formula as herein described comprises cis-and trans-isomer.Although this paper described cis or trans (cis of trans) configuration chemical structure (such as, for example structure I I, II _a, V, V _a, VI and VI _a), the implication of two kinds of configurations is that every kind of general formula includes.

In certain embodiments, compound of the present invention can exist with several tautomeric forms.Therefore, chemical structure as herein described comprises the tautomeric form of all possible compound of enumerating.

Compound of the present invention is generally by being purchased the preparation of raw material and known chemical technology.Can following synthetic embodiment of the present invention.Medicine or synthetic chemistry those skilled in the art are easy to know synthesizing mean requisite operation steps of institute and the technology as described below implemented.

Can by Gineinah etc. p.562 (Arch.Pharm.Pharm.Med.Chem.2002,11,556-562) make suitable hydrazine compound such as A and suitable aldehyde under the described similar condition, such as the compound of B prepared in reaction general formula I I.

For example, at protonic solvent, such as C ₁-C ₆In the alcohol A was heated 1-24 hour with 1.1 normal B,, can obtain C with postcooling and collecting precipitation.Perhaps, can be by evaporating solvent and by using silica gel, aluminum oxide or C ₄-C ₁₈The chromatography purification separated product C of inverted medium.Similar approach goes for " aryl " quilt as R ⁵The situation that other group of middle definition replaces.

Can be by making suitable hydrazine compound, such as D and activatory carboxylic acid, such as the compound of E prepared in reaction general formula III, wherein LG is a leavings group, such as halogen, 1-oxygen base benzotriazole, penta fluoro benzene oxygen base, right-nitro-phenoxy etc.; Or compd E can also be asymmetric carboxylic acid anhydride, wherein can use and Nair and Mehta p.408 (Indian J.Chem.1967 5,403-408) described in similar condition.

For example, under the suitable temperature of 0 ℃-solvent boiling point, alkali is being arranged, exist and choose wantonly such as pyridine or another kind of tertiary amine in that catalyzer is arranged, such as 4-N, the inert solvent under the N-Dimethylamino pyridine exists, such as methylene dichloride, 1,2-ethylene dichloride or N use active ester in the dinethylformamide, such as aryl-C (O)-OC ₆F ₅Handle D and can obtain F, can pass through evaporating solvent, use silica gel, aluminum oxide or C subsequently ₄-C ₁₈The chromatography of inverted medium is separated it.Be easy to by corresponding carboxylic acid and Pentafluorophenol, use carbodiimide, prepare the above-mentioned active ester example of E such as dicyclohexylcarbodiimide as condensation reagent.

Can be by making suitable nucleophile, for example, hydrazine derivative and with the nitrogen-atoms consecutive position on have the heteroaromatics prepared in reaction precursor of halogenic substituent, such as A and D.For example, can use and (J.Heterocyclic Chem.1990,27 such as Wu, 1559-1563), (J.Am.Chem.Soc.1959 such as Breshears, 81,3789-3792) or (Arch.Pharm.Med.Chem.2002,11 such as Gineinah, 556-562) described similar method, by for example, 2,4-dihalo pyrimidine derivatives is the example of feedstock production compd A and D, many for being obtained commercially in the described raw material perhaps are easy to be prepared by those skilled in the art.Therefore, with amine or other nucleophile (Z), choose wantonly have in the presence of the alkali of interpolation handle suitable 2,4-dihalo pyrimidine derivatives G, the 4-halogenic substituent on the selectivity substituted pyrimidines ring.Use second kind of nucleophilic reagent subsequently, choose wantonly at solvent such as hydrazine or hydrazine derivative, such as C ₁-C ₆Pure and mild choosing wantonly having in the presence of the alkali of interpolation handled product, the 2-halogenic substituent on the substituted pyrimidines ring, thus obtain compound into the example of said structure A and D.

Can be by synthesizing embodiment, wherein R such as these class methods following or that it is directly modified ²For-NR ²²And R ³For-C (=R ³³).Can be that raw material synthesizes with suitable ring A derivative J, described ring A derivative J has the leavings group (LG) adjacent with essential ring nitrogen.As mentioned above, the reaction product of said structure G and structure G and nucleophile Z is the example of the suitable ring A derivative J of this class.Suitable LG ' group is halogen, alkylthio, alkyl sulphonyl, alkyl sulfonic ester or aromatic yl sulphonate.Use amine R ¹²NH ₂Handling J replaces LG ' and obtains intermediate K.The example of this chemical conversion by Capps etc. at J.Agric.Food Chem.1993,41, report among the 2411-2415, wherein R ¹²For H and LG ' are CH ₃SO ₂-, and R ¹²For H and LG ' are reported in the J.Chem.Soc.1951 of Marshall etc. for the example of Cl, among the 1004-1015.

By simultaneously or introduce R successively ³, R ⁴And R ⁵Element, the intermediate of structure K changes into compound of the present invention.For example, use isocyanic ester R ⁶-N=C=O deals with the intermediate of structure K separately and obtain the compound of structure M in one step, and it is a compound of the present invention, wherein R ²=-NR ²²-, R ³=-C=O-, R ⁴=-NH-and R ⁵=-chemical bond-R ⁶The alternative approach of compound that the compound of structure K is changed into structure M is well-known for those skilled in the art, wherein, at first introduces R ³With leavings group (for example right-nitro-phenoxy or chlorine), use for example amine R subsequently ⁶-NH ₂Replace leavings group so that introduce R ⁵And R ⁶

Perhaps, generally with choosing wantonly solvent is being arranged, existing such as ethyl acetate Huo diox and use reagent down at heating condition, such as, cyanamide (NH ₂-CN) Processing Structure K intermediate and obtain intermediate N.The alternatives of cyanamide is nitroguanidine or amidino groups sulfonic acid (NH ₂-C (=NH)-SO ₃H).Use cyanamide carry out example that this class transforms by Latham etc. at J.Org.Chem.1950, report in 15,884.At Proc.Natl.Acad.Sci.USA1925,11,72 report the example of use nitroguanidine by Davis.The application of amidino groups sulfonic acid is by Bioorg.Med.Chem.Lett.1997 such as Shearer, and 7,1763 report.

According to intermediate A or D are changed into the similar mode of the embodiment of being represented by C or F, intermediate K is changed into the compound of being represented by P or Q respectively, they are the further embodiment of the present invention.

In such scheme, handle A or K so that replace B with ketone S, wherein R and obtains the compound of structure T or U respectively as mentioned above, and they are the further embodiment of the present invention.

With appropriate reductant such as metal (boron, aluminium, silicon etc.) hydride reagent, the two keys of non--guanidine radicals carbon-nitrogen of preferred a kind of reductive agent selective reduction U with alkalescence and obtain compound V of the present invention.

Can be prepared as follows embodiment of the present invention, wherein R ²=CO, R ³=-NR ^32-, R ⁴=N-and R ⁵=ZR ⁷, wherein Z is hydrocarbon chain and R ⁷As mentioned above.Work as R ³²=hour the time, by change into corresponding chloride of acid change into active ester or similarly activated derivatives activate ring A-deutero-carboxylic acid W, many in the said process are that this area is well-known.Handle the activatory carboxylic acid with hydrazine and obtain corresponding hydrazides Y.Handle Y (if necessary, under heating condition and/or gentle acid catalysis) and obtain required end product Z with aldehydes or ketones.

If not being obtained commercially, so can be by handling above-mentioned raw materials J with cyanide ion, optional heating or transition metal-catalyzed, the preparation ring A-deutero-carboxylic acid W of passing through so that replace leavings group LG ' with the cyano group residue.The alkalescence of cyano group or acidic hydrolysis obtain tart carboxylic acid intermediate W.

Work as R ³²When being not H, the protected form of mono-substituted hydrazine can be used for such scheme so that substitute hydrazine.Therefore, use R ³²NHNH-PG, wherein PG is a nitrogen-protecting group group, handles activating carboxy acid from W such as carbobenzoxy-(Cbz) or uncle-butoxy carbonyl, subsequently deprotection and as mentioned above with suitable aldehydes or ketones processing and obtain Z ', it is another embodiment of the invention.

The organic synthesis those skilled in the art are apparent, and above-mentioned reaction process is the representative of one group of extensive method of logical expansion in the method for enumerating.Therefore, the conspicuous modification by aforesaid method can prepare the extra R of the claimed introducing of the present invention ², R ³, R ⁴And R ⁵The further embodiment of the present invention of version.

Generally acknowledge as those skilled in the art, advantageously in obtaining end product, use interim blocking group.Term ＂ blocking group ＂ used herein refers to the interim modification of possible reactive functional group group, and described reactive functional group group can prevent unwanted chemical conversion.The example of this class blocking group comprises that the ester class of carboxylic acid, silyl ethers and aldehyde and the ketone of alcohol divide other acetals and ketals.Field (Greene, the T.W. of blocking group chemistry have been summarized; Wuts, P.G.M.Protective Groups in Organic Synthesis, 2 ^NdEd.; Wiley:New York, 1991).

One embodiment of the invention relate to the Mammals target protein of any endogenous existence of those skilled in the art's selection, and it is significant for the compound of identifying and/or optimize as described proteinic inhibitor or activator.Nosetiology or morbidity machine that the protein of common known described selection relates to human diseases cause.In another embodiment, the invention still further relates to the mutant form of described mammalian proteins matter." mutain " is for having the protein (Weigel etc., 1989) of the aminoacid sequence that changes as the conduct sudden change result who occurs in its corresponding gene.This class sudden change can cause one or more changes in the encoded protein matter characteristic.For example, the enzyme variants with catalytic chemistry of the modification that produces because of one or more amino acid changes is a mutain.

The present invention relates to the protein that implicit at least a amino-acid residue changes (term ＂ aminoacid sequence changes (amino acid sequence change) " or the ＂ aminoacid sequence change (amino acid sequence alteration) ＂ and comprise change, disappearance or the interpolation of at least a amino-acid residue; or disappearance, the arbitrary combination of adding, changing), so that the gained mutain with respect to described proteinic-mutant form for the susceptibility of therapeutical agent become (as results of mutation) the known treatment agent is produced resistance.The mutain of this particular type is called theramutein hereinafter, and the corresponding proteins matter of shortage sudden change is called prototheramutein in this article.

＂ prototheramutein ＂ used herein refers to the protein that endogenous occurs in cell, described cell is to giving the sudden change sensitivity of the treatment relative insensitivity of compound (being resistance), otherwise described treatment compound suppresses or activates described protein.Therefore, ＂ theramutein ＂ refers to protein or the protein portion that endogenous occurs in containing the cell that at least a aminoacid sequence changes for proteinic endogenous form, wherein behind the material that makes at least one individual's known inhibition of contact or activation prototheramutein, described aminoacid sequence change obtains or is identified or become and can identify, and through showing or shown that generation or development for specifying disease have clinical meaning.With regard to the purpose in determine going up sentence, unique is, and material need not be limited to is used for defining first the chemical active agent that there is purpose in theramutein.Therefore, as definition, theramutein is the protein that has implied sudden change in its corresponding endogenous gene, wherein said sudden change and patient to generally can activate or suppress not-the medicine generation clinical tolerance of mutain is relevant.With regard to specified theramutein, term used herein " corresponding prototheramutein " refers to the prototheramutein that produces described theramutein by sudden change.Similarly, with regard to specified prototheramutein, corresponding theramutein " refer to by theramutein by described prototheramutein sudden change generation.

Therefore, those skilled in the art are apparent, and when the gene of coding theramutein was limited to the gene of endogenous appearance, the definition of theramutein did not comprise by the pathogenic agent that causes disease, such as the protein of virus and bacterial identification.Term ＂ endogenous gene ＂ used herein refer to after taking at least with its not mutant form be present in gene in the karyomit(e) of organism.Term ＂ cell ＂ used herein refers to eukaryotic cell alive, no matter is organism, still maintains under organism outer suitable the laboratory tissue or organ culture condition.

In one embodiment of the invention, target protein (POI) can be the mammalian proteins matter of any endogenous coding.In another aspect of this invention, POI is theramutein, it is a kind of protein (that is, wild-type protein is prototheramutein, and theramutein is produced by it) that the described proteinic ＂ wild-type ＂ form of common existence is changed first.In the present invention on the other hand, theramutein is itself to be the protein variant (that is, mutain is prototheramutein, and theramutein is produced by it) of mutain.In another embodiment, theramutein compares and can further be suddenlyd change with the theramutein of preexist.In this class situation, can (the T315I mutant (vide infra) such as p210 BCR-ABL be considered as the elementary ＂ theramutein of ＂, and the sudden change of the T315I variant that (suddenlyd change) subsequently can be called secondary theramutein, three grades of theramutein etc. with first kind of theramutein.As hereinafter typical, mutain of the present invention is the variant of the Bcr-Abl Tyrosylprotein kinase of disengaging " wild-type " Bcr-Abl inhibitor inhibition.This class Bcr-Abl mutain obtains changing (being also referred to as mutain) for the more common of Bcr-Abl or " wild-type " form, changes proteinic characteristic in this class mode.

The protein (POI) that should understand concern is the mammalian proteins matter of endogenous coding.The specific activity that the mutain that also is appreciated that main concern is identical for having for its prototheramutein, increase or reduce, and be not subjected to or be difficult to be subjected to be used to suppress the theramutein that the promoting agent of prototheramutein suppresses.Equally, the another kind of main theramutein that pays close attention to has identical, specific activity (for its prototheramutein) that increases or reduce and the promoting agent activatory theramutein that is not subjected to or is difficult to be subjected to be used to activate prototheramutein.Other version is apparent to those skilled in the art.Further understand theramuteins and can comprise natural existence or common observed protein variant, for example, the variant of expressing by the not isoallele of specific gene.In some cases, this class variant may be not remarkable with regard to its normal cell function, and wherein function difference only becomes obvious in the presence of the promoting agent that difference inhibition or activation variant cell function are arranged.For example, the naturally occurring variant of certain enzyme can have different basically activity profiles, may be invalid to regulating another kind of variant but regulate a kind of therapeutical agent of variant.

Be appreciated that it is active reagent that one aspect of the present invention is to identify at selected POI, the cell function of POI promotes the appointment morbid state to make that the activator of the described POI of expection or inhibitor are that treatment is effective in the described lysis of treatment.Instruct albumen type that target regulates without limits for any kind of of treatable disease type or character and according to this paper, as long as satisfy the every other restriction (comprise the fact: any described protein of selecting to be used for target must be endogenous protein) of this paper statement.The nucleic acid of non--endogenous existence that obviously, those skilled in the art can use for example cDNA needs only the POI that aminoacid sequence exists corresponding to endogenous to implement the method for this paper instruction.

It is also understood that, one aspect of the present invention be to identify at before lysis is specified in treatment or treatment specify and produce in the lysis or the preponderate protein or the theramutein of (by any mechanism) of becoming has active promoting agent, another aspect is to identify at mutain common in being encroached on population of individuals to have active promoting agent, but wherein said mutain is lower to the susceptibility of the adjusting of the medicine ratified, and wherein the activity profile of mutain changes become important (and being accredited as theramutein thus first) in morbid state, such as, in described morbid state, this mutain obtains overexpression or participates in the signal conductive process, otherwise becomes unusual adjusting.For example, neoplastic disease can be because of due to the unusual adjusting of the cellular constituent of non-theramutein or its prototheramutein, and still can use the prototheramutein inhibitor for treating, and identical treatment is lower or invalid to the situation validity that has theramutein.This may be a kind of result, wherein observes the specific tumors type being reflected in the individuality of anticarcinogen changed, the different variants (Lynch etc., 2004) of the enzyme that described individual expression anticarcinogen is oriented to.Herein, variant can not produce or become and preponderates in the process of treatment disease, but preexists in the health population and only detect according to the reactivity that changes in its particular procedure to the treatment of setting up.

" agonist " and " activator " of term protein used herein can exchange use.Activator (agonist) is limited to the material of combination and activation appointment protein function.Except as otherwise noted, ＂ activator ＂, ＂ agonist ＂ are identical on implication with the proteinic activator ＂ of ＂.Activating by activator can be partially or completely.Equally, " antagonist " and " inhibitor " of term protein used herein can exchange use.Inhibitor (antagonist) is limited to combination and suppresses to specify the material of proteinic function.The implication that description material " inhibition " protein refers to is meant this material conjugated protein and reduction activity of proteins in cell, but does not reduce proteinic amount in the cell in fact.Similarly, describe material " activation " protein, be meant that such as prototheramutein or theramutein this material increases the function of proteinic qualification in the cell, but do not change proteinic level in the cell basically.Except as otherwise noted, the proteinic inhibitor ＂ of ＂ inhibitor ＂, ＂ antagonist ＂ and ＂ also is a synonym.The inhibition of inhibitor can be for partially or completely.Conditioning agent is activator or inhibitor.As an example, ＂ PKC _{β 1}Activator ＂ should be construed as denoting in conjunction with and PKC activation _{β 1}Material.Similarly, " p210 ^Bcr-AblInhibitor " be in conjunction with and suppress p210 ^Bcr-AblThe material of function.Describe material ＂ arrestin matter ＂ require this material in conjunction with described protein so that bring into play its restraining effect.Similarly, describe material ＂ activation of protein X ＂ and be meant this material combination and activation of protein X.Term " in conjunction with (bind (s)) ", " in conjunction with (binding) " and " in conjunction with (binds to) " have its implication common in biochemical field at (for example enzyme-substrate, protein-DNA, receptor-ligand etc.) aspect two kinds of interaction between substances of description.The context neutralization of term used herein " in conjunction with (binds to) " dependency between discussion material and its respective target albumen " with ... interact " be synonym.Description material used herein to protein " work ", " influence " protein, " bringing into play it " etc. and all these class relational language implication unanimities (fully understanding) as those skilled in the art to proteinic effect, promptly described material activation or suppress described protein.

Defined first and the mutant form of endogenous protein suppressed or be activated to notion, and be called positive ＂ specificity breach ＂ in this article greater than the degree of corresponding-corresponding albumen that suddenlys change (counterpart protein).In general, and with the situation of using inhibitor as an example, the specificity breach refers under the comparable condition that suppresses theramutein in the present invention is based on the pilot system of cell, the difference between designated substance and the ability that one of following situation is compared:

A) ability of same substance inhibition prototheramutein under comparable condition; Or

B) ability of second kind of material (being generally the known inhibitor of prototheramutein) inhibition theramutein under comparable condition; Or

C) ability of second kind of material inhibition prototheramutein under comparable condition.

When two kinds of different substancess (testing respectively) are compared between the effect to theramutein separately, the result is called homology specificity breach measures.

Perhaps, when comparing between the effect to two kinds of different substancess (general, but not always), respectively one of them is used for test to theramutein, and the another kind of test that is used for prototheramutein is called heterology specificity breach (SG) with the result and measures.Therefore, the example of measuring for heterology specificity breach (SG) as above-mentioned (a) that provides and (c) (although in two kinds of situations, all using identical material), and (b) be the example of specificity specificity breach mensuration.

The content that accompanying drawing 3 relates to is for understanding and illustrating information in these notions.

Result like the application class when situation relates to activator.Those skilled in the art be it is evident that at once, term used herein " comparable condition " comprises two kinds of different compounds of test, for example under same concentrations (such as two kinds of compounds that are closely related relatively so that measure relative potency), or by with two kinds at its corresponding IC ₅₀The different compounds that value is tested down compare the effect of corresponding prototheramutein and theramutein.Those skilled in the art are easy to approve version that other is useful and comparable condition.

Therefore, in the embodiment that this method is used, the more efficiently material of theramutein had the positive specificity breach of ＂＂." zero; invalid or do not have ＂ specificity breach be illustrated in material to the effect of theramutein with its to do not exist between the effect of prototheramutein the difference that can measure significantly (but; this compounds may be quite useful aspect the ability of its inhibition or activation theramutein and corresponding prototheramutein thereof), and the material that the negative specificity breach of ＂＂ is illustrated under the prescribed concentration is lower than the corresponding prototheramutein form of theramutein or the validity of another kind of suitable form (such as implying a kind of different sudden change) the validity of specifying theramutein.Other degree of concern of latter's compounds generally is lower than the classification of the former compound, but except following situation: wherein compound is so effective, makes it there is no actual worry to the relatively low effect of theramutein from the prospect of therapeutic efficiency.Those skilled in the art are easy to discern to be suitable for his or she mode of demand and quantize the various means that the specificity breach is estimated.This analysis can help those skilled in the art that all cpds is divided into independently class, and it is instructing further guide's optimization or helpful to the biological characterization research of described compound.

The present invention also provides the mode that is used to identify the compound that shows required specificity breach.Identify this compounds and use external pilot system to measure the ability of its inhibition or activation theramutein based on cell, wherein with material to the effect of the cell function of this proteinic sudden change endogenous form and same medicine to this protein non--effect of the cell function of the endogenous form of suddenling change compares.

Therefore, described system can find this compounds, described compound can in conjunction with theramutein and to the regulating effect of the cell function of described theramutein performance greater than regulating effect to the cell function performance of its corresponding prototheramutein.In addition, this system can find this compounds, described compound can in conjunction with theramutein and to the regulating effect of the cell function of theramutein performance at least greater than or can be greater than above-mentioned known compound to the regulating effect of the cell function performance of corresponding prototheramutein.In a specific embodiment of the present invention, be following result's screening and authenticating compound: 1) validity to theramutein is identical to the validity of prototheramutein with original medicine at least; And/or 2) to the validity of prototheramutein and validity similar (promptly showing specificity breach little or that be substantially zero) to theramutein.

In one embodiment of the invention, the cell of the theramutein that pays close attention to of overexpression is used to be accredited as the inhibitor of the theramutein that selects at least or the chemical active agent of activator (being combination and inhibition or combination and activation).These chemical active agents can also be inhibitor or the activator of other theramuteins of prototheramutein and even identical prototheramutein.Term ＂ chemical active agent ＂ used herein and ＂ compound ＂ can exchange use, and two terms only refer to have at the most, but not necessarily comprise the material of the molecular weight of 2000 atomic mass units (dalton).This class material is called " small molecules " sometimes.Except as otherwise noted, term material used herein only refers to chemical active agent/compound, and does not refer to biologically active agent.＂ biologically active agent ＂ used herein comprises the molecule of protein, polypeptide class and nucleic acid and has the molecule that is equal to or greater than 2000 atomic mass units (dalton).

In one embodiment of the invention, select theramutein and be used to the present invention is based on the raji cell assay Raji system of phenotypic response, design this system and be for the inhibitor that is accredited as theramutein or the promoting agent of activator.If known two or more different theramuteins derive from identical prototheramutein, the utilized theramutein that so preferential selection has resistance most is used for pilot system.In general, use at first give and known inhibition or activation prototheramutein and the drug determination theramutein that occurs ＂ at theramutein ＂ with its not-the corresponding body (prototheramutein) that suddenlys change compares to the resistance level of appointment chemical active agent.For example, by analyzing IC ₅₀Or AC ₅₀The method of this class resistance level of pH-value determination pH is well-known and is standard in the art, and do not repeat in this article.Yet cause-effect relationship is not to be absolutely necessary or should to infer using between self specified therapeutical agent treatment patient and the appearance of theramutein subsequently.And when relevant with theramutein, be suitably to select correct theramutein according to the instruction of this paper for implementing required for the present invention.

Therefore, for example, in the laboratory, produce, but the site-directed mutant that produces at random of the known protein matter that is not confirmed on clinical correlation as yet is not the suitable mutain that is used in the scope of the invention.Certainly, this class mutain can be divided into theramuteins yet.

For example, obtaining p210 ^Bcr-AblIn the trial of the potential inhibitor of mutant, Huron etc. (2003) have used reorganization c-abl goods and have screened the compound of a series of known inhibition c-src tyrosine kinase activities.The author has carried out the c-abl kinase assay to their compound and has identified when 8nM to the compounds effective of c-abl.Yet as the anti-various p210 of this compound of test (PD166326) ^Bcr-AblDuring theramuteins, it shows in the mutant some, such as p210 ^{Bcr-Abl-E255K}Activity, but find p210 ^{Bcr-Abl-T315I}Theramutein has kept the resistance more than 10 times (Huron etc. 2003, table 3).In addition, in each case, compound is to p210 ^Bcr-AblThe effect of theramuteins still significantly is lower than it to wild-type p210 ^Bcr-AblEffect.When test compounds to p210 ^{Bcr-Abl-T315I}During mutant active, it can not be suppressed to activity any appreciable degree (p.1270, left hand column, second section; In addition, referring to accompanying drawing 4).Therefore the compound that discloses can suppress STI-571 is produced the theramutein of partial resistance, but to the T315I mutant non-activity of Bcr-Abl, at known described theramutein at that time for STI-571 being shown the theramutein of maximum resistance.Therefore, accurately and briefly, the fubaritic p210 of the method for Huron ^Bcr-AblT315IEffective inhibitor of theramutein.

In fact, before disclosing the present invention, comprising the detailed description method and composition provided herein that this paper describes first, still nobody successfully identifies a kind of chemical active agent anywhere in the world, let alone a kind of can the evaluation p210 ^Bcr-AblT315IThe effective inhibition of theramutein can be to wild-type p210 to being equal to or higher than STI-571 ^Bcr-AblThe method of the chemical active agent of the degree that protein suppressed is (referring to Shah etc., Science, in August, 2004; O ' Hare etc., Blood, 2004; Tipping etc., Leukemia, 2004; Weisberg etc., Leukemia, 2004).

Can not overemphasize this compounds may be very useful, because still useless at present in developing into p210 ^{Bcr-Abl-T315I}The patient's of imatinib mesylate (the imatinib)-resistance state of theramutein-mediation alternative approach.In case this class resistance takes place the patient, then do not have available other effective backup means, and death is sure.Method as herein described provides and has been used for identifying, characterizing and chemosynthesis p210 from the pharmacology mode ^{Bcr-Abl-T315I}The method of first report of effective inhibitor of theramutein.In addition, those skilled in the art have approved the applicability of this means in any height drug resistance theramutein and meeting generalization immediately.At last, those skilled in the art further recognize under proper condition and specific POI in the phenotypic response of this paper definition and the cell to be existed and the increase of functionally active couples together any appointment endogenous target protein that allows people that active compound is treated in searching and uses this method.

In the present invention, use shows the test cell of the phenotypic characteristic (as described below) of careful selection, and existence and the functionally active of specific albumen of paying close attention to (POI) or the theramutein (TOI) that paid close attention to are relevant in described feature and the cell under proper condition.For theramutein, on qualitative, this result is identical with the phenotypic characteristic that the cell display of expressing prototheramutein goes out.Phenotypic characteristic (being the non--yielding characteristics of cell) is for observing (mensuration), select and/or be used for subsequently the characteristic that test method as described herein limits.The expression of phenotypic characteristic is proteinic overall active reaction in the pair cell, and is the result of proteinic absolute magnitude and specific activity thereof.If phenotypic characteristic can be used as usually that result that the protein active level raises observes and expressing a small amount of protein or described protein when also being theramutein, phenotypic characteristic is usually in the cell of expressing a spot of theramutein or its corresponding prototheramutein and not obvious so.In addition, usually can confirm to regulate phenotypic characteristic by the specific activity of regulating theramutein with proteinic inhibitor or activator, but, this is not to be uniform situation, because the inhibitor of TOI or activator may not be can get all the time when those skilled in the art are engaged in this intermediate item.Yet (, obviously specify the known inhibitor of prototheramutein or activator always to exist because of the inherence definition of the character of theramutein own.) therefore, purpose for the phenotypic characteristic of the nominative testing cell of determining to be used to subsequently test objective, those skilled in the art can also use to increase or to reduce and express the material that coding is specified the gene of POI (for example theragene under the theramutein situation), make the level of corresponding theramutein increase or reduce thus.This makes those skilled in the art can simulate the theramutein activator of some type or inhibitor (such as the suicide inhibitor of theramutein, but in fact the chemical active agent type of giving its lasting non-activity for irreversible fixation and covalent modification TOI), for accurate understanding be used to subsequently to establish usefulness the test cell line system suitable phenotypic characteristic purpose and can use this compounds.Help this classification example well known by persons skilled in the art comprise and utilize antisense DNA oligonucleotide, little intervening rna s, other method of interfering based on RNA and the vector construction body that contains the inducible promoter system.In this mode, the phenotypic characteristic of selection is relevant with the activity of theramutein in the test cell.The phenotypic characteristic that theramuteins be it should be noted that selection is usually also by the cell display of overexpression prototheramutein, and wherein phenotypic characteristic is regulated by inhibitor or the activator of known prototheramutein.

Phenotypic characteristic is merely the cell characteristic of acellular yielding characteristics.Except that the phenotypic characteristic of suitably determining as disclosed herein the instruction according to certain embodiments of the invention produces real needs in the useful test cell line system purpose, the phenotypic characteristic that is used for or is suitable for suitably and effectively implement term any type of the present invention or character is not had other restriction.In fact, the arbitrary characteristics of the maximized cell of application of those skilled in the art's suitable test based on cell that must be able to select foundation is used for his or she demand.But phenotypic characteristic can be for quantitatively or qualitatively and direct viewing or mensuration (for example available bore hole or use microscope), but modal being to use well known to a person skilled in the art standard automated lab equipment and the described feature of test operation step indirect measurement.Term ＂ can be observed ＂ and refers to and can measure feature, otherwise be exactly under proper condition can by no matter which kind of mode, comprise the available instrumentation survey in the laboratory of using any type.It is different with the implication of the ＂ of ＂ mensuration that term ＂ can measure ＂.Feature be to those skilled in the art can detect but the specified timing of meaning not in office, this depends on that how those skilled in the art select the design experiment system.For example, seeking POI for example in the process of prototheramutein (or theramutein) activator, only needing after adding the known activator that can activate POI or test substances, to detect relevant phenotypic characteristic.This provides the maximized ability of strength of signal that is produced by test cell in test that makes.

Phenotypic characteristic includes but not limited to the interior chemical species of ionic current (calcium, sodium, muriate, potassium, hydrogen ion etc.), pH change, second messenger molecule or other born of the same parents of growth characteristics, conversion conditions, differentiation state, substrate phosphorylation state, catalytic activity, cross-cell membrane, such as the change of cAMP, phosphoinositide class, cyclic nucleotide, the adjusting of genetic expression etc.(for example cell growth rate) continuously, or at certain hour after the time limit (for example final densities of cell culture) or instantaneous (for example proteic adjusting causes the instantaneous change of protein substrate phosphorylation, or the instantaneous delivery of ionic current strides film, or the cAMP level raises or reduces in the born of the same parents) observe or measure cell characteristic.In certain embodiments, can only the phenotypic characteristic that detects selection in the presence of the protein modulators arranged.To can there not being specified restriction for the feature that mensuration is selected.Term used herein " feature of cell " and " phenotypic characteristic " and simple " feature " in order to the subcellular fraction of intact cell after referring to the mass treatment test cell or cell specifically can measure characteristic the time implication be identical.For example, phenotypic characteristic can be kitchen range formation, cultivate when having at the proteinic cell that overexpression is selected in the presence of this protein activation agent, or born of the same parents' intracellular metabolite thing or ion, such as the instantaneous increase of level of cAMP, calcium, sodium, muriate, potassium, lithium, phosphatidylinositols, cGMP, supercarbonate etc. or when reducing, this kitchen range formation becomes and can observe.Those skilled in the art are apparent, and after the cells contacting test substances, subcellular fraction that can pair cell is measured the feature of so measuring (detection).Yet, must be to intact cell but not subcellular fraction carries out initial processing with material, make this material contact described cell thus.

Differ and be decided to be inherent physics of protein (or theramutein or prototheramutein) itself or chemical property (such as the proteinic amount (quality) that only is cell interior) for measuring the feature select in the cell, and must be to produce the feature that influence thus is different from the cell characteristic of theramutein itself because of cell interior protein (or theramutein or prototheramutein) is active, as what above go through.For example, if theramutein is for carrying out the protein kinase of autophosphorylation, described autophosphorylation promptly this endonuclease capable the process of catalysis autophosphorylation may be not suitable for selecting the suitable phenotypic characteristic of the phosphorylation state of TOI as the cell that is used to measure so by shifting the terminal phosphate part the ATP on himself.This is because this category feature can not reflect the activity of TOI to other cellular constituent.As well known by persons skilled in the art, autophosphorylation must not reflect the activity of protein kinase in the cell, because the kinase whose mutant of known protein has kept the enzymic activity that is enough to carry out autophosphorylation, but has lost the ability of carrying out signal transduction in cell.The classical paper (1988) of White etc. had both had instruction in this respect, merited attention again.

The reactive phenotypic characteristic ＂ of term ＂ refers to the cell characteristic that the inhibitor of specifying protein (for example, comprising prototheramutein or theramutein) or activator are reacted.The known therapeutical agent ＂ of term ＂ is defined as any promoting agent that in worldwide the country people is administered for the treatment disease.

As herein with p210 ^Bcr-AblAnd the relevant typical useful phenotypic characteristic of theramuteins is cell growth and propagation imbalance.Notice that same or similar test goes for the protein that many differences are paid close attention to.For example, growth, propagation and/or the common phenotypic characteristic of differentiation imbalance for producing because of various different cell protein overexpressions.Important instruction of the present invention is to cause this class phenotypic characteristic to occur by the protein that overexpression is selected, it is relevant with proteinic existence, amount and the specific activity selected that described feature becomes under appropriate condition, and the inhibitor or the activator of this dependency protein (POI) that can make those skilled in the art identify as required to be paid close attention to.Therefore, phenotypic characteristic is to the proteinic level of selection and/or the reaction of specific activity change.This phenotypic characteristic of replying is when also confirming to be called " phenotypic response " when the known conditioning agent of described POI replied in this article.Under the particular case of the theramuteins that does not have known conditioning agent, must use the prototheramutein conditioning agent to set up the phenotypic response that uses with theramutein.This height useful properties of described notion and recognizing cells represents the present invention than one of substantial advantage of prior art, prior art comprises that the applicant is based on the previous work (U.S.Pat.Nos.4 in the general field of raji cell assay Raji, 980,281; 5,266,464; 5,688,655; 5,877,007).With regard to it identifies POI inhibitor or activator ability, the evaluation of phenotypic response and be chosen as experienced investigator very sensitive raji cell assay Raji system is provided, therefore with prior art in disclosed any other related assays method compare and can identify these chemical reagent with much higher degree of certainty.

Although be not necessary all the time, the phenotypic characteristic that advantageously uses the cell of expressing high-level POI usually and select to produce because of the POI overexpression.This is because the phenotypic characteristic relevant with the POI function generally can be distinguished (being easier to mensuration) than POI to more the time by overexpression more.In addition, when the functional level of POI increases, the observed phenotypic response of the reaction of POI conditioning agent is obtained enlarging usually.If express in another way, the phenotypic response of the selection of arriving at the cell observation of overexpression protein (or theramutein) is responsive especially to the conditioning agent of protein (or theramutein) so.

Preferred protein obtains stably express in test cell.Stably express causes the protein level in the cell to keep can not changing relatively in process of the test.For example, be refractory phase behind the composition in stimulation or the activation signals pathway, in this process, the signal conduction is regulated because of the decrement of described composition and is suppressed.With regard to protein of the present invention, this class decrement is regulated to be enough to overcome by artificial overexpression protein usually.If express in another way, so fully keep expression, promptly observed these phenotypic characteristics changes mainly are because of protein suppresses or activation in process of the test, but not due to its level change, generation also is like this even proteinic decrement is regulated thereupon.Owing to these reasons, although so stably express of preferred protein, but can use proteinic transient expression after transfection, condition is that the phenotypic characteristic of selection can be measured and the time limit of pilot system lacks than carrying out property of proteinic level decline of its transient expression of estimating in time in this type systematic.Owing to these reasons, the clone (U.S. Pat 4,980,281) of preferred stably express.

Term ＂ cell-specific ＂ refers to that compound is regulated the ability of the phenotypic response of test cell selection under prescribed concentration, and does not influence control cells to same degree (if not influencing fully).Term ＂ cell-specific breach ＂ (＂ CSG ＂) refers to measure the ability that the selected compounds adjusting is regulated same phenotypic response corresponding to the ability (being not limited to theramutein) of the proteinic phenotypic response of specified target in the test cell with respect to compound described in the control cells.For the CGS technology being applied to non--theramutein endogenous target protein, selected phenotypic response must define with the known inhibitor or the activator of target protein before.

The mensuration of CGS provides the method for the potential relatively treatment value of more different compounds in compound group (two or more) to those skilled in the art, by with control cells its relative intersection-reactivity relatively, have nothing to do with effectiveness to the target protein of organizing interior any appointed compound.The relative comparison cell represents the compound that the compound of maximum ＂ specificity ＂ is normally wanted most in its activity to test cell, because compare with other compounds that have the narrow ＂ CSG of ＂ in aforementioned group, the wide ＂ CSG of ＂ helps to select reasonably to be considered to have the compound of minimum potential side effect in the patient.When relatively based on the whole metering response curve that produces of the test of cell, the effect that CSG measures is the most obvious, yet following hypothetical example also is irradiative.

Consider the following table of supposition compound and the corresponding IC of the acellular test macro of employing ₅₀Value.This embodiment uses protein kinase as target protein.When handle to attempt selected compound or compound group carried out the guide optimize be used for evaluation next clinical before during the problem of the potential guide of optimization candidate compound of (animal) and clinical study, this is that class situation that those skilled in the art face every day.

The standard method of this area at present is to identify this compound, and it highly is renderd a service and to different but closely-related protein kinase does not show significant inhibition activity suppressing to represent aspect the kinase whose enzymic activity of target protein in acellular test macro.The result who is shown in the acellular test macro of table 1 shows, compd A be in this series compound (A, B, C, D) the most virtuous and also show its at target protein with respect to its IC to non-target protein effect ₅₀Between with the biggest gap.For example, if interested in identifying the Abl kinase inhibitor, for example EGF acceptor, c-kit or c-Src contrast kinases as the negative ＂ of ＂ can to adopt other protein kinases in described test.The same with c-Abl, these enzymes of all back all are tyrosine protein kinase.Really, use the protein kinase (comprising serine/threonine kinase, Tyrosylprotein kinase and dual-specificity kinase) that is called ＂ panel ＂ to identify the compound (except that target protein itself) that suppresses the least possible protein kinase at present in the art usually.The reason of this method is that the designated compound of kinases few more in cell free system suppresses, and this compound is more little to the possibility that the patient produces adverse side effect.Yet, almost do not have clinical evidence to support this viewpoint really.

In addition, other people think that target surpasses a kinase whose compound and compares with those compounds to single target protein high degree of specificity and may have the other treatment effect in some cases.Under the situation of imatinib, there are some evidences to confirm that this is right, in the patient of carcinoma of small intestine, produce beneficial effect as previous imatinib of discussing of this paper and the cross reaction of c-kit with some types of organization.Though with this cross reaction of c-kit, yet imatinib is showed the height cell-specific in test macro of the present invention, this and its highly clinical efficacy and relative moderate side effect characteristics unanimity in 3 years of treatment.Yet, along with appointed compound specific reduction in cell system of the present invention, with other targets for example (in this embodiment) other protein kinases family member's cross reaction in the patient, cause disadvantageous side effect.Further go through below.

The conclusion that the acellular test result that the technician shows from last table 1 obtains is that compd A is the most virtuous compound, shows 50% inhibition concentration (IC ₅₀) value is 0.2 to receive rub (0.2nM).Yet, show as table 2 and Figure 14, with respect to this compound of control cells test cell is not shown specificity, because it is to the IC of control cells ₅₀Also be 1 to receive and rub.Similarly, compd B and D be the ten minutes effective force still, and both all have the IC of 10nM at test cell ₅₀, and Compound D is higher because the IC of its control cells than the specificity of B ₅₀Higher (200nM).The CSG of compd B and D measures and is reflected in that Compound D is a preferred compound in these two compounds at once, and every other Consideration equates.Yet, the most important thing is that (table 2 Figure 15) shows that Compound C is a best compound in this group compound with regard to its CSG of 40 in this embodiment.This refers to respect to the control cells Compound C test cell be shown maximum specificity, and be expected at and induce the possibility of not wishing side effect minimum among the patient, because this discovery derives from the direct test of compound in the viable cell system, the stone wall limit of the pharmaceutical effect of most drug.The emphasis of present embodiment is that CSG measure to allow those skilled in the art to be independent of its potential therapeutic value of rendeing a service some compounds in cell free system to sort.Therefore, although Compound C is to render a service minimum compound, its specificity in suppressing the test cell ability is the highest, and allows control cells relatively uninfluenced in wide concentration range.This is reflected among its CSG of 40, and confirms that Compound C (rather than compd A) is the compound that gives highest priority in further pre-clinical and clinical development is made great efforts.

Use method of the present invention that ordering and the preferred ability of considering drug discovery and development method are provided by this way, its be before impossible mode.Although the repeated application of aforesaid method, those skilled in the art and medical chemistry man cooperation can be synthesized in test macro described herein scoring and be positive compound analogue, the described compound of test in testing method of the present invention, according to its CSG value described compound is sorted, selection is used for the further optimizing compound of research and development, and repeats this method as required as far as possible repeatedly so that produce fully and optimize the compound that the nominative testing cell is represented high degree of specificity with respect to control cells.In case use this paper system that appointed compound is optimized, described system is often referred to CSG between control cells and the test cell (if use above-mentioned cell IC ₅₀The method of ratio) be 3 to 5 times at least, those skilled in the art can continue to finish guide's optimization method and test for example correlation parameter of plasma half-life, bioavailability and the suitable animal model of use of character by other chemically modifieds to the compound selected by this way then.Certainly, think that in fact target protein in the described compound pair cell environment has the possibility that needs effect for optimizing the result of the method for described compound according to method described herein, rather than with the result of older acellular method.

Be apparent that very much for a person skilled in the art, similar method can also be used to specify the activator of target protein.Also be apparent that for a person skilled in the art, have the additive method of determining CSG.For example, use the embodiment of the above-mentioned inhibitor that provides, another determines that the process useful of CSG is to measure compound to cause control cells be 50% growth inhibiting maximum concentration suppresses the minimum concentration of at least 90% test cell system divided by compound ratio.Also can utilize other tangible changes of present method, comprise the logarithm that calculates the active concentration of compound exhibits prescribed percentage, the contrast or the test cell of proofreading and correct are relative to each other replied etc.In order to determine CSG, do not plan the control cells of calculating or observe is replied and test cell relatively limiting between replying.

If people use the IC of aforesaid control cells/test cell ₅₀The method of ratio it has been generally acknowledged that so it is good clinical candidate compound that the CSG value is less than or equal to 1 compound, and CSG value is that ten minutes is hopeful and is worth further considering greater than about 10 compound.

Present embodiment also emphasize cell free system and the present invention medically and on the physiology more relevant based on the system of cell in difference between the effect of appointed compound.

In one embodiment of the invention, compound identification is used to identify and/or the side effect relevant with giving patient's administered compound is minimized.Compare with acellular method, allow the early stage evaluation of potential side effect based on guide's optimization method of cell.For example, according to method of the present invention described herein, imatinib shows wide cell-specific breach.This is consistent with the significant advantage of imatinib in the anticarcinogen field.Yet it is not free from side effects.Nearest evidence confirms in the small portion patient imatinib relevant with cardiac toxic people such as (, 2006) Kerkela.This group is taken the longer time of imatinib along with the patient and is increased.

The method of the application of the invention, accompanying drawing 16 show that the imatinib of testing with different concns is at the apparent Cytotoxic IC that is starkly lower than control cells system on wild-type Ba/F3 clone ₅₀Concentration (about 10 μ M) time show slight but significant growth-inhibiting effect, imatinib is showed this cytotoxicity under much higher concentration.When other compounds relatively the effect of control cells system and imatinib are done the time spent to what control cells was, described result becomes more obvious.

In another of this method implemented, think that the compound (as mentioned above) that shows promising activity but have a little CSG value in cell free system has higher potential side effect in the patient, especially treatment period to growing.Other people have reported some target p210Bcr-Abl for example ^T3151Sudden change has compound people such as (, 2005) Carter of low CSG value, and other groups even make these compounds enter clinical trial in addition.Yet,, can expect that the possibility of described compound adverse side effect in the patient increases based on instruction of the present invention.

The preferred drug screening method of the present invention comprises the following steps:

1) identifies concern albumen (POI), for example need the new inhibitor or the theramutein of activator.Usually, relate to POI establishing or keep to relate in the morbid state or suspects, may be because unsuitable expression or the variation of sudden change inductive specific activity.For example, can use standard technique to suitable theramutein identify (referring to, Gorre etc., Science, 2001; In addition, referring to PCT/US02/18729).Briefly, identify activator that uses known or suspicious prototheramutein or the patient that inhibitor is treated effective therapeutic process and shown clinical symptom and the symptom consistent with palindromia subsequently, and obtain to derive from this class patient's cell or tissue.Use the standard laboratory technology, measure the sequence of prototheramutein and compare with the nucleotide sequence of measuring in advance of known prototheramutein gene or cDNA sequence such as RT-PCR.If exist, identify sudden change so and reuse standard method with relevant based on the function resistance foundation of cell or function based on prototheramutein in the pilot system of cell more commonly used.In case confirmed the sudden change of induction of resistance, so described one or more attested mutant comprise can be used for the theramutein that method subsequently as described herein limits.

2) provide and express described POI and show the test cell that can be observed (can the measure) phenotypic characteristic relevant with the expression of described POI.For theramutein, phenotypic characteristic has normally confirmed described phenotypic characteristic in advance to theramutein, or more commonly inhibitor or the activator of corresponding prototheramutein respond.Relevant with the expression of described POI, and will be confirm in advance the inhibitor of the POI (or prototheramutein that is paid close attention to (pTOI)) that paid close attention to or this specific specificity phenotypic characteristic that activator responds are defined as " phenotypic response (phenoresponse) " in this article.One embodiment of the invention are the definite application of described phenotypic response in the purpose of identifying the compound that can become TOI inhibitor or activator.This process can be undertaken by using the high throughput of using the excessive specified TOI of generation and having identified and characterized the clone of suitable phenotypic response to screen.Perhaps, can use the elementary screening of high throughput of the more general clone phenotypic characteristic of application (instruction according to this paper is not decided to be phenotypic response) and use postsearch screening according to the instruction of this paper then, so that distinguish the compound of positive really " hitting " the false positive compound of the inhibitor of the theramutein that pays close attention to from not being or activator, i.e. the inhibitor of the theramutein that is paid close attention to or activator.In one embodiment, select the cell of natural expression theramutein, so that reactive phenotypic characteristic exists under the conspicuous suitable culture condition to those skilled in the art.In other embodiments, theramutein obtains overexpression in some cases in host cell, otherwise this host cell is not expressed theramutein fully.This process generally includes construction of expression vector, this expression vector can be imported proper host cell and use standard vector system and method overexpression (Gorre etc., 2001; Hour ousey etc., 1988).In one embodiment, the theramutein level that produces of overexpression is present in amount in the cell at least about 3 times usually to this protein.Perhaps, this amount is at least about 10 times of the amount that is present in usually in the cell.In another embodiment, this amount is at least about 20 times of the amount that is present in usually in the cell or more preferably is at least about 50 times.

3) if control cells than low degree or do not express POI (for example, the host cell of unmodified or implicit host cell of not expressing the expression vector of POI) fully.For the theramutein that pays close attention to, control cells also can be the cell of expressing corresponding to the prototheramutein of the theramutein that is paid close attention to.

4) when some mutain as herein described also is enzyme, they kept usually catalytic activity and thus control cells show identical with test cell basically phenotypic characteristic usually.But, this phenotypic characteristic does not need to carry out as two kinds of cells quantitatively.For example, the mutain that makes the prototheramutein reactivate also can increase, reduces and even influence its specific activity with regard to it with regard in the substrate in the cell one or more.As a result of, it can show the phenotypic characteristic of selecting big or lesser extent.Therefore, in some cases, need to adjust one of prototheramutein and theramutein or their both expression, so that test and control cells show phenotypic characteristic to approximately uniform degree.For example, all can use standard method,, wherein can adjust the activity (for example, referring to Sambrook etal.1989 and 2001) of promotor by the amount of adjusting the inductor that exists by reaching this purpose by promoter expression protein.

Those skilled in the art are apparent, suitably the phenotypic response of definition in the clone of expressing prototheramutein and theramutein, can exist quantitative difference as its corresponding prototheramutein between the result of specific activity difference (if any).Induce the sudden change of Theramutein can increase or reduce the specific activity of described theramutein for corresponding prototheramutein.When the clone that will express theramutein compared with the clone of expressing prototheramutein, the preferred phenotypic response of selecting was identical qualitatively in two kinds of cell types.Therefore, those skilled in the art can select to express theramutein clone activity and the clone of expressing prototheramutein the activity calibration or vice versa.This class normalization method is standard in the art.For example, referring to (2003) such as Bolstad.

Perhaps, those skilled in the art can also wish to use the host cell of unmodified or only imply expression vector as the host cell that is used for the control cells of some experimental implementation step (host cell produces the cell of test cell for the expression vector that has imported coding theramutein).This may be exactly that the researchist is only interested in specific inhibitor or the activator of the theramutein that paid close attention to, and with described compound the effectively irrelevant situation [replacement work activity] of prototheramutein (pTOI) whether also to being paid close attention to.

4) under appropriate condition, will test then and control cells is kept or made its propagation (but not necessarily at the same time) in growth medium (and even in intact animal body), so that can express and detect phenotypic response.Can handle the control cells of expressing prototheramutein with the known conditioning agent of prototheramutein or with test substances, and with test compounds handle test cell in case determine they whether as described material with what the expectation mode regulated that the ability of phenotypic response measured theramutein is had activity.Perhaps, can also not express the control cells of prototheramutein according to the difference replacement that the particular phenotype that those skilled in the art's selection is used to study is reacted.Detection material is to the effect of test cell then, and randomly simultaneously or in the effect of another time test to control cells, and comparative result.

In one embodiment of the invention, according to regulating the ability Rapid identification of the phenotypic response of test cell test cell is had active material for example to change the identical mode of the phenotypic response of the control cells of expressing prototheramutein with the known conditioning agent of prototheramutein.In another embodiment, can be by the activity of regulating theramutein in the test cell to the control cells of unmodified (not expressing prototheramutein and/or theramutein) almost or the ability that not have to act on come the identified activity material.For example, many versions of these means of those skilled in the art's easy to understand can be used to be accredited as more effective or to the conditioning agent of the corresponding concrete theramuteins equivalences of prototheramutein and one or more to theramutein.

Can observe and/or measure other phenotypic response, and comprise: for example, detect the substrate of prototheramutein and detect the change of passing through the active genetic expression of regulating of theramutein.In the simplest term, the arbitrary characteristics of those skilled in the art's cell with functionally active dependency theramutein that set up in advance are applicable to this class methods.Yet in the process of selecting specific characteristic, those skilled in the art must at first verify that according to the instruction that provides in describing in detail as this paper described feature satisfies the standard as phenotypic response.Those skilled in the art can also wish to use the phenotypic response of the cell of expressing theramutein and the phenotypic response of the cell of expressing prototheramutein to calibrate.

Can measure the feature that is suitable for detecting by the well-known the whole bag of tricks of those skilled in the art.These class methods include but not limited to: the proteinic fluorescence (FACS) that detects suitable mark; Be used to detect the immunohistochemistry (IHC) of protein expression; The competition radioligand is in conjunction with mensuration; The solid-phase matrix engram technology is such as RNA trace, southern blotting technique and the western blotting of cell extract; Reverse transcriptase-polymerase chain reaction (RT-PCR); Enzyme-linked immunosorbent assay (ELISA); Phosphorylation assay; The gel detention is measured; Membrane potential interference etc.Can be at the relevant phenotypic characteristic that detects after the use test mass treatment on the intact cell, the perhaps relevant phenotypic characteristic on the subcellular fraction that detects behind the use test mass treatment intact cell at cell.

In case identified the compound that the test cell of expressing theramutein is had required effect, by direct binding mechanism theramutein is brought into play its effect with regard to verifying (but not necessarily) institute's compounds identified independently, promptly in accordance with the teachings of the present invention, these compounds satisfy the standard (term ＂ activator ＂ that the reader relates to as mentioned above and the definition of ＂ inhibitor ＂) as inhibitor or the activator (as ideal) of theramutein.Can use to well known to a person skilled in the art that a large amount of standard binding assays reach this purpose, comprise the protein example of purifying or use with suitable prototheramutein or theramutein cells transfected and intact cell binding assay as being undertaken by the described suitable reference substance of acoustic science method.Because these class methods are fully to establish in this area, so do not repeat them herein.A large amount of reference text comprehensive discussions this class technology (for example, referring to Foreman and Johansen, 2002; (1991) Current Protocols in Pharmacology such as EnnaS.J., Wiley ﹠amp; Sons, Incorporated; Bonifacino, J.S. etc. (1999) CurrentProtocols in Cell Biology, Wiley ﹠amp; Sons, Incorporated). also referring to Housey, G.M.1988, Chapter 4 and reference wherein; In addition, referring to Horowitz etc., 1981.

In a specific embodiment of the present invention, this method is used to be accredited as p210 ^{Bcr-Abl-T315I}The material of the inhibitor of theramutein.Use standard method in Ba/F3 (mouse) cell, to express prototheramutein and theramutein separately, and observed phenotypic response is growth characteristics (the terminal cell density of careful definite cell culture and in the growth that does not have in the presence of the interleukin 3 (IL-3)).Can also choose the host cell that uses unmodified wantonly or only contain the host cell of expression vector or they both.In another embodiment, with test cell separately with or do not use with the known inhibitor or the activator that relate to.

Another kind of useful assay method is for measuring p210 ^{Bcr-Abl-T315I}The phosphorylation state of direct substrate.A kind of this class substrate is Crkl (Gorre etc., Science 293:876-80 (2001)), the adaptin that is connected between promptly a kind of Bcr-Abl of mediation and the Ras.The phosphorylation state of CRKL is p210 in the cell ^Bcr-AblThe representative of signaling activity.Another kind of downstream substrate is p62DOK.For these purposes, this class substrate all can satisfy arbitrarily, and certainly, condition is that the phosphorylation of verified described substrate takes place at cell interior, and not non-be the autophosphorylation activity of TOI or PTOI simply as mentioned above.Other signal transduction cascade composition be can also monitor, src family kinases, STAT5, PI3 kinases, raf kinases, RAS, MEK, ERK1 and ERK2, JNK1,2 and 3, MLK1,2 and 3, MKK4, MKK7, AKT, mTOR, HSP90 etc. comprised.

As typical case herein, identified the inhibitor of T315I theramutein.In addition, these inhibitor are also to wild-type prototheramutein p210 ^Bcr-Abl-wtHas activity in various degree.

According to the present invention, the Mammals of needs is treated the adjusting p210 of significant quantity ^Bcr-AblOne or more compounds of the functionally active of theramutein.Any means that term used herein " gives " to refer to by the result that can realize seeking is delivered to Mammals with compound of the present invention.For example, can give them by oral, non-enteron aisle (intravenously or intramuscular), part, transdermal or by suction.Term used herein " Mammals " is in order to include, but are not limited to people, laboratory animal, to raise and train pet and farm-animals." treatment significant quantity " refers to and effectively produce required therapeutic action to the Mammals administration time, such as the compound amount that suppresses kinase activity, anticancer growth and division etc.

The invention provides the method for treatment mammalian diseases, undertaken by the conditioning agent that this Mammals is given the theramutein of significant quantity.Include but not limited to administered agents is in advance produced recurrent tumor or other proliferative disorders of resistance according to the suitable disease of the present invention treatment.This method also is used for overcoming the version that there is the susceptibility aspect of the pharmacological agent that the allelotrope difference in the etiological treatment target produces in individuality.For example, extensively confirmed p210 in CML ^Bcr-AblThe effect of tyrosine kinase signal conduction is because p210 ^Bcr-AblTheramuteins in the recurrence of the drug resistance of CML, have effect.In addition, different p210 ^Bcr-AblMutain show p210 ^Bcr-AblThe variable susceptibility of inhibitor.Although some theramuteins occurs in the pharmacotherapy process, other may preexist in the colony.These examples of back are not known as theramuteins, up to morbid state take place thereupon and during subsequently with the treatment of the therapeutical agent of known type till.Only after described treatment, the theramuteins that this class is pre-existing in demonstrates non-relatively-the clinical significance aspect reactive of himself disease progression in the patient who causes implicit theramutein.

In one embodiment of the invention, give theramutein conditioning agent and one or more antineoplastic agents.Can use the antineoplastic agent of any appropriate, such as chemotherapeutics, radiation or its combination.Antitumour drug can be alkylating agent or metabolic antagonist.The example of alkylating agent includes but not limited to cis-platinum, endoxan, melphalan and Dacarbazine.The example of metabolic antagonist includes but not limited to Dx, daunorubicin and taxol, gemcitabine and topoisomerase enzyme inhibitor irinotecan (CPT-11), amino camptothecin, camptothecine, DX-8951f, Hycamtin (topoisomerase I inhibitor) and Etoposide (VP-16; The topoisomerase II inhibitor) and teniposide (VM-26; The topoisomerase II inhibitor).When antitumour drug is when radiation, radioactive source can be at treatment patient's external (external beam radiotherapy-EBRT) or (brachytherapy-BT) in the body.The dosage of the antineoplastic agent that is given depends on many factors, comprising: for example, and the route of administration of the type of promoting agent, the tumor type of being treated and severity and promoting agent.Yet, should emphasize that the present invention is not limited to the combination that any specific dosage, route of administration or merging gives chemotherapeutics or other treatment plan of protein modulators.

Antineoplastic agent known in the art or that estimate can be divided into dissimilarly, comprise: for example, mitotic inhibitor; Alkylating agent; Metabolic antagonist; Embed microbiotic; Growth factor receptor inhibitors; Cell cycle inhibitor; Enzyme; Topoisomerase enzyme inhibitor; Anti-survival agent; Biological response modifier; Hormone antagonist and antiangiogenic agent can be with inhibitor or the activator administration of all these class promoting agents with theramuteins.

The conditioning agent of theramutein can be related to the antibody administration of other acceptor of tumor growth with neutralization.In addition, can be with the conditioning agent of theramutein and the composition of other conditioning signal transduction pathway, preferably with in one or more other signal transduction pathways, having activity and these approach being the composition administration in the common signal transduction pathway.In one embodiment of the invention, the theramutein conditioning agent is combined the receptor antagonist coupling of EGF-R ELISA (EGFR) with specificity.Special preferred antigens is conjugated protein, the extracellular domain of these protein binding EGFR and blocking-up in conjunction with in its part one or more and/or in and part-inductive EGFR activate.The EGFR antagonist can be in conjunction with EGFR or EGFR part and inhibition and EGFR and its part bonded antibody.The example of the part of EGFR comprises: for example, EGF, TGF-α, amphiregulin, heparin-in conjunction with EGF (HB-EGF) and β tunicin.Think that EGF and TGF-α are the main endogenic ligand that causes the stimulation of EGFR-mediation, but, confirmed that TGF-α is promoting blood vessel more effective in taking place.Should understand the EGFR antagonist can suppress externally in conjunction with born of the same parents' outside part of EGFR, also can not suppress the combination of part, or under the situation of chemical active agent, in inside in conjunction with tyrosine kinase domain.Example in conjunction with the EGFR antagonist of EGFR includes but not limited to biotechnological formulation, such as EGFR being had specific antibody (and functional equivalent); And chemical active agent (small molecules), the synthetic kinase inhibitor that directly works such as cytoplasm domain to EGFR.

Other example of the growth factor receptors that relates to during tumour takes place is the acceptor of vascular endothelial growth factor (VEGFR-1 and VEGFR-2), platelet-derived somatomedin (PDGFR), nerve growth factor (NGFR), fibroblast growth factor (FGFR) etc.

In conjoint therapy, before using another kind of promoting agent begin treatment, in the process or give theramutein inhibitor and combination arbitrarily thereof afterwards, promptly before use antitumour drug therapy begins and in the process, before and afterwards, during the course and afterwards or before, after the process neutralization.For example, can be before beginning radiotherapy 1-30 days, preferred 3-20 days, more preferably gave theramutein inhibitor in 5-12 days.In an embodiment preferred of the present invention, before using antibody therapy, with it simultaneously or more preferably give chemotherapy thereafter.

In the present invention, can be used to give theramutein inhibitor of the present invention with the method or the approach of any appropriate, and optional antineoplastic agent and/or other receptor antagonist of giving jointly.The antitumour drug scheme comprises and thinks any scheme that is suitable for treating the patient tumors state of an illness most used according to the present invention.Different malignant tumours may need to use specificity antineoplastic antibody and specificity antineoplastic agent, and this need decide with the different of patient based on the patient.Route of administration comprises: for example, and oral, intravenously, intraperitoneal, subcutaneous or intramuscular administration.The antagonist dosage that gives depends on many factors, comprising: for example, and the route of administration of the type of antagonist, the tumor type of being treated and severity and antagonist.Yet, should emphasize that the present invention is not limited to any specific method and route of administration.

Suitable carriers comprises: for example, and one or more in water, salt solution, phosphate-buffered saline, glucose, glycerine, the ethanol etc. and combination thereof.Carrier may further include minor amounts of auxiliary substances, and such as wetting agent or emulsifying agent, sanitas or buffer reagent, they can increase shelf-life or validity as the theramutein conditioning agent of active ingredient.As well-known in the art, composition can be mixed with making the formulation that active ingredient is quick, continue or delay to discharge after the Mammals administration.

Composition of the present invention can be various forms.They comprise: for example, and solid, semisolid and liquid dosage form, but such as tablet, pill, pulvis, liquor, dispersion liquid or suspension, liposome, suppository, injectable and infusion solution.Preferred formulation depends on specified administering mode and treatment application.

Can prepare this based composition of the present invention according to well-known mode in the pharmacy field.In the process of preparation composition, usually active ingredient is mixed with carrier or be encapsulated in the carrier with the carrier diluted composition and/or with composition, described carrier is as thinner, it can be solid, semisolid or liquid substance, and it plays vehicle, vehicle or the medium of active ingredient.Therefore, composition can be for the powder of tablet, lozenge, sachet, cachet, elixir, suspension, aerosol (as solid or in liquid medium), the ointment that contains the active compound that for example reaches 10% weight, soft hard gelatin capsule, suppository, injection solution, suspension, sterile packed with as topical patch.

Should understand can be with the Mammals administration of method and composition of the present invention to any appropriate, such as rabbit, rat or mouse.More preferably described Mammals is behaved.

Compound of the present invention can also exist as salt.In the context of the present invention, preferably give pharmaceutically acceptable salt.Pharmaceutically acceptable salt refers to the salt of sour addition of The compounds of this invention or the salt of alkali addition, wherein the gained counter ion is interpreted as to be acceptable for medicinal application generally in the art.Pharmaceutically acceptable salt can be the salt of The compounds of this invention and mineral acid or organic acid formation.Preferably obtain salt with mineral acid formation, described mineral acid such as: for example, hydrochloric acid, Hydrogen bromide, phosphoric acid or sulfuric acid, or preferably obtain the salt that forms with organic carboxyl acid or sulfonic acid, described organic carboxyl acid or sulfonic acid such as: for example, acetate, toxilic acid, fumaric acid, oxysuccinic acid, citric acid, tartrate, lactic acid, phenylformic acid or methylsulfonic acid, ethyl sulfonic acid, Phenylsulfonic acid, toluenesulphonic acids or naphthalene disulfonic acid.Pharmaceutically acceptable salt can also be the metal or the ammonium salt of The compounds of this invention.Especially preferably obtain: for example, the salt of sodium, potassium, magnesium or calcium, and especially preferably obtain deriving from the ammonium salt of ammonia or organic amine, described organic amine such as: for example, ethamine, two-or triethylamine, two-or trolamine, dicyclohexyl amine, dimethylaminoethanol, arginine, Methionin, quadrol or 2-phenylethylamine (referring to J.Pharm.Sci.1977 such as Berge, 66,1-19).

In the application's context, with reference to various open source literatures, reference text, textbook, technical manual, patent and patent publication us.With introducing the application in contents intact ground these open source literatures, patent, patent application and other reference teaches and that disclose as a reference, so that more completely describe the state of this area that the present invention relates to.

Should understand and estimate that those skilled in the art can implement the version of the principle of the present invention that this paper discloses and think that this class modification comprises within the scope of the invention.

The following example is further explained the present invention, but should not be considered as limiting by any way scope of the present invention.Common method, such as those be used for carrier and plasmid construction, with the gene of coded polypeptide class insert this class carrier and plasmid, plasmid is imported the detailed description of the expression of host cell and gene and gene product and method for measuring thereof can be available from a large amount of open source literatures, comprise: Sambrook, J etc., (1989) Molecular Cloning:A Laboratory Manual, 2 ^NdEd., Cold Spring hour arbor Laboratory Press; Coligan, J. etc. (1994) Current Protocols in Immunology, Wiley ﹠amp; Sons, Incorporated; Enna, S.J. etc. (1991) Current Protocols inPharmacology, Wiley ﹠amp; Sons, Bonifacino, J.S. etc. (1999) CurrentProtocols in Cell Biology, Wiley ﹠amp; Sons; With U.S.Patent 4,980,281.The reference that all this paper mention is intactly introduced.

Embodiment

Be appreciated that and expect to change under the disclosed herein principle of the present invention of those skilled in the art, and intention comprises these changes within the scope of the present invention.

Enumerating the following embodiment of the present invention should not be construed as to further describe the present invention and limits the present invention by any way.

Embodiment 1: identify protein modulators

P210 ^{Bcr-Abl-T315I}For the restraining effect of imatinib mesylate being produced the p210Bcr-Abl protein (p210 of resistance ^Bcr-Abl) theramutein (Gleevec, STI-571).Sudden change on 315 changes into the Isoleucine residue with Threonine, and is one of observed several sudden changes in resistance or recurrent patient.Yet this specific mutant is this class theramutein of the tool resistance identified.

For being used for overexpression p210 ^{Bcr-Abl-T315I}The Ba/F3 clone that theramutein transforms is measured phenotypic response.Mensuration is with respect to unconverted Ba/F3 cell and express p210 ^Bcr-Abl-wtThe phenotypic response of the Ba/F3 cell of prototheramutein.To be the T315I mutant similarly growing to the cell saturation density of the unconverted Ba/F3 clone that is higher than control group and keeping the ability that the required interleukin (IL-3) of the unconverted Ba/F3 clone of control group is not grown down under the culture condition phenotypic response.According to above-mentioned instruction definition that provides and sign phenotypic response.

The detection system of using is high speed cell imaging and number system, wherein the cell sample volume of 3 μ l is stored, scanned and counting then by 5 μ l optics microcell injections, digital imagery and in electric mode in proper order, all operations all carries out in based on the Controlling System of minicomputer.This system has and carries out direct Cytometric ability to coming from childhood to the sample of the culture of 500 μ l, and provides from the total cell count with statistical significance that contains few culture sample to 12,500 cells.All figure that show cell counting and survival rate test all use data to obtain and analyze this system of usefulness.Carrying out the Cytometric while, this system can also be by distinguishing the raji cell assay Raji general cell survival rate of counting, imaging, and the cell of described counting, imaging is got rid of Trypan Blue (counting ＂ survival ＂ cell) from the cell that absorbs Trypan Blue dyestuff (count ＂ do not survive ＂ cell).Trypan Blue is injected cell sample, after this successively sample is injected microcell so that carry out cell counting and imaging simultaneously at once.

System integration is gone into the workflow of high throughput screening plant so that sensitive and accurate cell counting and cell survival rate pilot system are provided, it more reliably and is not easy to take place the test cell line based on the metabolism survival rate, such as the effect that mixes of XTT or Alamar indigo plant.

At first, at about 113, the 000 kinds of compounds of the concentration screening of 10-20 μ M scope, can influence overexpression p210 by any-mode with general so that identify ^{Bcr-Abl-T315I}The subgroup of the Ba/F3 cell of theramutein (Ba/F3 T315I cell) growth.

Amount to about 11,760 kinds of compounds and show growth-inhibiting effect, think that this is equivalent to about 4500 kinds of different chemical types greater than 50%.Use same cell system to test these compounds again and produced the reactive database of compound, carry out ranking compositor with its classification and according to showing inhibiting those compounds of the highest overall growth then.Then use Ba/F3 T315I as test cell and wild-type Ba/F3 in contrast cell determine based on the pilot system of cell in according to method of the present invention screening 130 kinds of compounds (observed maximum growth-inhibiting effect under based on minimum concentration) of high scoring again from the database of this grade ordering in test compounds.The compound of being paid close attention to is compared difference for those and is suppressed to express p210 with unconverted wild-type Ba/F3 cell ^{Bcr-Abl-T315I}The compound of the Ba/F3 cell growth of theramutein.Evaluation is satisfied 6 kinds of compounds of required standard and is used Ba/F3 p210 ^Bcr-Abl-wtIn further these compounds of detailed analysis of clone (Ba/F3 P210 cell) some.For want of available from chemical supplier additional material and cause a kind of compound can not be used for further test.State in the use clone additionally based on the test of cell with use the people who separates from wild-type P210 Bcr-Abl and P210 T315I mutant kinase domain to recombinate to estimate remaining 5 kinds of compounds independently in the segmental not celliferous purifying protein kinase assay of 120Kd kinase domain of generation.

All 5 kinds of compounds inhibition p210 that all active inhibition is measured as autophosphorylation ^{Bcr-Abl-T315I}The 120Kd activity.Therefore, at 6 kinds in the compound more than 113,000 kinds of screening in the compound of high scoring, in these 6 kinds at least 5 kinds direct inhibition p210 ^{Bcr-Abl-T315I}Mutant.A kind of compound seems to make recombinant protein band drawout on sds page.This situation also is tangible (data not shown) on silver-colored stained gel.Possible situation be this compound in fact can for can covalent cross-linking POI so that suppress its active ＂ suicide ＂ inhibitor lastingly, but this result needs further research.

Instruction as herein described and result provide final evidence jointly, promptly this system can identify inhibitor or the activator of the theramutein of selection, and those skilled in the art recognize that immediately this type systematic just easily is applied to other theramutein or other albumen arbitrarily under the situation of only carrying out conspicuous minimum variant.

Confirm Growth Inhibition that Ba/F3 T315I clone compares with the unconverted Ba/F3 cell of wild-type based on the representational example of the test-results of cell as shown in attached Fig. 1 and 2.These compounds (are not expressed p210 at wild-type Ba/F3 no transformed cells ^Bcr-Abl-wtOr p210 ^{Bcr-Abl-T315I}) the relative impregnable concentration of growth with survival rate under suppress to express the cell growth of T315Itheramutein and reduce its survival rate, and the cell of expressing prototheramutein and theramutein is inhibited basically.In some cases, to the cell inhibiting degree of expressing T315I in addition greater than to the cell inhibiting degree of expressing P210 prototheramutein (for example, referring to accompanying drawing 3, right-hand side, the result of 3 couples of P210 of compound and T315I cell.

Put it briefly, but method provided herein provides the basic development of promotion method form that is used to produce or identifies the conditioning agent of any appointment theramutein.Confirmed to concluding the ability of this method aspect the compound of identifying crucial requirement as a result, these compounds are used for overcoming the acquired drug resistance that becomes to consistence particular type fatefulue and that can not treat at present in some patient colony.In addition, it will be apparent for a person skilled in the art that and to use conspicuous modification that the techniques described herein and method directly are widely used in any possible theramutein or other diseases related protein with clinical meaning.

It should be noted that, 100, more than 000 kind in the preliminary screening of compound, wherein about 10,000 kind of compound shows growth-inhibiting effect to a certain degree, when the method for using this paper to describe in detail was screened the most effective growth-inhibiting substance again, all compounds of having identified 6 kinds of different compounds and test had subsequently all shown in the not celliferous purifying protein kinase assay of using the T315I mutant and have suppressed active (a kind of compound can not be used for further test).Based on the noticeable result of this class, those skilled in the art are immediately clearly based on the suitable selection of the phenotypic response of the instruction in above-mentioned part and the documents that is incorporated herein by reference with determine that this method can be effectively applied to identify Arbitrary proteinInhibitor or activator.For example, those skilled in the art use above-mentioned knowledge easily the design analysis system identify the inhibitor of the theramuteins that derives from other prototheramuteins, known described other prototheramuteins demonstrates the sudden change of giving drug resistance, such as c-kit gene product or Urogastron (EGF) acceptor (EGFR) or platelet-derived somatomedin (PDGF) acceptor α and β.When using this method, being used at its corresponding phenotypic response with regard to method is with regard to the ability of detectable any mammalian cell types any appointment protein (comprising theramutein and prototheramutein) of expressing, and deduction should be without limits.

Embodiment 2: the protein conditioning agent based on phenotypic response is optimized

In this embodiment, to instruct previous compounds identified to carry out being optimized according to the present invention at the activity of selected target protein.Yet different with the method that adopts usually in the art technology, the optimizing process of this paper is also fully by using the cell tests system based on phenotypic response to carry out.Consider thoroughness and in order to confirm that this method optimizes the ability of (refine), adopt the acellular test macro of the target enzyme that reorganization produces also be used for independent confirm based on the cell tests system scoring of phenotypic response for positive described compound really in acellular test macro pattern (it is standard of this area and the enzyme that uses reorganization to produce) also scoring for just.

The Compound C 2 that is accredited as T3151 theramutein inhibitor is at first carried out following new guide's optimizer.Use standard medical chemistry synthetic method is introduced various chemically modifieds in the basic skeleton structure of Compound C 2.In case synthetic, the cell tests system based on phenotypic response in use the foregoing description 1 tests various analogues (chemical variant).

Based on the original structure of Compound C 2, analyzed of the contribution of the phenyl ring of brominated, chlorine and hydroxyl substituent to pharmacological activity.The analogue of synthetic initial series, it is by unsubstituted phenyl ring (C2-01), or is having various substituting groups (for example being positioned at bromine, chlorine and hydroxyl etc. on all places around the phenyl ring) to form on the phenyl ring.Detailed chemical structure is shown in table 3.

Getting these compounds of test in the cell tests system based on phenotypic response then, as shown in Figure 17.Also in the acellular albumen kinases autophosphorylation test of standard, with the concentration of 20 μ M each compound is tested, as shown in Figure 18.Accompanying drawing 17 and accompanying drawing 18 relatively be presented at based on exist significantly between compound activity in the test macro of phenotypic response (cell) and its corresponding activity in acellular purifying protein kinases autophosphorylation test, qualitative fully relevant in essence.

For various medical chemistry purposes and reason outside the scope of the invention, his chemically modified in the above-mentioned enterprising Xingqi of same phenyl ring, but described chemically modified with to p210 ^{Bcr-Abl-T315I}The effectiveness of target strengthen relevant restriction simultaneously and contrast wild-type Ba/F3 non--the transformant cell (do not express p210 ^Bcr-Abl-wtOr p210 ^{Bcr-Abl-T315I}) cross reaction, and improve selectivity, the potential side effect among the patient minimized etc.As accompanying drawing 17 and 18 and table 3 shown in, other compound synthetic and test comprises C2-109, C2-112, C2-122 and C2-128.Figure 17 and 18 be presented at more once more in detail that scoring also represents protein kinase inhibiting activity for all positive compounds in the cell tests system in cell free system, and be that those of non-activity also are non-activity in essence in cell free system in essence in based on the cell tests of phenotypic response.Last these results confirm to show that the result who suppresses active compound and obtain is in full accord in nature in the test of acellular albumen kinases autophosphorylation in based on the test macro of phenotypic response.

In addition, when aspect relative effectivenes, having moderate difference in acellular test result with between based on the test result of phenotypic response (referring to embodiment, Compound C 2-122 seems in acellular test than more effective in the cell system based on phenotypic response), this difference shows with the acellular test macro of classics to be compared, and the ability of effect strengthens in the cell tests system prediction appointed compound body.Use classical acellular screening, people may think that C2-122 is an important compound, yet at once it are got rid of because its effectiveness is lower than the effectiveness of several other compounds of testing based on the test of phenotypic response.In the art, do not report to adopt before and do not depend on and depend on acellular radioligand or other this class guide prioritization scheme in conjunction with the cell system of test.

In a word, the present invention is to those skilled in the art's be provided for that the guide optimizes strong and method fast, and it has replaced acellular repeatedly external confirmation compound to hit necessity of the ability of its respective target.Therefore, optimization method can place one's entire reliance upon in essence based on the test macro result of phenotypic response, has eliminated necessity of confirming acellular test result repeatedly.And described affirmation experiment can be carried out (if those skilled in the art select to do), and method normally there is no need hereto, because the above-mentioned result who provides is unequivocally established.

Those skilled in the art recognize that fully the test macro without any type or character does not have false positive results fully, no matter be radioligand in conjunction with test, ELISA, part in conjunction with test or cell tests.This test macro, although surprising powerful, not the possibility that does not have false positive results, the independence that those skilled in the art understand abnormal results is confirmed only to be good science and should to consider in suitable.

Embodiment 3: based on the sign of the protein conditioning agent of phenotypic response

For its inhibition or the ability that the activates a plurality of different proteins targets test macro that the present invention is based on phenotypic response biological activity of can be used for discerning appointed compound in various degree.For example, in some cases, those skilled in the art may be to identifying or to optimize the proteinic conditioning agent of specified target interested, wherein known other protein be different but with target POI height correlation.Described protein family is by two or more member compositions, and described member has the homology of high level on DNA and amino acid sequence level, yet described family member may have different functions in cell.By the application repeatedly based on the phenotypic response system described herein, people can produce the single test cell of expressing each different family members, and to use three of phenotypic response with corresponding definition or four or how different test cell then be to identify or optimize the compound selectively to a specific family member.

In another embodiment of the invention, those skilled in the art also can select to express two or three or even four different proteins targets and produce and be used for identifying at the single isozyme of the specifying protein family test macro based on phenotypic response of compound optionally not in a single test cell (or test cell system).In some treatment situation, it may be preferred not having selectivity in single family member.For example Ibuprofen BP/EP is low-cost safety and the effective non-steroidal antiinflammatory drugs of having set up, and it does not significantly distinguish cyclooxygenase 1 type (COX-I) and COX-2 family member.Describedly as broad as longly be useful in some cases and may reduce the possibility that some does not wish side effect, describedly do not wish that side effect may take place for the chemical reagent of excessive selection.

For its inhibition or activate the ability of some associated protein target, the biological action of identification appointed compound, no matter the member of the same protein family of described target yes or no also has significant values from molecule and the cytosis mechanism aspect of understanding appointed compound.For example, under the situation of imatinib, not only described compound suppresses P210 Bcr/Abl albumen (p210 ^Bcr-Abl-wt) the wild-type form, it is also with c-kit carcinogenic protein cross action and also can suppress the c-kit carcinogenic protein.As discussed above, under background of the present invention, it is serendipitous that the described cross reaction of kit carcinogenic protein suppresses, because gastrointestinal stromal tumor (GIST) (tumour that a class produces in small intestine) is subjected to the active driving of kit and therefore also treatment with imatinib is had response (NEJMpaper).Therefore, do not need relevant with drug toxicity always with the described cross reactivity of associated protein.In some cases, described cross reactivity can be that treatment is effective.

In the raji cell assay Raji system of other local description (referring to embodiment 1) of this paper, test, and the active classification of regulation as shown in Table I corresponding to the representational compound of the present invention of top specified various chemical formulas.The active classification of regulation is by following appointment representative, the wherein IC of given clone ₅₀Be meant that given compound suppresses this cell line growth and reaches 50% o'clock concentration in the test cell line system.The IC that test represents on the given clone ₅₀The compound of value＜300nM (less than 300 nmoles) is designated as class " A " compound.The IC that test represents on the given clone ₅₀The compound of value＜1 μ M (less than 1 micromole) is designated as class " B " compound.The IC that test represents on the given clone ₅₀The compound of value＜10 μ M (less than 10 micromoles) is designated as class " C " compound.The IC that test represents on the given clone ₅₀The compound of value 〉=10 μ M (more than or equal to 10 micromoles) is designated as class " D " compound.

Table 4

[0100]

Embodiment 4:

1. reaction icon:

Experimental detail: backflow compound 1 (25g) and N, accelerine (24.2g) is at POCl ₃Mixture (110mL) 5 hours.POCl is removed in decompression evaporation down ₃, and carefully pour into resistates in ice-water (500g) and stirred 1 hour.Filtering mixt and wash the compound 2 that solid obtains the yellow solid shape with water then.

2. reaction icon:

Experimental detail: under 10 ℃, in 15 minutes, in 15ml alcoholic acid solution, dropwise add 1.08g (2eq) morphine to compound 2 (1.04g).Stirred the mixture 0.5 hour and 50 ℃ of down heating 15 minutes.After cooling and water (50ml) dilution, filter and obtain the pulverous compound 3 of yellow solid.

3. reaction icon:

Experimental detail: in 1.1g compound 3, add 8mlNH ₂NH ₂H ₂ O.Backflow mixture 2 hours.After the cooling, filter and obtain crude product.Obtain the pure compound 4 of faint yellow solid shape by the column chromatography purifying.

4. reaction icon:

Experimental detail: to compound 5 (1.0g, 1.0eq) and DMF (0.05g, calculated amount) in the solution of 20mL methylene dichloride, dropwise add (COCl) ₂(0.81g, 1.1eq).Stirred reaction mixture concentrated the crude product that obtains 1.2g compound 6 in 2 hours then under the room temperature, used it for next step under not being further purified.

5. reaction icon:

Experimental detail: to the crude product of compound 6 (1.2g, 1.0eq) in the solution of 20mL methylene dichloride, add 3-trifluoromethyl-aniline (0.94g, 1.0eq) and triethylamine (0.71g, 1.2eq).Stirred reaction mixture spends the night under the room temperature, with 1N NaOH solution, 1N HCl solution and salt water washing.Collected organic layer is used Na ₂SO ₄Drying concentrates the crude product that obtains compound 7.Behind the column chromatography purifying, obtain 1.1g compound 7.

6. reaction icon:

Experimental detail: to compound 7 (0.3g, 1.0eq) and add in the mixture of 3mL trifluoroacetic acid vulkacit H (0.53g, 4.0eq).At once the sealed reaction mixture and be heated to 90 ℃ 20 hours.After the cooling, to pH8,, concentrate and obtain brown solid with dichloromethane extraction and dry organic phase with 1N NaOH solution conditioned reaction mixture.Obtain the compound 8 of yellow solid shape by preparation type TLC purifying.

7. reaction icon:

Experimental detail: (30mg is 1.0eq) with compound 8 (19mg, 1.0eq) mixture overnight in the 5mL methylene dichloride for agitate compounds 4 under the room temperature.Collecting precipitation is also used the methylene dichloride thorough washing, the dry compound that obtains needs under the vacuum.

Embodiment 5:

1. reaction icon:

Experimental detail: to compound 1 (1.0g, 1.0eq), compound 2 (0.92g, 1.0eq), Na ₂CO ₃(0.77g 1.5eq) adds Pd (PPh in the mixture of 15mL diox ₃) ₄(0.56g, 0.1eq), at N ₂Following reaction mixture refluxed 16 hours.After the cooling, filtering mixt also evaporates filtrate to doing, and obtains compound 3 by the column chromatography purifying.

2. reaction icon:

Experimental detail: to compound 3 (0.3g, 1.0eq) and add in the mixture of 3mL trifluoroacetic acid vulkacit H (0.62g, 4.0eq).At once the sealed reaction mixture and be heated to 90 ℃ 20 hours.After the cooling, to pH 8,, concentrate and obtain brown solid with dichloromethane extraction and dry organic phase with 1N NaOH solution conditioned reaction mixture.Obtain the compound 4 of yellow solid shape by preparation type TLC purifying.

3. reaction icon:

Experimental detail: (30mg is 1.0eq) with compound 5 (19mg, 1.0eq) mixture overnight in the 5mL methylene dichloride for agitate compounds 4 under the room temperature.Collecting precipitation is also used washed with dichloromethane, and drying obtains required compound under the vacuum.

Embodiment 6.

1. reaction icon:

Experimental detail: (0.6g 1.0eq) adds 4mL 1N NaOH solution in the solution of 10mL methyl alcohol, stir the mixture under the room temperature and spend the night to compound 1.Evaporating solvent and with 5% citric acid acidifying resistates to pH6, use dichloromethane extraction.Dry organic layer concentrates and obtains compound 2.

2. reaction icon:

Experimental detail: agitate compounds 2 under the room temperature (0.4g, 1.0eq), 5-trifluoromethylaniline (0.39g, 1.0eq), EDC (0.71g, 1.5eq), HOBt (33mg, 0.1eq) mixture overnight in the 10mL methylene dichloride.With 1N NaOH solution, water washing mixture, use dichloromethane extraction.Use Na ₂SO ₄Dry organic layer is concentrated into driedly, and obtains compound 3. by the column chromatography purifying

3. reaction icon:

Experimental detail: handle compound 3 (0.2g, the 1.0eq) solution in the 10mL diox, and with mixture heating up to 60 ℃ 2 hours with 4mL1N HCl.After the cooling, add NaHCO ₃With pH regulator to 8.Use the dichloromethane extraction mixture, wash organic layer with water, use Na ₂SO ₄Drying also is evaporated to dried.Obtain compound 4 by column chromatography purifying crude product.

4. reaction icon:

Experimental detail: (40mg is 1.0eq) with compound 5 (30mg, 1.0eq) mixture overnight in the 5mL methylene dichloride for agitate compounds 4 under the room temperature.Enriched mixture to dry doubling obtains required compound by the preparation HPLC purifying.

Embodiment 7.

1. reaction icon:

Experimental detail: agitate compounds 2 under the room temperature (0.3g, 1.0eq), 2-chloro-6-methyl-aniline (0.26g, 1.0eq), EDC (0.53g, 1.5eq), HOBt (25mg, 0.1eq) mixture overnight in the 10mL methylene dichloride.With 1N NaOH solution, water washing mixture, and use dichloromethane extraction.Use Na ₂SO ₄Dry organic layer is concentrated into driedly, obtains compound 3 by the column chromatography purifying.

2. reaction icon:

Experimental detail: handle compound 3 (0.2g, the 1.0eq) solution in the 10mL diox, and with mixture heating up to 60 ℃ 2 hours with 4mL 1N HCl.After the cooling, by adding NaHCO ₃With pH regulator to 8.With the dichloromethane extraction mixture and wash organic layer with water, use Na ₂SO ₄Drying also is evaporated to dried.Obtain compound 4 by column chromatography purifying crude product.

3. reaction icon:

Embodiment 8.

1. reaction icon:

Experimental detail: in the solution of 100ml toluene, add 3eqt-BuONa, 0.2eq BINAP and 0.1eq Pd to 1g (5.5mmol) 5-bromo-2-cyanopyridine, 0.97g (6.05mmol1.1eq) 3-(trifluoromethyl) aniline ₂(dba) ₃This solution of reflux spends the night then.By the LC/MS monitoring reaction.Remove volatile matter under the decompression.Obtain compound 2 by the purification by flash chromatography crude product.

2. reaction icon:

Experimental detail: 250mg (0.95mmol) compound 2 is added among the dense HCl of 20mL, and this solution of reflux disappears up to original material then.Do not having under the situation of purifying, the concentrating under reduced pressure mixture obtains the compound 3 of yellow solid shape.

3. reaction icon:

Experimental detail: the solution of 50mg (0.18mmol) compound 3 in the 3mL methylene dichloride is added in the 0.5mL thionyl chloride.Heated mixt also stirred 3 hours.Last reduction vaporization solution.Obtain compound 4 and do not having to be used for next step under the purifying.

4. reaction icon:

Experimental detail: stirred 50mg compound 4 and the solution of 43mg (1.2eq) hydrazine in 5mLDCM 3 hours down at 25 ℃.Concentrated reaction mixture also obtains required compound by preparation HPLC purifying resistates.

Embodiment 9.

1. reaction icon:

Experimental detail: reflux compound 1 (25g, 0.145mol) and SeO ₂(27.5g, 0.247mol) suspension in acetate (1200mL) is 12 hours.The concentrating under reduced pressure reaction mixture is to doing.The dissolving resistates is also by adding K in water ₂CO ₃Make pH=9.With EA (100mL * 3) extraction gained mixture.Use Na ₂SO ₄The dry EA that merges.Filter out Na ₂SO ₄After, decompression thickening filtration thing down obtains crude product 2, is not having to use it for next step under the purifying.

2. reaction icon:

Experimental detail: 2 solution 4 hours in ethanol triethyl orthoformate (10mL) of the above-mentioned preparation that refluxes.Except that after desolvating, separate resistates by post and obtain oily product 3.

3. reaction icon:

Experimental detail: at N ₂Under the atmosphere, to compound 3 (73mg, 0.28mmol) and compound 4 (50mg, 0.28mmol), and tBuONa (27mg, 0.56mmol) and BINAP (70.4mg 1.12mol) adds Pd in the mixture of the stirring of toluene (15mL) and the degassing ₂(dba) ₃(26mg 0.028mmol) and at 80 ℃ stirred 48 hours.After filtering out solid, the thickening filtration thing obtains crude product 5, is not having to use it for next step under the purifying.

4. reaction icon:

Experimental detail: at-30 ℃ and N ₂Under the atmosphere, use BBr ₃(146mg, (335mg, the 0.1mmol) solution in methylene dichloride (10mL) stirred 4 hours under the room temperature then 0.6mmol) to handle compound 5.Reactant poured into add Na in ice-water then ₂CO ₃With dichloromethane extraction (25mL * 3) gained mixture, use Na ₂SO ₄The dry organic layer that merges.Filter out Na ₂SO ₄After, the thickening filtration thing obtains crude product 6, is not having to use it for next step under the purifying.

5. reaction icon:

Experimental detail: (27.74mg, 0.1mmol) (21mg is 0.1mmol) at anhydrous CH with compound 7 for agitate compounds 6 under refluxing ₂Cl ₂Solution (300mL) 6 hours.Decompression removes down and desolvates.Separate resistates by preparation-TLC and obtain required compound.

Embodiment 10.

1. reaction icon:

Experimental detail: at N ₂Under the atmosphere, (5g, 25mmol) (3.4g is 27mmol) at DMF (50mL) and Na with compound 2 to compound 1 ₂CO ₃(20mL adds Pd in stirring 2M) and the mixture of the degassing to the aqueous solution ₂(dbbf) ₃(26mg 0.028mmol), and stirred 18 hours under 100 ℃.Cool to room temperature and filter out solid after, with EA (200mL) extraction filtrate.Concentrate organic layer to doing.Obtain crude product 3 by the column purification resistates.

2. reaction icon:

Experimental detail: reflux compound 3 (2g, 0.1mol) and Na ₂S ₂O ₄(5.2g is 0.3mol) at methyl alcohol (80mL) and H ₂Mixture among the O (20mL) 3 hours.Decompression concentration response thing is down extremely done.The dissolving resistates is used EA (150mL) extraction then in water.With twice of salt water washing organic layer and use Na ₂SO ₄Dry.Filter Na ₂SO ₄After, the thickening filtration thing obtains product 4.

3. reaction icon:

Experimental detail: at N ₂Under the atmosphere, to compound 5 (228mg, 88mmol) and compound 4 (150mg, 88mmol), t BuONa (170mg, 176mmol) and BINAP (210mg 176mmol) adds Pd in the mixture of the stirring of toluene (25mL) and the degassing ₂(dba) ₃(79mg 0.88mmol), and stirred 48 hours under 80 ℃.After filtering out solid, the thickening filtration thing obtains crude product 6.

4. reaction icon:

Experimental detail: at-30 ℃ and N ₂Under the atmosphere, use BBr ₃(600mg, (140mg, the 4mmol) solution in methylene dichloride (20mL) at room temperature stirred 4 hours then 10.6mmol) to handle compound 6.Reactant is poured in ice-water then by adding Na ₂CO ₃Make pH=9.With the mixture of dichloromethane extraction (25mL * 3) gained, use Na ₂SO ₄The dry organic layer that merges.Filter out Na ₂SO ₄After, the thickening filtration thing is to doing.Obtain product 7 by the column purification resistates.

5. reaction icon:

Experimental detail: (98mg, 0.36mmol) (64mg is 0.3mmol) at anhydrous CH with compound 8 for agitate compounds 7 under refluxing ₂Cl ₂Solution (300mL) 6 hours.Decompression removes down and desolvates.Separate resistates by preparation-TLC and obtain required compound.

Embodiment 11.

1. reaction icon:

Experimental detail: at N ₂Under the atmosphere, (5.9g, 25mmol) (3.3g is 27mmol) at DMF (50mL) and Na with compound 2 to compound 1 ₂CO ₃(20mL adds Pd in stirring 2M) and the mixture of the degassing to the aqueous solution ₂(dbbf) ₃(26mg 0.028mmol), and stirred 18 hours under 100 ℃.Cool to room temperature and filter out solid after, with EA (200mL) extraction filtrate.Concentrated organic layer obtains crude product 3, is not using it for next step under the purifying.

2. reaction icon:

Experimental detail: under room temperature and nitrogen atmosphere (20psi), stir 3 (6.5g, 27.9mmol) and Pd (OH) ₂(10%, 0.5g) mixture in ethanol (200mL) is 2 hours.Filter out catalyzer, remove filtrate under the vacuum and obtain colorless oil product 4.

3. reaction icon:

Experimental detail: at N ₂Under the atmosphere, to compound 4 (406mg, 20mmol) and compound 5 (520mg, 20mmol), and tBuONa (170mg, 176mmol) and BINAP (210mg 176mmol) adds Pd in the mixture of the stirring of toluene (25mL) and the degassing ₂(dba) ₃(79mg 0.88mmol), and stirred 48 hours under 80 ℃.After filtering out solid, the thickening filtration thing obtains crude product 6, is not using it for next step under the purifying.

4. reaction icon:

Experimental detail: at-30 ℃ and N ₂Under the atmosphere, use BBr ₃(600mg, (383mg, the 10mmol) solution in methylene dichloride (20mL) stirred 4 hours under the room temperature then 10.6mmol) to handle compound 6.Reactant is poured in ice-water, then by adding Na ₂CO ₃Make pH 9.With dichloromethane extraction (25mL * 3) gained mixture, and use Na ₂SO ₄The dry organic layer that merges.Filter out Na ₂SO ₄After, the thickening filtration thing is to doing.Obtain product 7 by the column purification resistates.

5. reaction icon:

Experimental detail: (60mg, 0.22mmol) (33mg, 0.15mmol) mixture in methylene dichloride (10mL) is 3 hours with nicotinoyl aldehyde for reflux 7.Except that after desolvating, obtain required compound by residue purified by chromatography.

Embodiment 12.

1. reaction icon:

Experimental detail: stir under the room temperature DMAP (9.3g, 0.077mol), compound 1 (10g, 0.051mol) and Boc ₂(12g, 0.051mol) solution in tBuOH (200mL) spends the night O.Decompression removes down desolvates and by flash chromatography purifying resistates on silica gel.(ethyl acetate/petroleum ether=10:1) obtains 2 of colorless oil.

2. reaction icon:

Experimental detail: stir 2 down in room temperature and nitrogen atmosphere (50psi) (6.17g, 27.9mmol) and Pd (OH) ₂(10%, 1g) mixture in ethanol (200mL) is 4 hours.Filter out catalyzer, remove the product 3 that filtrate obtains colorless oil under the vacuum.

3. reaction icon:

Experimental detail: at N ₂Following heating compound 3 (2.65g, 12mmol), compound 4 (2.6g, 10mmol), tBuONa (1.34g, 14mmol), Pd ₂(dba) ₃(46.5mg, 50mmol) and DCHPB (70mg, 0.2mmol) mixture in dry toluene (50mL) to 80-90 ℃ 24 hours.Filtering-depositing is also removed filtrate under vacuum, by chromatogram on silica gel the purifying resistates (ethyl acetate/petroleum ether=10:1) obtains 5 of yellow oily.

4. reaction icon:

Experimental detail: under 0 ℃, (0.9g is 2.24mmol) at CHCl to 5 ₃Add CF in the solution (50mL) ₃COOH (40mL).After addition is finished, at room temperature stir the mixture overnight of gained.Decompression removes down and desolvates to doing.The recrystallization resistates obtains pale solid from ether.This solid of dissolving in ammoniacal liquor (10mL).Make the pH=7.0 of mixture also precipitate by adding 1M HCl.The collecting precipitation thing is also used cold water (5mL) washing, and decompression drying down obtains 6 of black solid.

5. reaction icon:

Experimental detail: (60mg, 0.22mmol), (33mg, 0.15mmol) mixture in methylene dichloride (10mL) is 3 hours for nicotinoyl aldehyde for reflux 6.Except that after desolvating, obtain the required compound of yellow solid shape by residue purified by chromatography.

Embodiment 13.

1. reaction icon:

Experimental detail: at N ₂Under the atmosphere, to compound 1 (6.7g, 25mmol), (4.1g is 27mmol) at DMF (50mL) and Na for compound 2 ₂CO ₃(20mL adds Pd in stirring 2M) and the mixture of the degassing to the aqueous solution ₂(dbbf) ₃(26mg 0.028mmol), and stirred 18 hours under 100 ℃.Behind the cool to room temperature and filter out solid, with EA (200mL) extraction filtrate.Concentrated organic layer obtains crude product 3.

2. reaction icon:

Experimental detail: under room temperature and nitrogen atmosphere (20psi), stir 3 (3.2g, 10mmol) and Pd (OH) ₂(10%, 0.5g) mixture in ethanol (200mL) is 2 hours.Filter out catalyzer, remove filtrate under the vacuum and obtain colorless oil product 4.

3. reaction icon:

Experimental detail: at N ₂Under the atmosphere, to compound 4 (297mg, 10mmol) and compound 5 (259mg, 10mmol), and tBuONa (170mg, 17.6mmol) and BINAP (210mg 17.6mmol) adds Pd in the mixture of the stirring of toluene (25mL) and the degassing ₂(dba) ₃(79mg 0.88mmol), and stirred 48 hours under 80 ℃.After filtering out solid, the thickening filtration thing obtains crude product 6, is not having to use it for next step under the purifying.

4. reaction icon:

Experimental detail: at-30 ℃ and N ₂Under the atmosphere, use BBr ₃(600mg, (446mg, the 10mmol) solution in methylene dichloride (20mL) at room temperature stirred 4 hours then 10.6mmol) to handle compound 6.Reactant is poured in ice-water then by adding Na ₂CO ₃Make pH=9.With the mixture of dichloromethane extraction (25mL * 3) gained, use Na ₂SO ₄The dry organic layer that merges.Filter out Na ₂SO ₄After, the thickening filtration thing is to doing.Obtain product 7 by the column purification resistates.

5. reaction icon:

Experimental detail: (67mg, 0.15mmol), (33mg, 0.15mmol) mixture in methylene dichloride (10mL) is 3 hours for nicotinoyl aldehyde for reflux 7.Except that after desolvating, obtain the required compound of yellow solid shape by residue purified by chromatography.

Embodiment 14.

1. reaction icon:

Experimental detail: at N ₂Under the atmosphere, to compound 1 (3g, 0.02mol) and compound 2 (3.4g, 0.02mol), and KOH (5.28g, 0.1mol) and TBBA (6.44g 0.02mol) adds Pd (PPh in the mixture of the stirring of anhydrous THF (100mL) and the degassing ₃) ₄(2.31g 2mmol), and refluxes and stirred 12 hours down.After filtering out solid, the thickening filtration thing is to doing.Obtain product 3 by the column purification resistates, do not using it for next step under the purifying.

2. reaction icon:

Experimental detail: at N ₂Under the atmosphere, to compound 3 (334mg, 1.7mmol) and compound 4 (434mg, 1.7mmol), and t-BuONa (322mg, 3.4mmol) and BINAP (420mg 0.67mol) adds Pd in the mixture of the stirring of toluene (60mL) and the degassing ₂(dba) ₃(156mg 0.017mmol), and stirred 48 hours under 80 ℃.After filtering out solid, the thickening filtration thing is to doing.Obtain product 5 by the column purification resistates.

3. reaction icon:

Experimental detail: at-30 ℃ and N ₂Under the atmosphere, use BBr ₃(393mg, (100mg, the 0.3mmol) solution in methylene dichloride (10mL) stirred 4 hours under the room temperature then 0.6mmol) to handle compound 5.Reactant poured into add Na in ice-water then ₂CO ₃With the mixture of methylene dichloride (25mL * 3) extraction gained, use Na ₂SO ₄The dry organic layer that merges.Filter out Na ₂SO ₄After, the thickening filtration thing obtains crude product 6.

4. reaction icon:

Experimental detail: reflux 6 (39mg, 0.13mmol), the mixture of nicotinoyl aldehyde (29mg, 0.13m mol) in methylene dichloride (10mL) 3 hours.Except that after desolvating, obtain the required compound of yellow solid shape by residue purified by chromatography.

Embodiment 15.

1. reaction icon:

Experimental detail: (1.8g, (1.0g 5.9mmol) in the solution of acetate (40mL), adds NaCNBH down at 10 ℃ then 59.3mmol) to add compound 1 to Paraformaldehyde 96 ₃(1.8g, 28.8mmol).At room temperature stirred 16 hours, solution is poured in ice/water (100mL), regulate PH to 10 with dense NaOH.(3 * 100mL) extract this solution to DCM.Dry organic layer (the MgSO that merges ₄), filter and the concentrated compound 2 that obtains under vacuum.

2. reaction icon:

Experimental detail: under 1atm hydrogen, use Pd/C hydrogenase 10 .84g (4.3mmol) compound 216 hours.Be not further purified down, filter reaction mixture and thickening filtration thing obtain compound 3.

3. reaction icon:

Experimental detail: under 150 ℃ and microwave, make 250mg (1.51mmol) compound 3,0.2eq BINAP, 0.1eq Pd ₂(dba) ₃, 3eq Cs ₂CO ₃With 5-bromo-2-diethoxymethyl-pyridine (0.783g, 3.01mmol) at 10mL 1, the solution reaction of 4-diox 2 hours.By the LC-Ms monitoring reaction.Enriched mixture also obtains compound 4 by preparation type TLC purifying resistates.

4. reaction icon:

Experimental detail: in the solution of 5mL DCM, add 4mL TFA to 300mg (0.55mmol) compound 4.Stirred reaction mixture is 30 minutes under the room temperature.Add ice/water and pass through NaHCO to mixture ₃Be basified to PH=10, with DCM (15mL*3) extraction.The organic layer that water and salt water washing merge is used MgSO ₄Drying is filtered and the concentrated compound 5 that obtains.

5. reaction icon:

Experimental detail: (125mg, 0.59mmol) solution in 10mL DCM is 15 hours with (5-fluoro-4-morpholinyl-4-base-pyrimidyl-2-yl)-hydrazine to stir down 80mg (0.295mmol) compound 5 at 25 ℃.Concentrated reaction mixture also obtains compound 6 by preparation HPLC purifying resistates.

6. reaction icon:

Experimental detail: under 0 ℃, to compound 6 (40mg, 0.086mmol) in the solution of dry DCM (5mL), dropwise add BBr (22mg, 0.258mmol).Stirred reaction mixture is 3 hours under the room temperature.With methyl alcohol cancellation reaction, and enriched mixture.Obtain required compound by preparation HPLC purifying resistates.

Embodiment 16.

1. reaction icon:

Experimental detail: at N ₂Pd (the PPh that in the solution of 30ml toluene, adds 0.leq under the atmosphere to the 3-bromaniline (0.86g) that stirs ₃) ₄, the saturated Na of 5ml ₂CO ₃The aqueous solution and the solution of 3-anisole ylboronic acid (0.75g) in 10ml EtOH.Vigorous stirring mixture 15 hours and cooling add 10ml H under refluxing ₂O, and use CH ₂Cl ₂(20ml * 3) extraction mixture.Use Na ₂SO ₄The dry organic layer that merges also concentrates.Obtain pure compound 2 by column chromatography (PE/EA=10:1) purifying resistates.

2. reaction icon:

Reaction details: under 150 ℃, make compound 2 (59.5mg), 5-bromo-2-diethoxymethyl-pyridine (116.7mg), t-BuONa (86.4mg), BINAP (36.7mg) and Pd ₂(dba) ₃(27.4mg) the mixture in the Zai diox (2ml) carried out microwave treatment 2 hours, and filtering solution also concentrates.Obtain compound 3 by preparation type TLC purifying resistates.

3. reaction icon:

Reaction details: in the solution of 5ml DCM, add 1ml BBr to compound 3 (120mg) ₃, stirring reaction spends the night under the room temperature.In mixture, add 5ml H then ₂O is with EtOAc extraction and concentrated.Obtain compound 4 by preparation type TLC purifying crude product.

4. reaction icon:

Experimental detail: stir 43.5mg compound 4 and the mixture overnight of 32mg hydrazine in 5ml DCM and concentrated under the room temperature.Obtain required compound by preparation type TLC purifying crude product.

Embodiment 17.

1. reaction icon:

Experimental detail: under 100 ℃, make 2.0g (16.2mmol, 1.0eq) compound 1,0.05eq BINAP, 0.05eq Pd ₂(dba) ₃, (1.0eq) solution reaction in the 20mL dry toluene is 24 hours for 3.28g, 16.2mmol for 1.2eq t-BuONa and 1-bromo-3-nitro-benzene.By the LC-MS monitoring reaction.Enriched mixture also obtains compound 2 by column chromatography purifying resistates.

2. reaction icon:

Experimental detail: under 1atm hydrogen, use Pd/C (0.25g) hydrogenation 2.5g compound 216 hours.Filter reaction mixture, the thickening filtration thing obtains compound 3 not being further purified down.

3. reaction icon:

Experimental detail: under 100 ℃, make 500mg (2.33mmol) compound 3,5-bromo-2-diethoxymethyl pyridine (607mg, 2.33mmol), 0.05eq xantphos, 0.05eq Pd ₂(dba) ₃Refluxed 24 hours with the solution of 1.5eqt-BuONa in 10mL toluene.By the LC-MS monitoring reaction.Enriched mixture also obtains compound 4 by column chromatography purifying resistates.

4. reaction icon:

Experimental detail: handle 100mg compound 4 with 1mL HCl (the 1N aqueous solution) and 10mL diox.Stirred the mixture under the room temperature 4 hours, and be adjusted to pH 8-9 with 0.5N NaOH solution.After the DCM extraction, use Na ₂SO ₄Dry organic layer and the concentrated compound 5 that obtains.

5. reaction icon:

Experimental detail: (50mg, 0.23mmol) solution in 5mL DCM is 15 hours with (5-fluoro-4-morpholinyl-4-yl pyrimidines base-2-yl)-hydrazine to stir down 50mg (0.16mmol) compound 5 at 25 ℃.Concentrated reaction mixture also obtains compound 6 by preparation HPLC purifying resistates.

6. reaction icon:

Experimental detail: (50mg 0.09mmol) dropwise adds BBr in the solution of dry DCM (5mL) to compound 6 under 0 ℃ ₃(20mg, 0.25mmol).Stirred reaction mixture is 3 hours under the room temperature.With methyl alcohol cancellation reaction, enriched mixture then.Obtain required compound by preparation HPLC purifying resistates.

Embodiment 18.

1. reaction icon:

Experimental detail: heat 10g (70.92mmol) 3-fluoro-oil of mirbane, 1eq imidazoles and 2eqK down at 130 ℃ ₂CO ₃Solution in 100ml DMSO 5 hours.By the LC-MS monitoring reaction.Add 500mL water and filtering-depositing then, under not being further purified, wash this solid and drying with water and obtain compound 2.

2. reaction icon:

Experimental detail: under 1atm hydrogen with 2 half an hour of Pd/C hydrogenation 5g (31.4mmol) compound.Be not further purified down, filter reaction mixture and thickening filtration thing obtain compound 3.

3. reaction icon:

Experimental detail: under 130 ℃, backflow 500mg (3.14mmol) compound 3,0.1eqxantphose, 0.1eq Pd ₂(dba) ₃With the solution of 1.5eq t-BuONa in 10mL toluene 15 hours.By the LC-Ms monitoring reaction and wash with water, extract with EtOAc.The organic layer that merges with the salt water washing is also used MgSO ₄Dry.Filter and concentrate, obtain compound 4 by preparation type TLC purifying resistates.

4. reaction icon:

Experimental detail: at 5mL 1, add 8mL4N HCl in the solution of 4-diox and descend heating 2 hours at 80 ℃ to 200mg compound 4.Extract to ph=10 and with DCM (15mL*3) by 2N NaOH quaternization mixture.The organic layer that water and salt water washing merge is also used MgSO ₄Drying is filtered and the concentrated 250mg crude product that obtains.Obtain compound 5 by preparation type TLC purifying crude product.

5. reaction icon:

Experimental detail: stirred 80mg compound 5 and the solution of 1eq compound 6 in 5mLDCM 15 hours down at 25 ℃.Concentrated reaction mixture also obtains required compound by preparation HPLC purifying resistates.

Embodiment 19.

1. reaction icon:

Experimental detail: at 130 ℃ of following backflow 1.50g1-bromo-3-methyl-5-nitro-benzene and 1.60g (1.2eq) piperazine-1-t-butyl formate, 0.1eq xantphos, 0.1eq Pd ₂(dba) ₃With the solution of 1.5eq t-BuONa in 20mL toluene 4 hours.By the LC-Ms monitoring reaction, wash with water and extract with EtOAc.The organic layer that merges with the salt water washing is also used Na ₂SO ₄Dry.Filter and concentrate, by column chromatography purifying resistates (adopting 10:1 PA:EA) on silica gel as eluent.Merge suitable fraction and concentrating under reduced pressure and obtain intermediate 1.

2. reaction icon:

Experimental detail: under 1atm hydrogen, use Raney/Ni hydrogenation 1.6g intermediate 12 hours.Be not further purified down, filter reaction mixture and thickening filtration thing obtain intermediate 2.

3. reaction icon:

Experimental detail: at 130 ℃ of following backflow 1.20g intermediates 2,1.30g (1.20eq) 5-bromo-2-diethoxymethyl-pyridine, 0.1eq xantphose, 0.1eq Pd ₂(dba) ₃With the solution of 1.5eq t-BuONa in 20mL toluene 4 hours.By the LC-Ms monitoring reaction, wash with water and extract with EtOAc.The organic layer that merges with the salt water washing is also used Na ₂SO ₄Dry.Filter and concentrate, with column chromatography purifying resistates (adopting 4:1 PA:EA) on silica gel as elutriant.Merge suitable fraction and under reduced pressure concentrate and obtain intermediate 3.

4. reaction icon:

Experimental detail: dissolving 200mg intermediate 3 in 10ml DCM.130mg TFA dropwise joined under reaction mixture solution and the room temperature stirred 3 hours.Reaction mixture NaHCO ₃Solution is basified to neutrality, and Na is used in the salt water washing ₂SO ₄Drying is filtered and the concentrated 220mg crude product that obtains.Obtain intermediate 4 by preparation type TLC purifying crude product.

5. reaction icon:

Experimental detail: stirred 50mg intermediate 4 and 1.0eq (5-fluoro-4-morpholinyl-4-yl pyrimidines base-2-yl)-solution of hydrazine in 5mL DCM under the room temperature 3 hours.Water and salt solution washing reaction mixture concentrate and to obtain resistates and to obtain required compound by the preparation HPLC purifying.

Embodiment 20.

1. reaction icon:

Experimental detail: under 0 ℃ with 15ml1M BH ₃/ THF dropwise joins 3g (13.95mmol) 4-bromo-2-methyl-phenylformic acid in the solution of 20ml THF.Make reaction soln arrive that room temperature continued 1 hour and by dropwise adding 50ml50%THF aqueous solution cancellation reaction.Use Na ₂CO ₃Treating mixture also concentrates.Use Et ₂The O extracted residues.Dry organic layer obtains compound 2.

2. reaction icon:

Experimental detail: in the solution of 20ml DCM, add the soup compound of 5.1g (23.8mmol) PCC in 60ml DCM to 2.4g (11.9mmol) compound 2.Stirring reaction solution is 1 hour under the room temperature, uses 300ml Et ₂The O dilution is also filtered.The thickening filtration thing obtains compound 3.

3. reaction icon:

Experimental detail: 2.1g (14mmol) solution is joined in the ethanolic soln that contains 1.9g (9.55mmol) compound 3.Heating reflux reaction solution 3 hours concentrates then.Use NaHCO ₃The washing solid is also used ethyl acetate extraction.Dry organic layer obtains compound 4.

4. reaction icon:

Experimental detail: with 0.7g compound 4,0.41g 3-trifluoromethyl-aniline 0.32gPd ₂(dba) ₃, 0.21g binap and 0.02g t-BuONa add in the 35ml toluene.Heating reflux reaction solution spends the night, and concentrates.(ethyl acetate/hexane=1:1) obtains compound 5 by column chromatography purifying crude product.

5. reaction icon:

Experimental detail: handle compound 5 (0.2g, the 1.0eq) solution in the 10mL diox, and heated mixt to 60 ℃ 2 hours with 4mL 1N HCl.After the cooling, by adding NaHCO ₃Regulate pH to 8.Use the dichloromethane extraction mixture, wash organic layer with water, use Na ₂SO ₄Drying also is evaporated to dried.Obtain compound 6 by column chromatography purifying crude product.

6. reaction icon:

Experimental detail: stirred 80mg compound 6 and the solution of 1eq compound 7 in 5mLDCM 15 hours down at 25 ℃.Concentrated reaction mixture also obtains required compound by preparation HPLC purifying resistates.

Embodiment 21.

1. reaction icon:

Experimental detail: to compound 1 (0.5g, 1.0eq), the 3-trifluoromethyl phenyl boronic acid (0.63g, 1.0eq), Na ₂CO ₃(0.46g 1.5eq) adds Pd (PPh in the mixture of 15mL diox ₃) ₄(0.33g, 0.1eq), and at N ₂Following reaction mixture refluxed 16 hours.After the cooling, filtering mixt also evaporates filtrate to doing, and obtains compound 2 by the column chromatography purifying.

2. reaction icon:

Experimental detail: at N ₂Next time fluidisation compound 2 (0.2g, 1.0eq), SeO ₂(0.19g, 2.0eq) mixture in 10mL acetate is 48 hours.By evaporating except that desolvating and in water, dissolving resistates, with saturated NaHCO ₃Solution is adjusted to pH6, uses dichloromethane extraction.Collected organic layer, drying also is evaporated to dried.Obtain compound 3 by column chromatography purifying crude product.

3. reaction icon:

Experimental detail: (30mg is 1.0eq) with compound 4 (19mg, 1.0eq) mixture overnight in the 5mL methylene dichloride for agitate compounds 3 under the room temperature.Enriched mixture to dry doubling obtains required compound by the preparation HPLC purifying.

Embodiment 22.

1. reaction icon:

Experimental detail: agitate compounds 1 under the room temperature (0.5g, 1.0eq), 5-trifluoromethylaniline (0.58g, 1.0eq), EDC (1.05g, 1.5eq) and HOBt (50mg, 0.1eq) mixture overnight in the 15mL methylene dichloride.With 1N NaOH solution, water washing mixture, use dichloromethane extraction.Use Na ₂SO ₄Dry organic layer is concentrated into driedly, obtains compound 2 by the column chromatography purifying.

2. reaction icon:

Experimental detail: at N ₂Next time fluidisation compound 2 (0.2g, 1.0eq), SeO ₂(0.16g, 2.0eq) mixture in 10mL acetate is 48 hours.By evaporating except that desolvating and in water, dissolving resistates, with saturated Na HCO ₃Solution is adjusted to pH6 and uses dichloromethane extraction.Collected organic layer, drying also is evaporated to dried.Obtain compound 3 by column chromatography purifying crude product.

3. reaction icon:

Experimental detail: (40mg is 1.0eq) with compound 4 (30mg, 1.0eq) mixture overnight in the 5mL methylene dichloride for agitate compounds 3 under the room temperature.Collecting precipitation is also used washed with dichloromethane, and drying obtains required compound under the vacuum.

Embodiment 23.

1. reaction icon:

Experimental detail: backflow compound 1 (3.0g, 1.0eq) and 2mL98%H ₂SO ₄Solution in 10mL EtOH 4 hours is cooled to room temperature and is evaporated to driedly, and dilute with water is used Na HCO ₃Be adjusted to pH8, use dichloromethane extraction.Dry organic layer and the concentrated compound 2 that obtains.

2. reaction icon:

Experimental detail: at N ₂Next time fluidisation compound 2 (1.7g, 1.0eq), SeO ₂(2.29g is 2.0eq) in the mixture of 80mL acetate 48 hours.By evaporating except that desolvating and in water, dissolving resistates, with saturated Na HCO ₃Solution is adjusted to pH6, uses dichloromethane extraction.Collected organic layer, drying also is evaporated to dried.Obtain compound 3 by column chromatography purifying crude product.

3. reaction icon:

Experimental detail: backflow compound 3 (1.1g, 1.0eq), diethoxy methoxyl group-ethane (2.3g, 2.5eq) and TsOH.H ₂(0.12g, 0.1eq) solution in 20mL ethanol is 5 hours for O.Evaporating solvent and in EtOAc dissolved solids, wash with water.Use Na ₂SO ₄Dry organic layer and evaporation obtain compound 4.

4. reaction icon:

Experimental detail: (0.6g 1.0eq) adds 4mL 1N NaOH solution in the solution of 10mL methyl alcohol, at room temperature stir the mixture and spend the night to compound 4.Evaporating solvent and with 5% citric acid acidifying resistates to pH 6, use dichloromethane extraction.Dry organic layer concentrates and obtains compound 5.

5. reaction icon:

Experimental detail: agitate compounds 5 under the room temperature (0.4g, 1.0eq), 5-trifluoromethylaniline (0.29g, 1.0eq), EDC (0.51g, 1.5eq), HOBt (25mg, 0.1eq) mixture overnight in the 10mL methylene dichloride.With 1N NaOH solution, water washing mixture, use dichloromethane extraction.Use Na ₂SO ₄Dry organic layer is concentrated into driedly, obtains compound 6. by the column chromatography purifying

6. reaction icon:

Experimental detail: handle compound 6 (0.2g, the 1.0eq) solution in the 10mL diox, and heated mixt to 60 ℃ 2 hours with 4mL1N HCl.After the cooling, by adding NaHCO ₃Regulate pH to 8.Use the dichloromethane extraction mixture, wash organic layer with water, use Na ₂SO ₄Drying also is evaporated to dried.Obtain compound 7 by column chromatography purifying crude product.

7. reaction icon:

Experimental detail: (30mg is 1.0eq) with compound 8 (19mg, 1.0eq) mixture overnight in the 5mL methylene dichloride for agitate compounds 7 under the room temperature.Collecting precipitation is also used washed with dichloromethane, and drying obtains required compound under the vacuum.

Embodiment 24.

1. reaction icon:

Experimental detail: at N ₂Next time fluidisation compound 1 (2.0g, 1.0eq), SeO ₂(2.6g, 2.0eq) mixture in 80mL acetate is 36 hours.By evaporating except that desolvating and in water, dissolving resistates, with saturated NaHCO ₃Solution is adjusted to pH 6, uses dichloromethane extraction.Collected organic layer, drying also is evaporated to dried.Obtain compound 2 by column chromatography purifying crude product.

2. reaction icon:

Experimental detail: backflow compound 2 (0.5g, 1.0eq), diethoxy methoxyl group-ethane (1.0g, 2.5eq) and TsOH.H ₂(0.05g, 0.1eq) solution in 8mL ethanol is 3 hours for O.Evaporating solvent also dissolves this solid in EtOAc, wash with water.Use Na ₂SO ₄Dry organic layer and evaporation obtain compound 3.

3. reaction icon:

Experimental detail: at N ₂Down to compound 3 (0.6g, 1.0eq), 3-trifluoromethyl-aniline (0.37g, 1.0eq), (0.26g 1.2eq) adds Pd to t-BuONa in the mixture of 15mL toluene ₂(dba) ₃(42mg, 0.02eq) and xantphos (28mg, 0.02eq).At N ₂Following backflow mixture 16 hours, cooling is filtered.The thickening filtration thing also obtains compound 4 by the column chromatography purifying.

4. reaction icon:

Experimental detail: handle compound 4 (0.25g, the 1.0eq) solution in the 10mL diox, and heated mixt to 60 ℃ 2 hours with 4mL1N HCl.After the cooling, by adding NaHCO ₃Regulate pH to 8.Use the dichloromethane extraction mixture, wash organic layer with water, use Na ₂SO ₄Drying also is evaporated to dried.Obtain compound 5. by column chromatography purifying crude product

5. reaction icon:

Experimental detail: (30mg is 1.0eq) with compound 6 (19mg, 1.0eq) mixture overnight in the 5mL methylene dichloride for agitate compounds 5 under the room temperature.Collecting precipitation is also used washed with dichloromethane, and drying obtains required compound under the vacuum.

Embodiment 25.

1. reaction icon:

Experimental detail: under 0 ℃, (14g 0.1mol) adds NaNO in the solution of moisture HBr (30mL) to compound 1 in 30 minutes ₂(8.3g 0.15mol) is at H ₂Solution among the O (10mL).Stir after 60 minutes, (14g is 0.1mol) in the solution of moisture HBr (16mL) to add reaction mixture to CuBr under 80 ℃.After adding fully, stirred reaction mixture is 2 hours under uniform temp.Behind the cool to room temperature, with EA (100mL * 3) extractive reaction mixture.The organic layer that merges with the salt water washing is also used Na ₂SO ₄Dry.Filter out Na ₂SO ₄After, the thickening filtration thing is to doing.Obtain product 2 by the column purification resistates.

2. reaction icon:

Experimental detail: at N ₂Under the atmosphere to compound 2 (4.11g, 0.02mol) and compound 3 (4.74g, 0.02mol), and KOH (5.28g, 0.1mol) and TBBA (6.44g 0.02mol) adds Pd (PPh in the mixture of the stirring of anhydrous THF (100mL) and the degassing ₃) ₄(2.31g 2mmol), stirred 12 hours under refluxing.After filtering out solid, the thickening filtration thing is to doing.Obtain product 4 by the column purification resistates.

3. reaction icon:

Experimental detail: (1g, the 3mmol) solution in methylene dichloride (10mL) stirred 6 hours under the room temperature then to handle 4 with TFA (1mL).Decompression removes to desolvate down and obtains product 5, is not having to use it for next step under the purifying.

4. reaction icon:

Experimental detail: at N ₂Under the atmosphere to compound 5 (619mg, 2.4mmol) and compound 6 (520mg, 2.4mmol), and t BuONa (460mg, 4.8mmol) and BINAP (599mg 6.9mol) adds Pd in the mixture of the stirring of toluene (60mL) and the degassing ₂(dba) ₃(221mg 0.024mmol), stirred 48 hours down at 80 ℃.After filtering out solid, the thickening filtration thing is to doing.Obtain product 7 by the column purification resistates.

5. reaction icon:

Experimental detail: at-30 ℃ and N ₂Under the atmosphere, use BBr ₃(146mg, (396mg, the 0.1mmol) solution in methylene dichloride (10mL) stirred 4 hours under the room temperature then 0.6mmol) to handle compound 7.Reactant poured into add Na in ice-water then ₂CO ₃With the mixture of dichloromethane extraction (25mL * 3) gained, use Na ₂SO ₄The dry organic layer that merges. filter out Na ₂SO ₄After, the thickening filtration thing obtains crude product 8, is not having to use it for next step under the purifying.

6. reaction icon:

Experimental detail: (32.2mg, 0.1mmol) (21mg is 0.1mmol) at anhydrous CH with compound 9 for agitate compounds 8 under refluxing ₂Cl ₂Solution (300mL) 6 hours.Decompression removes down and desolvates.Separate resistates by preparation-TLC and obtain required compound.

Embodiment 26.

1. reaction icon:

Experimental detail: use POCl ₃(500mL) handle 5-fluoro-1H-pyrimidine-2, (113g, 0.5mol) at N, the solution in the accelerine (70mL) refluxed 2 hours the 4-diketone then.Behind the cool to room temperature, pour reaction mixture into ice-oedema.Mixture with ethyl acetate (100mL * 3) extraction gained.With saturated NaHCO ₃The organic layer that solution washing merges is used the salt water washing then.Decompression removes to desolvate down and obtains compound 2.

2. reaction icon:

Experimental detail :-10 ℃ in 15 minutes to compound 2 (20.8g, 0.194mol) in the solution of ethanol (300mL), dropwise add morpholine (21.6g, 0.25mol).Stirred this mixture under the room temperature 0.5 hour, be heated to then 50 ℃ 15 minutes.Behind the cool to room temperature and dilute with water, precipitated solid.By solid collected by filtration and wash with water and obtain compound 3.

3. reaction icon:

Experimental detail: reflux 3 (4.6g, 17.5mmol) and hydrazine (8.75g, 87.5mmol) solution in ethanol (40mL) is 6 hours.Cooling and post precipitation are by filtering collecting precipitation and obtaining compound 4 with washing with alcohol.

4. reaction icon:

Experimental detail: at-15 ℃ and N ₂Under the atmosphere, (37.5mL 60mmol) handles compound 5 (14g, 50mmol) solution in anhydrous THF (100mL) with BuMgCl.After the addition, under this temperature, stirred the mixture 1 hour fully.(0.54g 75mmol) joins reaction mixture, rises again then to room temperature 1 hour with dry DMF in following 30 minutes at 0 ℃.By adding 2M HCl (80mL) cancellation reaction mixture.Mixture with ethyl acetate (50mL * 3) extraction gained.Use Na ₂SO ₄The dry organic layer that merges.Concentrated solvent separates resistates by post and obtains compound 6 to doing.

5. reaction icon:

Experimental detail: (4.5g, 22.5mmol) solution in triethyl orthoformate (15mL) spends the night heating compound 6 in the presence of the TsOH of trace.With ethyl acetate (100mL) diluted reaction mixture and use 5%Na ₂CO ₃Solution washing.Separate organic layer and use Na ₂SO ₄Dry.Concentrated solvent obtains compound 7.

6. reaction icon:

Experimental detail: at N ₂Under the atmosphere to compound 7 (1.3g, 5mmol) and compound 8 (0.97g, 6mmol) and t BuONa (0.7g, 7mmol) and P (t-Bu) ₃(15mg) in the mixture of the stirring of toluene (60mL) and the degassing, add Pd ₂(dba) ₃(23mgl), and under refluxing stirred 12 hours.After filtering out solid, the thickening filtration thing is to doing.Obtain product 9 by the column purification resistates.

7. reaction icon:

Experimental detail: at-30 ℃ and N ₂Under the atmosphere, use BBr ₃(146mg, (200mg, the 0.58mmol) solution in methylene dichloride (10mL) stirred 4 hours under the room temperature then 0.6mmol) to handle compound 9.Reactant poured into add Na in ice-water then ₂CO ₃With the mixture of dichloromethane extraction (25mL * 3) gained, use Na ₂SO ₄The dry organic layer that merges.Filter out Na ₂SO ₄After, the thickening filtration thing obtains crude product 10.

8. reaction icon:

Experimental detail: (48.7mg, 0.2mmol) (63mg is 0.2mmol) at anhydrous CH with compound 4 for agitate compounds 10 under refluxing ₂Cl ₂Solution (300mL) 6 hours.Decompression removes down and desolvates.Separate resistates by preparation-TLC and obtain required compound.

Embodiment 27.

1. reaction icon:

Experimental detail: reflux 3 (2.4g, 11mmol) and methyl hydrazine (2g, 45mmol) solution in ethanol (40mL) is 6 hours.Cooling and post precipitation are by filtering collecting precipitation and obtaining compound 2 with washing with alcohol.

2. reaction icon:

Experimental detail: (28mg, 0.1mmol) (37mg is 0.1mmol) at anhydrous CH with compound 3 for backflow agitate compounds 2 ₂Cl ₂Solution (300mL) 6 hours.Decompression removes down and desolvates.Separate resistates by preparation-TLC and obtain required compound.

Embodiment 28.

1. reaction icon:

Experimental detail: at-20 ℃ down with MeMgCl (15mL.0.038mol) handle 1 (2g, the 0.011mol) solution in anhydrous THF (100mL), and under this temperature, stirring 2 hours.By adding saturated NH ₄Cl aqueous solution cancellation reaction mixture.Mixture with ethyl acetate (100mL * 3) extraction gained.Organic layer with salt water washing merging.Decompression removes to desolvate down and obtains compound 2.

2. reaction icon:

Experimental detail: heating compound 2 in the presence of the TsOH of trace (2g, 0.01mol) and ethylene glycol (3g, 0.048mol) solution in aniline (100mL) is 3 hours.With ethyl acetate (100mL) diluted reaction mixture and use 5%Na ₂CO ₃Solution washing.Separate organic layer and use Na ₂SO ₄Dry.Concentrated solvent obtains compound 3.

3. reaction icon:

Experimental detail: at N ₂Under the atmosphere, to compound 3 (0.4g, 1.6mmol) and compound 4 (0.3g, 1.9mmol), and t BuONa (0.22g, 2mmol) and P (t-Bu) ₃(59mg) in the mixture of the stirring of toluene (30mL) and the degassing, add Pd ₂(dba) ₃(29mgl), and under refluxing stirred 12 hours.After filtering out solid, the thickening filtration thing is to doing.Obtain product 5 by the column purification resistates.

4. reaction icon:

Experimental detail: at-30 ℃ and N ₂Use BBr under the atmosphere ₃(146mg, (100mg, the 0.3mmol) solution in methylene dichloride (10mL) stirred 4 hours under the room temperature then 0.6mmol) to handle compound 5.Reactant poured into add Na in ice-water then ₂CO ₃With the mixture of dichloromethane extraction (25mL * 3) gained, use Na ₂SO ₄The dry organic layer that merges.Filter out Na ₂SO ₄After, the thickening filtration thing obtains crude product 6.

5. reaction icon:

Experimental detail: (64mg, 0.2mmol) (45mg is 0.2mmol) at anhydrous CH with compound 7 for agitate compounds 6 under refluxing ₂Cl ₂Solution (300mL) 6 hours.Decompression removes down and desolvates.Separate resistates by preparation-TLC and obtain required compound.

Embodiment 29.

1. reaction icon:

Experimental detail: under refluxing with 1 (5.2g, 22mmol) and 2 (2.44g is 20mmol) at 2M Na ₂CO ₃Mixture and Pd (PPh in the aqueous solution (25mL) and the toluene (40mL) ₃) ₄(0.57g, 0.05mmol) stirring is spent the night.With ethyl acetate (100mL * 3) extractive reaction mixture.Organic layer with salt water washing merging.Decompression removes down and desolvates to doing.Obtain 3 by the column purification resistates.

2. reaction icon:

Experimental detail: at-60 ℃ and N ₂Under the atmosphere, (1.5mL, (0.78g is 3.3mmol) with triisopropyl boric acid ester (1mL, 4mmol) solution in dry toluene (50mL) 3.75mmol) to handle compound 3 with n-BuLi.After the addition, stirred this mixture 1 hour down fully at-10 ℃.By adding 2M HCl aqueous solution cancellation reaction mixture and using toluene wash.By adding Na ₂CO ₃Make and use ethyl acetate (50mL * 3) extraction then by water layer pH=8.Organic layer with salt water washing merging.Decompression removes down and desolvates to doing.Separate resistates by post and obtain 4.

3. reaction icon:

Experimental detail: (2.8g, 14mmol) (7g is 42mmol) at 2M Na with compound 5 with compound 4 under refluxing ₂CO ₃The stirring in the aqueous solution (250mL) and the toluene (40mL) and the mixture and the Pd (PPh of the degassing ₃) ₄(0.57g, 0.05mmol) stirring is spent the night.With ethyl acetate (100mL * 3) extractive reaction mixture.Organic layer with salt water washing merging.Decompression removes down and desolvates to doing.Separate resistates by post and obtain 6.

4. reaction icon:

Experimental detail: under refluxing, stir 6 (0.53g, 1.9mmol) and hydrazine (0.52g, 8.8mmol) solution in ethanol (50mL) is 6 hours.Cooling and post precipitation are by filtering collecting precipitation and obtaining compound 7 with washing with alcohol.

5. reaction icon:

Experimental detail: (53mg, 0.13mmol) (79mg is 0.13mmol) at anhydrous CH with compound 8 for agitate compounds 7 under refluxing ₂Cl ₂Solution (300mL) 6 hours.Decompression removes down and desolvates.Obtain required compound by preparation-TLC purifying resistates.

Embodiment 30.

1. reaction icon:

Experimental detail: under refluxing with 1 (3.1g, 13mmol) and 2 (1g is 12mmpl) at 2M Na ₂CO ₃Mixture and Pd (PPh in the aqueous solution (15mL) and the toluene (30mL) ₃) ₄(0.4g, 0.029mmol) stirring is spent the night.With ethyl acetate (100mL * 3) extractive reaction mixture.Organic layer with salt water washing merging.Decompression removes down and desolvates to doing.Separate resistates by post and obtain 3.

2. reaction icon:

Experimental detail: at-60 ℃ and N ₂Under the atmosphere, (12mL, (2.0g is 10mmol) with triisopropyl boric acid ester (7mL, 30mmol) solution in dry toluene (50mL) 30mmol) to handle compound 3 with n-BuLi.After the addition, stirred this mixture 1 hour down fully at-10 ℃.By interpolation 2m HCl aqueous solution cancellation reaction mixture, and use toluene wash.By adding Na ₂CO ₃Make and use ethyl acetate (50mL * 3) extraction then by water layer pH=8.Organic layer with salt water washing merging.Decompression removes down and desolvates to doing.Separate resistates by post and obtain 4.

3. reaction icon:

Experimental detail: (0.5g, 3mmol) (1.5g is 9mmol) at 2M Na with compound 5 with compound 4 under refluxing ₂CO ₃The stirring of the aqueous solution (3.5mL) and toluene (40mL) and the mixture of the degassing and Pd (PPh ₃) ₄(94mg, 0.003mmol) stirring is spent the night.With ethyl acetate (100mL * 3) extractive reaction mixture.Organic layer with salt water washing merging.

Decompression removes down and desolvates to doing.Obtain 6 by the column purification resistates.

4. reaction icon:

Experimental detail: under refluxing, stir 6 (0.24g, 1mmol) and hydrazine (0.3g, 4.7mmol) solution in ethanol (50mL) is 6 hours.Cooling and post precipitation are by filtering collecting precipitation and obtaining compound 7 with washing with alcohol.

5. reaction icon:

Experimental detail: (70mg, 0.29mmol) (83mg is 0.3mmol) at anhydrous CH with compound 8 for agitate compounds 7 under refluxing ₂Cl ₂Solution (300mL) 6 hours.Decompression removes down and desolvates.Obtain required compound by preparation-TLC purifying resistates.

Embodiment 31.

1. reaction icon:

Experimental detail: to compound 2 (0.83g, 5mmol) in the solution of ethanol (100mL), dropwise add benzylamine (0.54g, 5mmol).Stir after 2 hours the dilute with water reaction mixture.Mixture with ethyl acetate (50mL * 3) extraction gained.Use Na ₂SO ₄The dry organic layer that merges.Concentrated solvent obtains compound 3.

2. reaction icon:

Experimental detail: reflux 3 (1.18g, 5mmol) and the solution of hydrazine (5ml) in ethanol (40mL) 6 hours.Cooling and post precipitation are by filtering collecting precipitation and obtaining compound 4 with washing with alcohol.

3. reaction icon:

Experimental detail: (48.7mg, 2mmol) (63mg is 0.2mmol) at anhydrous CH with compound 5 for agitate compounds 4 under refluxing ₂Cl ₂Solution (300mL) 6 hours.Decompression removes down and desolvates.Separate resistates by preparation-TLC and obtain required compound.

Embodiment 32.

1. reaction icon:

Experimental detail: to compound 1 (0.83g, 5mmol) in the solution of ethanol (100mL), dropwise add compound 2 (0.35g, 5mmol).Stir after 2 hours the dilute with water reaction mixture.Mixture with ethyl acetate (50mL * 3) washing gained.Use Na ₂SO ₄The dry organic layer that merges.Concentrated solvent obtains compound 3.

2. reaction icon:

Experimental detail: reflux 3 (1.0g, 5mmol) and the solution of hydrazine (5mL) in ethanol (40mL) 6 hours.Cooling and post precipitation are by filtering collecting precipitation and obtaining compound 4 with washing with alcohol.

3. reaction icon:

Experimental detail: (480mg, 2mmol) (60mg is 0.2mmol) at anhydrous CH with compound 5 for agitate compounds 4 under refluxing ₂Cl ₂Solution (300mL) 6 hours.Decompression removes down and desolvates.Separate resistates by preparation-TLC and obtain required compound.

All any open source literatures that relate to, patent or other reference of quoting are incorporated herein by reference.

Reference

Adcock, I.M., Lane, S.J. " steroid effect and inflammation resistance mechanism "-Journal of Endocrinology, Volume 178 (in September, 2003) 347-355 page or leaf

Allen, P.B., Wiedemann, L.M. " the activation sudden change in the ATP-binding site of ABL kinase domain "-The Journal of Biological Chemistry, Volume 271 (on August 9th, 1996) 19585-19591 page or leaf

Barthe，C.，Cony-Makhoul，P.，Melo，J.V.，Reiffers，J.，Mahon，F，X.Roots?of?Clinical?Resistance?to?STI-571?CancerTherapy.Science，Volume?293(September?21，2001)Page?2163a

Branford, S., Rudzki, Z., Walsh, S., Grigg, A., Arthur, C., Taylor, K., Hermann, R., Lynch, K.P., Hughes, T.P. " the high frequency point mutation of suffering from chronic myeloid leukemia or taking place to cluster in the patient B CR/ABL adenosine triphosphate-land of the positive acute lymphoblastic leukemia of the chemical sproof Ph-of imatinib (STI571) "-Blood, Volume 99 (on May 1st, 2002) 3472-3475

Burbaum, JJ., Ohlmeyer, M.H., Reader, J.C., Henderson, L, Dillard, L.W., Li, G., Randle, T.L., Sigal, N.H., Chelsky, D., Baldwin, JJ. " adopt the example of the drug discovery of the combinatorial library of encoding "-Proceedings of the National Academy of Science U S A, Volume92 (June 20,1995) Pages 6027-6031

Carter, T.A., Wodicka, L.M., Shah, N.P., Velasco, A.M., Fabian, M.A., Tretber, O.K., Milanov, Z.V., Atteridge, C.E., Biggs, W.H.3rd, Edeen, P.T., Floyd, M..Ford, J.M., Grotzfeld, R.M., Herrgard, S., Insko, D.E., Mehta, S.A., Patel, H.K., Pao, W..Sawyers, C.L., Varaius, H., Zarrinkar, P.P., Lockhart, DJ. " suppress ABL; the resistance sudden change of KIT and EGF receptor kinase "-Proceedings of theNational Academy of Science U S A, Volume 102 (August 2,2005) Pages 11011-11016

Corbin, A.S., Buchdunger, E., Pascal, F., Druker, B.J. " STI571 suppresses the kinase whose specific architecture basics analysis of Abl "-The Journal ofBiological Chemistry, Volume 277 (on August 30th, 2002) 32214-32219 page or leaf

Cunningham, B.C., De Vos, A.M., Mulkerrin, M.G., Ultsch, M, Wells, J.A. " selective ligands agonist and antagonist "-U.S. Pat 5,506,107 (on April 9th, 1996)

Cunningham, B.C., Wells, J.A., Clark, R.G., Olson, K., Fuh, G.G. " are used to suppress the method for growth hormone effects "-U.S. Pat 6,004,931 (on December 21st, 1999)

Daley, G.Q., Van Etten, R.A., Baltimore, the D. " P210 of Philadelphia chromosome ^Brc/ablThe chronic granulocytic leukemia of gene induced mouse "-Science, Volume 247 (February 16 nineteen ninety) 824-830 page or leaf

Druker, B.J., M.D., Talpaz, M., M.D., Resta, D.J., R.N., Peng, B., Ph.D., Buchdunger, E., Ph.D., Ford, J.M., M.D., Lydon, N.B., Ph.D., Kantarjian, H., M.D., Capdeville, R., M.D., Ohno-Jones, S., B.S., Sawyers, C.L., M.D. " effect of the specific inhibitor of BCR-ABL Tyrosylprotein kinase and security in the chronic myeloid leukemia "-The New England Journal of Medicine, Volume 344 (April 5 calendar year 2001) 1031-1037 page or leaf

Druker, B.J., Tamura, S., Buchdunger, E., Ohno, S., Segal, G.M., Fanning, S., Zimmermann, J., Lydon, N.B. " selective depressant of Abl Tyrosylprotein kinase is to the effect of Bcr-Abl positive cell growth "-Nature Medicine, Volume 2 (in May, 1996) 561-566 page or leaf

Druker, B.J., M.D., Sawyers, C.L., M.D., Kantarjian, H., M.D., Resta, D.J., R.N., Reese, S.F., M.D., Ford, J.M., M.D., Capdeville, R., M.D., Talpaz, M., M.D. " the specific inhibitor activity that has BCR-ABL Tyrosylprotein kinase in the blast crisis of the chronic myeloid leukemia of Philadelphia chromosome and acute lymphoblastic leukemia "-The New EnglandJournal of Medicine, Volume 344 (April 5 calendar year 2001) 1038-1042 page or leaf

Faderl, S., M.D., Talpaz, M., M.D., Estrov, Z., M.D., O ' Brien, S., M.D., Kurzrock, R., M.D., Kantarjian, H.M., M.D. " chronic myeloid leukemia biology "-The New England Journal ofMedicine, Volume 341 (on July 15th, 1999) 164-172 page or leaf

Foreman, J.C. and Johansen, T.Textbook of ReceptorPharmacology.CRC Press, 2002; Boca Raton,

Gambacorti-Passerini, C., Barni, R., LeCoutre, P., Zucchetti, M., Cabrita, G., Cleris, L., Rossi, F., Gianazza, E., Brueggen, J., Cozens, R., Pioltelli, P., Pogliani, E., Corneo, G., Formelli, F., D ' Incalci, M. " people BCR-ABL ⁺The leukemia cell is to the effect of 1 acid glycoprotein of the α in the tolerance in the body of Abl inhibitor STI571 "-Journal of the National Cancer Institute, Volume 92 (on October 18th, 2000) 1641-1650 page or leaf

Goodnow, R.A., Jr., Guba, W., Haap, W. " successfully produce the principle of design in guide's library with the small molecules library "-Combinatorial Chemistry and HighThroughput Screening, Volume 6 (November 2003) Pages 649-660.

Gorre, M.E., Mohammed, M., Ellwood, K., Ksu, N., Paquette, R., Rao, P.N., Sawyers, C.L. " the clinical tolerance that BCR-ABL transgenation or amplification cause "-Science, Volume293 (August 3 calendar year 2001) 876-880 page or leaf to the STI-571 cancer therapy

Hanke, J.H., Gardner, J.P., Dow, R.L., Changelian, P.S., Brissette, W.H., Weringer, E.J., Pollok, B.A., Connelly, P.A. " new, effectively reach the discovery of Src family selectivity tyrosine kinase inhibitor.Lck-and the research of FynT-dependent T cell activatory "-Journal of Biological Chemistry, Volume 271 (January 1996) Pages 695-701

Hofmann, W.K., Jones, L.C., Lemp, N.A., DeVos, S., Gschaidmeier, hour., Hoelzer, D., Ottmann, O.G., Koeffler, H.P. " Ph ⁺Acute lymphoblastic leukemia has unique BCR-ABL transgenation to the tolerance of tyrosine kinase inhibitor STI571 "-Blood, Volume 99 (on March 1st, 2002) 1860-1862 page or leaf

Hou, Y.Y., Tan, Y.S., Sun, M.H., Wei, Y.K., Xu, J.F., Lu, S.H., A-Ke-Su, S.J., Zhou, Y.N., Gao, F., Zheng, A.H., Zhang, T.M., Hou, W.Z., Wang, J., Du, X., Zhu, X.Z. " the C-kit transgenation in human gastrointestinal tract's mesenchymoma "-World Journa lofGastroenterology, Volume 10 (on May 1st, 2004) 1310-1314 page or leaf

Housey GM. " is used to screen the method for protein inhibitor and activator "-U.S. Pat 4,980,281 (December 25 nineteen ninety)

Housey GM, Johnson MD, Hsiao WL, O ' Brian CA, Murphy JP, Kirschmeier P, Weinstein IB. " overexpression of protein kinase C causes growth control obstacle in the rat fibroblast "-Cell, Volume 52 (on February 12nd, 1988) 343-354 page or leaf

Kerkela, R., Grazette, L., Yacobi, R., Iliescu, C, Patten, R., Beahm, C, Walters, B., Shevtsov, S., Pesant, S., Clubb, F.J., Rosenzweig, A., Salomon, R.N., Van Etten, R.A., Alroy, J., Durand, J.B., Force, T. " cardiac toxic of cancer therapeutic agent imatinib mesylate "-Nature Medicine, Volume 12 (August 2006) Pages 908-916

Knight, Z.A., Shokat, K.M. " feature of selective kinase inhibitors "-Chemistry and Biology, Volume 12 (June2005) Pages 621-637

La Rosee, P., Corbin, A.S., Stoffregen, E.P., Deininger, M.W., Druker, B.J. " to clinical relevant to imatinib mesylate (Gleevec, STI-571) activity of the Bcr-Abl kinase inhibitor PD180970 of tolerific Bcr-Abl isotype "-Cancer Research, Volume 62 (2002 12 pages 15 days) 7149-7153 page or leaf

Le Coutre, P., Tassi, E., Varella-Garcia, M., Barni, R., Mologni, L., Cabrita, G., Marchesi, E., Supino, R., Gambacorti-Passerini, C. " induces the tolerance of Abelson inhibitor among the STI571 human leukemia cell "-Blood by gene amplification, Volume 95 (20003 months 1 day) 1758-1766 page or leaf

Leonard, G.D., Fojo, T., Bates, S.E. " the proteic effect of abc transport in the clinical practice "-The Oncologist, Volume 8 (2003) 411-424 pages or leaves

Loutfy, M.R., Walmsley, S.L. " salvage of antiretroviral therapy in the HIV infection "-Expert Opinion, Volume 3 (in February, 2002) 81-90 page or leaf

Lynch, T.J., M.D., Bell, D.W., Ph.D., Sordella, R., Ph.D., Gurubhagavatula, S., M.D., Okimoto, R.A., B.S., Brannigan, B.W., B.A., Harris, P.L., M.S., Haserlat, S.M., B.A., Supko, J.G., Ph.D., Haluska, F.G., M.D., Ph.D., Louis, D.N., M.D., Christiani, D.C., M.D., Settleman, J., Ph.D., Haber, D.A., M.D., Ph.D. " suddenlys change to the activation in reactive EGF-R ELISA of Gefitinib based on non--little-cell lung cancer "-The NewEngland Journal of Medicine, Volume 350 (on May 20th, 2004) 2129-2139 page or leaf

Mahon, F.X., Deininger, M.W.N., Schultheis, B., Chabrol, J., Reiffers, J., Goldman, J.M., Melo, J.V. " selection and sign that tyrosine kinase inhibitor STI571 are had the BCR-ABL positive cell line of difference susceptibility: different resistance mechanisms "-Blood, Volume 96 (20008 months 1 day) 1070-1079 page or leaf

Marx, J. " why new cancer drug acts in some patient fully "-Science, Volume 304 (on April 30th, 2004) 658-659 page or leaf

Melo JV, Myint H, Galton DA, Goldman JM. " P190BCR-ABL chronic myeloid leukemia: have the leukemic missing link of chronic Myelomonocyte sample? "-Leukemia, Volume 8 (in January, 1994) 208-211 page or leaf

Noble, M.E.M., Endicott, J.A., Johnson, L.N. " kinases inhibitor: see clearly medicinal design "-Science, Volume 303 (on March 19th, 2004) 1800-1805 page or leaf according to structure

O ' HareT, Pollock R, Stoffregen EP, Keats JA, AbdullahOM, Moseson EM, Rivera VM, TangH, Metcalf CA 3rd, BohacekRS, Wang Y, Sundaramoorthi R, Shakespeare WC, Dalgarno D, Clackson T, Sawyer TK, Deininger MW, Druker BJ. " a kind of effectively based on the carcinogenic protein kinase inhibitor AP23464 of ATP inhibition: as to relate to CML "-Blood to wild-type and mutant Bcr-Abl, Volume 104 (on October 15th, 2004) 2532-2539 page or leaf

Paez, J.G., , P.A., Lee, J.C., Tracy, S., Greulich, hour., Gabriel, S., Herman, P., Kaye, F.J., Lindeman, N., Boggon, T.J., Naoki, K., Sasaki, H., Fujii, Y., Eck, M.J., Sellers, W.R., Johnson, B.E., Meyerson, M. " the EGFR sudden change in the lung cancer: "-Sciencexpress (on April 29th, 2004) 1-4 page or leaf with dependency to Gefitinib therapy clinical response

Ravandi F, CortesJ, Albitar M, Arlinghaus R, Qiang Guo J, Talpaz M, KantarjianHM. " chronic granulocytic leukemia with p185 (BCR/ABL) expression: feature and clinical importance "-British Journal of Haematology, Volume 107 (in December, 1999) 581-586 page or leaf

Sambrook and Russell, Molecular Cloning:A Laboratory Manual.Cold Spring Harbor Laboratory Press, New York, 2001, Volumes1-3

Sawyers, C.L.M.D. " chronic myeloid leukemia "-The New EnglandJournal of Medicine, Volume 340 (19994 months 29 days) 1330-1340 page or leaf

Sawyers, C.L., Gorre, M.E., Shah, N.P., Nicoll, J. " in the BCR-ABL Tyrosylprotein kinase relevant sudden change "-WO 02/102976A2Published December 27,2002 (PCT/US02/18729 Filed June 14,2002) with the STI-571 resistance

Schindler, T., Bornmann, W., Pell ices na, P., Miller, W.T., Clarkson, B., Kuriyan, J. " structure mechanism that the STI-571 of Abelson Tyrosylprotein kinase suppresses "-Science, Volume 289 (on September 15th, 2000) 1938-1942 page or leaf

Senechal, K., Halpern, J., Sawyers, C.L. " the CRKL adaptin changes into fibrocyte and passes through the function of BCR-ABL oncogene in conversion "-The Journalof Biological Chemistry, Volume 271 (on September 20th, 1996) 23255-23261 page or leaf

Tipping AJ, Baluch S, Barnes DJ, Veach DR, Clarkson BM, Bornmann WG, Mahon FX, Goldman JM, Melo JV. " two-way specific b cr-Abl and Src-family kinase inhibitor to the cellular sensitivity of imatinib mesylate and the effect in the tolerance "-Leukemia, Volume 18 (in August, 2004) 1352-1356 page or leaf

Von Bubnoff, N., Schneller, F., Peschel, C., Duyster, J. " the BCR-ABL transgenation relevant to the clinical tolerance of STI571: perspective study "-The Lancet, Volume 359 (on February 9th, 2002) 487-491 page or leaf with Philadelphia-karyomit(e)-positive leukemia

Von BubnoffN, Veach DR, Van Der Kuip H, Aulitzky WE, Sanger J, Seipel P, Bornmann WG, Peschel C, Clarkson B, Duyster. " the positive leukemic resistance of Bcr-Abl based on the Screening and Identification of cell as the sudden change pattern of the PD166326 of alternative Abl kinase inhibitor "-Blood, Volume 105 (on February 15th, 2005) 1652-1659 page or leaf

Wakai, T., Kanda, T., Hirota, S., Ohashi, A., Shirai, Y.Hatakeyama, K. " tolerance in late period of imatinib therapy is relevant with secondary KIT sudden change in the transfevent gastrointestinal stromal tumor "-British Journal of Cancer, Volume 90 (on June 1st, 2004) 2059-2061 page or leaf

Warmuth, M., Mathes, R., Hallek, M. " selects the method for enzyme inhibitors "-U.S.

Weigel, U., Meyer, M., Sebald, W. " mutain of human interleukin-12: renaturation yield, proliferation activity and receptors bind "-European Journal ofBiochemistry, Volume 180 (on March 15th, 1989) 295-300 page or leaf

Weisberg E; Catley L; Kujawa J; Atadja P, Remiszewski S, Fuerst P; Cavazza C; Anderson K, Griffin JD. " histone deacetylase inhibitors NVP-LAQ824 has the significance activity to the myelocytic leukemia cell in vitro and in vivo "-Leukemia, Volume 18 (in December, 2004) 1951-1963 page or leaf

Weisberg, E., Griffin, J.D. " mechanism of the tolerance of ABL tyrosine kinase inhibitor STI 571 in the hematopoietic cell system that BCR/ABL-transforms "-Blood, Volume95 (on June 1st, 2000) 3498-3505 page or leaf

Weisberg E, Manley PW, Breitenstein W, Bruggen J, Cowan-Jacob SW, Ray A, Huntly B, Fabbro D, FendrichG, Hall-Meyers E, Kung AL, Mestan J, Daley GQ, Callahan L, Catley L, Cavazza C, Azam M, Neuberg D, Wright RD, GillilandDG, Griffin JD. " sign of the natural and sudden change Bcr-Abl inhibitor AMN107 of a kind of selectivity "-Cancer Cell, Volume 7 (in February, 2005) 129-141 page or leaf

White MF, Livingston JM, Backer, Lauris V, Dull TJ, Ullrich A, Kahn CR. " the insulin receptor sudden change on the tyrosine 960 suppresses the signal transmission, but can not influence its tyrosine kinase activity "-Cell, Volume 54 (on August 26th, 1988) 641-649 page or leaf.

Claims

1. identify with first compound and compare the method with the compound that improves cell-specific, described first compound is proteinic inhibitor and regulates corresponding phenotypic response, comprising:

A) measure the adjusting of the phenotypic response of the test cell of crossing with described first compound treatment;

B) measure the adjusting of the phenotypic response of the control cells of crossing with described first compound treatment

C) measure the adjusting of the phenotypic response of the test cell of handling with test-compound;

D) measure the adjusting of the phenotypic response of the control cells of handling with test-compound;

E) determine the cell-specific breach of first compound and the cell-specific breach of test-compound;

And f) if the cell-specific breach of described test-compound greater than the cell-specific breach of described first compound, identifies that then described test-compound has improved cell-specific.

2. the process of claim 1 wherein the IC of cell-specific breach by described control cells ₅₀IC divided by described test cell ₅₀Determine.

3. identify the method for comparing the compound with improved cell-specific with first compound, described first compound is proteinic activator and regulates corresponding phenotypic response, comprising:

B) measure the adjusting of the phenotypic response of the control cells of crossing with described first compound treatment;

4. the method for claim 3, wherein said cell-specific breach is by the IC of described test cell ₅₀IC divided by described control cells ₅₀Determine.

5. improve the method that first compound is optimized, described first compound is proteinic inhibitor and regulates corresponding phenotypic response, comprising:

A) measure the IC of described first compound to the phenotypic response of test cell ₅₀

B) measure the IC of described first compound to the phenotypic response of control cells ₅₀

C) measure and to have and the test-compound of the identical skeleton of described first compound IC the phenotypic response of described test cell ₅₀

D) measure the IC of described test-compound to the phenotypic response of described control cells ₅₀

And e) if the cell-specific breach of described test compounds greater than the cell-specific breach of described first compound, identifies that then described test-compound is the improvement of described first compound.

6. the method for claim 5, wherein protein is P210 ^{Bcr-Ab1-T315I}Or p190 ^Bcr-Ab1, it comprises the sudden change of corresponding Threonine to Isoleucine at corresponding amino acid sites.

7. improve the method that first compound is optimized, described first compound is proteinic activator and regulates corresponding phenotypic response, comprising:

And e) if the cell-specific breach of described test compounds greater than the cell-specific breach of described first compound, identifies that then test-compound is the improvement of first compound.

8. each method among the claim 5-7, it comprises optimization compound and the repeating step (a)-(d) of selecting step (e).

9. each method among the claim 5-7, wherein said protein is theramutein.

10. each method among the claim 5-7, the IC of wherein said test-compound ₅₀Be higher than described first compound.

11. each method among the claim 5-7, the IC of wherein said test-compound ₅₀Be lower than described first compound.

12. determine whether a kind of material is the method for proteinic specific inhibitor, and described albumen mass-energy causes detectable phenotypic response, and it comprises:

A) incubation test cell, described test cell are expressed described protein and can be caused and the relevant phenotypic response of proteinic existence and functionally active described in this cell with described material;

B) incubation control cells, described control cells are in low expression level more or do not express described protein and can cause or not cause fully with proteinic described in the cell with low degree more and have a detectable phenotypic response relevant with functionally active;

C) phenotypic response of the phenotypic response of the described test cell of relatively crossing with described mass treatment and the described control cells of crossing with described mass treatment;

And d), determines that then described material is described proteinic specific inhibitor if described material can be regulated the degree of described test cell phenotypic response greater than described control cells.

13. determine whether a kind of material is proteinic specific, activated dose method, and described albumen mass-energy causes detectable phenotypic response, and it comprises:

B) incubation control cells, described control cells are in low expression level more or do not express described albumen and can cause or not cause fully with proteinic described in this cell with low degree more and have a detectable phenotypic response relevant with functionally active;

And d), determines that then described material is described proteinic specific, activated dose if described material can be regulated the degree of described test cell phenotypic response greater than described control cells.