CN101389626A

CN101389626A - Fused ring heterocycle kinase modulators

Info

Publication number: CN101389626A
Application number: CNA2005800326791A
Authority: CN
Inventors: W·D·阿诺尔德; A·格斯博格; Z·李; R·W·斯廷斯麻; M·E·威尔逊
Original assignee: SGX Pharmaceuticals Inc
Current assignee: SGX Pharmaceuticals Inc
Priority date: 2004-07-27
Filing date: 2005-07-27
Publication date: 2009-03-18
Also published as: ZA200700314B

Abstract

The present invention provides novel fused ring heterocycle kinase modulators and methods of using the novel fused ring heterocycle kinase modulators to treat diseases mediated by kinase activity.

Description

Fused ring heterocycle kinase modulators

The cross reference of related application

The application requires the U.S. Provisional Patent Application submitted on July 27th, 2004 number 60/591,778, the U.S. Provisional Patent Application of submitting on July 27th, 2004 number 60/591, the U.S. Provisional Patent Application of submitting on May 11st, 886 and 2005 number 60/680,091 rights and interests are incorporated herein by reference them in this article in full.

Background of invention

Mammiferous protein kinase is the important conditioning agent of cell function.Because the dysfunction of protein kinase activity is relevant with multiple disease and illness, so protein kinase is the target of drug development.

Confirmed tyrosine kinase receptor, FMS-sample Tyrosylprotein kinase 3 (FLT3) and cancers, comprised leukemia, relevant such as acute myelogenous leukemia (AML), acute lymphoblastic leukemia (ALL) and myelodysplasia.About AML patient of 1/4th to 1/3rd has the FLT3 sudden change of the constitutive activation that can cause kinases and downstream signal passage.Although in the normal people, FLT3 mainly expresses by normal marrow and lymph progenitor cell, and FLT3 expresses in 70-80% AML and ALL patient's leukemia cell.Reported that target is deleterious in the inhibitor of FLT3 to the leukemia cell who expresses FLT3 sudden change and/or constitutive activation.Therefore, need exploitation to can be used for treating disease and illness such as leukemic FLT3 inhibitor.

Abelson nonreceptor tyrosine kinase (c-Abl) participates in the signal conductive process by the phosphorylation of its substrate protein.In cell, c-Abl shuttles back and forth between tenuigenin and nucleus, and the active of it regulated closely by multiple different mechanism usually.Found that Abl participates in signal conduction, cell cycle, cytodifferentiation and the neural generation of the control growing factor and integrin, apoptosis, cell adhesion, cytoskeletal structure and to the response of dna damage and oxidative stress.

C-Abl albumen contains 1150 amino-acid residues of having an appointment, and these amino-acid residues have formed the terminal cap structural domain of N-, SH3 and SH2 structural domain, tyrosine kinase domain, nuclear localization sequence, DNA-binding domains and actin binding structural domain.

Chronic lymphocytic leukemia (CML) is relevant with the Philadelphia chromosome transposition between karyomit(e) 9 and 22.This transposition produces unusual the fusion between the gene of bcr gene and coding c-Abl.Formed Bcr-Abl fusion rotein has the tyrosine kinase activity of constitutive activation.The kinase activity that raises it is reported it is the main paathogenic factor of CML, and relevant with the forfeiture and the cell proliferation of cell transformation, growth factor dependency.

It is Bcr-Abl and two kinds of other Tyrosylprotein kinases that 2-phenyl amino pyrimidine compounds imatinib (imatinib also is known as STI-571, CGP57148 or Gleevec) has been determined, be the specificity and the effective inhibitors of c-kit and platelet-derived growth factor receptors.Imatinib stops these proteinic tyrosine kinase activities.Imatinib is reported as effective therapeutical agent in all stages that are treatment CML.Yet, promptly use imatinib to continue to treat, recurrence also can appear in most of late period or blast cell crisis CML patient, and this is because of the resistance that has produced medicine.Usually, this drug-fast molecular basis is the imatinib-resistant mutant that the kinase domain of Bcr-Abl occurred.Cause the modal aminoacid replacement of this sudden change to comprise Glu255Lys, Thr315Ile, Tyr293Phe and Met351Thr.

MET is determined to be in transfering DNA in the human osteosarcoma cell line of handling with N-methyl-N '-nitro-nitroso-guanidine at first and resets (TPR-MET) people 1984 such as () Cooper.MET receptor tyrosine kinase (also being known as hepatocyte growth factor receptor, HGFR, MET or c-Met) and part pHGF thereof (＂ HGF ＂) have the various biological activity, comprise stimulate proliferation, survive, differentiation and form takes place, the formation (branching tubulogenesis) of take-off pipe, cell viability and invasive growth.On pathology, found that MET is relevant with growth, intrusion and the transfer of multiple multi-form cancer, comprise kidney, lung cancer, ovarian cancer, liver cancer and mammary cancer.Somatocyte, the activity sudden change of MET have been found in such as Papillary Renal Cell Carcinoma in the mankind's metastasis of cancer and sporadic cancer.More and more evidences shows that MET is the control always sought for a long time of people to one of oncogene of metastatic tumor development, is a very interesting target spot therefore.Except cancer, show on evidence that also suppressing MET can be used for treating various indications, comprising: the intrusion of listeria, osteolysis, malaria infection, diabetic retinopathy, psoriasis and the sacroiliitis relevant with multiple myeloma.

Tyrosylprotein kinase RON is the acceptor of macrophage-stimulating protein, and belongs to the MET family of receptor tyrosine kinase.The same with MET, RON relates to growth, intrusion and the transfer of multiple multi-form cancer, comprises cancer of the stomach and bladder cancer.

The Aurora family of serine/threonine kinase is absolutely necessary for mitotic.In the cell cycle process, kinase whose expression of Arurora and activity are subjected to tight adjusting.It is the kinase whose substrate of Aurora that the multiple proteins that works in fission process has been determined.Based on the kinase whose known function of Aurora, its active inhibition it is believed that can interrupt cell cycle and blocking-up propagation, thereby suppresses the viability of tumour cell.People such as Harrington, Nature Medicine, up-to-date online delivering (2004).

3-phosphoinositide dependent kinases 1 (PDK1) but be phosphorylation and activate many kinase whose Ser/Thr protein kinase in the agc kinase superfamily that described agc kinase superfamily comprises the kinases (SGK) that Akt/PKB, protein kinase C (PKC), PKC associated kinase (PRK1 and PRK2), p70 ribosome S 6-kinases (S6K1) and serum and glucocorticosteroid are regulated.At first the PDK1 substrate of Que Rening is proto-oncogene Akt.Many researchs have found that high-caliber activation Akt is present in the kinds of tumor type of high per-cent (30-60%), comprises melanoma and mammary cancer, lung cancer, cancer of the stomach, prostate cancer, blood cancer and ovarian cancer.Therefore, on behalf of exploitation, the PDK1/Akt signalling channel can be used for treating the attractive target spot of the micromolecular inhibitor of cancer.People such as Feldman, JBC Papers in Press.Publish with the form of manuscript M501367200 on March 16th, 2005.

Because kinases relates to multiple disease and situation, such as cancer, so need exploitation new, effectively can be used for the protein kinase modulators for the treatment of.The present invention has satisfied these and other needs of this area.Although specified some protein kinase in this article particularly, the invention is not restricted to these kinase whose conditioning agents, and in its scope, comprise the conditioning agent of related protein kinase and the conditioning agent of homologous protein.

The accompanying drawing summary

Fig. 1 represents the wild-type ABL numbering according to ABL exon Ia.

The invention summary

Be surprisingly found out that now fused ring heterocycle compound of the present invention can be used for regulating kinase activity, and can be used for treating the disease that kinase activity mediates.These new fused ring heterocycle kinase modulators have detailed description below.In addition, this paper also discloses the inhibition activity of selected compound.

On the one hand, the invention provides fused ring heterocycle kinase modulators with following formula:

In formula (I), L ¹And L ²Be independently key ,-S (O) n-,-O-,-NH-, unsubstituted C ₁-C ₅The assorted alkylidene group of alkylidene group or unsubstituted 2-5 unit.Symbol n is the integer of 0-2.R ¹And R ²Be to replace or unsubstituted cycloalkyl, replacement or unsubstituted Heterocyclylalkyl, replacement or unsubstituted heteroaryl or replacement or unsubstituted aryl independently.In certain embodiments, R ¹Not to replace or unsubstituted pyrryl.In other embodiments, work as R ¹And R ²When all being unsubstituted phenyl, L ¹It or not the assorted alkylidene group of unsubstituted 2-5 unit.In other embodiments, work as R ²When being unsubstituted piperazinyl, L ¹Not-S (O) ₂-.

On the other hand, the present invention also provides the fused ring heterocycle kinase modulators with following formula (also being known as ＂ The compounds of this invention ＂ in this article):

In formula (II), L ¹, L ², R ¹And R ²As above definition in formula (I).

In formula (III), L ¹, L ², R ¹And R ²As above definition in formula (I).

On the other hand, the present invention also provides the method for regulating protein kinase activity with fused ring heterocycle kinase modulators of the present invention.This method comprises protein kinase is contacted with fused ring heterocycle kinase modulators.

On the other hand, the invention provides the method for the disease (kinase mediated disease or illness) of treatment kinase activity mediation in the individuality in the described treatment of needs (for example Mammals, such as the people).This method comprises fused ring heterocycle kinase modulators of the present invention from significant quantity to described individuality that use.

On the other hand, the present invention also provides and has comprised and the pharmaceutically acceptable vehicle pharmaceutical composition of blended fused ring heterocycle kinase modulators mutually.

Detailed Description Of The Invention

Definition

Abbreviation used herein has the conventional sense in its chemistry and biology field.

Under the situation that substituting group is described by their the conventional chemical formula of from left to right writing, they equally also comprise writes the chemically identical substituting group that is produced from right to left with structural formula, for example-and CH ₂O-is equal to-OCH ₂-.

Except as otherwise noted, term " alkyl " itself or be meant straight chain (promptly not branch) or side chain or cyclic alkyl or its combination as another substituent part, they can be saturated fully, single-or polyunsaturated, and can comprise that having specified carbonatoms (is C ₁-C ₁₀Be meant 1 to 10 carbon) divalence and multivalence group.The example of saturated hydrocarbyl includes but not limited to following group, such as methyl, ethyl, n-propyl, sec.-propyl, normal-butyl, the tertiary butyl, isobutyl-, sec-butyl, cyclohexyl, (cyclohexyl) methyl, cyclopropyl methyl, and for example homologue and the isomer of n-pentyl, n-hexyl, n-heptyl, n-octyl etc.Unsaturated alkyl is to have one or more pairs of keys or triple-linked group.The example of unsaturated alkyl includes but not limited to vinyl, 2-propenyl, crot(on)yl, 2-isopentene group, 2-(butadienyl), 2,4-pentadienyl, 3-(1, the 4-pentadienyl), ethynyl, 1-and 3-proyl, 3-butynyl and high-grade homologue and isomer.The alkyl that will be limited to alkyl is called " same alkyl (homoalkyl) ".

Term " alkylidene group " itself or be meant as another substituent part and for example, be not limited to divalent group derived from alkyl-CH ₂CH ₂CH ₂CH ₂-,-CH ₂CH=CHCH ₂-,-CH ₂C ≡ CCH ₂-,-CH ₂CH ₂CH (CH ₂CH ₂CH ₃) CH ₂-.In the present invention, alkyl (or alkylidene group) has 1-24 carbon atom usually, have 10 or still less the group of carbon atom be preferred." low alkyl group " or " low-grade alkylidene " is short-chain alkyl or alkylidene group, has 8 or carbon atom still less usually.

Except as otherwise noted, term " assorted alkyl " itself or be meant stable straight or branched or cyclic hydrocarbon group or its combination with another term is combined, they are made up of the heteroatoms that at least one carbon atom and at least one are selected from O, N, P, Si and S, wherein nitrogen, p and s atom can be randomly oxidized and nitrogen heteroatom can be randomly by quaternized.Heteroatoms O, N, P, S and Si can be positioned at any interior location of assorted alkyl or be positioned at the position that this alkyl is connected with the rest part of molecule.The example includes but not limited to-CH ₂-CH ₂-O-CH ₃,-CH ₂-CH ₂-NH-CH ₃,-CH ₂-CH ₂-N (CH ₃)-CH ₃,-CH ₂-S-CH ₂-CH ₃,-CH ₂-CH ₂,-S (O)-CH ₃,-CH ₂-CH ₂-S (O) ₂-CH ₃,-CH=CH-O-CH ₃,-Si (CH ₃) ₃,-CH ₂-CH=N-OCH ₃,-CH=CH-N (CH ₃)-CH ₃, O-CH ₃,-O-CH ₂-CH ₃With-CN.Two or three successive heteroatomss can be arranged at most, for example-CH ₂-NH-OCH ₃With-CH ₂-O-Si (CH ₃) ₃Similarly, term " assorted alkylidene group " itself or be meant derived from the divalent group of assorted alkyl as another substituent part for example, is not limited to-CH ₂-CH ₂-S-CH ₂-CH ₂-and-CH ₂-S-CH ₂-CH ₂-NH-CH ₂-.For assorted alkylidene group, what heteroatoms can also be at chain end appoints one or both ends (for example, alkylidene group oxo, alkylidene dioxygen generation, alkylidene amino, alkylidene group diamino etc.).For alkylidene group was connected base with assorted alkylidene group for, the presentation direction that connects the based structures formula was for connecting basic direction without any hint.For example expression-C (O) OR ' of formula-C (O) OR '-simultaneously-and-R ' OC (O)-.As mentioned above, assorted alkyl used herein comprises those groups that are connected with the rest part of molecule by heteroatoms, for example-C (O) R ' ,-C (O) NR ' ,-NR ' R ＂ ,-OR ' ,-SR ' and/or-SO ₂R '.When mentioning " assorted alkyl " and mentioning concrete assorted alkyl such as-NR ' R ＂ etc. afterwards again, be to be understood that the assorted alkyl of term and-NR ' R ＂ is not a multiple or mutually exclusive.On the contrary, enumerating concrete assorted alkyl is in order to increase clearness.Therefore, in this article, term " assorted alkyl " should not be interpreted as not comprising concrete assorted alkyl such as-NR ' R ＂ etc.

Except as otherwise noted, term " cycloalkyl " and " Heterocyclylalkyl " itself or represent the annular form of " alkyl " and " assorted alkyl " respectively with the combination of other term.In addition, for Heterocyclylalkyl, heteroatoms can be positioned at the position that heterocycle is connected with the rest part of molecule.The example of cycloalkyl includes but not limited to cyclopentyl, cyclohexyl, 1-cyclohexenyl, 3-cyclohexenyl, suberyl etc.The example of Heterocyclylalkyl includes but not limited to 1-(1,2,5,6-tetrahydro pyridyl), piperidino, 2-piperidyl, 3-piperidyl, 4-morpholinyl, morpholinyl, tetrahydrofuran (THF)-2-base, tetrahydrofuran (THF)-3-base, tetramethylene sulfide-2-base, tetramethylene sulfide-3-base, 1-piperazinyl, 2-piperazinyl etc.Term " cycloalkylidene " and " inferior Heterocyclylalkyl " are meant the divalent derivative of cycloalkyl and Heterocyclylalkyl respectively.

Except as otherwise noted, term " halo " or " halogen " itself or be meant fluorine, chlorine, bromine or iodine atom as another substituent part.In addition, term comprises single haloalkyl and multi-haloalkyl such as " haloalkyl ".For example, term " halo (C ₁-C ₄) alkyl " include but not limited to trifluoromethyl, 2,2,2-trifluoroethyl, 4-chlorobutyl, 3-bromopropyl etc.

Except as otherwise noted, term " aryl " is meant polyunsaturated aromatic hydrocarbon substituent, and they can be monocycles or condense together or covalently bound many rings (preferred 1-3 ring).Term " heteroaryl " is meant the heteroatomic aryl (or ring) that contains 1-4 (in the polycyclic situation, being meant in each independent ring) and be selected from N, O and S, and wherein nitrogen and sulphur atom are randomly oxidized, and nitrogen-atoms is randomly by quaternized.Heteroaryl can be connected on the rest part of molecule by carbon or heteroatoms.The limiting examples of aryl and heteroaryl comprises phenyl, the 1-naphthyl, the 2-naphthyl, the 4-xenyl, the 1-pyrryl, the 2-pyrryl, the 3-pyrryl, the 3-pyrazolyl, the 2-imidazolyl, the 4-imidazolyl, pyrazinyl, the 2-oxazolyl, the 4-oxazolyl, 2-phenyl-4-oxazolyl, the 5-oxazolyl, the 3-isoxazolyl, the 4-isoxazolyl, the 5-isoxazolyl, the 2-thiazolyl, the 4-thiazolyl, the 5-thiazolyl, the 2-furyl, the 3-furyl, the 2-thienyl, the 3-thienyl, the 2-pyridyl, the 3-pyridyl, the 4-pyridyl, the 2-pyrimidyl, the 4-pyrimidyl, the 5-benzothiazolyl, purine radicals, the 2-benzimidazolyl-, the 5-indyl, the 1-isoquinolyl, the 5-isoquinolyl, the 2-quinoxalinyl, the 5-quinoxalinyl, 3-quinolyl and 6-quinolyl.The substituting group of all above-mentioned aryl and heteroaryl ring all can be selected from the acceptable substituting group of the following stated.Term " arylidene " and " inferior heteroaryl " are meant the divalent group of aryl and heteroaryl respectively.

For brevity, when when being used in combination, term " aryl " comprises aryl defined above and heteroaryl ring with other term (for example aryl oxide generation, aryl sulfo-, arylalkyl).Therefore, term " arylalkyl " comprises those groups (for example benzyl, styroyl, pyridylmethyl etc.) that aryl wherein is connected with alkyl, comprises that wherein carbon atom (for example methylene radical) is by for example displaced those alkyl of Sauerstoffatom (for example phenoxymethyl, 2-pyridyl oxygen ylmethyl, 3-(1-naphthyl oxygen base) propyl group etc.).Yet term used herein " halogenated aryl " only comprises the aryl that is replaced by one or more halogens.Comprise that at assorted alkyl, Heterocyclylalkyl or heteroaryl term " member " is meant carbon or heteroatoms under member's the situation of concrete number (for example " 3-7 unit ").

Term used herein " oxo " is meant with carbon atom and passes through double linked oxygen.

All above-mentioned terms (for example " alkyl ", " assorted alkyl ", " cycloalkyl " and " Heterocyclylalkyl ", " aryl ", " heteroaryl " and divalent group derivative thereof) include the replacement and the unsubstituted form of described group.The preferred substituents of every type group is provided below.

The substituting group of alkyl, assorted alkyl, cycloalkyl, Heterocyclylalkyl monovalence and divalent derivative group (comprise and be commonly referred to as alkylidene group, alkenyl, assorted alkylidene group, heterochain thiazolinyl, alkynyl, cycloalkyl, Heterocyclylalkyl, cycloalkenyl group and heterocycloalkenyl) can be one or more be selected from but be not limited to following group :-OR ' ,=O ,=NR ' ,=N-OR ' ,-NR ' R ＂ ,-SR ' ,-halogen ,-SiR ' R ＂ R ＂ ' ,-OC (O) R ' ,-C (O) R ' ,-CO ₂R ' ,-C (O) NR ' R ＂ ,-OC (O) NR ' R ＂ ,-NR ＂ C (O) R ' ,-NR '-C (O) NR ＂ R ＂＇ ,-NR ＂ C (O) OR ' ,-NR-C (NR ' R ＂)=NR ＂＇ ,-S (O) R ' ,-S (O) ₂R ' ,-S (O) ₂NR ' R ＂ ,-NRSO ₂R ' ,-CN and-NO ₂, its quantitative range is 0 to (2m '+1), wherein m ' is the sum of carbon atom in the described group.R ', R ＂, R ＂＇ and R ＂＂ all preferably are meant hydrogen, replacement or unsubstituted assorted alkyl, replacement or unsubstituted cycloalkyl, replacement or unsubstituted Heterocyclylalkyl, replacement or unsubstituted aryl (for example aryl that is replaced by 1-3 halogen), replacement or unsubstituted alkyl, alkoxyl group or thio alkoxy or arylalkyl independently.When The compounds of this invention comprise more than one R group, for example when having more than one these groups, all R groups all are independently selected from R ', R ＂, R ＂＇ and R ＂＂ group.When R ' was connected with identical nitrogen-atoms with R ＂, they can lump together the ring that forms 4-, 5-, 6-or 7-unit with nitrogen-atoms.For example ,-NR ' R ＂ also includes but not limited to 1-pyrrolidyl and 4-morpholinyl.Discuss more than substituent, it will be understood to those of skill in the art that term " alkyl " comprises the carbon atom that combines with group outside the hydrogen group, for example haloalkyl (for example-CF ₃With-CH ₂CF ₃) and acyl group (for example-C (O) CH ₃,-C (O) CF ₃,-C (O) CH ₂OCH ₃Deng).

With above similar about the described substituting group of alkyl, the exemplary substituting group of aryl and heteroaryl (and divalent derivative) is diversified and for example is selected from: halogen ,-OR ' ,-NR ' R ＂ ,-SR ' ,-halogen ,-SiR ' R ＂ R ＂＇ ,-OC (O) R ' ,-C (O) R ' ,-CO ₂R ' ,-C (O) NR ' R ＂ ,-OC (O) NR ' R ＂ ,-NR ＂ C (O) R ' ,-NR '-C (O) NR ＂ R ＂＇ ,-NR ＂ C (O) OR ' ,-NR-C (NR ' R ＂ R ＂＇)=NR ＂＂ ,-NR-C (NR ' R ＂)=NR ＂＇ ,-S (O) R ' ,-S (O) ₂R ' ,-S (O) ₂NR ' R ＂ ,-NRSO ₂R ' ,-CN and-NO ₂,-R ' ,-N ₃,-CH (Ph) ₂, fluoro (C ₁-C ₄) alkyl oxo and fluoro (C ₁-C ₄) alkyl, its quantitative range is the zero sum of fastening open valence link to aromatic ring; And wherein R ', R ＂, R ＂＇ and R ＂＂ preferably are independently selected from hydrogen, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl, replacement or unsubstituted cycloalkyl, replacement or unsubstituted Heterocyclylalkyl, replacement or unsubstituted aryl and replacement or unsubstituted heteroaryl.When The compounds of this invention comprise more than one R group, for example when having more than one these groups, all R groups all are independently selected from R ', R ＂, R ＂＇ and R ＂＂ group.

Two in the substituting group on the adjacent atom of aryl or heteroaryl ring can randomly form formula-T-C (O)-(CRR ') _qThe ring of-U-, wherein T and U be independently-NR-,-O-,-CRR '-or singly-bound, q is the integer of 0-3.Perhaps, two in the substituting group on the adjacent atom of aryl or heteroaryl ring can be randomly by formula-A-(CH ₂) _rThe substituting group of-B-replaces, wherein A and B be independently-CRR '-,-O-,-NR-,-S-,-S (O)-,-S (O) ₂-,-S (O) ₂NR '-or singly-bound, r is the integer of 1-4.One of singly-bound of formed new ring can randomly be replaced by two key.Perhaps, two in the substituting group on the adjacent atom of aryl or heteroaryl ring can be randomly by formula-(CRR ') _s-X '-(CR ＂ R ＂＇) _d-substituting group replace, wherein s and d are the integer of 0-3 independently, X ' is-O-,-NR '-,-S-,-S (O)-,-S (O) ₂-or-S (O) ₂NR '-.Substituent R, R ', R ＂ and R ＂＇ preferably are independently selected from hydrogen, replacement or unsubstituted alkyl, replacement or unsubstituted cycloalkyl, replacement or unsubstituted Heterocyclylalkyl, replacement or unsubstituted aryl and replacement or unsubstituted heteroaryl.

Term used herein " heteroatoms " or " ring hetero atom " comprise oxygen (O), nitrogen (N), sulphur (S), phosphorus (P) and silicon (Si).

" aminoalkyl group " used herein is meant and is connected basic covalently bound amino with alkylidene group.Amino is-NR ' R ＂ that wherein R ' and R ＂ are selected from hydrogen, replacement or unsubstituted alkyl, replacement or unsubstituted assorted alkyl, replacement or unsubstituted cycloalkyl, replacement or unsubstituted Heterocyclylalkyl, replacement or unsubstituted aryl or replacement or unsubstituted heteroaryl usually.

" substituting group " used herein is meant and is selected from following group:

(A)-OH ,-NH ₂,-SH ,-CN ,-CF ₃,-NO ₂, oxo, halogen, unsubstituted alkyl, unsubstituted assorted alkyl, unsubstituted cycloalkyl, unsubstituted Heterocyclylalkyl, unsubstituted aryl, unsubstituted heteroaryl and

(B) be selected from the following alkyl that substituting group replaced, assorted alkyl, cycloalkyl, Heterocyclylalkyl, aryl and heteroaryl by at least one:

(i) oxo ,-OH ,-NH ₂,-SH ,-CN ,-CF ₃,-NO ₂, halogen, unsubstituted alkyl, unsubstituted assorted alkyl, unsubstituted cycloalkyl, unsubstituted Heterocyclylalkyl, unsubstituted aryl, unsubstituted heteroaryl and

(ii) be selected from the following alkyl that substituting group replaced, assorted alkyl, cycloalkyl, Heterocyclylalkyl, aryl and heteroaryl by at least one:

(a) oxo ,-OH ,-NH ₂,-SH ,-CN ,-CF ₃,-NO ₂, halogen, unsubstituted alkyl, unsubstituted assorted alkyl, unsubstituted cycloalkyl, unsubstituted Heterocyclylalkyl, unsubstituted aryl, unsubstituted heteroaryl and

(b) be selected from the following alkyl that substituting group replaced, assorted alkyl, cycloalkyl, Heterocyclylalkyl, aryl or heteroaryl by at least one: oxo ,-OH ,-NH ₂,-SH ,-CN ,-CF ₃,-NO ₂, halogen, unsubstituted alkyl, unsubstituted assorted alkyl, unsubstituted cycloalkyl, unsubstituted Heterocyclylalkyl, unsubstituted aryl and unsubstituted heteroaryl.

" restriction size substituting group " used herein or " the substituting group group of restriction size " is meant and is selected from abovely about " substituting group " described all substituent groups that wherein all replacements or unsubstituted alkyl all are to replace or unsubstituted C ₁-C ₂₀Alkyl, all replacements or unsubstituted assorted alkyl all are to replace or the assorted alkyl of unsubstituted 2-20 unit, and all replacements or unsubstituted cycloalkyl all are to replace or unsubstituted C ₄-C ₈Cycloalkyl, and all replacements or unsubstituted Heterocyclylalkyl all are to replace or the Heterocyclylalkyl of unsubstituted 4-8 unit.

" rudimentary substituting group " used herein or " rudimentary substituting group group " is meant and is selected from abovely about " substituting group " described all substituent groups that wherein all replacements or unsubstituted alkyl all are to replace or unsubstituted C ₁-C ₈Alkyl, all replacements or unsubstituted assorted alkyl all are to replace or the assorted alkyl of unsubstituted 2-8 unit, and all replacements or unsubstituted cycloalkyl all are to replace or unsubstituted C ₅-C ₇Cycloalkyl, and all replacements or unsubstituted Heterocyclylalkyl all are to replace or the Heterocyclylalkyl of unsubstituted 5-7 unit.

The compounds of this invention can salt form exist.The present invention includes described salt.The example of applicable salt form comprise hydrochloride, hydrobromate, vitriol, mesylate, nitrate, maleate, acetate, Citrate trianion, fumarate, tartrate (for example (+)-tartrate, (-)-tartrate or its mixture comprise racemic mixture), succinate, benzoate and with the formed salt of amino acid such as L-glutamic acid.These salt can make by method known to those skilled in the art.Also comprise base addition salt such as sodium, potassium, calcium, ammonium, organic amine salt or magnesium salts or similar salt.When The compounds of this invention contained the functional group that is alkalescence relatively, acid salt can be by direct with the required acid of capacity with the neutral form of this compound or contact in suitable inert solvent and obtain.The example of acceptable acid salt comprises derived from the mineral acid example hydrochloric acid, Hydrogen bromide, nitric acid, carbonic acid, one hydrogen carbonic acid, phosphoric acid, one hydrogen phosphoric acid, dihydrogen phosphoric acid, sulfuric acid, one hydrosulphuric acid, those salt of hydroiodic acid HI or phosphorous acid etc. and derived from organic acid such as acetate, propionic acid, isopropylformic acid, toxilic acid, propanedioic acid, phenylformic acid, succsinic acid, suberic acid, fumaric acid, lactic acid, amygdalic acid, phthalic acid, Phenylsulfonic acid, tosic acid, citric acid, tartrate, the salt of methylsulfonic acid etc.Also comprise the salt of amino acid such as arginine etc. and the salt of organic acid such as glucuronic acid or galacturonic acid etc.Some particular compound of the present invention contains alkalescence and acidic functionality simultaneously, thereby both this compound can be changed into base addition salt, also can convert it into acid salt.

The neutral form of compound is preferably by contacting salt, separating parent compound then in a conventional manner and regenerate with alkali or acid.The parent form of compound on some physical properties such as the form that is different from various salt aspect the solubleness in polar solvent.

Some compound of the present invention can non-solvent compound form and solvate forms, comprise that the form of hydrate exists.Usually, solvate forms is equal to the form of non-solvent compound and comprises within the scope of the invention.Some compound of the present invention can polymorphic or the existence of unbodied form.Usually, all physical form all are equal to for the desired purposes of the present invention, and comprise within the scope of the invention.

Some compound of the present invention has asymmetric carbon atoms (optics or chiral centre) or two key; Enantiomorph, racemic modification, diastereomer, tautomer, geometrical isomer, according to the absolute stereo chemistry may be defined as (R)-or (S)-or for amino acid may be defined as (D)-or (L)-stereoisomeric forms in any ratio and independent isomer be also included within the scope of the present invention.The compounds of this invention does not comprise too instability known in the art and can not synthesize and/or isolating those compounds.The present invention includes the compound of respective pure form on racemize and the optics.(R) of opticity-and (S)-or (D)-and (L)-isomer can utilize chiral synthon or chiral reagent to make, perhaps utilize routine techniques to split.When compound as herein described contained ethylene linkage or other how much asymmetric centers, except as otherwise noted, this compound comprised E and Z geometrical isomer.

Term used herein " tautomer " is meant to exist and be easy to equilibrium form and changes into one of alternative two or more constitutional isomers from a kind of isomeric form.

It will be apparent to one skilled in the art that some compound of the present invention can tautomeric form exist, all tautomeric forms of this compound all within the scope of the invention.

Except as otherwise noted, structure as herein described also comprises all stereochemical forms of this structure; That is, the R of each asymmetric center and S configuration.Therefore, the enantiomorph of single three-dimensional chemical isomer and this compound and non-enantiomer mixture are all within the scope of the invention.

Except as otherwise noted, structure as herein described comprises that also their difference only is to exist the compound of the atom of one or more isotopic enrichments.For example, replaced or the carbon quilt by deuterium or tritium except hydrogen ¹³C-or ¹⁴Outside the carbon of C-enrichment replaced, the compound with this structure also within the scope of the invention.

The compounds of this invention also can contain the atom isotope of non-natural ratio on the one or more atoms that constitute this compound.For example, this compound can use radio isotope such as tritium ( ³H), iodine-125 ( ¹²⁵I) or carbon-14 ( ¹⁴C) carry out radio-labeling.No matter whether all isotopic variations of The compounds of this invention have radioactivity, all comprises within the scope of the invention.

Term " pharmacologically acceptable salt " comprises the salt with the active compound of nontoxic relatively acid or alkali (depending on the specified substituent part on the compound as herein described) preparation.When The compounds of this invention contained relative tart functional group, base addition salt can be by with the required alkali of the neutral form of this compound and capacity directly or contact in suitable inert solvent and obtain.The example of pharmaceutically acceptable base addition salt comprises sodium, potassium, calcium, ammonium, organic amine salt or magnesium salts or similar salt.When The compounds of this invention contained relatively the functional group of alkalescence, acid salt can be by direct with the needed acid of capacity with the neutral form of this compound or contact in suitable inert solvent and obtain.The example of pharmaceutically acceptable acid additive salt comprises derived from the mineral acid example hydrochloric acid, Hydrogen bromide, nitric acid, carbonic acid, one hydrogen carbonic acid, phosphoric acid, one hydrogen phosphoric acid, dihydrogen phosphoric acid, sulfuric acid, one hydrosulphuric acid, those salt of hydroiodic acid HI or phosphorous acid etc. and derived from nontoxic relatively organic acid such as acetate, propionic acid, isopropylformic acid, toxilic acid, propanedioic acid, phenylformic acid, succsinic acid, suberic acid, fumaric acid, lactic acid, tussol, phthalic acid, Phenylsulfonic acid, tosic acid, citric acid, tartrate, the salt of methylsulfonic acid etc.Also comprise the salt of amino acid such as arginine etc. and the salt of organic acid such as glucuronic acid or galacturonic acid etc. (referring to people such as for example Berge, ＂ Pharmaceutical Salts ＂, Journal of Pharmaceutical Science, 1977,66,1-19).Some particular compound of the present invention contains alkalescence and acidic functionality simultaneously, thereby both this compound can be changed into base addition salt, also can convert it into acid salt.

Except salt form, the present invention also provides the compound of prodrug forms.The prodrug of compound as herein described is to be easy to take place chemical transformation so that the compound of The compounds of this invention to be provided under physiological conditions.In addition, prodrug also can change into compound of the present invention by chemistry or biochemical method in the environment that exsomatizes.For example, when placing the transdermal patch bank with suitable enzyme or chemical reagent, prodrug can change into compound of the present invention lentamente.

In this article, when mentioning one group of substituting group, term " ", " one " or " a kind of " are meant at least one.For example, when a kind of compound was replaced by alkyl or aryl, this compound was randomly replaced by at least one alkyl and/or at least one aryl.In addition, when this part was replaced by the R substituting group, this group can be known as " R-replaces ".In this part is under the situation of R-replacement, and this part is replaced by at least one R substituting group and all R substituting groups are optional different.

Be subjected to the restriction of chemical bonding principle well known by persons skilled in the art about the description of The compounds of this invention.Therefore, under a group can be by the situation that one or more substituting group replaced, described substituent selection should meet the principle of chemical bonding, and in obtaining in stable and/or the known condition around of those of ordinary skills, such as moisture, neutral and several known physiological conditions under stable compound.For example, Heterocyclylalkyl or heteroaryl should meet chemical bonding principle well known by persons skilled in the art by ring hetero atom and being connected of molecule rest part, to avoid producing inherent unstable compounds.

When mentioning concrete disease, term " treatment " also comprises the prevention of this disease.

Symbol "

" tie point of rest part of expression group and molecule.

I. fused ring heterocycle kinase modulators

On the one hand, the invention provides fused ring heterocycle kinase modulators (also being known as ＂ The compounds of this invention ＂ in this article) with following formula:

In formula (I), L ¹And L ²Be independently key ,-S (O) _n-,-O-,-NH-, replacement or unsubstituted C ₁-C ₅The assorted alkylidene group of alkylidene group or replacement or unsubstituted 2-5 unit.In certain embodiments, L ¹And L ²Be independently key ,-S (O) _n-,-O-,-NH-, unsubstituted C ₁-C ₅The assorted alkylidene group of alkylidene group or unsubstituted 2-5 unit.Symbol n is the integer of 0-2.R ¹And R ²Be to replace or unsubstituted cycloalkyl, replacement or unsubstituted Heterocyclylalkyl, replacement or unsubstituted heteroaryl or replacement or unsubstituted aryl independently.

In certain embodiments, R ¹Not to replace or unsubstituted pyrryl.In other embodiments, work as R ¹And R ²When all being unsubstituted phenyl, L ¹It or not the assorted alkylidene group of unsubstituted 2-5 unit.In other embodiments, work as R ²When being unsubstituted piperazinyl, L ¹Not-S (O) ₂-.

In certain embodiments, R ¹Not to replace or the heteroaryl of unsubstituted 5-unit.In other embodiments, R ¹Be to replace or heteroaryl, replacement or unsubstituted Heterocyclylalkyl, replacement or unsubstituted aryl or the replacement or the unsubstituted heteroaryl of unsubstituted 6-unit.R ¹Can also be to replace or heteroaryl or the replacement or the unsubstituted aryl of unsubstituted 6-unit.

In other embodiments, work as R ¹And R ²When all being unsubstituted aryl, L ¹It or not the assorted alkylidene group of unsubstituted 2-5 unit.In other embodiments, work as R ¹And R ²When all being replacement or unsubstituted phenyl, L ¹It or not the assorted alkylidene group of unsubstituted 2-5 unit.In other embodiments, L ¹Be selected from key ,-S (O) _n-,-O-,-NH-and unsubstituted C ₁-C ₅Alkylidene group.In other embodiments, n is 0 or 1.In other embodiments, L ¹Be selected from key ,-O-,-NH-and unsubstituted C ₁-C ₅Alkylidene group.

In certain embodiments, work as R ²When being replacement or unsubstituted piperazinyl, L ¹Not-S (O) ₂-.In other embodiments, work as R ²When being unsubstituted Heterocyclylalkyl, L ¹Not-S (O) ₂-.In other embodiments, work as R ¹When being replacement or unsubstituted Heterocyclylalkyl, L ¹Not-S (O) ₂-.In other embodiments, n is 0 or 1.In other embodiments, work as L ²When being key, L ¹Not-S (O) ₂-.

In certain embodiments, R ²It or not the first Heterocyclylalkyl of unsubstituted 6-.In other embodiments, R ²Not to replace or the Heterocyclylalkyl of unsubstituted 6-unit.In other embodiments, R ²Be selected from and replace or Heterocyclylalkyl, replacement or unsubstituted aryl and the replacement or the unsubstituted cycloalkyl of unsubstituted heteroaryl, replacement or unsubstituted 5-unit.R ²Can also be Heterocyclylalkyl, replacement or unsubstituted heteroaryl or the replacement or the unsubstituted aryl of replacement or unsubstituted cycloalkyl, replacement.

In certain embodiments, work as R ²When being the unsubstituted pyridine base, R ¹Not to replace or unsubstituted isoxazolyl.In other embodiments, work as L ¹Be key or-CH ₂-time, R ¹Not to replace or unsubstituted isoxazolyl.In other embodiments, R ¹Not to replace or unsubstituted isoxazolyl.In other embodiments, R ¹Not that 4-replaces the De isoxazolyl.In other embodiments, R ¹Not 5-base-isoxazolyls.In other embodiments, R ¹Not 4-replacement-5-base-isoxazolyls.In other embodiments, R ¹The aryl that is replaced by fluoro-does not replace the De isoxazolyl.

L ¹And L ²Can be independently key ,-S (O) _n-,-O-,-NH-or unsubstituted C ₁-C ₅Alkylidene group.In certain embodiments, L ¹And L ²It is key.In other embodiments, L ¹Or L ²It is key.

R ¹Can be heteroaryl or the replacement or the unsubstituted aryl of replacement or unsubstituted cycloalkyl, replacement or unsubstituted Heterocyclylalkyl, replacement or unsubstituted 5-or 6-unit.R ¹Can also be to replace or heteroaryl or the replacement or the unsubstituted aryl of unsubstituted 6-unit.

In other embodiments, R ¹Be (1) unsubstituted C ₃-C ₇Cycloalkyl; (2) Heterocyclylalkyl of unsubstituted 3-7 unit; (3) unsubstituted heteroaryl; (4) unsubstituted aryl; (5) C of Qu Daiing ₃-C ₇Cycloalkyl; (6) Heterocyclylalkyl of the 3-7 unit of Qu Daiing; (7) heteroaryl of the aryl of Qu Daiing or (8) replacement.In certain embodiments, (5) and (6) are replaced by following substituting group: oxo ,-OH ,-CF ₃,-COOH, cyano group, halogen, R ¹¹-replace or unsubstituted C ₁-C ₁₀Alkyl, R ¹¹-replace or assorted alkyl, the R of unsubstituted 2-10 unit ¹¹-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ¹¹-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ¹²-replacement or unsubstituted aryl, R ¹²-replacement or unsubstituted heteroaryl ,-L ¹²-C (X ¹) R ⁷,-L ¹²-OR ⁸,-L ¹²-NR ⁹¹R ⁹²Or-L ¹²-S (O) _mR ¹⁰X ¹Be=S ,=O or=NR ¹⁵, R wherein ¹⁵Be H ,-OR ¹⁵¹, R ¹¹-replace or unsubstituted C ₁-C ₁₀Alkyl, R ¹¹-replace or assorted alkyl, the R of unsubstituted 2-10 unit ¹¹-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ¹¹-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ¹²-replace or unsubstituted aryl or R ¹²-replace or unsubstituted heteroaryl.R1 ⁵¹Be hydrogen or R ¹¹-replace or unsubstituted C ₁-C ₁₀Alkyl.Symbol m is the integer of 0-2.

In other embodiments, (7) and (8) are replaced by following substituting group :-OH ,-CF ₃,-COOH, cyano group, halogen, R ¹¹-replace or unsubstituted C ₁-C ₁₀Alkyl, R ¹¹-replace or assorted alkyl, the R of unsubstituted 2-10 unit ¹¹-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ¹¹-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ¹²-replacement or unsubstituted aryl, R ¹²-replacement or unsubstituted heteroaryl ,-L ¹²C (X ¹) R ⁷,-L ¹²-OR ⁸,-L ¹²-NR ⁹¹R ⁹²Or-L ¹²-S (O) _mR ¹⁰L ¹²Be key, unsubstituted C ₁-C ₁₀Alkylidene group or unsubstituted assorted alkylidene group.X ¹With m as defined above.

R ⁷Be hydrogen, R ¹¹-replace or unsubstituted C ₁-C ₁₀Alkyl, R ¹¹-replace or assorted alkyl, the R of unsubstituted 2-10 unit ¹¹-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ¹¹-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ¹²-replacement or unsubstituted aryl, R ¹²-replacement or unsubstituted heteroaryl ,-OR ⁷¹Or-NR ⁷²R ⁷³R ⁷¹, R ⁷²And R ⁷³Be hydrogen, R independently ¹¹-replace or unsubstituted C ₁-C ₁₀Alkyl, R ¹¹-replace or assorted alkyl, the R of unsubstituted 2-10 unit ¹¹-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ¹¹-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ¹²-replace or unsubstituted aryl or R ¹²-replace or unsubstituted heteroaryl.R ⁷²And R ⁷³Randomly the nitrogen that is connected with them lumps together and forms R ¹¹-replace or the Heterocyclylalkyl or the R of unsubstituted 3-7 unit ¹²-replace or unsubstituted heteroaryl.

R ⁸, R ⁷¹And R ⁹²Be independently hydrogen ,-CF ₃, R ¹¹-replace or unsubstituted C ₁-C ₁₀Alkyl, R ¹¹-replace or assorted alkyl, the R of unsubstituted 2-10 unit ¹¹-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ¹¹-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ¹²-replacement or unsubstituted aryl, R ¹²-replacement or unsubstituted heteroaryl ,-C (X ²) R ⁸¹Or-S (O) _wR ⁸¹X ²Be=S ,=O or=NR ¹⁶R ¹⁶Be R ¹¹-replace or unsubstituted C ₁-C ₁₀Alkyl, R ¹¹-replace or assorted alkyl, the R of unsubstituted 2-10 unit ¹¹-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ¹¹-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ¹²-replace or unsubstituted aryl or R ¹²-replace or unsubstituted heteroaryl.Symbol w is the integer of 0-2.R ⁹¹And R ⁹²Randomly the nitrogen that is connected with them lumps together and forms R ¹¹-replace or the Heterocyclylalkyl or the R of unsubstituted 3-7 unit ¹²-replace or unsubstituted heteroaryl.

R ⁸¹Be hydrogen, R ¹¹-replace or unsubstituted C ₁-C ₁₀Alkyl, R ¹¹-replace or assorted alkyl, the R of unsubstituted 2-10 unit ¹¹-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ¹¹-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ¹²-replacement or unsubstituted aryl, R ¹²-replace or unsubstituted heteroaryl or-NR ⁸¹¹R ⁸¹²

R ⁸¹¹And R ⁸¹²Be hydrogen, R independently ¹¹-replace or unsubstituted C ₁-C ₁₀Alkyl, R ¹¹-replace or assorted alkyl, the R of unsubstituted 2-10 unit ¹¹-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ¹¹-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ¹²-replace or unsubstituted aryl or R ¹²-replace or unsubstituted heteroaryl.R ⁸¹¹And R ⁸¹²Randomly the nitrogen that is connected with them lumps together and forms R ¹¹-replace or the Heterocyclylalkyl or the R of unsubstituted 3-7 unit ¹²-replace or unsubstituted heteroaryl.

In certain embodiments, R ⁸¹And R ¹⁶Randomly form and replace or unsubstituted Heterocyclylalkyl or replacement or unsubstituted heteroaryl with the atom that they connected.In other embodiments, R ⁸¹¹And R ¹⁶Randomly form and replace or unsubstituted Heterocyclylalkyl or replacement or unsubstituted heteroaryl with the atom that they connected.In other embodiments, R ⁸¹And R ⁹²Randomly form and replace or unsubstituted Heterocyclylalkyl or replacement or unsubstituted heteroaryl with the atom that they connected.In other embodiments, R ⁸¹¹And R ⁹²Randomly form and replace or unsubstituted Heterocyclylalkyl or replacement or unsubstituted heteroaryl with the atom that they connected.

R ¹⁰Be hydrogen, R ¹¹-replace or unsubstituted C ₁-C ₁₀Alkyl, R ¹¹-replace or assorted alkyl, the R of unsubstituted 2-10 unit ¹¹-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ¹¹-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ¹²-replacement or unsubstituted aryl, R ¹²-replace or unsubstituted heteroaryl or-NR ¹⁰¹R ¹⁰²R ¹⁰¹And R ¹⁰²Be hydrogen, R independently ¹¹-replace or unsubstituted C ₁-C ₁₀Alkyl, R ¹¹-replace or assorted alkyl, the R of unsubstituted 2-10 unit ¹¹-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ¹¹-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ¹²-replace or unsubstituted aryl or R ¹²-replace or unsubstituted heteroaryl.R ¹⁰¹And R ¹⁰²Randomly the nitrogen that is connected with them lumps together and forms R ¹¹-replace or the Heterocyclylalkyl or the R of unsubstituted 3-7 unit ¹²-replace or unsubstituted heteroaryl.

R ¹¹Be oxo ,-OH ,-COOH ,-CF ₃,-OCF ₃,-CN, amino, halogen, R ¹³-replace or alkyl, the R of unsubstituted 2-10 unit ¹³-replace or assorted alkyl, the R of unsubstituted 2-10 unit ¹³-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ¹³-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ¹⁴-replace or unsubstituted aryl or R ¹⁴-replace or unsubstituted heteroaryl.

R ¹²Be-OH ,-COOH, amino, halogen ,-CF ₃,-OCF ₃,-CN, R ¹³-replace or alkyl, the R of unsubstituted 2-10 unit ¹³-replace or assorted alkyl, the R of unsubstituted 2-10 unit ¹³-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ¹³-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ¹⁴-replace or unsubstituted aryl or R ¹⁴-replace or unsubstituted heteroaryl.

R ¹³Be oxo ,-OH ,-COOH, amino, halogen ,-CF ₃,-OCF ₃,-CN, unsubstituted C ₁-C ₁₀Assorted alkyl, the unsubstituted C of alkyl, unsubstituted 2-10 unit ₃-C ₇Heterocyclylalkyl, unsubstituted aryl, the unsubstituted heteroaryl of cycloalkyl, unsubstituted 3-7 unit.

R ¹⁴Be-OH ,-COOH, amino, halogen ,-CF ₃,-OCF ₃,-CN, unsubstituted C ₁-C ₁₀Assorted alkyl, the unsubstituted C of alkyl, unsubstituted 2-10 unit ₃-C ₇Heterocyclylalkyl, unsubstituted aryl, the unsubstituted heteroaryl of cycloalkyl, unsubstituted 3-7 unit.

In certain embodiments, R ¹Be that (1), (2), (4), (5), (6) or (7) (promptly are respectively unsubstituted C ₃-C ₇The C of the Heterocyclylalkyl of cycloalkyl, unsubstituted 3-7 unit, unsubstituted aryl, replacement ₃-C ₇The Heterocyclylalkyl of the 3-7 unit of cycloalkyl, replacement or the aryl of replacement).In certain embodiments, at R ¹Be under the situation of (3) or (8), heteroaryl is the heteroaryl of 6-unit.

At R ¹Be under the situation of (7) or (8) (i.e. the heteroaryl of aryl of Qu Daiing or replacement), (7) and (8) can be selected from following substituting group and be replaced :-OH ,-CF ₃,-OCF ₃, halogen, unsubstituted C ₁-C ₁₀Assorted alkyl, the unsubstituted C of alkyl, unsubstituted 2-10 unit ₃-C ₇The Heterocyclylalkyl of cycloalkyl, unsubstituted 3-7 unit, unsubstituted aryl, unsubstituted heteroaryl or-L ¹²-OR ⁸In relevant embodiment, L ¹²It is key.In other related embodiment, (7) and (8) can be by-OCH ₃,-OCF ₃,-CH ₃,-CF ₃,-OCH ₂CH ₃, halogen or the ring propoxy-replace.

R ²Can be: (1) unsubstituted C ₃-C ₇Cycloalkyl; (2) Heterocyclylalkyl of unsubstituted 3-7 unit; (3) unsubstituted heteroaryl; (4) unsubstituted aryl; (5) C of Qu Daiing ₃-C ₇Cycloalkyl; (6) Heterocyclylalkyl of the 3-7 unit of Qu Daiing; (7) heteroaryl of the aryl of Qu Daiing or (8) replacement.In some related embodiment, (5) and (6) are replaced by following substituting group: oxo ,-OH ,-CF ₃,-COOH, cyano group, halogen, R ²¹-replace or unsubstituted C ₁-C ₁₀Alkyl, R ²¹-replace or assorted alkyl, the R of unsubstituted 2-10 unit ²¹-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ²¹-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ²²-replace or unsubstituted aryl or R ²²-replacement or unsubstituted heteroaryl ,-L ²²-C (X ³) R ³,-L ²²-OR ⁴,-L ²²-NR ⁵¹R ⁵²Or-L ²²-S (O) _qR ⁶X ³Be=S ,=O or=NR ¹⁷, R wherein ¹⁷Be H ,-OR ¹⁷¹, R ²¹-replace or unsubstituted C ₁-C ₁₀Alkyl, R ²¹-replace or assorted alkyl, the R of unsubstituted 2-10 unit ²¹-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ²¹-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ²²-replace or unsubstituted aryl or R ²²-replace or unsubstituted heteroaryl.R ¹⁷¹Be H or R ²¹-replace or unsubstituted C ₁-C ₁₀Alkyl.Symbol q is the integer of 0-2.

In other related embodiment, (7) and (8) are replaced by following substituting group :-OH ,-CF ₃,-COOH, cyano group, halogen, R ²¹-replace or unsubstituted C ₁-C ₁₀Alkyl, R ²¹-replace or assorted alkyl, the R of unsubstituted 2-10 unit ²¹-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ²¹-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ²²-replacement or unsubstituted aryl, R ²²-replacement or unsubstituted heteroaryl ,-L ²²-C (X ³) R ³,-L ²²-OR ⁴,-L ²²-NR ⁵¹R ⁵²Or-L ²²-S (O) _qR ⁶L ²²Be key, unsubstituted C ₁-C ₁₀Alkylidene group or unsubstituted assorted alkylidene group.X ³With q as defined above.

R ³Be hydrogen, R ²¹-replace or unsubstituted C ₁-C ₁₀Alkyl, R ²¹-replace or assorted alkyl, the R of unsubstituted 2-10 unit ²¹-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ²¹-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ²²-replacement or unsubstituted aryl, R ²²-replacement or unsubstituted heteroaryl ,-OR ³¹Or-NR ³²R ³³R ³²And R ³³Randomly the nitrogen that is connected with them lumps together and forms R ²¹-replace or the Heterocyclylalkyl or the R of unsubstituted 3-7 unit ²²-replace or unsubstituted heteroaryl.

R ³¹, R ³²And R ³³Be hydrogen, R independently ²¹-replace or unsubstituted C ₁-C ₁₀Alkyl, R ²¹-replace or assorted alkyl, the R of unsubstituted 2-10 unit ²¹-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ²¹-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ²²-replace or unsubstituted aryl or R ²²-replace or unsubstituted heteroaryl.

R ⁴, R ⁵¹And R ⁵²Be independently hydrogen ,-CF ₃, R ²¹-replace or unsubstituted C ₁-C ₁₀Alkyl, R ²¹-replace or assorted alkyl, the R of unsubstituted 2-10 unit ²¹-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ²¹-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ²²-replacement or unsubstituted aryl, R ²²-replacement or unsubstituted heteroaryl ,-C (X ⁴) R ⁴¹Or-S (O) _vR ⁴¹R ⁵¹And R ⁵²Randomly the nitrogen that is connected with them lumps together and forms R ²¹-replace or the Heterocyclylalkyl or the R of unsubstituted 3-7 unit ²²-replace or unsubstituted heteroaryl.

X ⁴Be=S ,=O or=NR ¹⁸, R wherein ¹⁸Be R ²¹-replace or unsubstituted C ₁-C ₁₀Alkyl, R ²¹-replace or assorted alkyl, the R of unsubstituted 2-10 unit ²¹-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ²¹-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ²²-replace or unsubstituted aryl or R ²²-replace or unsubstituted heteroaryl.Symbol v is the integer of 0-2.

R ⁴¹Be hydrogen, R ²¹-replace or unsubstituted C ₁-C ₁₀Alkyl, R ²¹-replace or assorted alkyl, the R of unsubstituted 2-10 unit ²¹-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ²¹-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ²²-replacement or unsubstituted aryl, R ²²-replace or unsubstituted heteroaryl or-NR ⁴¹¹R ⁴¹²R ⁴¹¹And R ⁴¹²Be independently selected from hydrogen, R ²¹-replace or unsubstituted C ₁-C ₁₀Alkyl, R ²¹-replace or assorted alkyl, the R of unsubstituted 2-10 unit ²¹-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ²¹-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ²²-replace or unsubstituted aryl or R ²²-replace or unsubstituted heteroaryl.R ⁴¹¹And R ⁴¹²Randomly the nitrogen that is connected with them lumps together and forms R ²¹-replace or the Heterocyclylalkyl or the R of unsubstituted 3-7 unit ²¹-replace or unsubstituted heteroaryl.

In certain embodiments, R ⁴¹And R ¹⁸Randomly form and replace or unsubstituted Heterocyclylalkyl or replacement or unsubstituted heteroaryl with the atom that they connected.In other embodiments, R ⁴¹¹And R ¹⁸Randomly form and replace or unsubstituted Heterocyclylalkyl or replacement or unsubstituted heteroaryl with the atom that they connected.In other embodiments, R ⁴¹And R ⁵²Randomly form and replace or unsubstituted Heterocyclylalkyl or replacement or unsubstituted heteroaryl with the atom that they connected.In other embodiments, R ⁴¹¹And R ⁵²Randomly form and replace or unsubstituted Heterocyclylalkyl or replacement or unsubstituted heteroaryl with the atom that they connected.

R ⁶Be hydrogen, R ²¹-replace or unsubstituted C ₁-C ₁₀Alkyl, R ²¹-replace or assorted alkyl, the R of unsubstituted 2-10 unit ²¹-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ²¹-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ²²-replacement or unsubstituted aryl, R ²²-replace or unsubstituted heteroaryl or-NR ⁶¹R ⁶²R ⁶¹And R ⁶²Be hydrogen, R ²¹-replace or unsubstituted C ₁-C ₁₀Alkyl, R ²¹-replace or assorted alkyl, the R of unsubstituted 2-10 unit ²¹-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ²¹-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ²²-replace or unsubstituted aryl or R ²²-replace or unsubstituted heteroaryl.R ⁶¹And R ⁶²Randomly the nitrogen that is connected with them lumps together and forms R ²¹-replace or the Heterocyclylalkyl or the R of unsubstituted 3-7 unit ²²-replace or unsubstituted heteroaryl.

R ²¹Be oxo ,-OH ,-COOH ,-CF ₃,-OCF ₃,-CN, amino, halogen, R ²³-replace or alkyl, the R of unsubstituted 2-10 unit ²³-replace or assorted alkyl, the R of unsubstituted 2-10 unit ²³-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ²³-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ²⁴-replace or unsubstituted aryl or R ²⁴-replace or unsubstituted heteroaryl.

R ²²Be-OH ,-COOH, amino, halogen ,-CF ₃,-OCF ₃,-CN, R ²³-replace or alkyl, the R of unsubstituted 2-10 unit ²³-replace or assorted alkyl, the R of unsubstituted 2-10 unit ²³-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ²³-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ²⁴-replace or unsubstituted aryl or R ²⁴-replace or unsubstituted heteroaryl.

R ²³Be oxo ,-OH ,-COOH, amino, halogen ,-CF ₃,-OCF ₃,-CN, unsubstituted C ₁-C ₁₀Assorted alkyl, the unsubstituted C of alkyl, unsubstituted 2-10 unit ₃-C ₇Heterocyclylalkyl, unsubstituted aryl, the unsubstituted heteroaryl of cycloalkyl, unsubstituted 3-7 unit.

R ²⁴Be-OH ,-COOH, amino, halogen ,-CF ₃,-OCF ₃,-CN, unsubstituted C ₁-C ₁₀Assorted alkyl, the unsubstituted C of alkyl, unsubstituted 2-10 unit ₃-C ₇Heterocyclylalkyl, unsubstituted aryl, the unsubstituted heteroaryl of cycloalkyl, unsubstituted 3-7 unit.

In certain embodiments, R ²Be that (1), (3), (4), (5), (6), (7) or (8) (promptly are respectively unsubstituted C ₃-C ₇The C of cycloalkyl, unsubstituted heteroaryl, unsubstituted aryl, replacement ₃-C ₇The Heterocyclylalkyl of the 3-7 unit of cycloalkyl, replacement, the aryl of replacement or the heteroaryl of replacement).R ²Can also be (3), (4), (7) or (8).In other embodiments, R ²Be (7) or (8).

In certain embodiments, at R ²Be under the situation of (7) and (8), (7) and (8) quilt-L ²²-C (X ³) R ³,-L ²²-OR ⁴,-L ²²-NR ⁵¹R ⁵²,-L ²²-C (NH)-NR ³²R ³³Or-L ²²-S (O) _qR ⁶Replace.

In certain embodiments, R ³Be-NR ³²R ³³X ³Can be=O or=NR ¹⁷R ⁶Can be-NR ⁶¹R ⁶²R ⁴Can be-C (O) R ⁴¹Or-S (O) _vR ⁴¹R ⁴¹Can be-NR ⁴¹¹R ⁴¹²

In other embodiments, at R ²Be under the situation of (7) and (8), (7) or (8) can by-OH ,-CF ₃Assorted alkyl, the unsubstituted C of ,-COOH, amino, halogen, unsubstituted 2-10 unit ₃-C ₇The Heterocyclylalkyl of cycloalkyl, unsubstituted 3-7 unit, unsubstituted aryl, unsubstituted heteroaryl or-L ²²-C (X ³) R ³Replace.X ³Can be=O.

R ³Can be unsubstituted C ₁-C ₁₀Assorted alkyl, the unsubstituted C of alkyl, unsubstituted 2-10 unit ₃-C ₇The Heterocyclylalkyl of cycloalkyl, unsubstituted 3-7 unit, unsubstituted aryl, unsubstituted heteroaryl or-NR ³²R ³³R ³²And R ³³Be hydrogen, R independently ²¹-replace or unsubstituted C ₁-C ₁₀Alkyl, R ²¹-replace or assorted alkyl, the R of unsubstituted 2-10 unit ²¹-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ²¹-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ²²-replace or unsubstituted aryl or R ²²-replace or unsubstituted heteroaryl.R ³²And R ³³Randomly the nitrogen that is connected with them lumps together and forms R ²¹-replace or the Heterocyclylalkyl or the R of unsubstituted 3-7 unit ²²-replace or unsubstituted heteroaryl.

In another embodiment, at R ²Be under the situation of (7) or (8), (7) and (8) can by the assorted alkyl of unsubstituted 2-10 unit or-L ²²-C (O) R ³Replace.L ²²It can be key.R ³Can be-NR ³²R ³³R ³²And R ³³Be hydrogen, R independently ²¹-replace or unsubstituted C ₁-C ₁₀Alkyl, R ²¹-replace or assorted alkyl, the R of unsubstituted 2-10 unit ²¹-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ²¹-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ²²-replace or unsubstituted aryl or R ²²-replace or unsubstituted heteroaryl.R ³²And R ³³Randomly the nitrogen that is connected with them lumps together and forms R ²¹-replace or the Heterocyclylalkyl or the R of unsubstituted 3-7 unit ²²-replace or unsubstituted heteroaryl.

In certain embodiments, R ¹Be to replace or unsubstituted fused ring aryl or replacement or unsubstituted condensed ring heteroaryl.In other embodiments, R ²Be to replace or unsubstituted indyl, replacement or unsubstituted quinolines base or replacement or unsubstituted benzo dioxolyl.R ²Can be to replace or unsubstituted fused ring aryl or replacement or unsubstituted condensed ring heteroaryl.R ¹Can be to replace or unsubstituted indyl, replacement or unsubstituted quinolines base or replacement or unsubstituted benzo dioxolyl.

R ¹And R ²Be to replace or unsubstituted glycolylurea base independently, replace or unsubstituted dioxolanyl, replace or unsubstituted alkyl dioxin, replace or unsubstituted trioxa alkyl, replace or unsubstituted tetrahydro-thienyl, replace or unsubstituted tetrahydrofuran base, replace or unsubstituted tetrahydro-thienyl, replace or unsubstituted THP trtrahydropyranyl, replace or unsubstituted tetrahydro thiapyran base, replace or unsubstituted pyrrolidyl, replace or unsubstituted morpholinyl, replace or unsubstituted piperidyl, replace or unsubstituted pyrazolyl, replace or unsubstituted furyl, replace or unsubstituted imidazolyl, replace or unsubstituted isoxazolyl, replace or do not replace De oxadiazole base, replace or do not replace the De oxazolyl, replace or the unsubstituted pyridine base, replace or unsubstituted pyrazinyl, replace or unsubstituted pyrimidyl, replace or unsubstituted pyridazinyl, replace or unsubstituted thiazolyl, replace or unsubstituted isothiazolyl, replace or unsubstituted triazolyl, replace or unsubstituted thienyl, replace or unsubstituted triazinyl, replace or unsubstituted thiadiazolyl group or replacement or unsubstituted tetrazyl.

In another embodiment, The compounds of this invention be embodiment part table 1-18 or 20 and/or the compound of method 2-61 in any.

In formula (II), L ¹, L ², R ¹And R ²As above definition in formula (I).

In formula (III), L ¹, L ², R ¹And R ²As above definition in formula (I).

In certain embodiments, all replaced at formula (I)-(III) all described in compound substituting groups by at least one substituting group.More particularly, in certain embodiments, the alkylidene group of the heteroaryl of the aryl of the Heterocyclylalkyl of the cycloalkyl of the assorted alkyl of the alkyl of above-mentioned all replacements in formula (I)-(III) compound, replacement, replacement, replacement, replacement, replacement, replacement and/or the assorted alkylidene group of replacement are all replaced by at least one substituting group.In other embodiments, at least one or all these groups are replaced by the substituting group of at least one restriction size.In addition, at least one or these all groups can also be replaced by at least one rudimentary substituting group.

In other embodiment of formula (I)-(III) compound, all replacements or unsubstituted alkyl all are to replace or unsubstituted C ₁-C ₂₀Alkyl, all replacements or unsubstituted assorted alkyl all are to replace or assorted alkyl, all replacements or the unsubstituted cycloalkyl of unsubstituted 2-20 unit all are to replace or unsubstituted C ₄-C ₈Cycloalkyl, all replacements or unsubstituted Heterocyclylalkyl all are to replace or Heterocyclylalkyl, all replacements or the unsubstituted alkylidene group of unsubstituted 4-8 unit all are to replace or unsubstituted C ₁-C ₂₀Alkylidene group and/or all replacements or unsubstituted assorted alkylidene group all are to replace or the assorted alkylidene group of unsubstituted 2-20 unit.

Perhaps, all replacements or unsubstituted alkyl all are to replace or unsubstituted C ₁-C ₈Alkyl, all replacements or unsubstituted assorted alkyl all are to replace or assorted alkyl, all replacements or the unsubstituted cycloalkyl of unsubstituted 2-8 unit all are to replace or unsubstituted C ₅-C ₇Cycloalkyl, all replacements or unsubstituted Heterocyclylalkyl all are to replace or Heterocyclylalkyl, all replacements or the unsubstituted alkylidene group of unsubstituted 5-7 unit all are to replace or unsubstituted C ₁-C ₈Alkylidene group and/or all replacements or unsubstituted assorted alkylidene group all are to replace or the assorted alkylidene group of unsubstituted 2-8 unit.

Exemplary synthetic method

The compounds of this invention synthesizes by the appropriate combination of common known synthetic method.The technology that is used for synthetic The compounds of this invention is apparent and understandable for those skilled in the relevant art.Below discussion be used for explaining normally how to obtain compound of the presently claimed invention, and provided the details of some different methods that can be used for assembling The compounds of this invention.Yet described discussion is not to be used to define or limit be used to prepare the reaction of The compounds of this invention or the scope of reaction sequence.The compounds of this invention can make by the disclosed method of following examples part and technology and by known organic synthesis technology.In scheme 1,2 and 3, L ¹, R ¹, L ²And R ²As defined above.

Synthesize 3, the key intermediate of the dibasic 1H-pyrazolo of 5-[3,4-b] pyridine derivate is 5-bromo-1H-pyrazolo [3,4-b] pyridine and 5-bromo-3-iodo-1H-pyrazolo [3,4-b] pyridine.Iodine on the aromatic series carbon atom of the sp2-hydridization in being present in these members and/or bromine substituent provide many synthetic possibilities for arbitrary position functionalized.There are many described synthetic methods, these methods are normally known for a person skilled in the art and be familiar with, and comprise such as but not limited to: transition metal-catalyzed method, utilize the foremost method of palladium, iron, nickel or copper catalyst, and metal-halogen exchange reaction, foremost method be introduce lithium or magnesium, subsequently will instantaneous or isolated Organometallic derivatives with the electrophilic reagent direct reaction with appropriate reaction or by the commentaries on classics metallization reaction fine to regulate the reactivity of organo-metallic species.

Utilize this method, at 1H-pyrazolo [3,4-b] on the 3-of pyridine nucleus and the 5-position introducing of different substituents can realize by the following method: with 5-bromo-1H-pyrazolo [3,4-b] pyridine introduces selected substituting group as raw material on the 5-position, subsequently at 1H-pyrazolo [3,4-b] 3 of pyridine nucleus carry out halogenation, especially iodate, thereby can utilize aforesaid method to introduce selected another substituting group in this position.Perhaps, above-described some method also can by optionally with the iodine substitution reaction not with bromine substituent reaction and on the 3-position optionally functionalized 5-bromo-3-iodo-1H-pyrazolo [3,4-b] pyridine.Known and familiar to those skilled in the art is, compare with aromatic bromine substituent, the various palladium catalysts that have higher speed of reaction with aromatic iodine substitution reaction are known and are easy to obtain or obtain, and described catalyzer can use under suitable condition to carry out the replacement of selectivity iodine.

5-bromo-1H-pyrazolo [3,4-b] pyridine or contain the derivative of due care base can also be on the 3-position by those skilled in the art usually known the and familiar electric aromatic series substitution reaction of various parents carry out functionalized such as the FRIEDEL-CRAFTS-acidylate.

Can represent the compound of making fully such as compound of the presently claimed invention according to the substituting group that this mode is introduced on arbitrary position; perhaps their functional groups that can contain free or have a suitable protecting group for example are not limited to amine, carboxylic acid or ester, nitrile, alkene or halogen, and they can be used as the raw material of known synthetic conversion reaction usually with synthetic compound of the presently claimed invention.

The suitable functionalized pyrazolo [3 that is used for synthetic The compounds of this invention, 4-b] pyridine derivate, especially 5-bromo-1H-pyrazolo [3,4-b] pyridine and 5-bromo-3-iodo-1H-pyrazolo [3,4-b] pyridine can make from the 5-bromo-2-fluorine pyridine that can buy according to scheme 1 described method.5-bromo-2-fluorine pyridine can carry out selectively functionalized on the 3-position by the common known selective metallization of 2-fluorine pyridine according to the described general method of following document: Schlosser, M., Organometallics in Synthesis, the 2nd edition, Wiley-VCH, 2002; Clayden, J., Organolithiums:Selectivity forSynthesis, Pergamon, 2002 and people such as Mongin, Tetrahedron (2001) 57,4059-4090.Therefore, metallization can be by with suitable non-nucleophilicity highly basic (di-isopropyl lithamide or 2 for example, 2,6,6-tetramethyl piperidine (piperidide) lithium) in aprotic solvent (for example THF, hexane, ether or its mixture) in low temperature, normally-78 ℃ or handle under the lower temperature and finish.

By handling and unpurified metallized intermediate can be changed into corresponding 3-formaldehyde 2 with formylating agent such as DMF, N-formyl radical-methylphenylamine, N-formyl morpholine, N-formyl piperidine or ethyl formate.Formaldehyde and hydrazine or suitable hydrazine derivative (for example hydrazine-tertiary butyl carbazates or derived from the solubility organic or inorganic salt of hydrazine such as the hydrazonium salt hydrochlorate) direct reaction or under the condition of utilizing due care base (for example acetal) protection aldehyde reaction will obtain 5-bromo-1H-pyrazolo [3,4-b] pyridine.In order further to process, the introducing of the suitable group on the 3-position can replace (for example bromination or iodate) by the electric aromatic series of the common known method in this area such as parent and realize.Therefore, iodide 4 can be from 3 by obtaining being convenient to handle under the condition of described conversion with suitable reagent such as N-iodine succinimide, iodine monochloride or iodine.Replace and other functionalized example for example is and be not limited to utilize functionalized carboxylic acid halides such as bromoacetyl chloride, acrylate chloride or trichoroacetic chloride in the FRIEDEL-CRAFTS-acidylate of carrying out under room temperature or lower temperature in methylene dichloride in the presence of the aluminum chloride by the electric aromatic series of parent.Those of skill in the art would recognize that the product of this reaction can be used as the raw material of synthetic some heterogeneous ring compound.

Perhaps, by the metallized intermediate that 5-bromo-2-fluorine pyridine deprotonation is formed also can change under suitable condition metallization with form organic cuprate reagent (referring to Lipshutz, B., Organometallics in Synthesis, the 2nd edition, Wiley-VCH, 2002).The cuprate that produces according to this mode and the reaction of carboxylic acid halides obtain the ketone of formula 5, it can be by cyclisation obtains 5-bromo-1H-pyrazolo [3, the 4-b] pyridine that the corresponding 3-of formula 6 replaces with hydrazine or derived from solubility organic or inorganic salt (for example hydrazonium salt hydrochlorate) reaction of hydrazine.

Scheme 1

Halogenide 3,4 or 6 processing can be by common known methods, for example following scheme 2 described methods realize at an easy rate.For example, can use the cross-coupling reaction of the metal catalytic that utilizes the various known transistion metal compounds compound of palladium, iron or nickel (for example derived from).The example of described conversion reaction can be referring to below with reference to document: Diederich, F., Stang, P.J.-Metal-catalyzedCross-coupling Reactions, Wiley-VCH, 1998; Beller, M., Transition metalsfor Organic Synthesis, Wiley-VCH, 1998; Tsuji, J., Palladium Reagentsand Catalysts, Wiley-VCH, 1st.﹠amp; 2nd ed.s, 1995,2004; Fuerstner, people such as A., J.Am.Chem.Soc. (2002) 124, and 13856; And Bolm, people such as C., Chem.Rev. (2004) 104, and 6217.Other useful method comprise with the common known method of bromine or iodine substituting group utilization (for example metal halogen exchange, and, suitably and in case of necessity, utilize the solubility of boron, magnesium, zinc, tin, silicon or copper and reactive compounds to change metallization reaction subsequently; The representative example of this method can be referring to Schlosser, M., Organometallics in Synthesis, the 2nd edition, Wiley-VCH, 2002.) change into metal or metalloid substituting group (for example organic boron, organolithium, organotin, organosilicon, organic zinc, organic copper or organo-magnesium compound).The Organometallic derivatives itself that obtains according to this mode can be used for transition metal-catalyzed and linked reaction aromatic series or olefinic halogenide or triflate, perhaps, if have enough reactive words, also can be directly and suitable electrophilic reagent such as some Organohalogen compounds, MICHAEL-acceptor, oxyethane, azacyclopropane, aldehyde, carboxylic acid halides or nitrile reaction.

According to the character that is used for introducing in arbitrary position the conversion reaction of functional group, particularly carry out functionalized order in arbitrary position, the selective functionalization in 3-or 5-position may need different strategies.Therefore, in some cases, realized that before 5-position functionalized the functionalized of 3-position may be favourable or essential, and may need opposite order in other cases, this depend on concrete group to be introduced character, finish the inherent selectivity of required method of described conversion or method therefor.For example, some reactant, (promptly such as some electron deficiency, contain one or more derivatives that draw electron substituent group or represent some heterocyclic system) and/or contain and one or morely may need to use highly active palladium catalyst (for example at Vilar at boron-substituent boric acid of carbon bond adjacent or its ester, R., Christman, U.Angew.Chem. (2005) 117,370; Littke, A.F., Fu, G.-Angew.Chem. (2002) 114, those palladium catalysts of mentioning in 4350) and more exacting terms, such as higher temperature and/or longer reaction times.In the reaction of 5-bromo-3-iodo-1H-pyrazolo [3,4-b] pyridine, described condition may not can help to realize obvious selectivity.Therefore, in this case, advantageously utilize the replacement of the above method of describing in detail by carrying out bromine in 5-bromo-1H-pyrazolo [3, the 4-b] pyridine successively, carry out iodate in the 3-position and introduce second substituting group at 3 subsequently and avoid optionally problem fully.Generally speaking, when relating to, the required condition of the replacement of arbitrary locational halogen atom is unfavorable for 5-bromo-3-iodo-1H-pyrazolo [3 usually, 4-b] high level between two halogen atoms existing in the pyridine is optionally when hyperergy catalyzer or reagent, advantageously adopts this method that replaces successively.

Should also be appreciated that with suitable protecting group protection L ¹, L ², R ¹And/or R ²And the reactive group (for example 1 on proton) in pyrazolo [3, the 4-b] pyridine skeleton is favourable or essential.For example, in some cross-coupling reaction, advantageously protect nitrogen on 1 of 1H-pyrazolo [3,4-b] pyridine skeleton by introduce (2-trimethylsilylethoxy))-methyl or (2-methoxyl group-oxyethyl group) methyl in this position.The introducing of these protecting groups and remove and to finish by known method in the chemical literature easily.Can contain by any resulting compound in the aforesaid method can be by the free or the protected functional group of the further processing of common known method.

In compound of the presently claimed invention synthetic, utilize the more detailed description of cross-coupling method to be documented in the scheme 2: X ¹And X ²Be selected from but be not limited to halogen, boric acid or ester, three fluoroborates, organic-magnesium, organic zinc or organotin.About independent residue-L ¹-R ¹Or-L ²-R ²Introducing, aforesaid conversion reaction can be finished by the halogen cross-coupling method of standard.

Scheme 2

Corresponding bromide or iodide (X ¹, X ²=Br, I) can in the presence of suitable transition-metal catalyst (for example palladium compound), carry out with the coupling of suitable reagent such as boric acid and borate, organo-borane, organic stannane, organic zinc compound, organo-magnesium compound, alkene or terminal alkyne (purchase or obtain by common known method).Described coupling can randomly be carried out in the presence of part such as phosphine, diphosphine, Arduengo-type heterocyclic carbene or arsine.Can use organic or inorganic alkali (for example uncle or secondary amine, alkaline carbonate, supercarbonate, fluorochemical or phosphoric acid salt) and/or other known additives (for example lithium chloride, copper halide or silver salt) to help or to promote described conversion reaction.

These cross-coupling reactions can carry out under 25 ℃-200 ℃ in such as THF, diox, glycol dimethyl ether, diglyme, methylene dichloride, ethylene dichloride, acetonitrile, DMF, N-Methyl pyrrolidone, water or its mixture in appropriate solvent.Temperature is randomly kept with heating, conventional heating or microwave irradiation.At 3-iodo-5-bromo-1H-pyrazolo [3,4-b] under the situation of pyridine, the iodine substituting group can be under exacting terms more not with respect to the selectivity of bromine substituent or preferential replacement, such as lower temperature and short reaction times and utilize suitable transition-metal catalyst to realize.Dihalo or low halogenated compound are on the books in chemical literature by the selective functionalization of transition metal-catalyzed conversion reaction: Ji for example, and people-Org.Lett such as J. (2003) 5, and 4611; Bach, people-J.Org.Chem such as T. (2002) 67, and 5789, Adamczyk, people-Tetrahedron such as M. (2003) 59, and 8129.

It not is nucleophilic reagent (for example alcohol, mercaptan, uncle or secondary amine) based on carbon that this method also can be used for introducing, and described nucleophilic reagent can randomly contain the protecting group of suitable alcohol, mercaptan or amine.Described examples of groups can be referring to Greene, T. etc., Protective Groups in Organic Synthesis, the 3rd edition, John Wiley ﹠amp; Sons, 1999.Exemplary guard method has description: Ley in following document, people such as S., and Angew.Chem. (2003) 115, and 5558; Wolfe, people such as J., Acc.Chem.Res. (1998) 31, and 805; Hartwig, Acc.Chem.Res. (1998) 31, and 852; Navarro, people such as O., J.Org.Chem. (2004) 69, and 3173; Ji, people Org.Lett such as J. (2003) 5,4611.Can further process by common known method by the compound that this method obtains to obtain other compound of the present invention.

In some cases, advantageously realize cross-coupling with carbon or non-carbon atom by at first various halogen derivatives being changed into corresponding Organometallic derivatives (for example boric acid or ester, three fluoroborates, organic-magnesium, organic zinc or organo-tin compound).Described compound can partly obtain by replacing halogenide with proper metal or metalloid.Any functional group that exists (for example 1 of pyrazolo [3,4-b] pyridine on ring nitrogen) all may need to protect by suitable protecting group (＂ PG ＂).Referring to Greene, people such as T., 1999.

Described metal or metalloid introducing can be by common known methods, realize such as utilizing metal or metal-halogen exchange reaction to metallize.Be used for the activated form that metallized useful metal comprises basic metal or alkaline-earth metal or described metal.The suitable reagent that is used for metal-halogen exchange reaction comprises organolithium or organo-magnesium compound (for example n-Butyl Lithium, tert-butyl lithium or isopropylmagnesium chloride or magnesium bromide).The commentaries on classics metallization reaction subsequently of organo-metallic intermediate can utilize suitable solubility and reactive metal compound such as magnesium chloride, magnesium bromide, three normal-butyl chlorination tin, trimethyltin chloride, trimethyl borate, triethyl borate, triisopropyl borate ester, trifluoromethanesulfonic acid zinc or zinc chloride to carry out when needed.The introducing of boric acid pinacol ester can be easily realizes by halogen derivative is directly reacted down in 80-160 ℃ in solvent such as DMSO, DMF, DMA or N-Methyl pyrrolidone under the existence of [1,1 '-two (diphenylphosphine) ferrocene] palladium chloride (II) and suitable alkali (for example potassium acetate or sodium) with two (pinacols), two boron.Conventional heating or microwave irradiation can be used for the temperature (the document precedent about similar conversion reaction can be referring to Ishiyama, and people-J.Org.Chem. such as T. (1995) 60,7508.) that keeps suitable.

It is normally well-known to change into the boratory method of other boric acid derivatives such as boric acid, boric acid ester or trifluoro by the boric acid pinacol ester that this method obtains.It will be apparent to one skilled in the art that described Organometallic derivatives can be used for cross-coupling reaction, this response class is similar to the reaction under the situation of above-described halogen-containing derivative at pyrazolo [3,4-b] pyridine.Described coupling can utilize suitable linked reaction thing such as aromatic series, heteroaromatic halogenide or olefinic reagent to carry out under identical with aforesaid method or obviously similar and/or relevant condition.

Other method can be utilized from the Halogen derivative of pyrrolo-[3,4-b] the pyridine reactivity by the Organometallic derivatives that any produced of aforesaid method.For example, the derivative of alkali metal containing or alkaline-earth metal (for example organolithium, organic-magnesium or organic zinc compound) can be used for directly being coupled on a series of other Electron Affinities linked reaction thing such as activated form alkene (MICHAEL-acceptor), aldehyde, nitrile, aromatic nitro compound, carboxylic acid derivative, oxyethane, azacyclopropane, organic disulfide or the Organohalogen compounds.Described conversion reaction normally known in the art (about with the reaction of aromatic nitro compound can be referring to for example Sapountzis, people such as I., J.Am.Chem.Soc. (2002) 124,9390.).

Be used to obtain 3, the synthesis strategy of the dibasic 1H-pyrrolo-of 5-[2,3-b] pyrazines derivatives and above closely related about 1H-pyrazolo [3,4-b] the described strategy of pyridine derivate, the key distinction relates to the synthetic of 1H-pyrrolo-[2,3-b] pyrazine skeleton itself.Used key intermediate is 5-iodo-1H-pyrrolo-[2,3-b] pyrazines derivatives and 5-bromo-1H-pyrrolo-[2, the 3-b] pyrazine itself that 3-replaces.

Above-described from 5-bromo-1H-pyrazolo [3,4-b] pyridine obtains 3, the dibasic 1H-pyrazolo [3 of 5-, 4-b] the general synthesis strategy of pyridine derivate also is suitable for from 5-bromo-1H-pyrrolo-[2,3-b] the 5-iodo-1H-pyrrolo-[2 that replaces of pyrazine and 3-, 3-b] pyrazines derivatives obtains 3, the dibasic 1H-pyrrolo-of 5-[2,3-b] pyrazines derivatives.Yet for 1H-pyrrolo-[2,3-b] pyrazines derivatives, the actual conditions of the conversion reaction that others are similar or identical may be very different, and may need to be optimized according to used skeleton.

5-bromo-1H-pyrrolo-[2; 3-b] pyrazine can obtain by the following method: trimethyl silyl acetylene and 3-amino-2; the regioselectivity SONOGASHIRA-coupling of 6-two bromo-pyrazines is (referring to Adamczyk; M. wait people-Tetrahedron (2003) 59; 8129.); the N-acidylate is used n-butyl ammonium fluoride cyclisation (precedent of this reaction sees also WO2004/032874A2) subsequently.With the 3-amino-2 that can buy, 6-two bromo-pyrazines are as raw material, and 5-bromo-3-TMS ethynyl-pyrazine-2-base amine can obtain by reacting under the temperature of rising in the mixture of DMF and alkaline trimethylamine such as triethylamine in the presence of copper co-catalyst such as the cupric iodide (I) of palladium catalyst such as tetrakis triphenylphosphine palladium (0) and catalytic amount with trimethyl silyl acetylene.In pyridine, under 20-60 ℃, carry out acetylize with Acetyl Chloride 98Min. and obtain N-(5-bromo-3-TMS ethynyl-pyrazine-2-yl)-ethanamide, subsequently with the tetra-n-butyl Neutral ammonium fluoride in THF under refluxing cyclisation obtain 5-bromo-1H-pyrrolo-[2,3-b] pyrazine.

In order further to process, the introducing of the suitable group on the 3-position can replace (for example bromination or iodate) by the electric aromatic series of the common known method in this area such as parent and realize.Therefore, 5-bromo-3-iodo-1H-pyrrolo-[2,3-b] pyrazine can be from 5-bromo-1H-pyrrolo-[2,3-b] pyrazine by obtaining being convenient to handle under the condition of described conversion with suitable reagent such as N-iodine succinimide, iodine monochloride or iodine.

Replace and other functionalized example is such as but not limited to utilize functionalized carboxylic acid halides such as bromoacetyl chloride, acrylate chloride or trichoroacetic chloride in the FRIEDEL-CRAFTS-acidylate of carrying out under room temperature or lower temperature in methylene dichloride in the presence of the aluminum chloride by the electric aromatic series of parent.The product that those of skill in the art would recognize that this reaction can be represented compound of the presently claimed invention, perhaps can be used as the raw material of synthetic described compound, particularly some heterogeneous ring compound.

Halogenide D (X ²=Br, I) further processing and 5-bromo-3-iodo-1H-pyrrolo-[2,3-b] the selectivity of two halogenic substituents in the pyrazine replaces successively and can realize by common known method at an easy rate, such as the cross-coupling reaction that can use the metal catalytic successively that utilizes the various known transistion metal compounds compound of palladium, iron or nickel (for example derived from).The example of described conversion reaction can be referring to following document: Diederich, F., Stang, P.J.-Metal-catalyzed Cross-couplingReactions, Wiley-VCH, 1998; Beller, M., Transition metals for OrganicSynthesis, Wiley-VCH, 1998; Tsuji, J., Palladium Reagents and Catalysts, Wiley-VCH, 1st.﹠amp; 2nd ed.s, 1995,2004; Fuerstner, people such as A., J.Am.Chem.Soc. (2002) 124, and 13856; And Bolm, people such as C., Chem.Rev. (2004) 104, and 6217.General method known and that those skilled in the art are familiar with is to react described identical method with above about the similar or same conversion of utilizing 1H-pyrazolo [3,4-b] pyridine derivate to carry out basically in the chemical literature.

As about 1H-pyrazolo [3,4-b] pyridine is described like that, one of ordinary skill in the art would recognize that, according to be used for introducing the character of the conversion reaction of functional group in arbitrary position, particularly in the functionalized order of arbitrary position, the selective functionalization in 3-or 5-position needs different strategies.Therefore, in some cases, realized that before 5-position functionalized the functionalized of 3-position may be favourable or essential, and may need opposite order in other cases, this depend on concrete group to be introduced character, finish the inherent selectivity of required method of described conversion or method therefor.

At 3-iodo-5-bromo-1H-pyrrolo-[2,3-b] under the situation of pyrazine, the iodine substituting group can be under exacting terms more not with respect to the selectivity of bromine substituent or preferential replacement, such as lower temperature and short reaction times and utilize suitable transition-metal catalyst to realize.Dihalo or low halogenated compound are on the books in chemical literature by the selective functionalization of transition metal-catalyzed conversion reaction: Ji for example, and people-Org.Lett such as J. (2003) 5, and 4611; Bach, people-J.Org.Chem such as T. (2002) 67, and 5789, Adamczyk, people-Tetrahedron such as M. (2003) 59, and 8129.

At halogenide D (X ²=Br, I) under the situation, other useful method comprise with the common known method of bromine or iodine substituting group utilization (for example metal halogen exchange, and, with in case of necessity, utilize the solubility of boron, magnesium, zinc, tin, silicon or copper and reactive compounds to change metallization reaction suitably subsequently; The representative example of this method can be referring to Schlosser, M., Organometallics in Synthesis, the 2nd edition, Wiley-VCH, 2002.) change into metal or metalloid substituting group (for example organic boron, organolithium, organotin, organosilicon, organic zinc, organic copper or organo-magnesium compound).The Organometallic derivatives itself that obtains according to this mode can be used for transition metal-catalyzed and linked reaction aromatic series or olefinic halogenide or triflate, perhaps, if have enough reactive words, also can be directly and suitable electrophilic reagent such as some Organohalogen compounds, MICHAEL-acceptor, oxyethane, azacyclopropane, aldehyde, carboxylic acid halides or nitrile reaction.Equally, known general method also is to react described identical method with above about the similar or same conversion of utilizing 1H-pyrazolo [3,4-b] pyridine derivate to carry out basically in the chemical literature.

In some described conversion reaction, advantageously maybe essential by known method in the chemical literature (referring to T.W.Greene, P.G.M.Wuts-Protective Groups in Organic Synthesis, the 3rd edition, John Wiley ﹠amp; Sons, 1999) introduce one or more suitable protecting groups with temporary transient replacement acid proton, for example be connected to the hydrogen atom on nitrogen or the oxygen, especially the hydrogen atom on 1 of 1H-pyrrolo-[2,3-b] pyrazine skeleton.

It not is nucleophilic reagent (for example alcohol, mercaptan, uncle or secondary amine) based on carbon that this cross-coupling method also can be used for introducing, and described nucleophilic reagent can randomly contain the protecting group of suitable alcohol, mercaptan or amine.Described examples of groups can be referring to Greene, T. etc., Protective Groups in Organic Synthesis, the 3rd edition, John Wiley ﹠amp; Sons, 1999.Exemplary guard method has description: Ley in following document, people such as S., and Angew.Chem. (2003) 115, and 5558; Wolfe, people such as J., Acc.Chem.Res. (1998) 31, and 805; Hartwig, Acc.Chem.Res. (1998) 31, and 852; Navarro, people such as O., J.Org.Chem. (2004) 69, and 3173; Ji, people Org.Lett such as J. (2003) 5,4611.Can further process by common known method by the compound that this method obtains to obtain other compound of the present invention.In some cases, 5-iodine in 1H-pyrrolo-[2, the 3-b] pyrazine or 5-bromine substituent by amine, alcohol or mercaptan direct substitution can be successfully under the temperature of room temperature or rising at weak acid such as acetate or non-nucleophilicity highly basic such as in amine, alcohol or mercaptan or in suitable aprotic solvent such as DMF, NMP, DMSO or acetonitrile, finishing respectively in the presence of the sodium hydride.

Scheme 3

Developed another kind of synthetic 3, the dibasic 1H-pyrrolo-[2 of 5-, 3-b] method of pyrazines derivatives, this method, can realize such as reacting under refluxing in ethanol with N-iodine succinimide by various known method to obtain 2-amino-5-iodo-3-pyrazine carboxylic acid methyl esters at introducing iodine atom on the 5-position as raw material with 2-amino-3-pyrazine carboxylic acid methyl esters.The halogen ester that will obtain by this method is by the standard method hydrolysis then.For example in the THF-water mixture, under room temperature, obtain corresponding acid with the lithium hydroxide processing.

The synthetic of ketone intermediate B can obtain by for example utilizing organic-magnesium or organolithium compound to handle with corresponding WEINREB-acid amides A (3-amino-6-iodo-pyrazine-2-formic acid methoxyl group-methyl-acid amides) or its hydrochloride and suitable organo-metallic material.Example for N-methoxyl group-N-methyl nitrosourea (Weinreb acid amides) used in the ketone building-up process can be referring to S.Nam, S.M.Weinreb-Tetrahedron Lett.1981,22,3815.).3-amino-6-iodo-pyrazine-2-formic acid methoxyl group-methyl-acid amides (A) is by utilizing the standard method of acid amides synthetic, by in advance or on the spot with acid activation or by direct condensation, with parent acid and N, the condensation of O-dimethyl hydroxyl amine obtains.Be used for the method for these two kinds of conversions and reagent is recorded in chemical literature and be that those skilled in the art are well-known, for example, under the situation of direct method, can use suitable coupling agent such as but be not limited to PyBOP, HBTU or HATU.

Ketone residue L among the B ¹R ¹The required organometallic reagent of introducing can be commercially available, perhaps obtain by the whole bag of tricks described in the document is synthetic, for example, but the Grignard reaction that is not limited to organic chloride, bromide or iodide and magnesium is (referring to J.March-Advanced OrganicChemistry, the 3rd edition, John Wiley ﹠amp; Sons, 1992), utilize metal-halogen exchange reaction (J.Clayden-Organolithiums:Selectivity for Synthesis for example of suitable organolithium or organo-magnesium compound, the organic bromide that carries out such as but not limited to n-Butyl Lithium, tert-butyl lithium or isopropylmagnesium chloride or magnesium bromide or iodide, Pergamon, 2002; A.Boudier, L.O.Bromm, M.Lotz, P.Knochel-Angew.Chem.Int.Ed. (2000) 39,4414.) or utilize suitable alkali, such as N, N-di-isopropyl lithamide or 2,2,6,6-tetramethyl piperidine lithium make have enough tart compounds such as pyrimidine, pyrazine, 2-chloro-or 2-fluorine pyridine deprotonation be (referring to J.Clayden-Organolithiums:Selectivity for Synthesis, Pergamon, 2002; A.Turck, N.Pl é, F.Mongin, G.Qu é guiner-Tetrahedron (2001) 57,4489; F.Mongin, G.Qu é guiner-Tetrahedron (2001) 57,4059).In some described conversion reaction, advantageously maybe need by known method in the chemical literature (referring to T.W.Greene, P.G.M.Wuts-Protective Groups in Organic Synthesis, the 3rd edition, John Wiley ﹠amp; Sons, 1999) introduce one or more suitable protecting groups, temporarily to replace acid proton (for example being connected to the hydrogen atom on nitrogen or the oxygen) when needed.

The ketone intermediate B generates the conversion of methoxy-ethylene radical derivative C and can finish by multiple known method, but the most frequently used is (referring to B.E.Maryanoff by the WITTIG-reaction, A.B.Reitz-Chem.Rev. (1989) 89,863) utilize, carry out such as, but not limited to the ylide that lithium, sodium or sylvite produced of non-nucleophilicity amide such as two (trimethyl silyl) amine from the methoxymethyl methyl triphenyl phosphonium chloride that can buy and suitable alkali, such as but not limited to strong organo-metallic alkali.

The cyclization subsequently of formed alkene C (alkene can use with the form of the mixture of E-or Z-type or these two kinds of configurations) can be finished under the acid catalytic condition of routine to obtain 1H-5-iodo-pyrrolo-[2, the 3-b] pyrazine that 3-replaces.Described method can be utilized strong inorganic or organic acid, carry out under 0 ℃-160 ℃ in appropriate solvent (such as THF, diox, diethyl ether, glycol dimethyl ether, diglyme, methylene dichloride, ethylene dichloride or chloroform, water, methyl alcohol or ethanol or its mixture) such as sulfuric acid, perchloric acid, hydrochloric acid, trifluoromethanesulfonic acid or trifluoroacetic acid.People such as Sakamoto are in Heterocycles (1992), and 34 (12), similar cyclization has been described among the 2379-84.Wherein the author has described the conversion that is generated parent pyrrolo-[2,3-b] pyridine by 2-nitro-3-(2-vinyl ethyl ether base) pyridine.The formation of vinyl realizes by the STILLE-coupling of 3-bromine analogue and tributyl-2-vinyl ethyl ether base stannane.

According to above-described method, the purposes of 1H-5-iodo-pyrrolo-[2, the 3-b] pyrazine that 3-replaces in compound of the presently claimed invention synthetic it will be apparent to those skilled in the art that.Those skilled in the art are readily appreciated that, can utilize and/or improve synthetic method as herein described, comprise the following examples part, with preparation formula (I), (II) and/or compound (III).

Protecting group

Term " protecting group " but be meant some or all reactive part of closing compound and before this protecting group is removed, can prevent the chemical part of described subparticipation chemical reaction, for example at T.W.Greene, P.G.M.Wuts, Protective Groups in Organic Synthesis, the 3rd edition John Wiley ﹠amp; Listed and those chemical parts of describing among the Sons (1999).Advantageously, under the situation of using different protecting groups, each (different) protecting group can be removed by diverse ways.The protecting group of removing under diverse reaction conditions can allow the difference of having removed of described protecting group.For example, protecting group can be removed by acid, alkali and hydrogenolysis.Group such as trityl, dimethoxytrityl, acetal and t-butyldimethylsilyl are unsettled to acid, and can be used for protecting carboxyl and hydroxyl reactive part in the presence of by the amino of Cbz group (can remove by hydrogenolysis) and Fmoc group (is unsettled to alkali) protection.Use unsettled group such as t-butyl carbamate to acid or use but the carbamate sealing that can by hydrolysis remove all stable to bronsted lowry acids and bases bronsted lowry amine in the presence of; carboxylic acid and hydroxyl reactive part are available to alkali labile group, seal such as but not limited to methyl, ethyl and ethanoyl.

Carboxylic acid and hydroxyl reactive part can also be with protecting such as benzyl by the protecting group that hydrolysis is removed, can be with alkali labile group is protected such as Fmoc and can carry out hydrogen bonded amine groups with acid.The available protecting group that can remove by oxidation of carboxylic acid reaction part is such as 2, and the 4-dimethoxy-benzyl is protected, and the amino available of coexistence protected the unsettled silyl carbamate of fluorochemical.

The allyl group blocking groups can use under the condition that the bronsted lowry acids and bases bronsted lowry protecting group exists, because the former is stable and can removes by metal or pi-acid catalyst subsequently.For example, the carboxylic acid of allyl group protection can be reflected to the unsettled t-butyl carbamate of acid or to carrying out deprotection under the existence of alkali labile acetate amine protecting group with palladium (0) is catalytic.The another kind of form of protecting group is the resin that compound or intermediate can be coupled.As long as residue is connected with resin, this functional group just is closed and can not reacts.In case discharge from resin, this functional group just can react.

Sealing/protecting group commonly used includes but not limited to following part:

Suppress kinase whose method

On the other hand, the invention provides the method for utilizing fused ring heterocycle kinase modulators of the present invention to regulate protein kinase activity.Term used herein " adjusting kinase activity " is meant for the activity when not having fused ring heterocycle kinase modulators, and the activity of protein kinase raises or reduces when contacting with fused ring heterocycle kinase modulators of the present invention.Therefore, the invention provides by protein kinase is contacted and regulates the method for protein kinase activity with fused ring heterocycle kinase modulators of the present invention (for example any compound in the formula (I)-(III)).

In exemplary, fused ring heterocycle kinase modulators suppresses kinase activity.Be meant that at some real term " inhibition " that relates to kinase activity used herein kinase activity reduces when contacting with fused ring heterocycle kinase modulators for the activity that does not have fused ring heterocycle kinase modulators.Therefore, the present invention also provides by protein kinase and fused ring heterocycle kinase modulators of the present invention being contacted the method for arrestin kinase activity.

In certain embodiments, protein kinase is a protein tyrosine kinase.Protein tyrosine kinase used herein is meant that but tyrosine residues in the catalytic proteins is by the enzyme of phosphodonor (for example the phosphoric acid nucleoside acid donors is such as ATP) phosphorylation.Protein tyrosine kinase comprises for example Abelson Tyrosylprotein kinase (＂ Abl ＂) (for example c-Abl and v-Abl), Ron receptor tyrosine kinase (＂ RON ＂), Met receptor tyrosine kinase (＂ MET ＂), Fms sample Tyrosylprotein kinase (＂ FLT ＂) (for example FLT3), src-Family Tyrosine Kinases (lyn for example, CSK) and p21-activatory kinases-4 (＂ PAK ＂), FLT3, the aurora kinases, B-lymph Tyrosylprotein kinase (＂ Blk ＂), cell cycle protein dependent kinase (＂ CDK ＂) (for example CDK1 and CDK5), the protein tyrosine kinase (for example Fyn kinases) that src-family is relevant, glycogen synthase kinase (＂ GSK ＂) (for example GSK3 α and GSK3 β), lymphocyte protein Tyrosylprotein kinase (＂ Lck ＂), ribosome S 6 kinases (Rsk1 for example, Rsk2 and Rsk3), sperm Tyrosylprotein kinase (for example Yes) and their hypotype that shows tyrosine kinase activity and autoploid.In certain embodiments, protein tyrosine kinase is Abl, RON, MET, PAK or FLT3.In other embodiments, protein tyrosine kinase is the member of FLT3 or Abl family.

In certain embodiments, kinases is selected from Abelson Tyrosylprotein kinase, Ron receptor tyrosine kinase, Met receptor tyrosine kinase, Fms sample Tyrosylprotein kinase-3, Aurora kinases, p21-activatory kinases-4 and 3-phosphoinositide dependent kinases-1.

In another embodiment, kinases is the mutant kinases, such as mutant Bcr-Abl kinases, FLT3 kinases or aurora kinases.Useful mutant Bcr-Abl kinases comprises those kinases with at least a following isolated sudden change clinically: M244V, L248V, G250E, G250A, Q252H, Q252R, Y253F, Y253H, E255K, E255V, D276G, F311L, T315I, T315N, T315A, F317V, F317L, M343T, M351T, E355G, F359A, F359V, V379I, F382L, L387M, H396P, H396R, S417Y, E459K and F486S.In certain embodiments, mutant Abl kinases has the T315I sudden change.The number system of the amino acid mutation position that expression is above is the known wild-type ABL numbering according to ABL exon Ia.Referring to Deininger, people such as M., Blood 105 (7), and 2640 (2005).This number system is reappeared in Fig. 1.In certain embodiments, mutant Bcr-Abl kinases comprises at least a above listed sudden change, and has at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with the sequence of Fig. 1.In certain embodiments, mutant Bcr-Abl kinases comprises at least a above listed sudden change, have the sequence identical, and comprise at least 50,100,150,200,250,300,350,400,450.500,550,600,650,700,750,800,850,900,950,1000,1050 or 1100 amino acid with above-described Fig. 1.

In certain embodiments, kinases is selected from Abelson Tyrosylprotein kinase, Ron receptor tyrosine kinase, Met receptor tyrosine kinase, Fms sample Tyrosylprotein kinase-3, Aurora kinases, p21-activatory kinases-4 and 3-phosphoinositide dependent kinases-1.In certain embodiments, formula (I) compound is contacted with kinases.

In certain embodiments, kinases and known kinases are homologous (also being known as ＂ homology kinases ＂ herein).The compound and the composition that can be used for suppressing the kinase whose biologic activity of homology can screen in for example in conjunction with test at first.The homology enzyme comprises the aminoacid sequence of equal length, the aminoacid sequence at least 50%, at least 60%, at least 70%, at least 80% or at least 90% of the known kinase of itself and total length is equal to, and perhaps has 70%, 80% or 90% homology with known kinase activity structural domain.Homology for example can utilize PSI BLAST research, such as but not limited to people such as Altschul, the described research of Nuc.Acids Rec.25:3389-3402 (1997) is determined.In certain embodiments, at least 50% or at least 70% sequence is compared in this analysis.Other instrument of comparing comprises for example DbClustal and ESPript, and they can be used for producing the PostScript version of comparison.Referring to people such as Thompson, Nucleic Acids Research, 28:2919-26,2000; Gouet waits the people, Bioinformatics, 15:305-08 (1999).Any functional domain of homologue and FLT3, Abl or another kind of known kinase or FLT3, Abl or another kind of known kinase can for example have 1 * 10 at least 100 amino acid ^-6BLAST E-value (people such as Altschul, Nucleic Acids Res., 25:3389-402 (1997).

Homology can also be by comparing the avtive spot binding pocket of enzyme and the avtive spot binding pocket of known kinase to determine.For example, in the homology enzyme, at least 50%, 60%, 70%, 80% or 90% amino acid of molecule or homologue has the amino acid structure coordinate with the sizable structural domain of kinase domain, and the root-mean-square deviation of its alpha-carbon atom is to the maximum approximately Approximately

Approximately

Approximately Approximately

Or approximately

Compound of the present invention and composition can be used for suppressing kinase activity, and can be used for suppressing and other enzyme of ATP bonded.Therefore, they can be used for treating can be by suppressing active disease and the illness that is eased of described ATP-desmoenzyme.The method of determining described ATP desmoenzyme comprises method known to those skilled in the art, the method for selecting the homology enzyme that relates to as herein described, and application data base PROSITE, from wherein identifying the enzyme of the feature, sequence pattern, primitive or the distribution plan that comprise protein families or structural domain.

Compound of the present invention and derivative thereof be the useful as kinase wedding agent also.As wedding agent, described compound and derivative can combine with the stabilized resins as the mooring substrate to be used for affinity chromatography and use.Compound of the present invention and derivative thereof can also be modified in (for example radio-labeling or affinity mark etc.) research for use in feature, structure and/or the function of enzyme or polypeptide.

In a kind of exemplary embodiment, fused ring heterocycle kinase modulators of the present invention is a kinase inhibitor.In certain embodiments, the IC of the inhibition constant (Ki) of kinase inhibitor ₅₀Less than 1 micromole.In another embodiment, the IC of kinase inhibitor ₅₀Or suppress constant (Ki) less than 500 micromoles.In another embodiment, the IC of kinase inhibitor ₅₀Or Ki is less than 10 micromoles.In another embodiment, the IC of kinase inhibitor ₅₀Or Ki is less than 1 micromole.In another embodiment, the IC of kinase inhibitor ₅₀Or Ki is less than 500 nmoles.In another embodiment, the IC of kinase inhibitor ₅₀Or Ki is less than 10 nmoles.In another embodiment, the IC of kinase inhibitor ₅₀Or Ki is less than 1 nmole.

Methods of treatment

On the other hand, the invention provides the method for the disease (kinase mediated disease or illness) of treatment kinase activity mediation in the individuality in the described treatment of needs (for example Mammals, such as the people)." kinase mediated " or " relevant with kinases " disease is meant that wherein disease or symptom can be by suppressing the disease that kinase activity is eased (for example, wherein kinases has participated in signal transmission, mediation, adjusting or the adjustment of lysis)." disease " is meant the symptom of disease or disease.This method comprises fused ring heterocycle kinase modulators of the present invention (for example each described compound in the formula (I)-(III)) from significant quantity to individuality that use.

With the example of kinases diseases associated comprise cancer (for example leukemia, tumour and metastatic tumor), allergy, asthma, obesity, inflammation (for example inflammatory diseases is such as airway inflammatory disease), blood disease, obstructive airway diseases, asthma, autoimmune disease, metabolic trouble, infection (for example bacterium, virus, yeast, fungi), CNS disease, cerebral tumor, degenerative neurological diseases, cardiovascular disorder and with vasculogenesis, neovascularization and blood vessel generation diseases associated.In exemplary embodiment, this compound is used for the treatment of cancer, comprises leukemia, and other relate to the disease of abnormal cell proliferation or illness, such as myeloproliferative disease.

The example more specifically of the cancer of available The compounds of this invention treatment comprises mammary cancer, lung cancer, melanoma, colorectal carcinoma, bladder cancer, ovarian cancer, prostate cancer, kidney, squamous cell carcinoma, glioblastoma, carcinoma of the pancreas, the Kaposi sarcoma, multiple myeloma and leukemia (myelomatosis for example, chronic lymphocytic leukemia, acute lymphoblastic leukemia, chronic lymphoblastic leukemia, Hodgkins sick and other leukemia and hematology cancer).

Other disease of available compound of the present invention or combination treatment or prevention or the object lesson of illness include but not limited to that transplant rejection (for example; kidney; liver; heart; lung; islet cells; pancreas; marrow; cornea; small intestine; skin heteroplastic transplantation or xenotransplantation and other transplanting); graft versus host disease (GVH disease); osteoarthritis; rheumatic arthritis; multiple sclerosis; diabetes; diabetic retinopathy; inflammatory bowel disease (Crohn disease for example; ulcerative colitis and other intestinal disease); ephrosis; emaciation; septic shock; lupus; myasthenia gravis; psoriasis; dermatitis; eczema; stearrhea; Alzheimer's; Parkinson's disease; stem cell protection in the chemotherapy process; external selection or purging in vitro from body or simplified marrow transplanting thing; ophthalmic diseases; retinopathy (for example; macular degeneration; diabetic retinopathy and other retinopathy); keratopathy; glaucoma; infect (bacterium for example; virus or fungi infestation); heart trouble includes but not limited to restenosis.

Test

Can test to determine the ability of their adjusting protein kinases, bindin kinase and/or growth of prevention cell or propagation compound of the present invention at an easy rate.Be some example of the test that to adopt below.

A. kinase inhibition and combination test

Various kinase whose restraining effect are measured the method described in the UpstateKinaseProfiler Assay Protocols that publishes all the whole bag of tricks as mentioned in this article and in June, 2003 by the known method of those of ordinary skills.

For example, under the situation of carrying out in vitro tests, usually kinase dilution is become suitable concentration to form kinase solution.Kinase substrate and phosphate donor such as ATP are joined in this kinase solution.Kinases with transphosphorylation to the kinase substrate to form the substrate of phosphorylation.The formation of the substrate of phosphorylation can by any appropriate means such as radioactivity (for example [γ- ³²P-ATP]) or utilize detectable secondary antibodies (for example ELISA) directly to detect.Perhaps, the formation of phosphorylated substrate can utilize any suitable technology to detect, such as the detection of ATP concentration (Kinase-Glo for example

Pilot system (Promega)).Kinase inhibitor is differentiated (referring to the following examples part) by the formation that the existence that is in or be not in testing compound detects phosphorylated substrate down.

Compound suppresses the interior kinase whose ability of cell and also can utilize methods known in the art to test.For example, will contain kinase whose cell and can activate kinase whose activator (such as somatomedin) and contact.The amount of formed endocellular phosphorus acidifying substrate can and detect existing phosphorylated substrate by any appropriate means (for example ELISA) and determine by dissolved cell under the condition that does not have and exist testing compound.When the amount that is produced under with respect to the condition that does not have testing compound when the amount of the phosphorylated substrate that is produced reduces, demonstrate kinase whose restraining effect in the presence of testing compound.More detailed cell kinase test embodiment part is below discussed to some extent.

Combine with kinase whose in order to measure compound, can use the known any method of those of ordinary skills.For example, can use Discoverx (Fremont, the CA) test kit of Sheng Chaning, ED-Staurosporine NSIP ^TMEnzyme is in conjunction with detection kit (referring to US 5,643,734).Kinase activity also can be tested according to US 6,589, the 950 described methods that on July 8th, 2003 authorized.

Suitable kinase inhibitor can be selected from compound of the present invention by the albumen crystallization screening, and described method is disclosed in for example people such as Antonysamy, among the PCT publication number WO03087816A1, in this article the document is incorporated herein by reference in full.

Compound of the present invention can screen to measure and to show their combinations and/or suppress various kinase whose abilities by computer.Available a large amount of compound of the present invention carries out the computer screening of structure, to determine the kinases bonded ability at they and various positions.The target spot of these compound useful as drug chemical research or guide's thing are to find for example to have the inhibitor (Travis, Science, 262:1374,1993) of potential treatment importance.The three-dimensional structure of described compound can be added to and spatially whether can match with the assessment compound on the 3-D view of kinases or its avtive spot or binding pocket with image and then with albumen.In this screening, the identical quality of described entity or compound and binding pocket can be judged people such as (, J.Comp.Chem.13:505-24,1992) Meng by the interaction energy of shape complementarity or estimation.

According to the present invention, screening can in conjunction with and/or when regulating the compound of the present invention of kinases (for example suppress or activate kinases), need to consider two factors usually.At first, this compound must produce with kinases and physically combine with structural, no matter be covalency or non-covalent.For example, covalent interaction may be very important for the irreversible or suicidal protein inhibitor of design.Very important non-covalent interaction of molecules for compound combines comprises hydrogen bond, ionic interaction, Van der Waals force and hydrophobic interaction for kinases.Secondly, this compound must be able to present conformation and the orientation that conforms to binding pocket, thereby it can be combined with kinases.Though some part of compound is not participated in directly with kinase whose and combined, these parts still might influence the whole conformation of molecule and may produce obviously influence to rendeing a service.Requirement on the conformation comprises total three-dimensional structure and chemical group or the compound orientation with respect to all or part of binding pocket, or comprise a plurality of directly and the interval between the functional group of the compound of the chemical group of kinase interactions.

Docking procedure as herein described (for example DOCK or GOLD) be used for differentiating can with avtive spot and/or binding pocket bonded compound.The more than one binding pocket of compound at protein structure can be screened, or screen, consider proteic differing molecular kinetics conformation simultaneously at the above coordinate of a same proteic cover.Available then consistence is marked and is differentiated the best compound (Charifson, people such as P.S., J.Med.Chem.42:5100-9 (1999)) that meets with albumen.Also the data that derive from an above protein molecular structure can be marked according to the described method of people such as Klingler (U.S. utility model application, May 3 2002 applying date, denomination of invention " computer system and method for virtual screening compound ").Synthetic then or obtain the best compound that meets and use it in conjunction with test and biological assay from the manufacturer in chemical storehouse.

The available computers modelling technique is assessed a kind of compound kinase whose potential is regulated or keying action.If computer model shows intensive and interacts, then can synthesize this molecule and test the ability that it combines with kinases and influences kinase activity (inhibition or activation).

Kinase whose adjusting or other binding compounds can be assessed with computer by a series of step, wherein, chemical group or fragment are screened and select according to itself and kinase whose each binding pocket and other regional bonded ability.This method can be from coming according to kinase whose coordinate range estimation avtive spot on computer screen.Then (Blaney, J.M. and Dixon, J.S., Perspectives inDrug Discovery and design, 1:301,1993) are placed or docked to fragment or the chemical group selected with a plurality of different directions in kinase whose binding pocket.Manually butt joint available software such as Insight II (Accelrys, San Diego, CA), MOE (Chemical Computing Group, Inc., Montreal, Quebec, Canada) and SYBYL (Tripos, Inc., St.Louis, MO, 1992) finish, use field of force of molecule such as the CHARMM (people such as Brooks of standard then, J.Comp.Chem.4:187-217,1983), AMBER (people such as Weiner, J.Am.Chem.Soc.106:765-84,1984) and C ²MMFF (Merck Molecular Force Field; Accelrys, SanDiego CA) carries out energy minimization and/or molecular dynamics.More the butt joint of automatization can be finished with for example following program: DOCK (people such as Kuntz, J.Mol.Biol., 161:269-88,1982; DOCK can derive from University of California, San Francisco, CA); AUTODOCK (Goodsell ﹠amp; Olsen, Proteins:Structure, Function, andGenetics 8:195-202,1990; AUTODOCK can derive from Scripps Research Institute, La Jolla, CA); GOLD (Cambridge crystallization Data centre (Cambridge CrystallographicData Centre, CCDC); People such as Jones, J.Mol.Biol.245:43-53,1995); And FLEXX (Tripos, St.Louis, MO; Rarey, people such as M., J.Mol.Biol.261:470-89,1996).Other suitable program for example is recorded in people such as Halperin.

Selecting by aforesaid method in the process of compound, can by computer evaluation measure and optimize compound may with kinases bonded efficient.For example, the occupied non-overlapping volume of volume when the compound that is designed to or has selected the kinase inhibitor function may occupy and combine with natural substrate with the avtive spot residue, but, one of ordinary skill in the art will appreciate that, main chain and side chain in this respect certain handiness can be arranged, so that can be reset.In addition, those of ordinary skills can design in conjunction with the time can utilize albumen to reset compound, thereby cause induced fit.A kind of effective kinase inhibitor under its combination and unbound state, should show relative less capacity volume variance (that is, it in conjunction with the time must have less in conjunction with deformation energy and/or lower conformation tension force).Therefore, the most effective kinase inhibitor should be designed to for example be not more than 10kcal/mol, be not more than 7kcal/mol, be not more than 5kcal/mol or be not more than 2kcal/mol in conjunction with deformation energy.Kinase inhibitor can be under the conformation of the similar total binding energy of having of more than one and protein-interacting.In this case, the bonded deformation energy should be poor between the average energy of the energy of free cpds and inhibitor viewed multiple conformation when combining with enzyme.

Can adopt certain computer software known in the art to assess the deformation energy and the electrostatic interaction of compound.The example that is designed to the program of these application comprises: Gaussian94, C revised edition (Frisch, Gaussian, Inc., Pittsburgh, PA.

1995); AMBER, the 7th edition (Kollman, University of California at San Francisco, 2002); QUANTA/CHARMM (Accelrys, Inc., San Diego, CA, 1995); InsightII/Discover (Accelrys, Inc., San Diego, CA,

1995); DelPhi (Accelrys, Inc., San Diego, CA,

1995); And AMSOL (University of Minnesota) (QuantumChemistry Program Exchange, Indiana University).These programs can for example LINUX, SGI or sun station move with computer workstation known in the art.Other hardware systems and software package are well known by persons skilled in the art.

Those of ordinary skills can utilize methods known in the art and method disclosed herein to express kinase protein.Kinase polypeptide natural and sudden change as herein described can utilize technology known in the art carry out the complete synthesis or partial synthesis of chemistry (referring to, Creighton for example, Proteins:Structures and Molecular Principles, W.H.Freeman ﹠amp; Co., NY, 1983).

Can come the polypeptide of synthesis of natural and sudden change with gene expression system.Can make up the polypeptid coding sequence and the suitable expression vector of transcribing/translating control signal well known by persons skilled in the art that comprise natural or sudden change.These methods comprise reorganization/gene recombination in extracorporeal recombinant DNA technology, synthetic technology and the body.Referring to, for example, people such as Sambrook are at Molecular Cloning:A LaboratoryManual, Cold Spring Harbor Laboratory, NY, 2001 and people such as Ausubel at Current Protocols in Molecular Biology, Greene Publishing Associates andWiley Interscience, NY, the technology described in 1989.

Can adopt the host expresses carrier system to express kinases.Described expression system includes but not limited to that microorganism is such as the bacterium that transforms with the recombinant phage dna that comprises encoding sequence, plasmid DNA or cosmid DNA expression vector; Yeast with the recombinant yeast expression vector conversion that comprises encoding sequence; Insect cell system with the recombinant virus expression vector that comprises encoding sequence (for example baculovirus) infection; With the recombinant virus expression vector that comprises encoding sequence (cauliflower mosaic virus for example, CaMV; Tobacco mosaic virus (TMV), TMV) infect or with the recombinant plasmid expression vector that comprises encoding sequence (for example Ti-plasmids) plant transformed cell system; Or zooblast system.Albumen can also be expressed in the human gene therapy system, comprises that for example expressing protein is so that proteic content increases in individual or expressing gene engineering treatment albumen.The Expression element of these systems is different on its intensity and specificity.

Specially designed carrier allows DNA shuttling back and forth between such as bacterium-yeast or bacterium-zooblast between the host.Suitably the expression vector that makes up can contain: be used for copy source, one or more selectable marks, the available restriction endonuclease sites of limited quantity, the possibility and the active promotor of high copy number at the host cell self-replicating.Promotor is meant that the indication RNA polymerase combines with DNA and causes RNA synthetic dna sequence dna.Strong promoter is the promotor that mRNA is initiated with high frequency.

Expression vector can also contain the influential various elements of transcribing and translating, and comprises for example constitutive promoter and inducible promoter.These elements normally host and/or carrier are dependent.For example, when in bacterial system, cloning, can use pL, plac, ptrp, the ptac (ptrp-lac hybrid promoter) etc. of inducible promoter such as T7 promotor, phage; When in insect cell system, cloning, can use baculovirus polyhedrin body protein promotor etc.; When in the vegetable cell system, cloning, can use promotor derived from the plant cell chromosome group (heat-shocked promotor for example; The promotor of the small subunit of RUBISCO; The protein-bonded promotor of chlorophyll a/b) or derived from the promotor of plant virus (the 35S RNA promotor of CaMV for example; The coat protein promotor of TMV); When in mammal cell line system, cloning, can use mammalian promoter (for example metallothionein promoter) or mammalian disease virus promoter (adenovirus late promoter for example; Vaccinia virus 7.5K promotor; The SV40 promotor; The bovine papilloma virus promotor; With the Epstein-Barr virus promotor).

Can adopt the whole bag of tricks that carrier is incorporated in the host cell, for example conversion, transfection, infection, protoplastis merge and electroporation.The cell clone breeding that will contain expression vector is also analyzed individually to determine whether they have produced suitable polypeptide.The host cell that can adopt various systems of selection to differentiate to transform, described method comprises for example antibiotic resistance.Can differentiate the host cell clone of express polypeptide by several different methods, described method includes but not limited to and the immunological response of antikinase antibody and the relevant active existence with host cell.

The expression of cDNA can also be carried out with the synthetic mRNA of external generation.Synthetic mRNA can be in various acellular systems, include but not limited in wheat germ extract and the skein cell extract translation effectively, and can be in system based on cell (comprise but do not sink into microinjection in the frog ovocyte) translation effectively.

In order to determine to produce the active and/or proteic cDNA sequence of optimum level, made up the cDNA molecule of modification.The limiting examples of the cDNA of modification is that the host cell that the codon of wherein cDNA is selected will express therein at cDNA is optimized.Host cell is transformed and measures kinase RNA and/or proteic level with the cDNA molecule.

The level of kinase protein quantitatively separates with kinases specificity affinity pearl or specific antibody by several different methods, for example immunoaffinity and/or part affinity technology in the host cell ³⁵S-methionine(Met) mark or unlabelled albumen.Come evaluation of markers or unlabelled albumen by SDS-PAGE.Unlabelled albumen is by Western blotting, ELISA or adopt the RIA of specific antibody to detect.

Kinases can reclaim polypeptide so that the protein of activity form to be provided after expressing in the host cell of reorganization.Can adopt multiple purification process.Can by the various combinations of fractional separation known in the art or chromatographic step or should be used for separately from lysate or from conditioned medium the kinases of purification of Recombinant.

In addition, the kinases of reorganization can also utilize by the nascent protein of total length or its polypeptide fragment being had immunoaffinity post and other cell proteins that specific mono-clonal or polyclonal antibody make and be separated.Also can adopt other technology based on affinity.

Perhaps, polypeptide can reclaim with form that is folding, non-activity from host cell, for example reclaims from the inclusion body of bacterium.The albumen that reclaims with this form can utilize method known to those skilled in the art for example to dialyse and be folded into activity form more then with denaturing agent guanidine hydrochloride dissolution for example.

B. cell growth test

Various cell growth tests are known in this area and can be used for differentiating the pyrrolopyridine compounds (i.e. " testing compound ") that can suppress (for example reducing) cell growth and/or propagation.

For example, known have various kinds of cell to need specific kinases to grow and/or breed.The energy for growth of described cell in the presence of testing compound can estimate, the energy for growth when not having testing compound compares then, confirms the antiproliferative properties of testing compound thus.A kind of common method in the type method is the degree of measuring in marker such as tritium mixes the cell in the division for thymidine the DNA.Perhaps, the restraining effect of on cell proliferation can determine that the total metabolism activity of cell tests by using the surrogate markers thing relevant with cell number.Cell can be handled under the condition that has and do not exist testing compound with the metabolism indicator.Great-hearted cell meeting metabolism metabolism indicator, forming thus can detected meta-bolites.When can detected meta-bolites level in the presence of testing compound, reducing under with respect to the condition that does not have testing compound, just represent cell growth and/or inhibition of proliferation effect.Exemplary metabolism indicator for example comprise tetrazolium salts and

(referring to the following examples part).

V. pharmaceutical composition and administration

On the other hand, the invention provides pharmaceutical composition, said composition comprises and pharmaceutically acceptable vehicle blended fused ring heterocycle kinase modulators mutually.It will be understood by those skilled in the art that this pharmaceutical composition comprises the pharmacologically acceptable salt of above-described fused ring heterocycle kinase modulators.

In treatment and/or diagnostic use, The compounds of this invention can be mixed with various form of medication, comprises whole body and part or site-specific delivery of drugs.Various technology and preparation usually can be referring to Remington:TheScience and Practice of Pharmacy (the 20th edition) Lippincott, Williams ﹠amp; Wilkins (2000).

The compounds of this invention is effective in wide dosage range.For example, in adult treatment, the example of used dosage is 0.01-1000mg, 0.5-100mg, 1-50mg/ days and 5-40mg/ days.Most preferred dosage is 10-30mg/ days.Exact dosage desired will change along with the body weight of route of administration, the form of giving drug compound, individuality to be treated, individuality to be treated and attending doctor's preference and experience.

Pharmacologically acceptable salt is normally known to those skilled in the art, and can comprise such as but not limited to acetate, benzene sulfonate, benzoate, supercarbonate, bitartrate, bromide, Ca-EDTA salt, carnsylate, carbonate, Citrate trianion, edetate, ethanedisulphonate, estolate, esilate, fumarate, gluceptate, gluconate, glutaminate, glycollylarsanilate, hexylresorcinate, breathe out amine, hydrobromate, hydrochloride, Hydroxynaphthoate, iodide, isethionate, lactic acid salt, lactobionate, malate, maleate, mandelate, mesylate, mucate, naphthalenesulfonate, nitrate, embonate, pantothenate, phosphoric acid salt/diphosphate, polygalacturonate, salicylate, stearate, subacetate, succinate, vitriol, tannate, tartrate or chloro theophylline salt.Other pharmacologically acceptable salt can be referring to for example Remington:The Science and Practice of Pharmacy (the 20th edition) Lippincott, Williams ﹠amp; Wilkins (2000).Preferred pharmacologically acceptable salt for example comprise acetate, benzoate, bromide, carbonate, Citrate trianion, gluconate, hydrobromate, hydrochloride, maleate, mesylate, naphthalenesulfonate, embonate, vitriol, salicylate, succinate, vitriol or tartrate.

According to concrete situation to be treated, described reagent can be mixed with liquid or solid formulation and whole body or topical.This reagent can be with for example form administration of timing well known by persons skilled in the art or slowly-releasing.The method of preparation and administration can be referring to Remington:The Science and Practice ofPharmacy (the 20th edition) Lippincott, Williams ﹠amp; Wilkins (2000).That suitable approach can comprise is oral, contain clothes, by inhalation spraying, hypogloeeis, rectum, through skin, vagina, through mucous membrane, intranasal or through enteral administration; Parenterai administration, comprise in intramuscular, subcutaneous, intramedullary injection and the sheath, directly in the ventricle, in the intravenously, intraarticular, breastbone, in the synovial membrane, in the liver, in the damage, in the encephalic, intraperitoneal, nose or intraocular injection or other administering mode.

For injection, reagent of the present invention can be at the aqueous solution such as preparing in such as Hank solution, Ringer solution or normal saline buffer solution at the damping fluid of physical compatibility and diluting.For described mucosal, in preparation, use the permeate agent that is applicable to the barrier that will penetrate.Described permeate agent is normally known in the art.

In order to implement the present invention, utilize pharmaceutical acceptable inert carriers with compound disclosed herein be mixed be suitable for the whole body administration formulation also within the scope of the invention.By selecting carrier and suitable production practice condition rightly, but composition of the present invention, especially with solution form composition prepared parenterai administration, such as by intravenous injection.Utilize pharmaceutically acceptable carrier known in the art this compound to be mixed with at an easy rate the formulation that is suitable for oral administration.Described carrier can make The compounds of this invention be mixed with to be used for tablet, pill, capsule, liquid, gelifying agent, syrup, paste, suspensoid of the oral absorption of individuality to be treated (for example patient) etc.

For in the nose or inhalation, reagent of the present invention also can be prepared by method known to those skilled in the art, and can comprise such as but not limited to solubilizing agent, thinner or dispersed substance such as salt solution, sanitas such as benzylalcohol, absorption enhancer and fluorocarbon.

Being applicable to that pharmaceutical composition of the present invention comprises wherein comprises activeconstituents to realize its intended purposes composition with significant quantity.Significant quantity fixes in those skilled in the art's the limit of power, particularly according in the detailed scope of disclosure provided herein really.

Except activeconstituents, these pharmaceutical compositions also can contain suitable pharmaceutically acceptable carrier, and described carrier comprises vehicle and is convenient to active compound is processed into the auxiliary agent of pharmaceutically acceptable preparation.The preparation that is used for oral administration can be the form of tablet, drageeing, capsule or solution.

Being used for oral pharmaceutical preparation can obtain by the following method: active compound is mixed, randomly grinds formed mixture mutually with solid excipient; then; if necessary, after adding proper assistant, granular mixture is processed to obtain tablet or drageeing label.Specifically, appropriate excipients be weighting agent such as carbohydrate, comprise lactose, sucrose, N.F,USP MANNITOL or Sorbitol Powder; Cellulosics is W-Gum, wheat starch, rice fecula, yam starch, gelatin, tragacanth gum, methylcellulose gum, HPMC, Xylo-Mucine (CMC) and/or polyvinylpyrrolidone (PVP: polyvidone) for example.If necessary, can add disintegrating agent such as crosslinked polyvinylpyrrolidone, agar or Lalgine or its salt such as sodium alginate.

Provide suitable dressing to the drageeing label.Thus; can use dense sugar soln, it randomly contains gum arabic, talcum, polyvinylpyrrolidone, carbopol glue (carbopol gum), polyoxyethylene glycol (PEG) and/or titanium dioxide, lacquer and suitable organic solvent or solvent mixture.Can in tablet or drageeing coating, add dyestuff or pigment various combination with difference or sign active compound doses.

The pharmaceutical preparation that can orally use comprises fastening (push-fit) capsule made by gelatin and the soft seal capsule of being made by gelatin and softening agent such as glycerine or Sorbitol Powder.Fastening capsule contains and weighting agent such as lactose, tackiness agent such as starch and/or lubricant such as talcum or Magnesium Stearate and optional solubilizing agent blended activeconstituents mutually.In soft capsule, active compound dissolves in or is suspended in suitable liquid such as fatty oil, whiteruss or the liquid macrogol (PEG).In addition, also can add stablizer.

According to particular condition or morbid state to be treated or prevention, also can give to be used to usually treat or prevent other therapeutical agent of described situation with inhibitor of the present invention.For example, chemotherapeutic or other antiproliferative reagent and inhibitor of the present invention can be united and treat proliferative disease and cancer.The example of known chemotherapeutic includes but not limited to Zorubicin, dexamethasone, vincristine(VCR), endoxan, Ro 2-9757, Hycamtin, taxol, interferons and platinum derivatives.

Also can include but not limited to anti-inflammatory agent such as corticosteroid, TNF blocker, IL-1RA, azathioprine, endoxan and sulfasalazine with other example of the reagent of inhibitor combined utilization of the present invention; Immunomodulator and inhibition immunizing agent such as S-Neoral, tacrolimus, rapamycin, mycophenolate mofetile, Interferon, rabbit, corticosteroid, endoxan, azathioprine and sulfasalazine; The sick reagent of neural factor such as acetylcholinesterase depressant, MAO inhibitor, Interferon, rabbit, anticonvulsive agent, ion channel blocking agent, Riluzole and Kang Pajinsenshi; Reagent such as the beta blocker, ACE inhibitor, diuretic(s), nitric ether, calcium channel blocker and the statins that are used for the treatment of cardiovascular disorder; Reagent such as the corticosteroid, QUESTRAN, Interferon, rabbit and the antiviral property reagent that are used for the treatment of hepatopathy; Reagent such as the corticosteroid, leukemia reagent and the somatomedin that are used for the treatment of hematologic disease; Be used for the treatment of reagent such as Regular Insulin, insulin analog, α alpha-glucosidase inhibitors, the biguanides and the insulin sensitizer of diabetes and be used for the treatment of immunity and lack the reagent of limit illness such as gamma Globulin.

These additional reagent can be used as the part and the composition that comprises inhibitor administration respectively of multiple dose administration scheme.Perhaps, these reagent can also mix in single composition with inhibitor mutually as the part of single formulation.

The present invention is not used to explain the restriction of scope of the exemplary embodiment of all respects of the present invention.In fact, except content as herein described, various improvement of the present invention it will be apparent to those skilled in the art that from the description of front.Described improvement also falls within the scope of the present invention.In addition, any one of any embodiment of the present invention or a plurality of feature all can be combined and do not depart from scope of the present invention with any one or a plurality of further feature of any other embodiment of the present invention.For example, can be applied to methods of treatment as herein described with being equal to and suppress kinase whose method at the described fused ring heterocycle kinase modulators of " fused ring heterocycle kinase modulators " part.The reference of being quoted is the example of prior art level in this application, in this article they is incorporated herein by reference in full.

Embodiment

Following examples are to be used for explaining rather than restriction the present invention for required protection.The preparation of embodiments of the present invention is described in following examples to some extent.One of ordinary skill in the art will appreciate that the chemical reaction and the synthetic method that are provided can improve to make many other compounds of the present invention.Under the situation that does not illustrate The compounds of this invention, it will be appreciated by those of ordinary skill in the art that these compounds can be by improving synthetic method as herein described and by utilizing synthetic method known in the art to make.

Synthesizing of compound:

Method 1:

Synthesizing of step 1:5-bromo-2-fluoro-pyridine-3-formaldehyde

With the di-isopropyl lithamide (5mL, anhydrous THF (40mL) solution 35mmol) is cooled to-78 ℃ under nitrogen, add then n-Butyl Lithium (hexane solution of 2.5M, 12mL, 30mmol).Then the mixture that forms was stirred 15 minutes down at-78 ℃, add then 5-bromo-2-fluoro-pyridine (5g, 28mmol).Then the mixture that forms was stirred 90 minutes down at-78 ℃.(4mL 36mmol) very rapidly joins under-78 ℃ in the suspension and with 60 seconds of this mixture vigorous stirring with the N-formyl piperidine.By adding 10% (w/v) aqueous citric acid solution termination reaction immediately.Mixture is warming up to room temperature and between water and methylene dichloride, distributes.With water dichloromethane extraction three times, organic phase is merged, use dried over sodium sulfate, filter also concentrated.Crude product is obtained 5-bromo-2-fluoro-pyridine-3-formaldehyde (2.993g, 52% yield) with the hexanaphthene crystallization, be the oldlace plate crystal. ¹H-NMR(500MHz，d ₆-DMSO)δ 10.07(s，1H)，8.70(dd，1H)，8.55(dd，1H)。MS：m/z 236，238[MNa ⁺]，204，206[MH ⁺]，176，178[MH-CO ⁺]。

Synthesizing of step 2 and 3:5-bromo-1H-pyrazolo [3,4-b] pyridine

With 5-bromo-2-fluoro-pyridine-3-formaldehyde (13.66g, 66.96mmol), tetramethyl ethylene ketone (8.75g, 74.0mmol) and the tosic acid monohydrate (1.50g 7.89mmol) places the flask that is equipped with the DEAN-STARK-prolong, is dissolved in dry-out benzene (400mL) then.Mixture heating up is refluxed, steam solvent and keep clarification to overhead product, residual volume is about 200ml.Mixture with ethyl acetate (300mL) dilution, with saturated sodium bicarbonate aqueous solution and salt water washing, is used dried over sodium sulfate then, filter and concentrate.The resistates that forms is dissolved in the mixture of ethanol (400mL) and diisopropyl ethyl amine (25mL).(15ml 0.48mol) and with the mixture that forms under refluxad stirred 4 hours to add anhydrous hydrazine then.Then the mixture that forms is concentrated into driedly, the resistates that forms is distributed between water and toluene.With organic phase salt solution washed twice, use dried over sodium sulfate, filter and concentrate.Resistates is dissolved in anhydrous diethyl ether (700mL), and (2M 70mL) slowly joins in the solution of vigorous stirring with the anhydrous ether solution of hydrogenchloride.Leach throw out, with ether and hexane wash, vacuum-drying then. ¹H-NMR(500MHz，d ₆-DMSO)δ 10.31(s，br，1H)，8.86(s，1H)，8.37(d，1H)，7.88(d，1H)，6.08(s，1H)，3.56(s，br)，1.27(s，6H)，1.19(s，6H)。MS：m/z 198，200[MH ⁺]。

Mixture with above solid water-soluble (500mL), ethanol (200mL) and concentrated hydrochloric acid aqueous solution (50mL) under 50-65 ℃.Then the mixture that forms was at room temperature stirred 16 hours, be neutralized to pH=8 with sodium bicarbonate then.The throw out that forms is leached, with water ethyl acetate extraction three times.With the organic phase salt water washing that merges, use dried over sodium sulfate, filter and concentrate.Resistates that forms and the throw out that obtains are obtained 5-bromo-1H-pyrazolo [3,4-b] pyridine (6.615g, 50% yield) with alcohol crystal, be the cream-coloured extremely shallow pausiaceous solid of crystalloid. ¹H-NMR(500MHz，d ₆-DMSO)δ 13.91(s，1H)，8.60(d，1H)，8.54(d，1H)，8.16(s，br，1H)。MS：m/z 198，200[MH ⁺]。

Synthesizing of step 4:5-bromo-3-iodo-1H-pyrazolo [3,4-b] pyridine

(3.00g, 15.2mmol) (3.60g 16.0mmol) is dissolved in anhydrous ethylene dichloride (100mL) with N-iodine succinimide with 5-bromo-1H-pyrazolo [3,4-b] pyridine.The mixture that forms was under refluxad stirred 6 hours, be cooled to room temperature, with THF (300mL) dilution.With solution saturated aqueous sodium thiosulfate (100mL) and the salt water washing that forms, use dried over mgso then, filter also concentrated.With 1:1 mixture and the ether washing of resistates with methylene dichloride and ether, vacuum-drying obtains 5-bromo-3-iodo-1H-pyrazolo [3,4-b] pyridine (3.795g, 77% yield) then, is cream-coloured-brown solid. ¹H-NMR(500MHz，d ₆-DMSO)δ 14.31(s，1H)，8.65(d，1H)，8.20(d，1H)。MS：m/z 323，325[MH ⁺]。

Synthesizing of step 5:5-bromo-3-iodo-1-(2-TMS-ethoxyl methyl)-1H-pyrazolo [3,4-b] pyridine

(2.68g 8.27mmol) is dissolved in dry DMF (40mL) with 5-bromo-3-iodo-1H-pyrazolo [3,4-b] pyridine under nitrogen.The continuation adding solution is cooled to 0-5 ℃, adds excessive dry sodium hydride, till also can not form hydrogen.In the suspension that forms in 0-5 ℃ drip down 2-TMS-ethoxyl methyl chlorine (2.5ml, 14mmol).The mixture that forms was stirred 1 hour down at 0 ℃, then by adding methyl alcohol and saturated aqueous ammonium chloride termination reaction.Under 50 ℃, be evaporated to the mixture that forms dried then.The resistates that forms is distributed between water, salt solution and methylene dichloride.With the water dichloromethane extraction,, filter and concentrate then the organic phase dried over sodium sulfate that merges.Crude product is passed through the flash chromatography on silica gel purifying, hexane solution gradient elution with ethyl acetate obtains 5-bromo-3-iodo-1-(2-TMS-ethoxyl methyl)-1H-pyrazolo [3,4-b] pyridine (2.929g, 78% yield), be cream-coloured extremely brown solid. ¹H-NMR(500MHz，d ₆-DMSO)δ 8.85(d，1H)，8.40(d，1H)，5.85(s，2H)，3.69(t，2H)，0.92(t，2H)，0.11(s，9H)。

Synthesizing of step 6:5-bromo-3-(2-methoxyl group-phenyl)-1-(2-TMS-ethoxyl methyl)-1H-pyrazolo [3,4-b] pyridine

With 5-bromo-3-iodo-1-(2-TMS-ethoxyl methyl)-1H-pyrazolo [3,4-b] pyridine (1.606g, 3.537mmol), 2-methoxyl group-phenyl-boric acid (575mg, 3.78mmol) and 1,1-two (diphenylphosphine) ferrocene palladium chloride (II) methylene dichloride adducts (145mg, 0.178mmol) (2M, 8mL) mixture in stirred 100 minutes down in 85 ℃ in sealed vial at acetonitrile (8mL) and aqueous sodium carbonate.Then the mixture that forms is distributed between saturated sodium bicarbonate aqueous solution and methylene dichloride, with water dichloromethane extraction three times.With the organic phase dried over sodium sulfate that merges, filter and concentrate.Crude product is passed through the flash chromatography on silica gel purifying, hexane solution gradient elution with ethyl acetate obtains 5-bromo-3-(2-methoxyl group-phenyl)-1-(2-TMS-ethoxyl methyl)-1H-pyrazolo [3,4-b] pyridine (1.002g, 65% yield), be rice white oil. ¹H-NMR(500MHz，d ₆-DMSO)δ 8.70(d，1H)，8.40(d，1H)，7.61(d，1H)，7.50(ddd，1H)，7.23(dd，1H)，7.10(ddd，1H)，5.81(s，2H)，3.85(s，3H)，3.66(t，2H)，0.84(t，2H)，-0.10(s，9H)。MS：m/z 456，458[MNa ⁺]。

Synthesizing of step 7:3-(2-methoxyl group-phenyl)-5-(4,4,5,5-tetramethyl--[1,3,2] two oxa-boron heterocycle pentane-2-yls)-1-(2-TMS-ethoxyl methyl)-1H-pyrazolo [3,4-b] pyridine

With two (pinacols), two boron (1.20g, 4.73mmol), 1,1 '-two (diphenylphosphine) ferrocene palladium chloride (II) methylene dichloride adducts (100mg, 0.122mmol) and anhydrous sodium acetate (625mg 7.62mmol) places the bottle of nitrogen wash.To wherein adding 5-bromo-3-(2-methoxyl group-phenyl)-1-(2-TMS-ethoxyl methyl)-1H-pyrazolo [3,4-b] pyridine (1.002g, dry DMF 2.307mmol) (15mL) solution.The mixture that forms was shone 60 minutes down in 130 ℃ in Personal Chemistry Optimizer, then at 50 ℃ of following concentrating under reduced pressure.The resistates that forms distributed between ether and salt solution and with the water extracted with diethyl ether.Organic phase is merged, use dried over sodium sulfate, filter and concentrate.Crude product is passed through the flash chromatography on silica gel purifying, hexane solution gradient elution with ethyl acetate obtains 3-(2-methoxyl group-phenyl)-5-(4,4,5,5-tetramethyl--[1,3,2] two oxa-boron heterocycle pentane-2-yls)-1-(2-TMS-ethoxyl methyl)-1H-pyrazolo [3,4-b] pyridine (1.370g, 123% yield), be shallow olive-green solid. ¹H-NMR(500MHz，d ₆-DMSO)δ 8.76(d，1H)，8.40(d，1H)，7.59(dd，1H)，7.51(ddd，1H)，7.25(m，1H)，7.12(ddd，1H)，5.84(s，2H)，3.82(s，3H)，3.67(t，2H)，1.33(s，12H)，0.84(t，2H)，-0.10(s，9H)。

Step 8:{2-hydroxyl-5-[3-(2-methoxyl group-phenyl)-1H-pyrazolo [3,4-b] pyridine-5-yl]-phenyl }-morpholine-4-base-ketone synthetic

With 3-(2-methoxyl group-phenyl)-5-(4,4,5,5-tetramethyl--[1,3,2] two oxa-boron heterocycle pentane-2-yls)-1-(2-TMS-ethoxyl methyl)-1H-pyrazolo [3,4-b] pyridine (100mg, 0.21mmol), (5-bromo-2-hydroxyl-phenyl)-morpholine-4-base-ketone (66mg (0.23mmol) and 1,1 '-two (diphenylphosphine) ferrocene palladium chloride (II) methylene dichloride adducts (9mg, 11 μ mol) acetonitrile (2mL) and aqueous sodium carbonate (2M, 2mL) mixture in Personal Chemistry Optimizer in 135 ℃ of irradiations 20 minutes down.Crude product mixture is distributed between methylene dichloride and saturated sodium bicarbonate aqueous solution.With the water dichloromethane extraction,, filter and concentrate then the organic phase dried over sodium sulfate that merges.Crude product is passed through the flash chromatography on silica gel purifying, hexane solution gradient elution with ethyl acetate obtains { 2-hydroxyl-5-[3-(2-methoxyl group-phenyl)-1-(2-TMS-ethoxyl methyl)-1H-pyrazolo [3,4-b] pyridine-5-yl]-phenyl }-morpholine-4-base-ketone (35mg, 30% yield), be colorless solid. ¹H-NMR(500MHz，d ₆-DMSO)δ 8.86(d，1H)，8.27(d，1H)，7.64(dd，1H)，7.62(dd，1H)，7.54(d，1H)，7.49(ddd，1H)，7.24(d，br，1H)，7.11(ddd，1H)，7.00(d，1H)，5.84(s，2H)，3.84(s，3H)，3.69(t，2H)，3.7-3.2(m，8H)，0.86(t，2H)，-0.08(s，9H)。MS：m/z 583[MNa ⁺]，561[MH ⁺]，443[MH ⁺-(Me ₃Si(CH ₂) ₂O)]。

With { 2-hydroxyl-5-[3-(2-methoxyl group-phenyl)-1-(2-TMS-ethoxyl methyl)-1H-pyrazolo [3,4-b] pyridine-5-yl]-phenyl }-morpholine-4-base-ketone (34mg, 61 μ mol) methylene dichloride (15mL) solution be cooled to 0-5 ℃ and add the boron trifluoride diethyl etherate compound (100 μ l, 0.8mmol).Then the mixture that forms was stirred 40 minutes down at 0-5 ℃, add 10ml 10% (w/v) potassium hydroxide solution then.Mixture was at room temperature continued to stir 1 hour.Then by add citric acid with pH regulator to being about 3-4, water is saturated with sodium sulfate.With the mixture dichloromethane extraction (3x) that forms.Organic phase is merged,, use dried over sodium sulfate with the saturated sodium bicarbonate aqueous solution washing, evaporation obtains { 2-hydroxyl-5-[3-(2-methoxyl group-phenyl)-1H-pyrazolo [3 then, 4-b] pyridine-5-yl]-phenyl }-morpholine-4-base-ketone (11.5mg, 44% yield), be colorless solid. ¹H-NMR(500MHz，d ₆-DMSO)δ 13.76(s，1H)，10.06(s，1H)，8.78(d，1H)，8.23(d，1H)，7.64(dd，1H)，7.62(dd，1H)，7.51(d，1H)，7.46(ddd，1H)，7.22(d，1H)，7.10(t，1H)，6.99(d，1H)，3.84(s，3H)，3.7-3.2(m，8H)。MS：m/z 431[MH ⁺]。

Other compound that makes by method 1:

Table 1

Method 2:

Step 1: morpholine-4-base-[3-(1H-pyrazolo [3,4-b] pyridine-5-yl)-phenyl]-ketone synthetic

With 5-bromo-1H-pyrazolo [3,4-b] pyridine (1.50g, 7.57mmol), 3-(morpholine-4-carbonyl) phenyl-boron dihydroxide (2.136g, 9.09mmol) and tetrakis triphenylphosphine palladium (0) (435mL, 0.376mmol) mixture in glycol dimethyl ether (8mL) and saturated sodium bicarbonate aqueous solution (8mL) in PersonalChemistry Optimizer in 175 ℃ of down irradiations 60 minutes.Crude product mixture is distributed between methylene dichloride and saturated sodium bicarbonate aqueous solution.Then with water methylene dichloride and ethyl acetate extraction, with the organic phase dried over sodium sulfate that merges, filter, concentrate and obtain the light green foam, this foam contains 80% morpholine-4-base-[3-(1H-pyrazolo [3,4-b] pyridine-5-yl)-phenyl]-ketone (2.30g, 80% yield) and 20% triphenylphosphine oxide. ¹H-NMR(500MHz，d ₆-DMSO)δ13.75(s，1H)，8.87(d，1H)，8.54(d，1H)，8.21(d，1H)，7.85(m，1H)，7.77(m，1H)，7.58(t，1H)，7.41(m，1H)。

Step 2:[3-(3-iodo-1H-pyrazolo [3,4-b] pyridine-5-yl)-phenyl]-morpholine-4-base-ketone synthetic

With morpholine-4-base-[3-(1H-pyrazolo [3,4-b] pyridine-5-yl)-phenyl]-ketone (2.30g, 80% purity ,～6mmol) (2.50g 11.1mmol) is dissolved in ethylene dichloride (180mL) with N-iodine succinimide.Mixture was under refluxad stirred 5 hours, be cooled to room temperature then and dilute with methylene dichloride.Solution with saturated aqueous sodium thiosulfate washing (1x), with saturated aqueous sodium bromide washing (2x), is used dried over sodium sulfate then, filter and concentrate.The resistates that forms is washed with ether (80mL), and drying obtains [3-(3-iodo-1H-pyrazolo [3,4-b] pyridine-5-yl)-phenyl]-morpholine-4-base-ketone then, is cream-coloured powder (2.881g, 88% yield is through two steps). ¹H-NMR(500MHz，d ₆-DMSO)δ 14.19(s，1H)，8.92(d，1H)，8.14(d，1H)，7.91(m，1H)，7.83(m，1H)，7.59(ddd，1H)，7.44(dt，1H)，3.75-3.35(m，8H)。

Step 3: morpholine-4-base-{ 3-[3-(1H-pyrazoles-4-yl)-1H-pyrazolo [3,4-b] pyridine-5-yl]-phenyl }-ketone synthetic

With [3-(3-iodo-1H-pyrazolo [3,4-b] pyridine-5-yl)-phenyl]-morpholine-4-base-ketone (25mg, 58 μ mol), 1,1-two (diphenylphosphine) ferrocene palladium chloride (II) methylene dichloride adducts (5mg, 6 μ mol) and the mixture of 1H-pyrazoles-4-ylboronic acid (11mg, 98 μ mol) in acetonitrile (2mL) and 2M sodium carbonate solution (1mL) in Personal Chemistry Optimizer in 175 ℃ of down irradiations 30 minutes.With crude product mixture water (1mL) and ethyl acetate (3mL) dilution, isolate organic phase then, filter and concentrate.Then with the reversed-phase HPLC purifying of crude mixture that forms by gathering based on quality, obtain morpholine-4-base-{ 3-[3-(1H-pyrazoles-4-yl)-1H-pyrazolo [3 with the acetonitrile solution gradient elution that contains 0.1% formic acid, 4-b] pyridine-5-yl]-phenyl }-ketone (6.2mg, 29% yield), be colourless powder. ¹H-NMR(500MHz，d ₆-DMSO)δ 13.59(s，1H)，13.17(s，1H)，8.87(d，1H)，8.73(d，1H)，8.60(s，br，1H)，8.17(s，br，1H)，7.95(ddd，1H)，7.89(t，1H)，(t，1H)，7.59(t，1H)，7.43(ddd，1H)，3.80-3.35(m，8H).MS：m/z 397[MNa ⁺]，375[MH ⁺]。

Other compound that makes by method 2:

Table 2

Method 3:

Step 1:{3-[3-iodo-1-(2-TMS-ethoxyl methyl)-1H-pyrazolo [3,4-b] pyridine-5-yl]-phenyl }-morpholine-4-base-ketone synthetic

To [3-(3-iodo-1H-pyrazolo [3,4-b] pyridine-5-yl)-phenyl]-morpholine-4-base-ketone (2.12g, in dry DMF 4.88mmol) (30mL) solution in 0-5 ℃ add down sodium hydride (60% mineral oil solution, 750mg, 30mmol).Mixture is stirred several minutes, under identical temperature, drip then trimethylsilylethoxymethyl chlorine (2.0ml, 11mmol).Mixture was stirred 4 hours to room temperature at 0 ℃, be cooled to 0-5 ℃ then also by adding the methyl alcohol termination reaction.Then the suspension that forms is distributed between water, saturated aqueous ammonium chloride and ether.With water extracted with diethyl ether three times,, filter also concentrated with the organic phase dried over sodium sulfate that merges.Then crude product is passed through the silica gel chromatography purifying, hexane solution gradient elution with ethyl acetate obtains { 3-[3-iodo-1-(2-TMS-ethoxyl methyl)-1H-pyrazolo [3,4-b] pyridine-5-yl]-phenyl }-morpholine-4-base-ketone, be cream-coloured-brown foam (1.806g, 66%, by ¹H-NMR confirms, by product is morpholine-4-base-{ 3-[2-(2-TMS-ethoxyl methyl)-2H-pyrazolo [3,4-b] pyridine-5-yl]-phenyl }-ketone). ¹H-NMR(500MHz，d ₆-DMSO)δ 9.08(d，1H)，8.29(d，1H)，8.01(m，1H)，7.95(t，br，1H)，7.69(t，1H)，7.55(d，br，1H)，5.88(s，2H)，3.71(t，2H)，3.85-3.45(m，8H)，0.94(t，2H)，-0.2(s，9H)。MS：m/z 565[MNa ⁺]，537[MH ⁺]，447[MH ⁺-(Me ₃Si(CH ₂) ₂O)]。

Step 2:{3-[3-(2-methoxyl group-pyridin-3-yl)-1H-pyrazolo [3,4-b] pyridine-5-yl]-phenyl }-morpholine-4-base-ketone synthetic

With { 3-[3-iodo-1-(2-TMS-ethoxyl methyl)-1H-pyrazolo [3,4-b] pyridine-5-yl]-phenyl }-morpholine-4-base-ketone (33mg, 66% purity, 38 μ mol), 1,1 '-two (diphenylphosphine) ferrocene palladium chloride (II) methylene dichloride adducts (5mg, 6 μ mol) and the mixture of 3-trifluoromethyl phenyl boronic acid (14mg, 92 μ mol) in acetonitrile (2mL) and 2M sodium carbonate solution (1mL) in PersonalChemistry Optimizer in 175 ℃ of down irradiations 20 minutes.Crude product mixture is diluted with saturated aqueous sodium bromide (1mL) and ethyl acetate (4mL), isolate organic phase then, be adsorbed onto on the silica gel also by the flash chromatography on silica gel purifying, { the 3-[3-(2-methoxyl group-pyridin-3-yl)-1-(2-TMS-ethoxyl methyl)-1H-pyrazolo [3 that utilizes the hexane solution gradient elution of ethyl acetate to obtain, 4-b] pyridine-5-yl]-phenyl }-morpholine-4-base-ketone (24mg, 116% yield), be the rice white resistates.MS：m/z568[MNa ⁺]，546[MH ⁺]，428[MH ⁺-(Me ₃Si(CH ₂) ₂O)]。

This resistates is dissolved in THF (2ml) and with activatory 4 Molecular sieve joins in the mixture.(1M solution, 0.5ml 0.5mmol) and with mixture stirred 26 hours down at 70 ℃ the THF solution of adding tetra-n-butyl Neutral ammonium fluoride.Mixture is cooled to room temperature, adds 1ml Zeo-karb (Amberlyst, Na ⁺Type) and with mixture vibrated 40 minutes.Leach resin and molecular sieve then,, the filtrate that obtains is concentrated with methylene dichloride and methanol wash.Resistates is dissolved in ethyl acetate and by the flash chromatography on silica gel purifying, uses the ethyl acetate gradient elution of the ethyl acetate solution that contains 15% (v/v) methyl alcohol.The product fraction is merged, concentrate, then by reversed-phase HPLC purifying based on the quality collection, obtain { 3-[3-(2-methoxyl group-pyridin-3-yl)-1H-pyrazolo [3 with the acetonitrile solution gradient elution that contains 0.1% formic acid, 4-b] pyridine-5-yl]-phenyl }-morpholine-4-base-ketone (3.2mg, 20% yield), be colorless solid. ¹H-NMR(500MHz，d ₆-DMSO)δ 14.01(s，1H)，8.91(d，1H)，8.51(d，1H)，8.31(dd，1H)，8.11(dd，1H)，7.87(ddd，1H)，7.80(t，1H)，7.59(t，1H)，7.43(dt，1H)，7.18(dd，1H)，3.97(s，3H)，3.70-3.35(m，8H)。MS：m/z 416[MH ⁺]。

Other compound that makes by method 3:

Table 3

Method 4:

Step 1:N, N-dimethyl-3-(5-(3-(morpholine-4-carbonyl) phenyl)-1-((2-(trimethyl silyl) oxyethyl group) methyl)-1H-pyrazolo [3,4-b] pyridin-3-yl) benzamide synthetic

With ((3-iodo-1-((2-(trimethyl silyl) oxyethyl group) methyl)-1H-pyrazolo [3 for 3-; 4-b] pyridine-5-yl) phenyl) (morpholine-4-yl) ketone (50mg; 0.089mmol), 3-(formyl-dimethylamino) phenyl-boron dihydroxide (34mg; 0.177mmol), [1; 1-two (diphenylphosphine) ferrocene] palladium chloride (II) methylene dichloride complex compound (3.6mg; 0.0045mmol) and yellow soda ash (the 2M aqueous solution; 0.134mL 0.267mmol) mixture in acetonitrile (1mL) heated 30 minutes down at 90 ℃ in the Personal microwave.With the mixture dilute with water that forms and use ethyl acetate extraction.Organic layer is merged, use dried over sodium sulfate, filter and be concentrated into dried.Crude product is obtained N by the silica gel chromatography purifying, and N-dimethyl-3-(5-(3-(morpholine-4-carbonyl) phenyl)-1-((2-(trimethyl silyl) oxyethyl group) methyl)-1H-pyrazolo [3,4-b] pyridin-3-yl) benzamide is clarifying oil.MS：m/z 586.2[M+H ⁺]。

Step 2:N, N-dimethyl-3-(5-(3-(morpholine-4-carbonyl) phenyl)-1H-pyrazolo [3,4-b] pyridin-3-yl) benzamide synthetic

With N, N-dimethyl-3-(5-(3-(morpholine-4-carbonyl) phenyl)-1-((2-(trimethyl silyl) oxyethyl group) methyl)-1H-pyrazolo [3, the 4-b] pyridin-3-yl) solution of benzamide in the acetic acid solution (1mL) of 5% perchloric acid at room temperature stirred 1 hour.Then saturated sodium bicarbonate solution is slowly joined in this solution and at room temperature stirred 24 hours to pH～8 and with mixture.Use ethyl acetate extraction then, organic layer is merged, use dried over sodium sulfate, filter and be concentrated into dried.Reversed-phase HPLC purifying based on the quality collection obtains N, and N-dimethyl-3-(5-(3-(morpholine-4-carbonyl) phenyl)-1H-pyrazolo [3,4-b] pyridin-3-yl) benzamide (7.4mg, 18% yield is through two steps) is light yellow solid. ¹H NMR(500MHz，CDCl ₃)δ 2.99(s，3H)，3.10(s，3H)，3.46(br，2H)，3.61(br，2H)，3.76(br，4H)，7.40(d，J＝7.0Hz，1H)，7.47(d，J＝8.0Hz，1H)，7.52(m，2H)，7.64(d，J＝7.5Hz，1H)，7.68(s，1H)，7.98(m，2H)，8.62(s，1H)，8.85(br，1H)。MS：m/z 456.1(M+H ⁺)。

Other compound that makes by method 4:

Condition in the step 2 can change along with the compound in the following table.Sometimes need to replace sodium bicarbonate with yellow soda ash, the variation range in reaction times is 30 minutes to 24 hours.

Table 4

Method 5:

Synthesizing of step 1:3-(1H-pyrazolo [3,4-b] pyridine-5-yl) methyl benzoate

With 5-bromo-1H-pyrazolo [3,4-b] pyridine (2.00g, 10.10mmol), 3-(methoxycarbonyl) phenyl-boron dihydroxide (2.20g, 12.12mmol), sodium bicarbonate (2.2g, 6.00mmol) and tetrakis triphenylphosphine palladium (0) (0.250g, 0.202mmol) the mixture in Zai diox/water (40mL/10mL) stirred 15 hours down at 110 ℃.The mixture that forms is poured in the frozen water also with ethyl acetate extraction (3X) then.Organic layer is merged, use dried over sodium sulfate, filter and be concentrated into dried.Crude product is carried out silica gel chromatography obtain 3-(1H-pyrazolo [3,4-b] pyridine-5-yl) methyl benzoate (8) (1.65g, 65% yield), be yellow solid.MS：m/z 254.0(M+H ⁺)。

Synthesizing of step 2:3-(3-iodo-1H-pyrazolo [3,4-b] pyridine-5-yl) methyl benzoate

(1.65g, (1.81g 8.04mmol) and with mixture stirred 6 hours down at 70 ℃ to add NIS in ethylene dichloride 6.52mmol) (40mL) solution to 3-(1H-pyrazolo [3,4-b] pyridine-5-yl) methyl benzoate.Removal of solvent under reduced pressure obtains 3-(3-iodo-1H-pyrazolo [3,4-b] pyridine-5-yl) methyl benzoate (9) (988mg, 40% yield) with crude product by the silica gel chromatography purifying.MS：m/z 379.9(M+H ⁺)。

Step 3:3-(3-iodo-1-((2-(trimethyl silyl) oxyethyl group) methyl)-1H-pyrazolo [3,4-b] pyridine-5-yl) is benzoic synthetic

To 3-(3-iodo-1H-pyrazolo [354-b] pyridine-5-yl) methyl benzoate (988mg, in DMF solution 2.61mmol)-40 ℃ add down sodium hydrides (60% mineral oil solution, 525mg, 13.03mmol).Mixture was stirred 60 minutes, add then SEMCl (920 μ l, 5.22mmol).Reaction solution is heated to room temperature, with first alcohol and water termination reaction.Regulate pH to 4-5 with acetate then.With the mixture ethyl acetate extraction (3x) that forms, organic layer is merged then, use dried over sodium sulfate, filter and be concentrated into dried.The crude product that forms is obtained 3-(3-iodo-1-((2-(trimethyl silyl) oxyethyl group) methyl)-1H-pyrazolo [3, the 4-b] pyridine-5-yl) phenylformic acid (200mg, 15% yield) of solid state by the silica gel chromatography purifying.MS：m/z 517.9(M+Na ⁺)。

Step 4:(4-(2-(dimethylamino) ethyl) piperazine-1-yl) (3-(3-iodo-1-((2-(trimethyl silyl) oxyethyl group) methyl)-1H-pyrazolo [3,4-b] pyridine-5-yl) phenyl) ketone is synthetic

With crude product 3-(3-iodo-1-((2-(trimethyl silyl) oxyethyl group) methyl)-1H-pyrazolo [3,4-b] pyridine-5-yl) phenylformic acid (200mg, 0.404mmol), N, N-dimethyl-2-(piperazine-1-yl) ethamine (76mg, 0.485mmol), O-(7-azepine benzo triazol-1-yl)-N, N, N ', N '-tetramethyl-urea hexafluorophosphate (185mg, 0.485mmol), triethylamine (0.700ml, 0.485mmol) and the mixture of DMF (2ml) in Personal Microwave, stirring 1 hour under 90 ℃.Water is joined in the mixture also with ethyl acetate extraction (3x).Organic layer is merged, use dried over sodium sulfate, filter and be concentrated into dried.Crude product is carried out silica gel chromatography obtain (4-(2-(dimethylamino) ethyl) piperazine-1-yl) (3-(3-iodo-1-((2-(trimethyl silyl) oxyethyl group) methyl)-1H-pyrazolo [3,4-b] pyridine-5-yl) phenyl) ketone (110mg, 43% yield), be the rice white solid.MS：m/z 635.1(M+H ⁺)。

Step 5:(4-(2-(dimethylamino) ethyl) piperazine-1-yl) (3-(3-(5-fluoro-2-p-methoxy-phenyl)-1-((2-(trimethyl silyl) oxyethyl group) methyl)-1H-pyrazolo [3,4-b] pyridine-5-yl) phenyl) ketone is synthetic

((3-iodo-1-((2-(trimethyl silyl) oxyethyl group) methyl)-1H-pyrazolo [3 for 3-with (4-(2-(dimethylamino) ethyl) piperazine-1-yl), 4-b] pyridine-5-yl) phenyl) ketone (50mg, 0.079mmol), 5-fluoro-2-anisole ylboronic acid (20mg, 0.118mmol), [1,1-two (diphenylphosphine) ferrocene] palladium chloride (II) methylene dichloride complex compound (7.9mg, 0.10mmol) and yellow soda ash (the 2M aqueous solution, 0.119mL 0.237mmol) mixture in acetonitrile (1mL) heated 30 minutes down at 90 ℃ in Personal microwave.With the mixture dilute with water that forms and use ethyl acetate extraction.Organic layer is merged, use dried over sodium sulfate, filter and be concentrated into dried.Crude product is obtained (4-(2-(dimethylamino) ethyl) piperazine-1-yl) (3-(3-(5-fluoro-2-p-methoxy-phenyl)-1-((2-(trimethyl silyl) oxyethyl group) methyl)-1H-pyrazolo [3 by the silica gel chromatography purifying, 4-b] pyridine-5-yl) phenyl) ketone, be light yellow oil.MS：m/z 633.3(M+H ⁺)。

Step 6:(4-(2-(dimethylamino) ethyl) piperazine-1-yl) (3-(3-(5-fluoro-2-p-methoxy-phenyl)-1H-pyrazolo [3,4-b] pyridine-5-yl) phenyl) ketone is synthetic

(4-(2-(dimethylamino) ethyl) piperazine-1-yl) (3-(3-(5-fluoro-2-p-methoxy-phenyl)-1-((2-(trimethyl silyl) oxyethyl group) methyl)-1H-pyrazolo [3, the 4-b] pyridine-5-yl) phenyl) solution of ketone in the methanol solution (1mL) of 5% perchloric acid that step 5 is obtained at room temperature stirred 45 minutes.Then sodium hydroxide solution (2M) is slowly joined in this solution to pH～8.Use ethyl acetate extraction then, organic layer merged, be concentrated into dried, then it is dissolved in again methyl alcohol (1mL) and yellow soda ash (2M, 1mL).Mixture was at room temperature stirred 15 hours, and dilute with water is also with ethyl acetate extraction (3x) then.Organic layer is merged, use dried over sodium sulfate, filter and be concentrated into dried.Reversed-phase HPLC purifying based on the quality collection obtains (4-(2-(dimethylamino) ethyl) piperazine-1-yl) (3-(3-(5-fluoro-2-p-methoxy-phenyl)-1H-pyrazolo [3,4-b] pyridine-5-yl) phenyl) ketone (25.6mg, 64% yield calculates based on 12), be white solid. ¹H NMR(500MHz，CD ₃OD)δ 2.42(s，6H)，2.49(br，2H)，2.59(m，4H)，2.69(m，2H)，3.54(br，2H)，3.81(br，2H)，3.85(s，3H)，7.17(m，2H)，7.40(dd，J＝3.0，9.5Hz，1H)，7.44(d，br，J＝7.5Hz，1H)，7.59(t，J＝7.5Hz，1H)，7.72(br，1H)，7.77(d，J＝8.5Hz，1H)，8.39(d，J＝1.5Hz，1H)，8.80(d，J＝2.5Hz，1H)。MS：m/z 503.2(M+H ⁺)。

Method 6:

Step 1:(3-(3-(2, the 6-Dimethoxyphenyl)-1-((2-(trimethyl silyl) oxyethyl group) methyl)-1H-pyrazolo [3,4-b] pyridine-5-yl) phenyl) (4-(2-(dimethylamino) ethyl) piperazine-1-yl) ketone is synthetic

((3-iodo-1-((2-(trimethyl silyl) oxyethyl group) methyl)-1H-pyrazolo [3 for 3-with (4-(2-(dimethylamino) ethyl) piperazine-1-yl), 4-b] pyridine-5-yl) phenyl) ketone (50mg, 0.079mmol), 2,6-dimethoxy benzene ylboronic acid (22mg, 0.118mmol), tetrakis triphenylphosphine palladium (0) (9.1mg, 0.10mmol) and yellow soda ash (the 2M aqueous solution, 0.119mL 0.237mmol) mixture in acetonitrile (1mL) heated 30 minutes down in 120 ℃ in Personal microwave.With the mixture dilute with water that forms and use ethyl acetate extraction.Organic layer is merged, use dried over sodium sulfate, filter and be concentrated into dried.Crude product is obtained clarifying buttery by the silica gel chromatography purifying, and ((3-(2 for 3-, the 6-Dimethoxyphenyl)-and 1-((2-(trimethyl silyl) oxyethyl group) methyl)-1H-pyrazolo [3,4-b] pyridine-5-yl) phenyl) (4-(2-(dimethylamino) ethyl) piperazine-1-yl) ketone.MS：m/z 645.3(M+H ⁺)。

Step 2:(3-(3-(2, the 6-Dimethoxyphenyl)-1H-pyrazolo [3,4-b] pyridine-5-yl) phenyl) (4-(2-(dimethylamino) ethyl) piperazine-1-yl) ketone is synthetic

With ((3-(2 for 3-, the 6-Dimethoxyphenyl)-and 1-((2-(trimethyl silyl) oxyethyl group) methyl)-1H-pyrazolo [3,4-b] pyridine-5-yl) phenyl) (4-(2-(dimethylamino) ethyl) piperazine-1-yl) ketone solution in the acetic acid solution (1mL) of 5% perchloric acid at room temperature stirred 45 minutes.Then sodium hydroxide solution (2M) is slowly joined in this solution to pH～8.Use ethyl acetate extraction then, organic layer merged, be concentrated into dried, then it is dissolved in again methyl alcohol (1mL) and yellow soda ash (2M, 1mL).Mixture was at room temperature stirred 15 hours, and dilute with water is also with ethyl acetate extraction (3x) then.Organic layer is merged, use dried over sodium sulfate, filter and be concentrated into dried.Based on the reversed-phase HPLC purifying of quality collection obtain white solid ((3-(2 for 3-, the 6-Dimethoxyphenyl)-1H-pyrazolo [3,4-b] pyridine-5-yl) phenyl) (4-(2-(dimethylamino) ethyl) piperazine-1-yl) ketone (22.70mg, the yield of two steps are 56%). ¹H NMR(500MHz，CD ₃OD)δ 2.48(br，2H)，2.52(s，6H)，2.60(m，4H)，2.69(m，2H)，3.51(br，2H)，3.74(s，6H)，3.80(br，2H)，6.80(d，J＝8.5Hz，2H)，7.43(m，2H)，7.56(t，J＝8.0Hz，1H)，7.69(s，1H)，7.75(d，J＝8.0Hz，1H)，8.09(d，J＝2Hz，1H)，8.81(d，J＝2Hz，1H)。MS：m/z 515.2(M+H ⁺)。

Other compound that makes by method 6:

Table 5

^* Step 1 Suzuki coupling condition: 150 ℃, 1 hour, μ w.

^* Step 1 Suzuki coupling condition: 120 ℃, 1 hour, μ w.

^{* *} Step 1 Suzuki coupling condition: Pd ₂(dba) ₃, K ₃PO ₄With dicyclohexyl Phenylphosphine, CH ₃CN, 150 ℃, 4 hours, μ w.

Method 7:

Step 1:5-[3-(2-methoxyl group-phenyl)-1-(2-TMS-ethoxyl methyl)-1H-pyrazolo [3,4-b] pyridine-5-yl]-Nikithan synthetic

To 3-ethoxy carbonyl-5-pyridyl boric acid (529mg, 1.91mmol), 1,1 '-two (diphenylphosphine) ferrocene palladium chloride (II) methylene dichloride adducts (66mg, 0.09mmol) and 5-bromo-3-iodo-1-(2-TMS-ethoxyl methyl)-1H-pyrazolo [3,4-b] pyridine (780mg, 1.80mmol) mixture in add acetonitrile (5mL) and 2M aqueous sodium carbonate (5mL), then with mixture in PersonalChemistry Optimizer in 90 ℃ of down irradiations 30 minutes.Crude product mixture is distributed between ethyl acetate and water.With water ethyl acetate extraction three times,, filter also concentrated with the organic phase dried over sodium sulfate that merges.Crude product is passed through the flash chromatography on silica gel purifying, hexane solution gradient elution with ethyl acetate obtains 5-[3-(2-methoxyl group-phenyl)-1-(2-TMS-ethoxyl methyl)-1H-pyrazolo [3,4-b] pyridine-5-yl]-Nikithan (552mg, 61% yield), be yellow oil. ¹H-NMR(500MHz，d ₆-DMSO)δ 9.24(d，1H)，9.12(d，1H)，9.03(d，1H)，8.60(t，1H)，8.56(d，1H)，7.66(dd，1H)，7.51(ddd，1H)，7.25(dd，1H)，7.12(dt，1H)，5.88(s，2H)，4.40(q，2H)，3.87(s，3H)，3.71(t，2H)，1.37(t，3H)，0.87(t，2H)，-0.073(t，9H)。MS：m/z 505[MH ⁺]。

Step 2:5-[3-(2-methoxyl group-phenyl)-1H-pyrazolo [3,4-b] pyridine-5-yl]-nicotinic acid synthetic

To 5-[3-(2-methoxyl group-phenyl)-1-(2-TMS-ethoxyl methyl)-1H-pyrazolo [3,4-b] pyridine-5-yl]-Nikithan (494mg, 0.98mmol) THF (20mL) solution in add the tetra-n-butyl Neutral ammonium fluoride THF solution (1M, 10ml, 10mmol) and activatory 4 Molecular sieve.The mixture that forms was stirred 7 hours down at 70 ℃.Leach molecular sieve,, then the filtrate that forms is concentrated with the ethyl acetate washing.Resistates is distributed between methylene dichloride and water.With water dichloromethane extraction three times, organic phase is merged, use dried over sodium sulfate, filter and concentrate and obtain 5-[3-(2-methoxyl group-phenyl)-1H-pyrazolo [3,4-b] pyridine-5-yl]-nicotinic acid (654mg, 62% purity, 405mg, 119% yield), be brown solid. ¹H-NMR(500MHz，d ₆-DMSO)δ 13.98(s，1H)，8.99(d，1H)，8.95(s，1H)，8.88(d，1H)，8.43(t，1H)，8.40(d，1H)，7.67(dd，1H)，7.47(ddd，1H)，7.23(d，1H)，7.10(dt，1H)，3.86(s，3H)。MS：m/z 345(96％)[MH ⁺]。

Step 3:{5-[3-(2-methoxyl group-phenyl)-1H-pyrazolo [3,4-b] pyridine-5-yl]-pyridin-3-yl }-tetramethyleneimine-1-base-ketone synthetic

With (5-[3-(2-methoxyl group-phenyl)-1H-pyrazolo [3,4-b] pyridine-5-yl]-nicotinic acid (350mg, 62% purity, 0.63mmol) under 50-60 ℃, be dissolved in dry DMF (20mL), then solution is cooled to room temperature, adds PS-HOBt resin (Argonaut Technologies) (0.9mmolg this moment ^-1Charge capacity, 2.20g, 1.98mmol), DMAP (32mg, 0.26mmol) and EDCI (375mg, 1.95mmol).Mixture was at room temperature vibrated 16 hours.Resin is leached, with DMF washing six times, subsequently with ether washing three times and dry.With resin and (5-[3-(2-methoxyl group-phenyl)-1H-pyrazolo [3,4-b] pyridine-5-yl]-nicotinate) (460mg, theoretical negative carrying capacity 105 μ mol) (110 μ l form suspension in dry DMF 1.3mmol) (3mL), vibrated then 22 hours containing tetramethyleneimine.Resin is leached, with methylene dichloride, ether and DMF washing.Merge filtrate and washings concentrated then.With the reversed-phase HPLC purifying of resistates that forms by gathering based on quality, obtain { 5-[3-(2-methoxyl group-phenyl)-1H-pyrazolo [3 with the acetonitrile solution gradient elution that contains 0.1% formic acid, 4-b] pyridine-5-yl]-pyridin-3-yl }-tetramethyleneimine-1-base-ketone (2.4mg, 6 μ mol, 6% yield), be the light brown solid. ¹H-NMR(500MHz，d ₆-MeOH)δ 8.98(d，1H)，8.87(d，1H)，8.74(d，IH)，8.50(d，1H)，8.31(t，1H)，7.67(dd，1H)，7.48(ddd，1H)，7.21(d，1H)，7.11(dt，1H)，3.89(s，3H)，3.65(t，2H)，3.58(t，2H)，2.02(m，2H)，1.96(m，2H)。MS：m/z 400[MH ⁺]。

Other compound that makes by method 7:

Table 6

^*With 3-[3-(2-methoxyl group-phenyl)-1H-pyrazolo [3,4-b] pyridine-5-yl]-phenylformic acid is synthetic as raw material, then by the flash chromatography on silica gel purifying, with the hexane solution gradient elution of the ethyl acetate that contains 20%v/v methyl alcohol

^*With 3-[3-(2-methoxyl group-phenyl)-1H-pyrazolo [3,4-b] pyridine-5-yl]-phenylformic acid is synthetic as raw material

Method 8:

Step 1:[4-(2-dimethylamino-ethyl)-piperazine-1-yl]-{ 3-[3-(2-methoxyl group-phenyl)-1H-pyrazolo [3,4-b] pyridine-5-yl]-phenyl }-ketone synthetic

With 3-[3-(2-methoxyl group-phenyl)-1H-pyrazolo [3,4-b] pyridine-5-yl]-phenylformic acid (338mg, 0.79mmol) and O-(7-azepine benzo triazol-1-yl)-N, N, N ', (300mg 0.79mmol) is dissolved in 20ml acetonitrile and 10ml methanol mixture to N '-tetramethyl-urea hexafluorophosphate.Adding 131mg (0.83mmol) 1-(2-dimethyl aminoethyl)-piperazine also at room temperature stirred this mixture 6 hours.The mixture that forms is distributed between methylene dichloride and 2M aqueous sodium carbonate.Separate each mutually and with water layer with dichloromethane extraction three times.Organic layer is merged, with saturated aqueous sodium bromide washing, with dried over sodium sulfate and evaporation.Crude product is passed through the flash chromatography on silica gel purifying, 4:4:1 solvent mixture (containing 2%v/v35%wt ammoniacal liquor) substep gradient elution with ethyl acetate and ethyl acetate, methylene dichloride and methyl alcohol obtains buttery [4-(2-dimethylamino-ethyl)-piperazine-1-yl]-{ 3-[3-(2-methoxyl group-phenyl)-1H-pyrazolo [3,4-b] pyridine-5-yl]-phenyl }-ketone (160mg, 42%). ¹H-NMR(d ₆-CDCl ₃)δ 8.87(d)[1H]，8.36(d)[1H]，7.75(dd)[1H]，7.68(t)[1H]，7.67(m)[1H]，7.53(m)[1H]，7.46(mt)[1H]，7.42(md)[1H]，7.13(dt)[1H]57.10(d)[1H]，3.89(s)[3H]，3.81-3.86(m)[2H]，3.48-3.55(m)[2H]，2.58-2.64(m)[2H]，2.56(m)[2H]，2.50(m)[2H]，2.44-2.52(m)[2H]。MS：m/z 485(M+H ⁺)。

Method 9:

Synthesizing of step 1:5-bromo-3-iodo-1-(2-methoxyl group-ethoxyl methyl)-1H-pyrazolo [3,4-b] pyridine

To 5-bromo-3-iodo-1H-pyrazolo [3,4-b] pyridine (470mg, 1.45mmol), the mineral oil dispersion (104mg of 60% sodium hydride, 4.35mmol) and tetrabutylammonium iodide (134mg, 0.36mmol) DMF (10mL) solution at room temperature add methoxy ethoxy methyl chloride (248 μ l, 2.18mmol) and mixture stirred 4 hours under identical temperature, subsequently by adding the methyl alcohol termination reaction.Then the mixture that forms is distributed between ether and salt solution,, filter and concentrate the organic layer dried over sodium sulfate.Crude product is passed through the flash chromatography on silica gel purifying, hexane solution gradient elution with ethyl acetate obtains 5-bromo-3-iodo-1-(2-methoxyl group-ethoxyl methyl)-1H-pyrazolo [3,4-b] pyridine (254mg, 0.69mmol, 74% yield), be colorless solid (with the 1:1 mixture of regional isomer 5-bromo-3-iodo-2-(2-methoxyl group-ethoxyl methyl)-1H-pyrazolo [3,4-b] pyridine). ¹H-NMR (500MHz, d ₆-DMSO) isomer A (50%) δ 8.75 (d, 1H), 8.29 (d, 1H), 5.78 (s, 1H), 3.61-3.63 (m, 2H), 3.37-3.39 (m, 2H), 3.17 (s, 3H); Isomer B (50%) δ 8.74 (d, 1H), 8.28 (d, 1H), 5.77 (s, 1H), 3.61-3.63 (m, 2H), 3.37-3.39 (m, 2H), 3.16 (s, 3H).

Synthesizing of step 2:5-bromo-1-(2-methoxyl group-ethoxyl methyl)-3-(2-methoxyl group-phenyl)-1H-pyrazolo [3,4-b] pyridine

With 5-bromo-3-iodo-1-(2-methoxyl group-ethoxyl methyl)-1H-pyrazolo [3,4-b] pyridine (180mg, 0.44mmol), 1,1-two (diphenylphosphine) ferrocene palladium chloride (II) methylene dichloride adducts (18mg, 25 μ mol) and 2-anisole ylboronic acid (82mg, 0.51mmol) mixture in acetonitrile (3mL) and 2M aqueous sodium carbonate (3ml) stirred 2 hours down in 60 ℃ in air-tight bottle.Crude mixture is distributed between ethyl acetate and salt solution.Water with ethyl acetate extraction (3x), with the organic phase dried over sodium sulfate that merges, is filtered and concentrates.Then crude product is passed through the flash chromatography on silica gel purifying, hexane solution gradient elution with ethyl acetate obtains 5-bromo-1-(2-methoxyl group-ethoxyl methyl)-3-(2-methoxyl group-phenyl)-1H-pyrazolo [3,4-b] pyridine (80mg, 0.2mmol, 46% yield), be yellow oil. ¹H-NMR(500MHz，d ₆-DMSO)δ 8.70(d，1H)，8.40(d，IH)，7.62(dd，1H)，7.49(ddd，1H)，7.22(d，1H)，7.09(dt，1H)，5.85(s，2H)，3.85(s，3H)，3.68-3.70(m，2H)，3.39-3.41(m，2H)，3.18(s，3H)。MS：m/z 316，318[MH ⁺]。

Step 3:3-[3-(2-methoxyl group-phenyl)-1H-pyrazolo [3,4-b] pyridine-5-yl]-benzoic synthetic

With 5-bromo-3-iodo-1/2-(2-methoxyl group-ethoxyl methyl)-1H-pyrazolo [3,4-b] pyridine (560mg, 1.36mmol, the mixture of regional isomer), 2-anisole ylboronic acid (217mg, 1.4mmol) and 1, the mixture of 1-two (diphenylphosphine) ferrocene palladium chloride (II) methylene dichloride adducts (50mg, 68 μ mol) in acetonitrile (4mL) and 2M aqueous sodium carbonate (2mL) stirred 105 minutes down in 70 ℃ in air-tight bottle.Then crude product is distributed between ethyl acetate and water.With water ethyl acetate extraction (3x), with the organic phase salt water washing that merges, use dried over sodium sulfate, filter, concentrate then and obtain 5-bromo-1-(2-methoxyl group-ethoxyl methyl)-3-(2-methoxyl group-phenyl)-1H-pyrazolo [3,4-b] pyridine (780mg, 75% purity, 1.12mmol, 82% yield), be the crude product dark oil. ¹H-NMR(500MHz，d ₆-DMSO)δ 8.70(d，1H)，8.40(d，1H)，7.62(dd，1H)，7.49(ddd，1H)，7.22(d，1H)，7.09(dt，1H)，5.85(s，2H)，3.85(s，3H)，3.68-3.70(m，2H)，3.39-3.41(m，2H)，3.18(s，3H)。MS：m/z 316，318[MH ⁺]。

With above crude product oil (1.12mmol), 3-carboxyl phenyl boric acid (259mg (1.56mmol) and 1, the mixture of 1-two (diphenylphosphine) ferrocene palladium chloride (II) methylene dichloride adducts (54mg, 75 μ mol) in acetonitrile (5mL) and 2M aqueous sodium carbonate (5mL) shone 20 minutes down in 165 ℃ in Personal ChemistryOptimizer.With the crude product dilution in acetonitrile, isolate organic phase, concentrate then.Resistates is dissolved in the aqueous solution of potassium hydroxide, and (10%w/v 15mL), with ethyl acetate washing (3x), passes through diatomite filtration then.By adding concentrated hydrochloric acid aqueous solution filtrate is acidified to pH3-4 and collecting precipitation thing then.Then methylene dichloride is joined in the throw out, leach insolubles, then filtrate concentrating obtained 3-[1-(2-methoxyl group-ethoxyl methyl)-3-(2-methoxyl group-phenyl)-1H-pyrazolo [3, the 4-b] pyridine-5-yl of dark solid shape]-phenylformic acid (369mg, 0.85mmol, 57% yield). ¹H-NMR(500MHz，d ₆-DMSO)δ 8.95(d，1H)，8.42(d，1H)，8.26(t，1H)，8.02(dt，1H)，7.98(dt，1H)，7.68(dd，1H)，7.64(t，1H)，7.51(ddd，1H)，7.25(d，1H)，7.12(dt，1H)，5.91(s，2H)，3.87(s，3H)，3.73-3.75(m，2H)，3.43-3.45(m，2H)，3.21(s，3H)。MS：m/z 432[M-H ^-]。

The solid that forms is dissolved in methylene dichloride and PS-thiophenol (Argonaut Technologies) (1.4mmolg ^-1, 1.2g, 1.7mmol) and add trifluoroacetic acid (6ml).The mixture that forms was leniently stirred under 50 ℃ 8.5 hours.Resin is leached and wash with methylene dichloride and ether.The filtrate that merges is concentrated and between saturated sodium bicarbonate solution and methylene dichloride, distribute.With water washed with dichloromethane 3 times, be acidified to pH3-4 by adding concentrated hydrochloric acid then.With water ethyl acetate extraction three times that form.The ethyl acetate that merges is used the salt water washing mutually, use dried over sodium sulfate, filter and concentrate 3-[3-(2-methoxyl group-phenyl)-1H-pyrazolo [3 that obtains the yellow solid shape, 4-b] pyridine-5-yl]-phenylformic acid (117mg, 0.34mmol, 40% yield, three steps are 25%). ¹H-NMR(500MHz，d ₆-DMSO)δ 8.87(d，1H)，8.37(d，1H)，8.24(t，1H)，8.02(dt，1H)，7.98(dt，1H)，7.68(dd，1H)，7.64(t，1H)，7.47(ddd，1H)，7.23(d，1H)，7.11(dt，1H)，3.86(s，3H)。MS：m/z 346[MH ⁺]。

Step 4:{3-[3-(2-methoxyl group-phenyl)-1H-pyrazolo [3,4-b] pyridine-5-yl]-phenyl }-(4-methyl-piperazine-1-yl)-ketone synthetic

To 3-[3-(2-methoxyl group-phenyl)-1H-pyrazolo [3,4-b] pyridine-5-yl]-phenylformic acid (25mg, 72 μ mol) add PS-DCC resin (ArgonautTechnologies) (180mg in dry DMF (1.5mL) solution, 0.22mmol, 1.21 rnmol-g-1) and N methyl piperazine (9.6 μ l, 86 μ mol).The mixture that forms was stirred 16 hours down at 60 ℃.Resin is leached, with methylene dichloride and ether washing and filtrate is concentrated.Resistates is dissolved in methylene dichloride and uses PS-three polyimide resins (Argonaut Technologies) (20mg) to handle.With resin once more by removing by filter and with the washing of methylene dichloride and ether.Filtrate is concentrated, the crude product that forms is also obtained { 3-[3-(2-methoxyl group-phenyl)-1H-pyrazolo [3 with the acetonitrile solution gradient elution that contains 0.1% formic acid by the reversed-phase HPLC purifying based on the quality collection, 4-b] pyridine-5-yl]-phenyl }-(4-methyl-piperazine-1-yl)-ketone (1.7mg, 4.0 μ mol, 6% yield). ¹H-NMR(500MHz，d ₆-DMSO)δ 8.87(d，1H)，8.37(d，1H)，8.24(t，1H)，8.02(dt，1H)，7.98(dt，1H)，7.68(dd，1H)，7.64(t，1H)，7.47(ddd，1H)，7.23(d，1H)，7.11(dt，1H)，3.86(s，3H)。MS：m/z 346[MH ⁺]。

Other compound that makes by method 9:

Table 7

Method 10:

Step 1:3-[1-(2-methoxyl group-ethoxyl methyl)-3-(2-methoxyl group-phenyl)-1H-pyrazolo [3,4-b] pyridine-5-yl]-methyl benzoate synthetic

With 5-bromo-1-(2-methoxyl group-ethoxyl methyl)-3-(2-methoxyl group-phenyl)-1H-pyrazolo [3,4-b] pyridine (535mg, 1.36mmol), 3-methoxycarbonyl phenyl-boron dihydroxide (258mg, 1.43mmol) and 1, the mixture of 1-two (diphenylphosphine) ferrocene palladium chloride (II) methylene dichloride adducts (50mg, 68 μ mol) in acetonitrile (7mL) and 2M aqueous sodium carbonate (7mL) shone 10 minutes down in 90 ℃ in Personal ChemistryOptimizer.The mixture that forms is distributed between ethyl acetate and water.Water with ethyl acetate extraction twice, with the organic phase salt water washing that merges, is used dried over sodium sulfate, filter and concentrate.Crude product is passed through the flash chromatography on silica gel purifying, obtain 3-[1-(2-methoxyl group-ethoxyl methyl)-3-(2-methoxyl group-phenyl)-1H-pyrazolo [3,4-b] pyridine-5-yl with ethyl acetate and hexane gradient wash-out]-methyl benzoate (612mg, 1.36mmol, 100% yield), be yellow oil. ¹H-NMR(500MHz，d ₆-DMSO)δ 8.96(d，1H)，8.43(d，1H)，8.28(t，1H)，8.07(td，1H)，7.99(td，1H)，7.67-7.69(m，2H)，7.51(ddd，1H)，7.25(d，1H)，7.12(dt，1H)，5.92(s，2H)，3.90(s，3H)，3.87(s，3H)，3.73-3.75(m，2H)，3.43-3.45(m，2H)，3.21(s，3H)。MS：m/z 372[MH ⁺]。

Step 2:3-[3-(2-methoxyl group-phenyl)-1H-pyrazolo [3,4-b] pyridine-5-yl]-methyl benzoate synthetic

With 3-[1-(2-methoxyl group-ethoxyl methyl)-3-(2-methoxyl group-phenyl)-1H-pyrazolo [3,4-b] pyridine-5-yl]-methyl benzoate (573mg, 1.28mmol) methylene dichloride (25mL) solution be cooled to 0-5 ℃ and add the boron trifluoride etherate (0.8ml, 6.4mmol).This mixture slowly is warming up to room temperature and stirred 16 hours.The yellow mercury oxide that forms leached obtains 3-[3-(2-methoxyl group-phenyl)-1H-pyrazolo [3,4-b] pyridine-5-yl]-methyl benzoate (110mg (0.29mmol; 23% yield). ¹H-NMR(500MHz，d ₆-DMSO)δ 8.88(d，1H)，8.39(d，1H)，8.27(t，1H)，8.06(td，1H)，7.99(td，1H)，7.65-7.68(m，2H)，7.48(ddd，1H)，7.23(d，1H)，7.11(dt，1H)，3.90(s，3H)，3.87(s，3H)。MS：m/z 360[MH ⁺]。

Step 3:{3-[3-(2-methoxyl group-phenyl)-1H-pyrazolo [3,4-b] pyridine-5-yl]-phenyl }-tetramethyleneimine-1-base-ketone synthetic

With 3-[3-(2-methoxyl group-phenyl)-1H-pyrazolo [3,4-b] pyridine-5-yl]-methyl benzoate (30mg, 83 μ mol) is dissolved in tetramethyleneimine, and (0.35ml 4.15mmol) and with this mixture stirred 16 hours down at 90 ℃.Then the mixture that forms is concentrated, crude product is passed through the flash chromatography on silica gel purifying, obtain { the 3-[3-(2-methoxyl group-phenyl)-1H-pyrazolo [3 of yellow solid shape with the ethyl acetate solution gradient elution of 10%v/v methyl alcohol, 4-b] pyridine-5-yl]-phenyl }-tetramethyleneimine-1-base-ketone (22mg, 55 μ mol, 67% yield). ¹H-NMR(500MHz，d ₆-MeOH)δ 8.82(d，1H)，8.39(d，1H)，7.83(t，IH)，7.79(td，1H)，7.66(dd，1H)，7.55-7.59(m，2H)，7.48(ddd，1H)，7.20(d，1H)，7.11(dt，1H)，3.88(s，3H)，3.63(t，2H)，3.52(t，2H)，2.02(m，2H)，1.92(m，2H)。MS：m/z 399[MH ⁺]。

Method 11:

Step 1:{2-hydroxyl-5-[3-(2-methoxyl group-phenyl)-1H-pyrazolo [3,4-b] pyridine-5-yl]-pyridin-3-yl }-morpholine-4-base-ketone synthetic

With 122mg (0.25mmol) 3-(2-methoxyl group-phenyl)-5-(4,4,5,5-tetramethyl--[1,3,2] two oxa-boron heterocycle pentane-2-yls)-and 1-(2-TMS-ethoxyl methyl)-1H-pyrazolo [3,4-b] pyridine, 150mg (0.52mmol) (5-bromo-2-fluoro-pyridin-3-yl)-morpholine-4-base-ketone and 15mg (18 μ mol) 1,1-two (diphenylphosphine) ferrocene palladium chloride (II) methylene dichloride adducts places the Smith bottle.Add 2mL acetonitrile, 1ml water and 1ml saturated sodium bicarbonate aqueous solution and the mixture that forms is descended irradiation 30 minutes in 100 ℃ in Personal Chemistry Optimizer.The resistates that forms is distributed between the saturated aqueous solution of methylene dichloride and sodium bicarbonate.Separate each phase, with twice of dichloromethane extraction of water.Organic phase is merged, use dried over sodium sulfate, filter and concentrate.Crude product by the flash chromatography on silica gel purifying, is obtained the cream-coloured oil of 137mg with the ethyl acetate gradient elution that contains 15% v/v methyl alcohol and hexane.

The 1:1 mixture that described oil is dissolved in 24mL glycol dimethyl ether and concentrated hydrochloric acid aqueous solution also heats this mixture 1 hour down at 55 ℃, neutralizes by adding sodium bicarbonate then.The mixture that forms is distributed between ethyl acetate and water,,, evaporate then with dried over sodium sulfate with the organic phase salt water washing that merges with water ethyl acetate extraction three times.Crude product is carried out purifying and uses 12.3mg (the 28 μ mol that contain the acetonitrile solution gradient elution of 0.1% formic acid, obtain the rice white solid state through freeze-drying by the reversed-phase HPLC based on the quality collection, 11% yield) 2-hydroxyl-5-[3-(2-methoxyl group-phenyl)-1H-pyrazolo [3,4-b] pyridine-5-yl]-pyridin-3-yl }-morpholine-4-base-ketone. ¹H-NMR(500MHz，d ₆-DMSO)δ 13.79(s，1H)，12.25(s，1H)，8.77(d，1H)，8.26(d，1H)，8.00(d，1H)，7.94(d，1H)，7.62(dd，1H)，7.46(ddd，1H)，7.22(d，1H)，7.09(ddd，1H)，3.83(s，3H)，3.7-3.2(m，8H)。MS：m/z 432[MH ⁺]。

Method 12:

Step 1:(2-amino-5-bromo-phenyl)-morpholine-4-base-ketone synthetic

In the screw-cap bottle of 8mL, add the 5-bromoisatin acid anhydrides (0.200g, 0.826mmol), anhydrous THF (5mL) and morpholine (101mg, 1.16mmol).With the sealing of this bottle and place heating component in 60 ℃ of heating 1.5 hours down, vacuum concentration then.With crude product Et ₂The development of O/ hexane obtains 111mg (94%) (2-amino-5-bromo-phenyl)-morpholine-4-base-ketone of tawny solid state.m/z285/287[MH ⁺]。

Step 2:{2-amino-5-[3-(2-methoxyl group-phenyl)-1H-pyrazolo [3,4-b] pyridine-5-yl]-phenyl }-morpholine-4-base-ketone synthetic

In the Personal of 5mL Chemistry microwave reaction bottle, add 3-(2-methoxyl group-phenyl)-5-(4,4,5,5-tetramethyl--[1,3,2] two oxa-boron heterocycle pentane-2-yls)-1-(2-TMS-ethoxyl methyl)-1H-pyrrolo-[2,3-b] pyridine (0.0498g, 0.103mmol), (2-amino-5-bromo-phenyl)-morpholine-4-base-ketone (0.0378g, 0.132mmol), 1,1 '-two (diphenylphosphine) ferrocene palladium chloride (II) methylene dichloride adducts (13.4mg, 0.017mmol), acetonitrile (1mL) and saturated NaHCO ₃The aqueous solution (1mL).With this bottle sealing, use N ₂Purify, in Personal ChemistryOptimizer, shone 5 minutes down then in 90 ℃.Separate each layer, water is extracted 3 times with EtOAc.With the organic phase brine treatment that merges, dry (Na ₂SO ₄), filter and concentrate.Crude product is dissolved in 5mL by 1 part of HClO ₄(70%, ACS) and the solution formed of 20 parts of glacial acetic acids, then this solution was at room temperature stirred 8 hours.With the reaction mixture vacuum concentration, use saturated NaHCO ₃With solid NaHCO ₃Be neutralized to pH7.The reaction mixture of termination reaction is distributed between EtOAc and water, separate each layer, water is extracted 2 times with EtOAc.With the organic phase brine treatment that merges, dry (Na ₂SO ₄), filter and concentrate.By LC (the positive sexual norm of gathering based on quality, ESI) through C-18 reversed-phase column (Thomson Instrument Co.ODS-A100A, 5 μ, 50 x 21.3mm) purifying, obtain title compound with the flow velocity of 20mL/min with the 5-95% gradient elution of acetonitrile (containing 0.1% formic acid) and water (containing 0.1% formic acid), obtain brown thick oil (12.9mg, 29%) after its freeze-drying. ¹H-NMR(500MHz，d ₆-DMSO)δ 13.71(br.s，1H)，8.74(d，J＝2.0Hz，1H)，8.16(d，J＝2.0Hz，1H)，7.62(d，J＝7.5Hz，1H)，7.49(dd，J＝2.5，8.0Hz，1H)，7.45(m，1H)，7.37(d，J＝2.0Hz，1H)，7.20(d，J＝8.0Hz，1H)，7.08(t，J＝8.5Hz，1H)，6.81(d，J＝8.0Hz，1H)，5.37(s，2H)，3.83(s，3H)，3.60(m，4H)，3.49(m，4H)；MS：m/z 430.1[MH ⁺]。

Other embodiment that is prepared by method 12:

Table 8

Method 13:

Step 1:2-[3-(2-methoxyl group-phenyl)-1H-pyrazolo [3,4-b] pyridine-5-yl]-thiazole-5-formic acid synthetic

In the Personal of 20mL Chemistry microwave reaction bottle, add 3-(2-methoxyl group-phenyl)-5-(4,4,5,5-tetramethyl--[1,3,2] two oxa-boron heterocycle pentane-2-yls)-1-(2-TMS-ethoxyl methyl)-1H-pyrrolo-[2,3-b] pyridine (0.4992g, 1.038mmol), 2-bromo-thiazole-5-methyl-formiate (0.2592g, 1.167mmol), 1,1 '-two (diphenylphosphine) ferrocene palladium chloride (II) methylene dichloride adducts (95.4mg, 0.117mmol), acetonitrile (5mL) and 2M Na ₂CO ₃The aqueous solution (5mL).With this bottle sealing, use N ₂Purify, in Personal Chemistry Optimizer, shone 30 minutes down then in 130 ℃.With reaction mixture with EtOAc dilution and be acidified with acetic acid to pH5.Separate each layer, water is extracted 5 times with EtOAc.With the organic phase brine treatment that merges, dry (Na ₂SO ₄), filter and concentrate.Crude product is dissolved in 10mL by 1 part of HClO ₄(70%, ACS) and the solution formed of 20 parts of glacial acetic acids, then this solution was at room temperature stirred 4 hours.With the reaction mixture vacuum concentration, use saturated NaHCO ₃With solid NaHCO ₃Be neutralized to pH7.The reaction mixture of termination reaction is distributed between EtOAc and water, and each layer separates, and water is extracted 2 times with EtOAc.Then water is acidified with acetic acid to pH4 and with EtOAc extraction 5 times.With the organic extract liquid brine treatment that merges, dry (Na ₂SO ₄), filter and concentrate.Use Et ₂The O development obtains green-brown ceramic powder shape title compound (0.296g, 81% yield). ¹H-NMR(500MHz，d ₆-DMSO)δ 13.73(br.s，1H)，9.16(s，1H)，8.71(s，1H)，8.40(s，1H)，7.68(d，J＝8.0Hz，1H)，7.48(t，J＝8.0Hz，2H)，7.24(d，J＝8.0Hz，1H)，7.10(t，J＝7.5Hz，1H)，3.87(s，3H)；MS：m/z 353.1[MH ⁺]。

Other embodiment that is prepared by method 13:

Table 9

Method 14:

Step 1:3-bromo-N, N-dimethyl-benzsulfamide synthetic

In the scintillation vial of 20mL, add the 3-bromobenzene sulfonyl chloride (0.301g, 1.179mmol) and anhydrous pyridine (5mL).Drip the 2M dimethyl amine THF solution (1.0mL, 2.0mmol), then with reaction mixture at room temperature and N ₂Under stirred vacuum concentration then 5 hours.Thick resistates is distributed between EtOAc and 1M citric acid.Each layer separates, and organic phase with 1M citric acid washing 3 times, is used brine treatment then, dry (Na ₂SO ₄), filter and concentrate.Use Et ₂The O development obtains the 3-bromo-N of white powder, N-dimethyl-benzsulfamide (0.297g, 96%).MS：m/z 263.9/265.9[MH ⁺]。

Step 2:3-[3-(2-methoxyl group-phenyl)-1H-pyrazolo [3,4-b] pyridine-5-yl]-N, N-dimethyl-benzsulfamide synthetic

In the Personal of 5mL Chemistry microwave reaction bottle, add 3-(2-methoxyl group-phenyl)-5-(4,4,5,5-tetramethyl--[1,3,2] two oxa-boron heterocycle pentane-2-yls)-1-(2-TMS-ethoxyl methyl)-1H-pyrrolo-[2,3-b] pyridine (0.0496g, 0.103mmol), 3-bromo-N, N-dimethyl-benzsulfamide (0.0417g, 0.143mmol), 1,1 '-two (diphenylphosphine) ferrocene palladium chloride (II) methylene dichloride adducts (13.9mg, 0.017mmol), acetonitrile (1mL) and saturated NaHCO ₃The aqueous solution (1mL).With this bottle sealing, use N ₂Purify, in Personal Chemistry Optimizer, shone 15 minutes down then in 90 ℃.Each layer separates, and water is extracted 3 times with EtOAc.With the organic phase brine treatment that merges, dry (Na ₂SO ₄), filter and concentrate.With crude product be dissolved in 5mL by 1 part of HClO4 (70%, ACS) and the solution formed of 20 parts of glacial acetic acids, then this solution was at room temperature stirred 1 hour.With the reaction mixture vacuum concentration, use saturated NaHCO ₃With solid NaHCO ₃Be neutralized to pH7.The reaction mixture of termination reaction is distributed between EtOAc and water, and each layer separates, and water is extracted 2 times with EtOAc.With the organic phase brine treatment that merges, dry (Na ₂SO ₄), filter and concentrate.By LC (the positive sexual norm of gathering based on quality, ESI) through C-18 reversed-phase column (Thomson Instrument Co.ODS-A100A, 5 μ, 50 x 21.3mm) purifying, obtain title compound with the flow velocity of 20mL/min with the 5-95% gradient elution of acetonitrile (containing 0.1% formic acid) and water (containing 0.1% formic acid), obtain buff powder (10.4mg, 25%) after its freeze-drying. ¹H-NMR(500MHz，d ₆-DMSO)δ＝13.91(br.s，1H)，8.92(d，J＝2.0Hz，1H)，8.44(d，J＝2.0Hz，1H)，8.12(m，1H)，8.01(br.s，1H)，7.76(m，2H)，7.67(dd，J＝2.0，7.5Hz，1H)，7.45(m，1H)，7.22(d，J＝8.0Hz，1H)，7.09(t，J＝8.0Hz，1H)，3.85(s，3H)，2.66(s，6H)；MS：m/z 409.1[MH ⁺]。

Other compound that makes by method 14:

Table 10

Method 15:

Step 1:3-[3-(2-methoxyl group-phenyl)-1H-pyrazolo [3,4-b] pyridine-5-yl]-benzoic synthetic

(997mg is 2.30mmol) at acetonitrile (15mL) and saturated NaHCO for 5-bromo-3-(2-methoxyl group-phenyl) in the microwave reaction bottle-1-(2-TMS-ethoxyl methyl)-1H-pyrazolo [3,4-b] pyridine ₃Add in the solution in the aqueous solution (10mL) 3-carboxyl phenyl boric acid, pinacol ester (625mg, 2.52mmol) and [1,1 '-two (diphenylphosphine) ferrocene] palladium chloride (II) methylene dichloride complex compound (1:1) (94mg, 0.12mmol).With this bottle capping, use N ₂Flushing, decompression is found time, and heats for 1500 seconds down in 90 ℃ in microwave then.Remove acetonitrile by rotary evaporation.Add ethyl acetate, from water layer, separate then.Organic layer is dark-brown, and LC/MS shows that product is retained in this layer.Be concentrated into dark-brown oil, be dissolved in the acetic acid solution (10mL) of 5% perchloric acid then again.Reaction soln was at room temperature stirred 4.5 hours.Remove perchloric acid by rotary evaporation, add ethyl acetate and H then ₂O.Add sodium bicarbonate powder to pH=3.Isolate organic layer, use Na ₂SO ₄Drying, vacuum concentration obtains brown ceramic powder (580mg, 62% yield) then. ¹H NMR(500MHz，d ₆-OMSO)δ 13.81(br s，1H)，8.81(d，J＝2.5Hz，1H)，8.31(d，J＝2.5Hz，1H)，8.18(s，1H)，7.95(d，J＝7.5Hz，1H)，7.90(d，J＝7.5Hz，1H)，7.59(m，2H)，7.41(t，J＝7.0Hz，1H)，7.17(d，J＝8.0Hz，1H)，7.04(t，J＝7.5Hz，1H)，3.80(s，3H)。MS：m/e 346.1(M+H ⁺)。

Step 2:{3-[3-(2-methoxyl group-phenyl)-1H-pyrazolo [3,4-b] pyridine-5-yl]-phenyl }-(4-pyrimidine-2-base-piperazine-1-yl)-ketone synthetic

To 3-[3-(2-methoxyl group-phenyl)-1H-pyrazolo [3,4-b] pyridine-5-yl]-phenylformic acid (18mg, add in DMF 0.05mmol) (1mL) solution HATU (20mg, 0.05mmol) and 1-(2-pyrimidyl) piperazine (11 μ L, 0.08mmol).Reaction soln was at room temperature stirred 16 hours.With crude product with ethyl acetate extraction and use H ₂The O washing.With organic layer Na ₂SO ₄Drying is filtered, and is adsorbed onto on the silica gel then.By purification by flash chromatography, obtain white powder title compound (7.2mg, 28% yield) with ethyl acetate (containing 10% MeOH) and hexane gradient wash-out. ¹H NMR(500MHz，CD ₃OD)δ 8.85(d，J＝2Hz，1H)，8.42(d，J＝2Hz，1H)，8.35(d，J＝4.5Hz，2H)，7.83(d，J＝8Hz，1H)，7.79(s，1H)，7.64(m，2H)，7.50(m，2H)，7.20(d，J＝8.5Hz，1H)，7.11(t，J＝8Hz，1H)，6.64(t，J＝4.5Hz，1H)，3.96(br s，2H)，3.89(s，3H)，3.86(br s，4H)，3.60(br s，2H)。MS：m/z 492.1(M+H ⁺)。

Other compound that makes by method 15:

Table 11

Method 16:

Synthesizing of step 1:5-bromo-3-(2-methoxyl group-phenyl)-1H-pyrazolo [3,4-b] pyridine

To 5-bromo-3-(2-methoxyl group-phenyl)-1-(2-TMS-ethoxyl methyl)-1H-pyrazolo [3,4-b] pyridine (260mg, 0.60mmol) THF (3mL) solution in add tetrabutyl ammonium fluoride (the THF solution of 6mL1M, 6.00mmol) and molecular sieve.This vlil (70 ℃) 4 are little, do not stir.Solution is cooled to room temperature and is acidified to pH=5 by the MeOH dilute solution that drips acetate.Solution is filtered, filtrate is concentrated by rotary evaporation.This material is extracted and uses H with EtOAc ₂O washing 3 times.With organic layer Na ₂SO ₄Drying concentrates 5-bromo-3-(2-methoxyl group-phenyl)-1H-pyrazolo [3, the 4-b] pyridine (177mg, 97% yield) that obtains the orange solids shape then.MS：m/z 303.9，305.9[M+H ⁺]。This material can be directly used in step 2 without being further purified.

Step 2:4-[3-(2-methoxyl group-phenyl)-1H-pyrazolo [3,4-b] pyridine-5-yl]-N-methyl-benzamide synthetic

5-bromo-3-(2-methoxyl group-phenyl) in the microwave reaction bottle-1H-pyrazolo [3,4-b]] (26mg is 0.085mmol) at acetonitrile (1mL) and saturated NaHCO for pyridine ₃Add in the solution in the aqueous solution (1mL) 4-(N-methylamino carbonyl) phenyl-boron dihydroxide (17mg, 0.094mmol) and [1,1-two (diphenylphosphine) ferrocene] palladium chloride (II) methylene dichloride complex compound (1:1) (3.5mg, 0.004mmol).With this bottle capping, use N ₂Flushing, vacuum-evacuate heated for 1800 seconds down in 130 ℃ in microwave then.With this material with EtOAc extraction and with organic layer Na ₂SO ₄Dry.This material is adsorbed onto SiO ₂Go up and by purification by flash chromatography, with EtOAc (containing 10% MeOH) and hexane gradient wash-out.Clarifying fraction concentrated by rotary evaporation obtain white powder title compound (5.9mg, 20% yield). ¹H NMR(500MHz，CD ₃OD)δ 8.85(s，1H)，8.41(s，1H)，7.94(d，J＝8.5Hz，2H)，7.78(d，J＝8.5Hz，2H)，7.67(d，J＝7.5Hz，1H)，7.48(t，7.0Hz，1H)，7.21(d，J＝8.5Hz，1H)，7.11(t，J＝7.0Hz，1H)，3.89(s，3H)，2.95(s，3H)。MS：m/z 359.1[M+H ⁺]。

Other compound that makes by method 16:

Table 12

Method 17:

Synthesizing of step 1:3-(2-methoxyl group-phenyl)-5-(3-tetramethyleneimine-1-ylmethyl-phenyl)-1H-pyrazolo [3,4-b] pyridine

To 3-[3-(2-methoxyl group-phenyl)-1-(2-TMS-ethoxyl methyl)-1H-pyrazolo [3,4-b] pyridine-5-yl]-phenyl aldehyde (23mg, 0.050mmol) and tetramethyleneimine (5 μ l, adding 3 μ l AcOH in 1.5ml dichloroethane solution 0.082mmol).Mixture was at room temperature stirred 30 minutes, then in this mixture disposable adding three oxygen base ethanoyl sodium borohydrides (22mg, 0.10mmol).At room temperature continue reaction 2 hours, the mixture that forms is concentrated obtain the SEM product then, this product was at room temperature handled 1 hour with acetate (2mL) solution of 5% perchlorate.Evaporating solvent neutralizes resistates with sodium bicarbonate powder, then by the flash chromatography on silica gel purifying, with ethyl acetate and EtOAc/DCM/MeOH/NH ₄The mixture wash-out of OH (4/4/1/0.05) obtains 3-(2-methoxyl group-phenyl)-5-(3-tetramethyleneimine-1-ylmethyl-phenyl)-1H-pyrazolo [3, the 4-b] pyridine (8.50mg, 44% yield) of white solid. ¹H NMR(500MHz，CD ₃OD)δ 1.89(d，J＝3Hz，4H)，2.76(d，J＝1.5Hz，4H)，3.89(s，2H)，3.9(s，2H)，7.11(t，J＝I Hz，1H)，7.22(d，J＝8Hz，1H)，7.42(t，J＝7Hz，1H)，7.47(d，J＝1.5Hz，1H)，7.48(d，J＝2Hz，1H)，7.51(d，J＝2.5Hz，1H)，7.65(t，J＝6.75Hz，1H)，7.72(s，1H)，8.39(d，J＝2.5Hz，1H)，8.83(d，J＝2Hz，1H)。MS：m/z 385[MH ⁺]。

Other compound that makes by method 17:

Table 13

Method 18:

Synthesizing of step 1:5-bromo-3-TMS ethynyl-pyrazine-2-base amine

To 3,5-two bromo-pyrazine-2-base amine (3.00g adds triethylamine (16ml) in DMF 11.86mmol) (35ml) solution, disposable then adding tetra-triphenylphosphine palladium (0) (685mg, 0.59mmol) and cupric iodide (I) (271mg, 1.42mmol).Drip at last trimethyl silyl acetylene (2.0ml, 14.3mmol).Reaction mixture was stirred 30 minutes down at 120 ℃, directly be adsorbed onto on the silica gel then.By the flash chromatography on silica gel purifying, obtain yellow oily title compound (2.30g, 71% yield) with the ethyl acetate/hexane gradient elution.MS：m/z 270.0/272.0[MH ⁺]。

Synthesizing of step 2:N-(5-bromo-3-TMS ethynyl-pyrazine-2-yl)-ethanamide

To 5-bromo-3-TMS ethynyl-pyrazine-2-base amine (2.30g, anhydrous THF (35ml) 8mmol) and pyridine (1.62ml, 20.0mmol) add in the solution Acetyl Chloride 98Min. (682 μ l, 9.6mmol).Mixture at room temperature stirred spend the night, stirred 5 hours down at 60 ℃ then.Solvent removed in vacuo, with the brown resistates that forms by the silica gel chromatography purifying, obtain with the ethyl acetate/hexane gradient elution light yellow-rice white solid state title compound (474mg, 1.52mmol).MS：m/z311.9/313.9[MH ⁺]。

Synthesizing of step 3:2-bromo-5H-pyrrolo-[2,3-b] pyrazine

To N-(5-bromo-3-TMS ethynyl-pyrazine-2-yl)-ethanamide (474mg, drip in THF 1.52mmol) (4ml) solution 1M tetra-n-butyl Neutral ammonium fluoride THF solution (3.3ml, 3.3mmol).After the stirring and refluxing 15 hours, with the reaction mixture vacuum concentration and add entry.With water layer dichloromethane extraction three times, the extraction liquid that merges directly is adsorbed onto on the silica gel.By the silica gel chromatography purifying, obtain yellow solid shape title compound (130mg, 43% yield) with the ethyl acetate/hexane gradient elution. ¹H-NMR(500MHz，d ₆-DMSO)δ 12.38(s br，1H)，8.38(s，1H)，7.95(d，3.5Hz，1H)，6.61(d，3.5Hz，1H)。MS：m/z 197.9/199.9[MH ⁺]。

Synthesizing of step 4:2-bromo-7-iodo-5H-pyrrolo-[2,3-b] pyrazine

To 2-bromo-5H-pyrrolo-[2,3-b] pyrazine (258mg, disposable adding N-iodine succinimide in acetone 1.3mmol) (5ml) solution (324mg, 1.44mmol).Reaction mixture was at room temperature stirred 45 minutes.The throw out that forms is leached, and with minimum washing with acetone, vacuum-drying obtains light brown solid state title compound then. ¹H-NMR(500MHz，d ₆-DMSO)δ 12.81(sbr，1H)，8.40(s，1H)，8.19(d，3.0Hz，1H)。MS：m/z 323.8/325.8[MH ⁺]。

Synthesizing of step 5:2-bromo-7-iodo-5-(toluene-4-alkylsulfonyl)-5H-pyrrolo-[2,3-b] pyrazine

To 2-bromo-7-iodo-5H-pyrrolo-[2,3-b] pyrazine (290mg, in THF 0.895mmol) (5ml) suspension in 0 ℃ of NaH of property adding next time (60%, 43mg, 1.08mmol).The mixture that forms was stirred 20 minutes, add Tosyl chloride (188mg, THF 0.98mmol) (2mL) solution then.Then reaction mixture was at room temperature stirred 3 hours.Remove and desolvate, with the dark-brown resistates KOH aqueous solution, the water washing that form, drying obtains light brown solid state title compound (423mg, 99% yield) then. ¹H-NMR(500MHz，d ₆-DMSO)δ 8.60(d，11.5Hz，1H)，7.99(d，11.5Hz，2H)，7.44(d，7.5Hz，2H)，2.34(s，3H)。MS：m/z477.8/479.8[MH ⁺]。

Synthesizing of step 6:2-bromo-7-(2-methoxyl group-phenyl)-5-(toluene-4-alkylsulfonyl)-5H-pyrrolo-[2,3-b] pyrazine

In the 50-ml round-bottomed flask, add 2-bromo-7-iodo-5-(toluene-4-alkylsulfonyl)-5H-pyrrolo-[2; 3-b] pyrazine (423mg; 0.885mmol), 2-anisole ylboronic acid (148mg (0.973mmol) and dichloro two (triphenylphosphine) palladium (II) (31mg, 0.04mmol).In this mixture, add acetonitrile (10mL) and 2M sodium bicarbonate aqueous solution (5mL).Reaction mixture was stirred 1 hour down at 40 ℃, stirred 1 hour down at 55 ℃ then.Crude product mixture is distributed between ethyl acetate and saturated sodium bicarbonate aqueous solution.With the water ethyl acetate extraction,, filter and concentrate then the organic phase dried over sodium sulfate that merges.Crude product by the flash chromatography on silica gel purifying, is obtained 2-bromo-7-(2-methoxyl group-phenyl)-5-(toluene-4-alkylsulfonyl)-5H-pyrrolo-[2, the 3-b] pyrazine (139mg, 34% yield) of light yellow solid shape with the hexane solution gradient elution of ethyl acetate; Also obtain two adducts 2,7-two-(2-methoxyl group-phenyl)-5-(toluene-4-alkylsulfonyl)-5H-pyrrolo-[2,3-b] pyrazine (213mg, 50% yield).MS:m/z 457.9/460.0[MH ⁺]; MS:m/z486.1[MH ⁺] (two adducts).Step 7:3-[7-(2-methoxyl group-phenyl)-5H-pyrrolo-[2,3-b] pyrazine-2-yl]-benzoic synthetic

With 2-bromo-7-(2-methoxyl group-phenyl)-5-(toluene-4-alkylsulfonyl)-5H-pyrrolo-[2; 3-b] pyrazine (70mg; 0.15mmol), 3-(4,4,5; 5-tetramethyl--[1; 3,2] two oxa-boron heterocycle pentane-2-yls)-(57mg is 0.23mmol) with dichloro two (triphenylphosphine) palladium (II) (5.4mg for phenylformic acid; 0.008mmol) acetonitrile (2mL) and aqueous sodium carbonate (2M, 2mL) mixture in Personal ChemistryOptimizer in 95 ℃ of irradiations 20 minutes down.Crude product mixture is distributed between methylene dichloride and saturated sodium bicarbonate aqueous solution.With the water dichloromethane extraction,, filter and concentrate then the organic phase dried over sodium sulfate that merges.Then thick brown resistates is dissolved in MeOH (2mL) and adds 5N KOH (150 μ L).Then the mixture that forms was stirred 2 hours down at 40 ℃, remove then and desolvate.With rare HCl (2ml), water washing, drying obtains crude acid 3-[7-(2-methoxyl group-phenyl)-5H-pyrrolo-[2,3-b] pyrazine-2-yl then with the yellow residue that forms]-phenylformic acid, it can be directly used in step 8.MS：m/z 346.0[MH ⁺]。

Step 8:[4-(2-dimethylamino-ethyl)-piperazine-1-yl]-{ 3-[7-(2-methoxyl group-phenyl)-5H-pyrrolo-[2,3-b] pyrazine-2-yl]-phenyl }-ketone synthetic

To 3-[7-(2-methoxyl group-phenyl)-5H-pyrrolo-[2,3-b] pyrazine-2-yl]-phenylformic acid (35mg, 0.1mmol), EDCI (77mg, 0.40mmol), HBTU (3.8mg, 0.01mmol) and diisopropyl ethyl amine (175 μ l, 1.0mmol) DMF (1mL) mixture in add 2-(dimethylamino) ethyl piperazidine (55 μ l, 0.3mmol).Mixture was stirred 5 hours down at 70 ℃, remove then and desolvate.The yellow oil that forms is washed with water, be dissolved in DMSO, obtain yellow solid shape title compound (8.6mg, 12%, through 2 steps) with the reversed-phase HPLC purifying then.MS：m/z 485.2[MH ⁺]。

Other compound that makes by method 18:

Table 14

Method 19:

Step 1:3-[7-(2-methoxyl group-phenyl)-5H-pyrrolo-[2,3-b] pyrazine-2-yl]-N, N-dimethyl-benzamide synthetic

With 2-bromo-7-(2-methoxyl group-phenyl)-5-(toluene-4-alkylsulfonyl)-5H-pyrrolo-[2; 3-b] pyrazine (30mg; 0.065mmol), 3-dimethylamino carbonyl phenyl boric acid (25.3mg; 0.130mmol) and dichloro two (triphenylphosphine) palladium (II) (2.5mg; 0.003mmol) acetonitrile (1mL) and sodium bicarbonate aqueous solution (2M, 1mL) mixture in Personal Chemistry Optimizer in 90 ℃ of irradiations 15 minutes down.Crude product mixture is distributed between methylene dichloride and saturated sodium bicarbonate aqueous solution.With the water dichloromethane extraction,, filter and concentrate then the organic phase dried over sodium sulfate that merges.Then the brown resistates of crude product is dissolved in MeOH (5mL), 5N KOH (50 μ L) and mixture was at room temperature stirred 75 minutes.Removing desolvates obtains yellow residue, then it is passed through the flash chromatography on silica gel purifying, utilize the EtOAc solution gradient wash-out of the hexane solution of ethyl acetate and 10%MeOH to obtain 3-[7-(2-methoxyl group-phenyl)-5H-pyrrolo-[2 of light yellow solid shape, 3-b] pyrazine-2-yl]-N, N-dimethyl-benzamide (13.0mg, 54% yield). ¹H-NMR(500MHz，CD ₃OD)δ 8.89(m，1H)，8.80(m，1H)，8.40(m，1H)，8.25(m，2H)，7.62(m，1H)，7.50(m，1H)，7.25(m，1H)，7.10(m，2H)，3.97(m，3H)，3.17(m，3H)，3.10(m，3H)。MS：m/z 373.1[MH ⁺]。

Other compound that makes by method 19:

Table 15

Method 20:

Step 1:2,7-two-(2-methoxyl group-phenyl)-5H-pyrrolo-[2,3-b] pyrazine synthetic

To 2, (200mg adds NaOH (66mg, water 1.65mmol) (200 μ L) solution to 7-two-(2-methoxyl group-phenyl)-5-(toluene-4-alkylsulfonyl)-5H-pyrrolo-[2,3-b] pyrazine in MeOH 0.412mmol) (5mL) solution.Mixture was at room temperature stirred 3.5 hours, remove then and desolvate.Then the yellow residue that forms is passed through the flash chromatography on silica gel purifying, utilize the hexane solution gradient elution of ethyl acetate to obtain 2 of light yellow solid shape, 7-two-(2-methoxyl group-phenyl)-5H-pyrrolo-[2,3-b] pyrazine (43mg, 32% yield). ¹H-NMR(500MHz，CD ₃OD)δ 12.21(s br，1H)，8.80(dd，6.0Hz，1.75Hz，1H)，8.69(s，1H)，7.76(dd，6.0Hz，1.75Hz，1H)，7.43(dt，7.5Hz，1.75Hz，1H)，7.20(m，2H)，7.13(m，2H)，7.04(t，7.3Hz，1H)，3.92(s，3H)，3.85(s，3H)。MS：m/z 332.1[MH ⁺]。

Method 21:

Synthesizing of step 1:3-amino-6-iodo-pyrazine-2-methyl-formiate

With 3-amino-2-pyrazine carboxylic acid methyl esters (10g, 65.3mmol) and N-iodine succinimide (24g 106.7mmol) is dissolved in dry DMF (150mL) and mixture stirred 15 hours down at 70 ℃ under nitrogen atmosphere.Then the mixture that forms is cooled to room temperature and adds saturated aqueous sodium thiosulfate (400mL).With suspension supersound process 15 minutes, vacuum concentration was dispersed in the water then.Crude product is leached and uses cold washing with alcohol.With the resistates alcohol crystal, obtain orange acicular 3-amino-6-iodo-pyrazine-2-methyl-formiate (11.2g, 61% yield) with the charcoal decolouring. ¹H-NMR(d ₆-DMSO)δ 8.57[1H]s，7.59[2H]s，br，3.93[3H]s。MS：m/z 280[MH ⁺]。

Synthesizing of step 2:3-amino-6-iodo-pyrazine-2-formic acid

(45g 161mmol) is dissolved in 750ml THF with 3-amino-6-iodo-pyrazine-2-methyl-formiate.Add 90ml water and 40ml 4M lithium hydroxide aqueous solution.Mixture was at room temperature stirred 2 hours, or the baseline material is only arranged to the TLC analysis revealed.Add 10% aqueous citric acid solution to regulate pH to about 3-4.Mixture is diluted with methylene dichloride, isolate organic phase.With water layer dichloromethane extraction three times, the organic phase that merges is also evaporated with dried over sodium sulfate.Resistates vacuum-drying is obtained the 37.0g (140mmol of yellow powder shape; 87% yield) 3-amino-6-iodo-pyrazine-2-formic acid. ¹H-NMR (d ₆-DMSO) δ 11.70s, a little less than, 8.44[1H] s, 7.50[2H] s, br.MS：m/z 266[MH ⁺]。

Synthesizing of step 3:3-amino-6-iodo-pyrazine-2-formic acid methoxyl group-methyl-acid amides

With 27.50g (0.104mol) 3-amino-6-iodo-pyrazine-2-formic acid, 63.0g (0.125mol) PyBOP (1-benzotriazole base oxygen base-three (tetramethyleneimine-1-base) Phosphonium hexafluorophosphate) and 18.70g (0.193mol) N, O-dimethyl hydroxyl amine hydrochlorate places the flask of nitrogen flushing, be dissolved in 100ml dry DMF and 27ml N then, the mixture of N-two-sec.-propyl ethylamine.Mixture was heated 16 hours down at 80 ℃.Obtain dark oil 50-60 ℃ of following solvent evaporated under reduced pressure.Should extract 3-4 time with 300ml toluene by oil.Toluene is merged mutually, then evaporation.The oil that forms is passed through the silica gel chromatography purifying, obtain the 18.40g (59.72mmol of yellow solid shape with the hexane solution gradient elution of ethyl acetate; 58%) 3-amino-6-iodo-pyrazine-2-formic acid methoxyl group-methyl-acid amides. ¹H-NMR(d ₆-DMSO)δ8.28[1H]s，6.78[2H]s，3.66[3H]s，3.25[3H]s。MS：m/z 309[MH ⁺]。

Step 4:(3-amino-6-iodo-pyrazine-2-yl)-phenyl-ketone synthetic

8.00g (25.97mmol) 3-amino-6-iodo-pyrazine-2-formic acid methoxyl group-methyl-acid amides is dissolved in the anhydrous THF of 100ml under nitrogen.Solution is cooled to 55 ℃, adds the diethyl ether solution of 27ml 3M phenyl-magnesium-bromide then.With mixture heating up to 10 ℃ and add 10% lemon aqueous acid.Mixture is diluted with methylene dichloride, separate each phase.With water dichloromethane extraction three times, the organic phase that merges is evaporated then with dried over sodium sulfate.The solid that forms is obtained yellow-orange crystalloid 5.74g (17.66mmol with alcohol crystal; 68%) (3-amino-6-iodo-pyrazine-2-yl)-phenyl-ketone. ¹H-NMR(d ₆-DMSO)δ：8.52[1H]s，7.94[2H]s，br，7.85[2H]d，7.62[1H]t，7.52[2H]t。MS：m/z 326[MH ⁺]。

Synthesizing of step 5:5-iodo-3-(2-methoxyl group-1-phenyl-vinyl)-pyrazine-2-base amine

2.52g (12.6mmol) two (trimethyl silyl) ammonification potassium is dissolved in the anhydrous THF of 150ml under nitrogen.Add 3.80g (11.1mmol) methoxymethyl triphenyl phosphonium chloride and mixture was at room temperature stirred 1 hour.In formed mixture, add 2.50g (7.69mmol) (3-amino-6-iodo-pyrazine-2-yl)-phenyl-ketone and reaction mixture was at room temperature stirred 1 hour.The mixture heating up that forms was refluxed 20 hours.After the cooling, mixture with the methylene dichloride dilution and with the saturated aqueous ammonium chloride washing, is used dried over sodium sulfate then.Evaporating solvent by the silica gel chromatography purifying, obtains the 1.942g (5.50mmol of light yellow solid shape with resistates with the hexane solution gradient elution of ethyl acetate; 72%) E-of part fractionation and Z-5-iodo-3-(2-methoxyl group-1-phenyl-vinyl)-pyrazine-2-base amine. ¹H-NMR (d ₆-DMSO) δ: isomer A8.12[1H] s, 7.33-7.26[4H] m, 7.20[1H] m, 6.66[1H] s, 6.00[2H] s, br, 3.81[3H] s; Isomer B8.09[1H] s, 7.27[2H] t, 7.17[1H] t, 7.11[2H] d, 6.98[1H] s, 6.24[2H] s, br, 3.76[3H] s.MS：m/z354[MH ⁺]。

Synthesizing of step 6:5-iodo-3-phenyl-1H-pyrrolo-[2,3-b] pyrazine

780mg5-iodo-3-(2-methoxyl group-1-phenyl-vinyl)-pyrazine-2-base amine (E-, Z-type or mixture) is formed suspension in the aqueous solution (about 1-2N) of 40ml dilute hydrochloric acid and alcoholic acid 1:1 mixture.Mixture heating up was refluxed 2 hours.Ice is joined in the formed suspension, then throw out is leached 480mg 5-iodo-3-phenyl-1H-pyrrolo-[2, the 3-b] pyrazine that obtains the yellow powder powder.Make filtrate be alkalescence by adding sodium bicarbonate, use dichloromethane extraction then 3 times.The extraction liquid that merges is also evaporated with dried over sodium sulfate.Resistates is obtained the green-acicular 76mg 5-of brown crystallization iodo-3-phenyl-1H-pyrrolo-[2,3-b] pyrazine with alcohol crystal, and the merging yield is 556mg (78%). ¹H-NMR(d ₆-DMSO)δ：12.56[1H]s，br，8.53[1H]s，8.46[1H]d，8.14[2H]d，7.45[2H]dd，7.25[1H]dd。MS：m/z 322[MH ⁺]。

Synthesizing of step 7:5-(3,4-dimethoxy-phenyl)-3-phenyl-1H-pyrrolo-[2,3-b] pyrazine

With 50mg (0.16mmol) 5-iodo-3-phenyl-1H-pyrrolo-[2,3-b] pyrazine, 38mg (0.20mmol) 3,4-dimethoxy benzene ylboronic acid and 6mg (5mol%) dichloride two (triphenylphosphine) palladium (II) places bottle, add 1ml acetonitrile and 1ml 2M aqueous sodium carbonate then and with mixture at Personal

Shone for 1200 seconds down in 165 ℃ in the microwave reactor.The mixture that forms is distributed between 15ml saturated sodium bicarbonate aqueous solution and 75ml methylene dichloride.Organic phase is also evaporated with dried over sodium sulfate.Crude product by the flash chromatography on silica gel purifying, is used the dichloromethane solution gradient elution of methyl alcohol.The product that separation is obtained obtains 14mg (43 μ mol, 27% yield) 5-(3,4-dimethoxy-phenyl)-3-phenyl-1H-pyrrolo-[2,3-b] pyrazine of orange powder shape with the hot ethanol crystallization. ¹H-NMR(d ₆-DMSO)δ 12.30[1H]s，8.92[1H]s，8.43[1H]s，8.35[2H]d，7.80[1H](m)，7.79[1H]d(m)，7.46[2H]dd，7.25[1H]dd(d)，7.13[1H]d，3.92[3H]s，3.84[3H]s。MS：m/z 332[MH ⁺]。

Other compound that makes by method 21:

Table 16

Method 22

Synthesizing of step 1:5-(morpholine-4-yl)-3-phenyl-1H-pyrrolo-[2,3-b] pyrazine

25mg (80 μ mol) 5-iodo-3-phenyl-1H-pyrrolo-[2,3-b] pyrazine is dissolved in the 1ml morpholine.Add 200 μ l glacial acetic acids and with mixture at Personal Chemistry Heat 2400-4800 second down at 250 ℃ in the microwave reactor.Crude product is not carried out aftertreatment in advance by the flash chromatography on silica gel purifying, utilize the hexane solution gradient elution of ethyl acetate to obtain 13mg (46 μ mol, 58% yield) 2-morpholine-4-base-7-phenyl-1H-pyrrolo-[2,3-b] pyrazine of beige solid shape. ¹H-NMR(d ₆-DMSO)δ 11.89[1H]s，8.19[1H]s，8.18[2H]d，7.39[2H]dd，7.17[1H]dd，3.80[4H]t，3.52[4H]t。MS，m/z：281[MH ⁺]。

Table 17

Method 23:

Synthesizing of step 1:4-(5-iodo-1H-pyrrolo-[2,3-b] pyrazine-3-yl)-phenol

With 680mg 5-iodo-3-{2-methoxyl group-1-[4-(tetrahydrochysene-pyrans-2-base oxygen base)-phenyl]-vinyl }-pyrazine-2-base amine forms suspension in 70ml rare (1-2N) aqueous hydrochloric acid.Add methyl alcohol and obtain settled solution with dissolving raw material (10-20% v/v).Add the 0.5ml concentrated hydrochloric acid and mixture heating up was refluxed 7 hours.With mixture with being cooled to room temperature and stirring 16 hours.Mixture is neutralized by adding sodium bicarbonate, add entry then when needed to keep salt in solution.With mixture dichloromethane extraction four times that form, with the organic phase dried over sodium sulfate that merges, evaporation obtains enough pure (85%) 428mg (1.27mmol of orange solids shape then, 85% yield) 4-(5-iodo-1H-pyrrolo-[2,3-b] pyrazine-3-yl)-phenol, it is obtained the purified 4-of yellow-orange crystalloid 195mg (578 μ mol, 39% yield) (5-iodo-1H-pyrrolo-[2,3-b] pyrazine-3-yl)-phenol with methylene dichloride-re-crystallizing in ethyl acetate. ¹H-NMR(d ₆-DMSO)δ：12.37[1H](d)，9.43[1H]s，8.49[1H]s，8.26[1H]d，7.92[2H]d，8.85[2H]d。MS：m/z 338[MH ⁺]。

Step 2:4-{5-[3-methoxyl group-4-hydroxy phenyl]-1H-pyrrolo-[2,3-b] pyrazine-3-yl }-phenol synthetic

With 84mg (0.25mmol) 4-(5-iodo-1H-pyrrolo-[2,3-b] pyrazine-3-yl)-phenol, 120mg (0.33mmol) 2-[3-methoxyl group-4-(4-methoxyl group-benzyloxy)-phenyl]-4,4,5,5-tetramethyl--[1,3,2] two oxa-boron heterocycle pentanes and 15mg (8mol%) dichloride two (triphenylphosphine) palladium (II) place bottle, add 1.5ml acetonitrile and 1.5ml 2M aqueous sodium carbonate then.With mixture at Personal

Shone for 1200 seconds down at 165 ℃ in the microwave reactor.The mixture that forms is distributed between methylene dichloride and saturated sodium bicarbonate aqueous solution.Water layer with dichloromethane extraction twice, is evaporated the organic phase that merges with dried over sodium sulfate then.Crude product by the flash chromatography on silica gel purifying, is used the hexane solution gradient elution of ethyl acetate.The intermediate that forms is dissolved in the 120ml methylene dichloride and adds 1.5g (2.12mmol) PS-thiophenol (Argonaut Technologies).To wherein adding the 2ml trifluoroacetic acid and mixture at room temperature being stirred 1 hour.Resin is leached and uses washed with dichloromethane.Filtrate is washed with saturated sodium bicarbonate aqueous solution.Separate each phase, water layer with ethyl acetate extraction twice, is merged all organic phases, evaporate then with dried over sodium sulfate.Resistates is heated with acetonitrile, be cooled to room temperature and remove supernatant.Resistates vacuum-drying is obtained 15mg (45 μ mol, 18% yield) 4-{5-[3-methoxyl group-4 hydroxy phenyl of cream-coloured powder shape]-1H-pyrrolo-[2,3-b] pyrazine-3-yl }-phenol. ¹H-NMR(d ₆-DMSO)δ：12.06[1H]d，9.35[1H]s，9.30[1H]s，8.83[1H]s，8.20[1H]d，8:12[2H]d(m)，7.75[1H]d，7.65[1H]dd，6.93[1H]d，6.85[2H]d(m)，3.91[3H]s.MS，m/z：334[MH ⁺]。

Method 24

Synthesizing of step 1:3-(3-(2-p-methoxy-phenyl)-1H-pyrazolo [3,4-b] pyridine-5-yl) imino-methyl benzoate

Under 0 ℃, HCl gas is passed through 3-[3-(2-methoxyl group-phenyl)-1-(2-TMS-ethoxyl methyl)-1H-pyrazolo [3,4-b] pyridine-5-yl]-benzonitrile (40mg, the anhydrous MeOH suspension of 2.5ml 0.088mmol) bubbling 3 minutes.After at room temperature stirring 23 hours, add ether (10mL), precipitation occurs.With solid collection, drying obtains 3-(3-(2-p-methoxy-phenyl)-1H-pyrazolo [3,4-b] pyridine-5-yl) the imino-methyl benzoate of yellow solid shape then after the filtration.

Step 2:C-{3-[3-(2-methoxyl group-phenyl)-1H-pyrazolo [3,4-b] pyridine-5-yl]-phenyl }-C-morpholine-4-base-benzylidene amino synthetic

3-(the 3-(2-p-methoxy-phenyl)-1H-pyrazolo [3 that obtains to above step, 4-b] pyridine-5-yl) add morpholine (15.3mg in MeOH (1.0mL) solution of imino-methyl benzoate, 0.176mmol) and triethylamine (90mg 0.88mmol), at room temperature stirred mixture 3 days.Remove then and desolvate, crude product is obtained C-{3-[3-(2-methoxyl group-phenyl)-1H-pyrazolo [3 of white solid by the reversed-phase HPLC purifying, 4-b] pyridine-5-yl]-phenyl }-C-morpholine-4-base-benzylidene amino (3.7mg, 10% yield is through two steps). ¹H-NMR(500MHz，CD ₃OD)δ 8.86(d，2Hz，1H)，8.45(d，2Hz，1H)，8.37(br s，1H)，8.02(m，1H)，7.96(t，1.8Hz，1H)，7.76(t，7.8Hz，1H)，7.66(dd，1.8Hz，7.8Hz，1H)，7.63(m，1H)，7.49(m，1H)，7.21(d，8Hz，1H)，7.12(dt，1Hz，7.8Hz，1H)，3.95(m，2H)，3.88(s，3H)，3.82(m，2H)3.78(m，2H)，3.59(m，2H)。MS：m/z 414.1[MH ⁺]。

Other compound that makes by method 24:

Table 18

Biological test

Available kinase assay well known by persons skilled in the art is tested the inhibition activity of The compounds of this invention and composition.Kinase assay includes but not limited to following examples.

Although in first embodiment, adopted the kinase domain (＂ Abl T315I KD ＂) of the Abl T315I of mutant form, but kinase assay can be utilized the enzyme of various forms of mutant and wild-type, comprises for example complete albumen, kinase domain or its part (for example Abl Y393F).Kinases used in the test can also be in various phosphorylation states.In the c-Abl example, use the mutant kinases that is in zero phosphorylation state.

C-Abl pyruvate kinase/serum lactic dehydrogenase link coupled enzyme test

In the test of c-Abl pyruvate kinase (PK)/serum lactic dehydrogenase (LDH) coupling, the protein kinase dependent form phosphorylation of peptide substrate is associated with the oxidation of NADH.Reduction by monitoring 340nm place absorbancy detects the oxidation from NADH to NAD+.

Material: Abl peptide substrate=EAIYAAPFAKKK-OH (Biopeptide, San Diego, CA); β NADH (Sigma catalog number (Cat.No.) N-8129, FW=709.4); 2M MgCl ₂1M HEPES damping fluid, pH7.5; Phosphoenolpyruvic acid (PEP) (Sigma catalog number (Cat.No.) P-7002, FW=234); Serum lactic dehydrogenase (LDH) (Worthington Biochemical catalog number (Cat.No.) 2756); Pyruvate kinase (PK) (Sigma catalog number (Cat.No.) P-9136); ATP (Sigma catalog number (Cat.No.) A-3377, FW=551); Greiner 384-hole UV star plate; With purifying and unphosphorylated T315I Abl kinase domain.

Storing solution: every day the freshly prepd 10mM NADH (miliQH of 7.09mg/ml ₂O solution); Be kept at the 10mM Abl peptide substrate (miliQH of 13.4mg/ml under-20 ℃ ₂O solution); 100mM HEPES damping fluid, pH7.5 (5ml1M storing solution+45ml miliQH ₂O); 100mMMgCl ₂(5ml 2M MgCl ₂+ 95ml dH ₂O); Be kept at the 100mM PEP (dH of 23.4mg/ml under-20 ℃ ₂O solution); Be kept at the 10mM ATP (dH of 5.51mg/ml under-20 ℃ ₂O solution) (be diluted to altogether 10ml miliQH with 50 μ l every day ₂Among the O=the 50 μ M ATP storage liquid of working); Under liquid nitrogen quick freezing and be kept under-80 ℃ 1000U/ml PK (U/mg, different between the different batches) and under liquid nitrogen quick freezing and be kept at 1000U/mlLDH (U/mg, different between the different batches) under-80 ℃.

The standard test setting of 384 hole patterns (50 μ l reaction solution): 300 μ M NADH; 10mMMgCl ₂2mM PEP; 45U/ml PK; 60U/ml LDH; 200 μ M Abl peptide substrates; 2.5 μ l testing compound (DMSO solution); 2 μ g/ml Abl kinase domains; 10 μ M ATP; The 100mMHEPES damping fluid.Positive control contains DMSO and does not have testing compound.Negative control contains 5 μ l 0.5M EDTA (being in test 50mM).The form of the dephosphorylation of c-Abl T315I mutant is used for the biological chemistry shaker test.When time t=0, begin kinase whose reaction by adding ATP.

Activity is measured according to the time-dependent manner loss of NADH by the absorption spectrum at the 340nm place.The linear portion of the progress curve that will obtain is then analyzed to obtain the activity with absorbance unit/time representation by linear regression, (mole/unit time can be with NADH at the molar extinction coefficient at 340nm place, 6250M to be reported as the collinear slope of best-fit ^-1Cm ^-1Calculate).

Data are estimated with following equation: Z '=1-[3* (σ ₊+ σ _-)/μ ₊-μ _-] (people such as Zhang, 1999 J Biomol Screening, 4 (2) 67-73), wherein μ represents that mean value and σ represent standard deviation.Subscript is represented the positive or negative contrast.For rough sizing test, the score value of Z ' should 〉=0.50.Threshold value=μ commonly used ₊-3* σ ₊Anyly all be designated as ＂ hit ＂ in this value below threshold value.

Dose response utilizes following equation to analyze: y=min+{ (max-min)/(1+10 ^{[chemical combination} ^{Thing]-logIC50}), wherein y is viewed initial slope, max=is at the slope that does not exist under the inhibitor, the slope of min=under unlimited inhibitor, IC ₅₀It is [compound] that be equivalent to 1/2 observed net amplitude (amplitude=max-min).

For adjusting, activation or the restraining effect of measuring Abl KD, the concentration of testing compound with certain limit is joined in this test.Inhibitor suppresses the active IC of Abl KD ₅₀Can be in micro-molar range, in nmole scope or for example inferior nmole (subnanomolar) scope.

Other kinase assay

Except c-Abl PK/LDH coupling test (above-mentioned), c-Abl, MET, AurA and PDK1 kinases etc. have also been developed based on luminous similar shaker test.These all tests all utilize ATP consumption trial (Kinase-Glo ^TM, Promega Corporation, Madison WI) comes quantitative kinase whose activity.Kinase-Glo ^TMPattern uses heat-staple luciferase to produce luminous signal with the ATP from be retained in solution after kinase reaction.This luminous signal becomes negative correlation with the amount of kinase activity.

CAbl based on luminous enzyme test

Material: Abl peptide substrate=EAIYAAPFAKKK-OH (Biopeptide, San Diego, CA), ATP (Sigma catalog number (Cat.No.) A-3377, FW=551), HEPES damping fluid, pH7.5, bovine serum albumin (BSA) (Roche 92423420), MgCl ₂, Staurosporine (streptomyces, Sigma catalog number (Cat.No.) 85660-1MG), white Costar 384 hole flat undersides (VWR catalog number (Cat.No.) 29444-088), Abl kinases (referring to following), Kinase-Glo ^TM(Promega catalog number (Cat.No.) V6712).

Storing solution: be kept at the 10mM Abl peptide substrate (miliQH of 13.4mg/ml under-20 ℃ ₂O solution); 100mM HEPES damping fluid, pH7.5 (5ml1M storing solution+45ml miliQH ₂O); Be kept at the 10mM ATP (dH of 5.51mg/ml under-20 ℃ ₂O solution) (be diluted to altogether 10ml miliQH with 50 μ l every day ₂Among the O=the 50 μ M ATP storage liquid of working); 1% BSA (100ml 0.1M HEPES solution, the pH7.5 of 1g BSA, be kept under-20 ℃), 100mM MgCl ₂200 μ M Staurosporines, 2X Kinase-Glo ^TMReagent (freshly prepd or be kept under-20 ℃).

The standard test setting of 384 hole patterns (20 μ l kinase reaction liquid, 40 μ l detection reaction liquid): 10mM MgCl ₂100 μ M Abl peptide substrates; 0.1% BSA; 1 μ l testing compound (DMSO solution); 0.4 μ g/ml Abl kinase domain; 10 μ M ATP; 100mM HEPES damping fluid.Positive control contains DMSO and does not have testing compound.Negative control contains 10 μ M Staurosporines.When time t=0, begin kinase whose reaction by adding ATP.Kinase whose being reflected at carried out under 21 ℃ 30 minutes, then with 20 μ l Kinase-Glo ^TMReagent joins in each hole to stop kinase whose reaction and to cause luminous reaction.Insulation is after 20 minutes down at 21 ℃, and detection is luminous on board-like reading photometer.

MET based on luminous enzyme test

Material: Poly Glu-Tyr (4:1) substrate (Sigma catalog number (Cat.No.) P-0275), ATP (Sigma catalog number (Cat.No.) A-3377, FW=551), the HEPES damping fluid, pH7.5, bovine serum albumin (BSA) (Roche92423420), MgCl ₂, Staurosporine (streptomyces, Sigma catalog number (Cat.No.) 85660-1MG), white Costar 384 hole flat undersides (VWR catalog number (Cat.No.) 29444-088), MET kinases (referring to following), Kinase-Glo ^TM(Promega catalog number (Cat.No.) V6712).

Storing solution: the aqueous solution that is kept at the 10mg/ml poly Glu-Tyr under-20 ℃; The 100mMHEPES damping fluid, pH7.5 (5ml 1M storing solution+45ml miliQH ₂O); Be kept at the 10mM ATP (dH of 5.51mg/ml under-20 ℃ ₂O solution) (be diluted to altogether 10mlmiliQH with 50 μ l every day ₂Among the O=the 50 μ M ATP storage liquid of working); 1% BSA (the 100ml 0.1MHEPES solution of 1g BSA, pH7.5 is kept under-20 ℃), 100mM MgCl ₂200 μ M Staurosporines, 2X Kinase-Glo ^TMReagent (freshly prepd or be kept under-20 ℃).

The standard test setting of 384 hole patterns (20 μ l kinase reaction liquid, 40 μ l detection reaction liquid): 10mM MgCl ₂0.3mg/ml poly Glu-Tyr; 0.1% BSA; 1 μ l testing compound (DMSO solution); 0.4 μ g/ml MET kinases; 10 μ M ATP; 100mM HEPES damping fluid.Positive control contains DMSO and does not have testing compound.Negative control contains 10 μ M Staurosporines.When time t=0, begin kinase whose reaction by adding ATP.Kinase whose being reflected at carried out under 21 ℃ 60 minutes, then with 20 μ l Kinase-Glo ^TMReagent joins in each hole to stop kinase whose reaction and to cause luminous reaction.Insulation is after 20 minutes down at 21 ℃, and detection is luminous on board-like reading photometer.

AurA based on luminous enzyme test

Material: Kemptide peptide substrates=LRRASLG (Biopeptide, San Diego, CA), ATP (Sigma catalog number (Cat.No.) A-3377, FW=551), the HEPES damping fluid, pH7.5,10% Brij35 (Calbiochem catalog number (Cat.No.) 203728), MgCl ₂, Staurosporine (streptomyces, Sigma catalog number (Cat.No.) 85660-lMG), white Costar 384 hole flat undersides (VWR catalog number (Cat.No.) 29444-088), the AurA kinases (referring to following) of autophosphorylation, Kinase-Glo ^TM(Promega catalog number (Cat.No.) V6712).

Storing solution: be kept at the 10mM Kemptide peptide (aqueous solution of 7.72mg/ml) under-20 ℃; 100mM HEPES damping fluid+0.015% Brij 35, pH 7.5 (5ml1M HEPES storing solution+75 μ L, 10% Brij 35+45ml miliQH ₂O); Be kept at the 10mM ATP (dH of 5.51mg/ml under-20 ℃ ₂O solution) (be diluted to altogether 10ml miliQH with 50 μ l every day ₂Among the O=the 50 μ M ATP storage liquid of working); 100mM MgCl ₂200 μ M Staurosporines, 2XKinase-Glo ^TMReagent (freshly prepd or be kept under-20 ℃).

The autophosphorylation reaction of AurA: with ATP and MgCl ₂Join among the 1-5mg/ml AurA with final concentration 10mM and 100mM respectively.Autophosphorylation is reflected at and carried out under 21 ℃ 2-3 hour.By adding the EDTA stopped reaction, final concentration is 50mM, with sample with the liquid nitrogen quick freezing and be kept under-80 ℃.

The standard test setting of 384 hole patterns (20 μ l kinase reaction liquid, 40 μ l detection reaction liquid): 10mM MgCl ₂0.2mM Kemptide peptide; 1 μ l testing compound (DMSO solution); 0.3 the AurA kinases of μ g/ml autophosphorylation; 10 μ M ATP; 100mM HEPES+0.015%Brij damping fluid.Positive control contains DMSO and does not have testing compound.Negative control contains 5 μ M Staurosporines.When time t=0, begin kinase whose reaction by adding ATP.Kinase whose being reflected at carried out under 21 ℃ 45 minutes, then with 20 μ l Kinase-Glo ^TMReagent joins in each hole to stop kinase whose reaction and to cause luminous reaction.Insulation is after 20 minutes down at 21 ℃, and detection is luminous on board-like reading photometer.

PDK1 based on luminous enzyme test

Material: PDKtide peptide substrates

=KTFCGTPEYLAPEVRREPRILSEEEQEMFRDFDYIADWC (Upstate catalog number (Cat.No.) 12-401), ATP (Sigma catalog number (Cat.No.) A-3377, FW=551), HEPES damping fluid, pH7.5,10% Brij 35 (Calbiochem catalog number (Cat.No.) 203728), MgCl ₂, Staurosporine (streptomyces, Sigma catalog number (Cat.No.) 85660-1MG), white Costar 384 hole flat undersides (VWR catalog number (Cat.No.) 29444-088), PDK1 kinases (referring to following), Kinase-Glo ^TM(Promega catalog number (Cat.No.) V6712).

Storing solution: be kept at the 1mM PDKtide substrate (the 200 μ l solution of 1mg are provided by Upstate) under-20 ℃; 100mM HEPES damping fluid, pH7.5 (5ml 1M HEPES storing solution+45ml miliQH ₂O); Be kept at the 10mM ATP (dH of 5.51mg/ml under-20 ℃ ₂O solution) (be diluted to altogether 10ml miliQH with 25 μ l every day ₂Among the O=the 25 μ M ATP storage liquid of working); 100mM MgCl ₂Be kept at 10% Brij 35 under 2-8 ℃; 200 μ M Staurosporines, 2XKinase-Glo ^TMReagent (freshly prepd or be kept under-20 ℃).

The standard test setting of 384 hole patterns (20 μ l kinase reaction liquid, 40 μ l detection reaction liquid): 10mM MgCl ₂0.01mM PDKtide; 1 μ l testing compound (DMSO solution); 0.1 μ g/mlPDK1 kinases; 5 μ M ATP; 10mM MgCl ₂100mM HEPES+0.01% Brij damping fluid.Positive control contains DMSO and does not have testing compound.Negative control contains 10 μ M Staurosporines.When time t=0, begin kinase whose reaction by adding ATP.Kinase whose being reflected at carried out under 21 ℃ 40 minutes, then with 20 μ l Kinase-Glo ^TMReagent joins in each hole to stop kinase whose reaction and to cause luminous reaction.Insulation is after 20 minutes down at 21 ℃, and detection is luminous on board-like reading photometer.

The preparation of the plasmid of coexpression

The plasmid of λ phosphoesterase coexpression makes up in accordance with the following methods.

The kinase whose opening code-reading frame of Aurora utilizes following primer to increase from the mankind's HepG2 cDNA library (ATCCHB-8065) by polymerase chain reaction (PCR):

Forward primer: TCAAAAAAGAGGCAGTGGGCTTTG

Reverse primer: CTGAATTTGCTGTGATCCAGG.

PCR product (expect 795 base pairs) is carried out gel-purified in accordance with the following methods.With the PCR product by electrophoresis with 1% sepharose purifying in the TAE damping fluid, the electrophoresis band of suitable size excised from gel and extract the test kit wash-out with standard gel.The DNA of wash-out is at room temperature handled 5 minutes to be connected in the pSB2-TOPO with topoisomerase.Carrier pSB2-TOPO is pET26b (Novagen, Madison, WI) topoisomerase-activatory, the modification of modification have wherein been inserted following sequence: CATAATGGGCCATCATCATCATCATCACGGTGGTCATATGTCCCTT and have been inserted following sequence: AAGGGGGATCC in the BamHI site in the NdeI site TAAACTGCAGAGATCC.As follows from the Shine-Dalgarno sequence through " original " NdeI site, the formed plasmid sequence of termination site and " original " BamHI site: AAGGAGGAGATATACATA ATGGGCCATCATCATCATCATCACGGTGGTCATATGTCCCTT[ORF] AAGGGGGATCC TAAACTGCAGAGATCC.Have 14 with the Aurora kinases of this vector expression and join the amino acid (MetGlyHisHisHisHisHisHisGlyGlyHisMetSerLeu) of N-end and the amino acid (GluGlyGlySer) that four join the C-end.

Then by will insert from the phosphatase gene of lambda particles phage above plasmid produce the phosphoesterase co-expression plasmid (people such as Matsui T, Biochem.Biophys.Res.Commun., 2001,284:798-807).Utilize following Oligonucleolide primers to increase with round pcr from template lambda bacteriophage dna (HinDIIIdigest, New England Biolabs) phosphatase gene:

Forward primer (PPfor): GCAGAGATCCGAATTCGAGCTCCGTCGACGGATGGAGTGAAAGAGATGCGC

Reverse primer (PPrev): GGTGGTGGTGCTCGAGTGCGGCCGCAAGCTTTCATCATGCGCCTTCTCCCTGTAC.

PCR product (expect 744 base pairs) is carried out gel-purified.Then the DNA of purifying and the plasmid DNA of non-coexpression are digested with SacI and XhoI restriction enzyme.Utilize standard method plasmid and the PCR product of digestion carried out gel-purified and under 16 ℃, to be connected 8 hours and to transfer in Top 10 cells then with the T4DNA ligase enzyme.The existence of phosphatase gene confirms by checking order in co-expression plasmid.Molecular biology method for the standard that this paper followed, also can be referring to for example described technology of following document: people such as Sambrook, Molecular Cloning:ALaboratory Manual, Cold Spring Harbor Laboratory, NY, 2001 and people such as Ausubel, Current Protocols in Molecular Biology, Greene PublishingAssociates and Wiley Interscience, NY, 1989.

The plasmid of this coexpression contains Aurora kinases and the λ phosphatase gene under the control of lac primer, and they all have the ribosome bind site of oneself.Be used for the suitable restriction site of phosphoesterase subclone in other plasmid by phosphoesterase being cloned into the middle part of multiple clone site, the downstream of target gene, can producing.These sites comprise the SalI between SacI, kinases and the phosphoesterase and HinDIII, NotI and the XhoI downstream of EcoRI and phosphoesterase.

Protein kinase is expressed

The opening code-reading frame of c-Abl increases by the following primer of round pcr utilization with the test kit (Invitrogen) that can buy from the mouse cDNA library that the mouse liver by fresh results prepares:

Forward primer: GACAAGTGGGAAATGGAGC

Reverse primer: CGCCTCGTTTCCCCAGCTC.

PCR product (expect 846 base pairs) is removed test kit (Qiagen) with PCR carry out purifying from the PCR reaction mixture.The DNA of purifying is at room temperature handled 5 minutes to be connected in the pSGX3-TOPO with topoisomerase.Carrier pSGX3-TOPO is pET26b (Novagen, Madison, Wisconsin) topoisomerase activatory, the modification of modification have wherein been inserted following sequence C ATATGTCCCTT and have been inserted following sequence A AGGGCATCATCACCATCACCACTGATCC in the BamHI site in the NdeI site.As follows from the Shine-Dalgarno sequence through the sequence of the formed plasmid of termination site and BamHI site: AAGGAGGAGATATACAT ATGTCCCTT[ORF] AAGGGCATCATCACCATCACCAC TGATCC.Utilize the c-Abl of this vector expression to have three amino acid (Met Ser Leu) that join its N-end and 8 amino acid (GluGlyHisHisHisHisHisHis) that join its C-end.

To in above-mentioned plasmid, produce c-Abl phosphoesterase co-expression plasmid from the phosphoesterase subclone of the Aurora co-expression plasmid of embodiment 1 then.Aurora co-expression plasmid and the non-co-expression plasmid of Abl were all digested 3 hours with limiting enzyme EcoRI and NotI.Dna fragmentation is carried out gel-purified, will under 16 ℃, be connected 8 hours with the c-Abl plasmid of digestion, transfer to then in Top 10 cells from the phosphatase gene of Aurora plasmid.Analyze the existence that confirms phosphatase gene in the formed construct by restriction digestion.

The coexpression of this plasmid-encoded c-Abl and λ phosphoesterase.Its additional advantage is restriction site XbaI and the NdeI with two uniquenesses, and they are positioned at and can be used for the upstream of other target protein subclone to the target group of this phosphoesterase co-expression plasmid.

The plasmid of Abl T315I makes by the Abl plasmid is modified according to the method for manufacturer suggestion and following oligonucleotide with Quick Change mutagenesis kit (Stratagene):

Mm05582dS4

5′-CCACCATTCTACATAATCATTGAGTTCATGACCTATGGG-3′

Mm05582dA4

5′-CCCATAGGTCATGAACTCAATGATTATGTAGAATGGTGG-3′。

The protein that will produce from the phosphoesterase co-expression plasmid is purifying in accordance with the following methods.The plasmid of non-coexpression has been transferred in chemically active BL21 (DE3) Codon+RIL (Stratagene) cell, and the plasmid of coexpression is transferred in BL21 (DE3) pSA0145 (express lambda particles phage the bacteriolyze gene and can be at freezing and dissolved bacterial strain (Crabtree S, Cronan JE Jr.JBacteriol 1984 Apr when melting; 158 (1): 354-6)), they are placed on the culture dish that comprises the LB agar that is added with kantlex then.Make isolated single colony growth to the mid-log phase, and under-80 ℃, be kept among the LB that contains 15% glycerine.This glycerine storing solution is being contained cut on the LB agar plate of kantlex, single bacterium colony is being used to inoculate the LB substratum that 10ml contains kantlex and paraxin, with its under 30 ℃ in the vibration under incubated overnight.This substratum is used to inoculate contains the 2L flask that 500ml contains the LB of kantlex and paraxin, make it grow to the mid-log phase under 37 ℃, the final concentration that adds IPTG to 0.5mM is then induced.After inducing, this flask is being incubated 18 hours under 21 ℃ under vibration.

With c-Abl T315I KD (kinase domain) purifying in accordance with the following methods.With cell by centrifugal collection, ultrasonic dissolution in rare decomposition damping fluid (500mM KCl, 0.1%Tween 20, the 20mM imidazoles for 50mM Tris HCl, pH7.5), centrifugation is to remove cell debris then.With IMAC post (Pharmacia, Uppsala, Sweden) purifying of dissolved fraction by being filled with nickel, under natural condition, use the imidazoles of 20mM-500mM at 50mM Tris solution, pH7.8,500mMNaCl, the solution gradient wash-out in 10mM methionine(Met), 10% glycerine.Then this protein is further purified by gel-filtration utilization equilibrated Superdex75 preparation scale post in GF5 damping fluid (10mM HEPES, pH7.5,10mM methionine(Met), 500mM NaCl, 5mM DTT and 10% glycerine).Collection contains the fraction of the c-Abl T315I KD kinase domain of purifying.Confirm that through sds polyacrylamide gel electrophoresis resulting protein has 98% purity.Proteinic mass spectroscopy behind the purifying shows that it mainly is single phosphorylation.(MBI Fermentas, Burlington Canada) carry out the dephosphorylation effect under the following conditions: 100U Shrimp alkaline phosphatase/mg c-Abl T315I KD, 100mM MgCl with the Shrimp alkaline phosphatase with this protein then ₂With the additional NaCl of 250mM.This is reflected under 23 ℃ and spends the night.This protein confirms it is non-phosphorylating through mass spectroscopy.All throw outs are separated, with soluble fraction and reactant by the gel-filtration utilization at GF4 damping fluid (10mM HEPES, pH7.5,10mM methionine(Met), 150mMNaCl, 5mM DTT and 10% glycerine) in equilibrated Superdex 75 preparation scale posts separate.

The purifying of Met:

To be suspended in again from the cell deposition thing that half produced of the 12L Sf9 insect cell nutrient solution of the kinase domain of expressing human Met and contain 50mM Tris-HCl pH7.7 and 250mM NaCl, volume are about the damping fluid of 40ml/1L initial incubation liquid.In every 1L initial incubation liquid, add a slice Roche Complete, do not contain the proteinase inhibitor mixture (catalog number (Cat.No.) 1873580) of EDTA.This suspension was stirred 1 hour down at 4 ℃.By under 4 ℃ with 39,800x g removed fragment in centrifugal 30 minutes.Supernatant liquor is inclined in the beaker of 500ml, add 10ml then at 50mMTris-HCl pH7.8,50mM NaCl, 10% glycerine, 50% slurries of the Qiagen Ni-NTA Agarose (catalog number (Cat.No.) 30250) of pre-balance in 10mM imidazoles and the 10mM methionine(Met), and under 4 ℃, stirred 30 minutes.Then this sample is poured under 4 ℃ in the instillation post (drip column) and with the 50mM Tris-HCl pH7.8 of 10 column volumes, 500mM NaCl, 10% glycerine, 10mM imidazoles and 10mM methionine(Met) wash.Protein utilization is the identical damping fluid substep gradient elution of two column volumes, and described damping fluid contains the imidazoles of 50mM, 200mM and 500mM successively.6x Histidine mark is spent the night with TEV proteolytic enzyme (Invitrogen catalog number (Cat.No.) 10127017)/1mg protein decomposition of 40 units, simultaneously at 50mM Tris-HCl pH7.8,500mMNaCl, 10% glycerine is dialysed down in 4 ℃ in 10mM imidazoles and the 10mM methionine(Met).With sample by Pharmacia 5ml IMAC post (catalog number (Cat.No.) 17-0409-01) to remove 6x Histidine mark, described column packed has nickel and at 50mM Tris-HCl pH7.8,500mM NaCl, 10% glycerine, balance in 10mM imidazoles and the 10mM methionine(Met).The protein that decomposes is attached on the nickel post with low affinity, carries out the substep gradient elution then.The substep gradient is 80% B-side (A-side=50mM Tris-HCl pH7.8,500mM NaCl, 10% glycerine, 10mM imidazoles and 10mM methionine(Met) with 15% then; B-side=50mM Tris-HCl pH7.8,500mM NaCl, 10% glycerine, 500mM imidazoles and 10mM methionine(Met)) carry out, be 4 column volumes.Met albumen is wash-out in the first step (15%), and the Histidine of undecomposed Met and decomposition is marked at wash-out in 80% fraction.Behind the Met that SDS-PAGE gel analysis alleged occurrence decomposes, 15% fraction is collected; Further purifying is being used 50mM Tris-HCl pH8.5 by gel filtration chromatography, 150mM NaCl finishes on 10% glycerine and 5mM DTT equilibrated Amersham Biosciences HiLoad 16/60Superdex 200 preparation scale (catalog number (Cat.No.) 17-1069-01).Purified fraction is merged, by at Amicon Ultra-15 10, carry out centrifugation on the 000Da MWCO centrifugal filter device (catalog number (Cat.No.) UFC901024) and be concentrated into about 10.4mg/ml then.

The purifying of AurA:

The cell preparation Sf9 insect cell settling (about 18g) of the expressing human Aurora-2 that cultivates from 6L also is suspended in 50mM sodium phosphate pH8.0 again with it, 500mM NaCl, 10% glycerine is in 0.2% n-octyl-β-D-glucopyranoside (BOG) and the 3mM beta-mercaptoethanol (BME).In every 1L initial incubation liquid, add a slice Roche Complete, do not contain proteinase inhibitor mixture (catalog number (Cat.No.) 1873580) and 85 Benzonase of unit (Novagen catalog number (Cat.No.) 70746-3) of EDTA).This settling is suspended in about 50ml/1 L initial incubation liquid again, uses the impact (100% under-stream period) of twice 30-45 second to carry out sonic treatment then on ice.Remove fragment by centrifugation,, then it is loaded to 5ml Ni the injection filter of supernatant liquor by 0.8 μ m ²⁺On the HiTrap post (Pharmacia).With the 50mM sodium phosphate pH8.0 of this post with 6 column volumes, 500mM NaCl, 10% glycerine, 3mM BME washing.With the linear gradient elution of protein with the same buffer that contains the 500mM imidazoles.Eluate (24ml) is being contained 50mM sodium phosphate pH8.0 under 4 ℃, 500mM NaCl, 10% glycerine decomposes in the damping fluid of 3mM BME and 10,000 TEV of unit (Invitrogen catalog number (Cat.No.) 10127-017) and spends the night.Protein is passed through second above-described nickel affinity post; Collect effluent.The protein fraction of decomposing is merged and utilize rotary concentrator to concentrate.Further purifying by gel filtration chromatography on the S75 separator column at 50mM sodium phosphate (pH8.0), 250mM NaCl, 1mM EDTA carries out among 0.1mM AMP-PNP or ATP damping fluid and the 5mM DTT.Will be the most purified fraction merge, be concentrated into about 8-11mg/ml then, perhaps under liquid nitrogen with 120 μ l aliquots containig quick freezing and be kept at-80 ℃ following or be kept under 4 ℃.

The purifying of PDK1:

Prepare the cell deposition thing and it is suspended in again from the Sf9 insect cell of 6L expressing human PDK1 and contain 50mM Tris-HCl pH 7.7 and 250mM NaCl, volume are about the damping fluid of 40ml/1 L initial incubation liquid.In every 1L initial incubation liquid, add a slice Roche Complete, do not contain the proteinase inhibitor mixture (catalog number (Cat.No.) 1873580) of EDTA and the Benzonase (Novagen catalog number (Cat.No.) 70746-3) of 85 units).This suspension was stirred 1 hour down at 4 ℃.By under 4 ℃ with 39,800x g removed fragment in centrifugal 30 minutes.Supernatant liquor is inclined in the beaker of 500ml, add 10ml then at 50mM Tris-HCl pH 7.8,50mM NaCl, 10% glycerine, 50% slurries of the Qiagen Ni-NTA Agarose (catalog number (Cat.No.) 30250) of pre-balance in 10mM imidazoles and the 10mM methionine(Met), and under 4 ℃, stirred 30 minutes.Then this sample is poured under 4 ℃ in the instillation post (drip column) and with the 50mM Tris-HCl pH7.8 of 10 column volumes, 500mM NaCl, 10% glycerine, 10mM imidazoles and 10mM methionine(Met) wash.Protein utilization is the identical damping fluid substep gradient elution of two column volumes, and described damping fluid contains the imidazoles of 50mM and 500mM successively.6x Histidine mark is spent the night with TEV proteolytic enzyme (Invitrogen catalog number (Cat.No.) 10127017)/1mg protein decomposition of 40 units, simultaneously at 50mMTris-HCl pH7.8,500mM NaCl, 10% glycerine is dialysed down in 4 ℃ in 10mM imidazoles and the 10mM methionine(Met).With sample by Pharmacia 5ml IMAC post (catalog number (Cat.No.) 17-0409-01) to remove 6x Histidine mark, described column packed has nickel and at 50mM Tris-HCl pH7.8,500mM NaCl, 10% glycerine, balance in 10mM imidazoles and the 10mM methionine(Met).The protein that has decomposed is eluted in the effluent, and undecomposed protein and His mark still be combined on the Ni post.The protein fraction of having decomposed is merged and utilize rotary concentrator to concentrate.Further purifying, is finished on 150mM NaCl and 5mM DTT equilibrated Amersham Biosciences HiLoad 16/60 Superdex, 200 preparation scale (catalog number (Cat.No.) 17-1069-01) with 25mM Tris-HCl pH7.5 by gel filtration chromatography.The most purified fraction is merged, by at Amicon Ultra-1510, carry out centrifugation on the 000Da MWCO centrifugal filter device (catalog number (Cat.No.) UFC901024) and be concentrated into about 15mg/ml then.

Test cell line

MV4-11 and THP cell are kept in the Dulbecco substratum of the Iscove modification that is supplemented with 10% foetal calf serum (FBS) and penicillin/streptomycin, the Ba/F3 cell is kept among the RPMI 1640 that is supplemented with 10% FBS, penicillin/streptomycin and 5ng/ml recombined small-mouse IL-3.

The cell survival test

To test in duplicate in the compound test below.

96 hole XTT test: cell was grown 72 hours down in 37 ℃ on 96 orifice plates in containing the compound of the various concentration growth medium of (in duplicate).The cell number of beginning is a 5000-8000 cells/well, and volume is 120 μ l.Terminal point insulation in 72 hours, with 40 μ l XTT mark mixtures (3 '-[1-(phenyl amino-carbonyl)-3,4-tetrazolium]-two (4-methoxyl group-6-nitro) benzene sulfonic acid sodium salt hydrate and electron coupling agent: PMS (the 50:1 solution of N-methyldiphenyl and pyrazine Methylsulfate) join in each hole of this plate.Continue insulation down after 2-6 hour at 37 ℃, utilize the absorbancy reading of spectrophotometric determination, carry out background correction at 650nm at 405nm.

384 hole AlamarBlue test: 90 μ l cell suspending liquids are placed into the DMSO solution that is added with 0.5 μ l compound in advance or only have on each hole of 384 orifice plates of DMSO.The cell number of beginning is 4000 cells/well.Be incubated after 72 hours, 10 μ l AlamarBlue solution (the PBS solution of 440 μ M resazurins) are joined on each hole of this plate.Continue insulation down after 2 hours at 37 ℃, utilize the board-like reading photofluorometer of TECAN to measure fluorescence, excite, launch at 591nm at 535nm.

BCR-ABL Phospho-ELISA test

Following table represents to be generally used for the reagent of BCR-ABL phospho-ELISA (＂ P-ELISA ＂) test.

The tabulation of table 40 BCR-ABL phospho-ELISA (p-ELISA) common agents

Describe	Supplier	Catalog number (Cat.No.)
Describe	Supplier	Catalog number (Cat.No.)	RPMI 1640	Invitrogen	11875-135
10% foetal calf serum, through identify, heated and inactivated	VWR	16777-014	RPMI 1640	Invitrogen	11875-135
	VWR	16777-014	Human plasma, antithrombotics=EDTA	Bioreclamation Inc.	HMPLEDTA
C-Abl (Ab-3) monoclonal antibody	VWR	80001-286	Human plasma, antithrombotics=EDTA	Bioreclamation Inc.	HMPLEDTA
C-Abl (Ab-3) monoclonal antibody	VWR	80001-286	The recombined small-mouse interleukin-3	Chemicon	IL015
The dull and stereotyped sealed strip of viscosity			The recombined small-mouse interleukin-3	Chemicon	IL015
The dull and stereotyped sealed strip of viscosity			96 hole PP, 325 μ l round bottom plate w/lidTC	Thompson Instrument Co	932465
96 hole Nunc Maxisorp plates (being used for colorimetric estimation)	Fisher Scientific	12-565-136	96 hole PP, 325 μ l round bottom plate w/lidTC	Thompson Instrument Co	932465
	Fisher Scientific	12-565-136	96 holes white flat underside (being used for luminous test)	Matrix	4923
The composition of dissolving damping fluid				Matrix	4923
The composition of dissolving damping fluid			Tris-Cl pH7.4(20mM)
NP-40(1％)			Tris-Cl pH7.4(20mM)
NP-40(1％)			EDTA(5mM)
Trisodium phosphate (NaPP; 5mM)			EDTA(5mM)
Trisodium phosphate (NaPP; 5mM)			NaF(5mM)
NaCl(150mM)			NaF(5mM)
NaCl(150mM)			The proteinase inhibitor mixture	Sigma	P2714

PMSF(1mM)
PMSF(1mM)			Vanadic acid sodium (NaVO ₄；2mM)
PBS is ice-cold			Vanadic acid sodium (NaVO ₄；2mM)
PBS is ice-cold			Anti--Tyrosine O-phosphate (4G10 ^TM)	Upstate	16-105 or 05-321
HRP bonded or unconjugated			Anti--Tyrosine O-phosphate (4G10 ^TM)	Upstate	16-105 or 05-321
HRP bonded or unconjugated			Goat anti-mouse IgG, HRP binding substances (if using unconjugated 4G10)	Upstate	12-349
BD OptELA test kit B	BD Biosciences	550534		Upstate	12-349
BD OptELA test kit B	BD Biosciences	550534	The coating damping fluid (0.1M yellow soda ash, pH9.5)
The test thinner			The coating damping fluid (0.1M yellow soda ash, pH9.5)
The test thinner			Lavation buffer solution (0.05% Tween/PBS)
Stop bath (2N sulfuric acid)			Lavation buffer solution (0.05% Tween/PBS)
Stop bath (2N sulfuric acid)			Substrate reagent A﹠B
SuperSignal ELISA Pico chemical luminous substrate (can be used for replacing substrate reagent A﹠B)	Pierce	37070	Substrate reagent A﹠B

Cell (is used the Ba/F of other mutant forms transfection of WT BCR-ABL, other kinases or T315I, Y253F or BCR-ABL ₃Cell) at least 1/2 week of growth under the condition that does not have IL-3, tests then.The test the day before yesterday, with this cell with fresh culture medium culturing so that cell is in logarithmic phase when testing.The Ba/F3 cell at least 1/2 week of growing under the condition that does not have IL-3 is suspended among the RPMI 1640 again, makes each hole of 96 orifice plates contain 200,000 cells of having an appointment.Cell is distributed in 96 orifice plates of the testing compound that contains serial dilution concentration.With cell usually with or not with testing compound at 5% CO ₂Down in 37 ℃ of insulations 60-120 minute.Be incubated under the existence that is in or be not in other additive such as 10% FCS or 50% human plasma and carry out.After with the compound insulation, add the dissolving damping fluid and be incubated 10-15 minute; Clarify by the centrifugal lysate that makes.

In order to prepare elisa plate, anti--ABL the antibody that can buy ((Ab-3 for example, Calbiochem OP20) with the concentration of 0.125 μ g/ml be formulated into the coating damping fluid (0.1M yellow soda ash, pH9.5) in, carry out making sheet (12.5 μ l, 100 μ g/ml Ab/10ml) with the 10ml/ plate then.In height bonded porous plate, in each hole, add the coating buffer soln of 100 μ l Ab, and each plate is covered with dull and stereotyped sealed strip, then 4 ℃ of following incubated overnight.

Excessive antibody removed and (the PBS solution of 0.05%Tween, pH7.4) washing is 3-4 time with 200 μ l lavation buffer solutions with elisa plate.150 μ l lysates (referring to top) are transferred on the elisa plate.This plate sealing also at room temperature is incubated 2 hours.(for example the HRP bonded resists-pTyr or unconjugated α-p-Y 4G10, Upstate) prepares in the test thinner to detect antibody.Antibody is diluted to 1:1000 (storing solution=2 μ g/ μ l, the 100 μ l solution of 200 μ g in the test thinner; F.c.=2 μ g/ml) and add the antibody/plate of 10ml dilution.Lysate is removed from elisa plate, each hole is washed 4 times with 200 μ l lavation buffer solutions/hole.In each hole, add 100 μ l and detect antibody; With this plate sealing, (21 ℃) insulation then at room temperature 1 hour.Excessive detection antibody is removed from elisa plate, each hole is washed 4 times with 200 μ l lavation buffer solutions/hole.

If necessary (that is, and for unconjugated anti--pTyr antibody), secondary antibodies (goat antirabbit HRP) be diluted to 1:3000 (3.33 μ l/10ml thinner) and add in the test thinner with the amount of the antibody/plate of 10ml dilution.Excessive secondary antibodies is removed from elisa plate, then this plate is washed 4 times with 200 μ l/ hole lavation buffer solutions.

Substrate reagent A and substrate reagent B (Pierce catalog number (Cat.No.) 37070 SuperSignal ELISAPico Chemiluminescent Substrate) are facing with preceding adding (the formed solution/plate of 10ml).Every hole adds 100 μ l substrates, mixes 1 minute, measures chemiluminescence signal with luminometer then.

Table 20: selected test-results

For last table 21, the IC of active symbolic representation ₅₀As follows: A〉10 μ M; B=1-10 μ M; C＜1 μ M.

Claims

1, following formula: compound:

Wherein

L ¹And L ²Be independently key ,-S (O) _n-,-O-,-NH-, unsubstituted C ₁-C ₅The assorted alkylidene group of alkylidene group or unsubstituted 2-5 unit, wherein n is the integer of 0-2, and

R ¹And R ²Be to replace or unsubstituted cycloalkyl, replacement or unsubstituted Heterocyclylalkyl, replacement or unsubstituted heteroaryl or replacement or unsubstituted aryl independently,

Condition is R ¹Not to replace or unsubstituted pyrryl, and work as R ¹And R ²When being unsubstituted phenyl, L ¹Not the assorted alkylidene group of unsubstituted 2-5 unit, and work as R ²When being unsubstituted piperazinyl, L ¹Not-S (O) ₂-, and work as R ²When being the unsubstituted pyridine base, R ¹Not to replace or unsubstituted isoxazolyl.

2. the described compound of claim 1, wherein L ¹And L ²Be independently key ,-S (O) _n-,-O-,-NH-or unsubstituted C ₁-C ₅Alkylidene group.

3, the described compound of claim 1, wherein L ¹And L ²It is key.

4, the described compound of claim 1, wherein L ¹Or L ²It is key.

5, the described compound of claim 1, wherein R ¹Be to replace or unsubstituted cycloalkyl, replacement or unsubstituted Heterocyclylalkyl, replacement or unsubstituted 5 or 6 yuan heteroaryl or replacement or unsubstituted aryl.

6, the described compound of claim 1, wherein R ¹Be to replace or unsubstituted 6 yuan heteroaryl or replacement or unsubstituted aryl.

7, the described compound of claim 1, wherein R ¹Be

(1) unsubstituted C ₃-C ₇Cycloalkyl;

(2) Heterocyclylalkyl of unsubstituted 3-7 unit;

(3) unsubstituted heteroaryl;

(4) unsubstituted aryl;

(5) C of Qu Daiing ₃-C ₇Cycloalkyl;

(6) Heterocyclylalkyl of the 3-7 unit of Qu Daiing;

(7) aryl of Qu Daiing; Or

(8) heteroaryl of Qu Daiing;

Wherein

(5) and (6) by oxo ,-OH ,-CF ₃,-COOH, cyano group, halogen, R ¹¹-replace or unsubstituted C ₁-C ₁₀Alkyl, R ¹¹-replace or assorted alkyl, the R of unsubstituted 2-10 unit ¹¹-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ¹¹-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ¹²-replacement or unsubstituted aryl, R ¹²-replacement or unsubstituted heteroaryl ,-L ¹²-C (X ¹) R ⁷,-L ¹²-OR ⁸,-L ¹²-NR ⁹¹R ⁹²Or-L ¹²-S (O) _mR ¹⁰Replace,

(7) and (8) quilt-OH ,-CF ₃,-COOH, cyano group, halogen, R ¹¹-replace or unsubstituted C ₁-C ₁₀Alkyl, R ¹¹-replace or assorted alkyl, the R of unsubstituted 2-10 unit ¹¹-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ¹¹-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ¹²-replacement or unsubstituted aryl, R ¹²-replacement or unsubstituted heteroaryl ,-L ¹²-C (X ¹) R ⁷,-L ¹²-OR ⁸,-L ¹²-NR ⁹¹R ⁹²Or-L ¹²-S (O) _mR ¹⁰Replace, wherein

(a) X ¹Be=S ,=O or=NR ¹⁵, R wherein ¹⁵Be H ,-OR ¹⁵¹, R ¹¹-replace or unsubstituted C ₁-C ₁₀Alkyl, R ¹¹-replace or assorted alkyl, the R of unsubstituted 2-10 unit ¹¹-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ¹¹-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ¹²-replace or unsubstituted aryl or R ¹²-replace or unsubstituted heteroaryl, wherein R ¹⁵¹Be hydrogen or R ¹¹-replace or unsubstituted C ₁-C ₁₀Alkyl,

(b) m is the integer of 0-2;

(c) R ⁷Be hydrogen, R ¹¹-replace or unsubstituted C ₁-C ₁₀Alkyl, R ¹¹-replace or assorted alkyl, the R of unsubstituted 2-10 unit ¹¹-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ¹¹-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ¹²-replacement or unsubstituted aryl, R ¹²-replacement or unsubstituted heteroaryl ,-OR ⁷¹Or-NR ⁷²R ⁷³, wherein

R ⁷¹, R ⁷²And R ⁷³Be hydrogen, R independently ¹¹-replace or unsubstituted C ₁-C ₁₀Alkyl, R ¹¹-replace or assorted alkyl, the R of unsubstituted 2-10 unit ¹¹-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ¹¹-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ¹²-replace or unsubstituted aryl or R ¹²-replace or unsubstituted heteroaryl,

R wherein ⁷²And R ⁷³Randomly the nitrogen that is connected with them lumps together and forms R ¹¹-replace or the Heterocyclylalkyl or the R of unsubstituted 3-7 unit ¹²-replace or unsubstituted heteroaryl;

(d) R ⁸, R ⁹¹And R ⁹²Be independently hydrogen ,-CF ₃, R ¹¹-replace or unsubstituted C ₁-C ₁₀Alkyl, R ¹¹-replace or assorted alkyl, the R of unsubstituted 2-10 unit ¹¹-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ¹¹-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ¹²-replacement or unsubstituted aryl, R ¹²-replacement or unsubstituted heteroaryl ,-C (X ²) R ⁸¹Or-S (O) _wR ⁸¹, R wherein ⁹¹And R ⁹²Randomly the nitrogen that is connected with them lumps together and forms R ¹¹-replace or the Heterocyclylalkyl or the R of unsubstituted 3-7 unit ¹²-replace or unsubstituted heteroaryl, wherein

(i) X ²Be=S ,=O or=NR ¹⁶, R wherein ¹⁶Be R ¹¹-replace or unsubstituted C ₁-C ₁₀Alkyl, R ¹¹-replace or assorted alkyl, the R of unsubstituted 2-10 unit ¹¹-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ¹¹-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ¹²-replace or unsubstituted aryl or R ¹²-replace or unsubstituted heteroaryl;

(ii) w is the integer of 0-2, and

(iii) R ⁸¹Be hydrogen, R ¹¹-replace or unsubstituted C ₁-C ₁₀Alkyl, R ¹¹-replace or assorted alkyl, the R of unsubstituted 2-10 unit ¹¹-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ¹¹-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ¹²-replacement or unsubstituted aryl, R ¹²-replace or unsubstituted heteroaryl or-NR ⁸¹¹R ⁸¹²,

R wherein ⁸¹¹And R ⁸¹²Be hydrogen, R independently ¹¹-replace or unsubstituted C ₁-C ₁₀Alkyl, R ¹¹-replace or assorted alkyl, the R of unsubstituted 2-10 unit ¹¹-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ¹¹-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ¹²-replace or unsubstituted aryl or R ¹²-replace or unsubstituted heteroaryl, wherein R ⁸¹¹And R ⁸¹²Randomly the nitrogen that is connected with them lumps together and forms R ¹¹-replace or the Heterocyclylalkyl or the R of unsubstituted 3-7 unit ¹²-replace or unsubstituted heteroaryl;

(e) R ¹⁰Be hydrogen, R ¹¹-replace or unsubstituted C ₁-C ₁₀Alkyl, R ¹¹-replace or assorted alkyl, the R of unsubstituted 2-10 unit ¹¹-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ¹¹-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ¹²-replacement or unsubstituted aryl, R ¹²-replace or unsubstituted heteroaryl or-NR ¹⁰¹R ¹⁰², wherein

(i) R ¹⁰¹And R ¹⁰²Be hydrogen, R independently ¹¹-replace or unsubstituted C ₁-C ₁₀Alkyl, R ¹¹-replace or assorted alkyl, the R of unsubstituted 2-10 unit ¹¹-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ¹¹-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ¹²-replace or unsubstituted aryl or R ¹²-replace or unsubstituted heteroaryl, wherein R ¹⁰¹And R ¹⁰²Randomly the nitrogen that is connected with them lumps together and forms R ¹¹-replace or the Heterocyclylalkyl or the R of unsubstituted 3-7 unit ¹²-replace or unsubstituted heteroaryl;

(f) L ¹²Be key, unsubstituted C ₁-C ₁₀Alkylidene group or unsubstituted assorted alkylidene group;

(g) R ¹¹Be oxo ,-OH ,-COOH ,-CF ₃,-OCF ₃,-CN, amino, halogen, R ¹³-replace or alkyl, the R of unsubstituted 2-10 unit ¹³-replace or assorted alkyl, the R of unsubstituted 2-10 unit ¹³-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ¹³-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ¹⁴-replace or unsubstituted aryl or R ¹⁴-replace or unsubstituted heteroaryl;

(h) R ¹²Be-OH ,-COOH, amino, halogen ,-CF ₃,-OCF ₃,-CN, R ¹³-replace or alkyl, the R of unsubstituted 2-10 unit ¹³-replace or assorted alkyl, the R of unsubstituted 2-10 unit ¹³-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ¹³-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ¹⁴-replace or unsubstituted aryl or R ¹⁴-replace or unsubstituted heteroaryl;

(i) R ¹³Be oxo ,-OH ,-COOH, amino, halogen ,-CF ₃,-OCF ₃,-CN, unsubstituted C ₁-C ₁₀Assorted alkyl, the unsubstituted C of alkyl, unsubstituted 2-10 unit ₃-C ₇Heterocyclylalkyl, unsubstituted aryl, the unsubstituted heteroaryl of cycloalkyl, unsubstituted 3-7 unit; And

(j) R ¹⁴Be-OH ,-COOH, amino, halogen ,-CF ₃,-OCF ₃,-CN, unsubstituted C ₁-C ₁₀Assorted alkyl, the unsubstituted C of alkyl, unsubstituted 2-10 unit ₃-C ₇Heterocyclylalkyl, unsubstituted aryl, the unsubstituted heteroaryl of cycloalkyl, unsubstituted 3-7 unit.

8, the described compound of claim 7, wherein R ¹Be to replace or unsubstituted 6-unit's heteroaryl or replacement or unsubstituted aryl.

9, the described compound of claim 8, wherein L ¹It is key.

10, the described compound of claim 7, wherein R ¹Be (7) or (8), wherein (7) and (8) quilt-OH ,-CF ₃, halogen, unsubstituted C ₁-C ₁₀Assorted alkyl, the unsubstituted C of alkyl, unsubstituted 2-10 unit ₃-C ₇The Heterocyclylalkyl of cycloalkyl, unsubstituted 3-7 unit, unsubstituted aryl, unsubstituted heteroaryl or-L ¹²-OR ⁸Replace L wherein ¹²It is key.

11, the described compound of claim 10, wherein R ⁸Be CF ₃

12, the described compound of claim 7, wherein R ¹Be (7) or (8), (7) and (8) quilt-OCH wherein ₃,-OCF ₃,-CH ₃,-CF ₃,-OCH ₂CH ₃, halogen or the ring propoxy-replace.

13, the described compound of claim 7, wherein L ¹And L ²It is key.

14, claim 1,7 or 13 described compound, wherein R ²Be

(1) unsubstituted C ₃-C ₇Cycloalkyl;

(2) Heterocyclylalkyl of unsubstituted 3-7 unit;

(3) unsubstituted heteroaryl;

(4) unsubstituted aryl;

(5) C of Qu Daiing ₃-C ₇Cycloalkyl;

(6) Heterocyclylalkyl of the 3-7 unit of Qu Daiing;

(7) aryl of Qu Daiing; Or

(8) heteroaryl of Qu Daiing;

Wherein

(5) and (6) by oxo ,-OH ,-CF ₃,-COOH, cyano group, halogen, R ²¹-replace or unsubstituted C ₁-C ₁₀Alkyl, R ²¹-replace or assorted alkyl, the R of unsubstituted 2-10 unit ²¹-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ²¹-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ²²-replace or unsubstituted aryl or R ²²-replacement or unsubstituted heteroaryl ,-L ²²-C (X ³) R ³,-L ²²-OR ⁴,-L ²²-NR ⁵¹R ⁵²Or-L ²²-S (O) _qR ⁶Replace,

(7) and (8) quilt-OH ,-CF ₃,-COOH, cyano group, halogen, R ²¹-replace or unsubstituted C ₁-C ₁₀Alkyl, R ²¹-replace or assorted alkyl, the R of unsubstituted 2-10 unit ²¹-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ²¹-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ²²-replacement or unsubstituted aryl, R ²²-replacement or unsubstituted heteroaryl ,-L ²²-C (X ³) R ³,-L ²²-OR ⁴,-L ²²-NR ⁵¹R ⁵²Or-L ²²-S (O) _qR ⁶Replace, wherein

(a) X ³Be=S ,=O or=NR ¹⁷, R wherein ¹⁷Be H ,-OR ¹⁷¹, R ²¹-replace or unsubstituted C ₁-C ₁₀Alkyl, R ²¹-replace or assorted alkyl, the R of unsubstituted 2-10 unit ²¹-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ²¹-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ²²-replace or unsubstituted aryl or R ²²-replace or unsubstituted heteroaryl, wherein R ¹⁷¹Be H or R ²¹-replace or unsubstituted C ₁-C ₁₀Alkyl;

(b) q is the integer of 0-2;

(c) R ³Be hydrogen, R ²¹-replace or unsubstituted C ₁-C ₁₀Alkyl, R ²¹-replace or assorted alkyl, the R of unsubstituted 2-10 unit ²¹-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ²¹-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ²²-replacement or unsubstituted aryl, R ²²-replacement or unsubstituted heteroaryl ,-OR ³¹Or-NR ³²R ³³, wherein

(i) R ³¹, R ³²And R ³³Be hydrogen, R independently ²¹-replace or unsubstituted C ₁-C ₁₀Alkyl, R ²¹-replace or assorted alkyl, the R of unsubstituted 2-10 unit ²¹-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ²¹-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ²²-replace or unsubstituted aryl or R ²²-replace or unsubstituted heteroaryl, wherein R ³²And R ³³Randomly the nitrogen that is connected with them lumps together and forms R ²¹-replace or the Heterocyclylalkyl or the R of unsubstituted 3-7 unit ²²-replace or unsubstituted heteroaryl;

(d) R ⁴, R ⁵¹And R ⁵²Be independently hydrogen ,-CF ₃, R ²¹-replace or unsubstituted C ₁-C ₁₀Alkyl, R ²¹-replace or assorted alkyl, the R of unsubstituted 2-10 unit ²¹-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ²¹-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ²²-replacement or unsubstituted aryl, R ²²-replacement or unsubstituted heteroaryl ,-C (X ⁴) R ⁴¹Or-S (O) _vR ⁴¹, R wherein ⁵¹And R ⁵²Randomly the nitrogen that is connected with them lumps together and forms R ²¹-replace or the Heterocyclylalkyl or the R of unsubstituted 3-7 unit ²²-replace or unsubstituted heteroaryl, wherein

(i) X ⁴Be=S ,=O or=NR ¹⁸, R wherein ¹⁸Be R ²¹-replace or unsubstituted C ₁-C ₁₀Alkyl, R ²¹-replace or assorted alkyl, the R of unsubstituted 2-10 unit ²¹-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ²¹-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ²²-replace or unsubstituted aryl or R ²²-replace or unsubstituted heteroaryl;

(ii) v is the integer of 0-2;

(iii) R ⁴¹Be hydrogen, R ²¹-replace or unsubstituted C ₁-C ₁₀Alkyl, R ²¹-replace or assorted alkyl, the R of unsubstituted 2-10 unit ²¹-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ²¹-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ²²-replacement or unsubstituted aryl, R ²²-replace or unsubstituted heteroaryl or-NR ⁴¹¹R ⁴¹²,

R wherein ⁴¹¹And R ⁴¹²Be independently selected from hydrogen, R ²¹-replace or unsubstituted C ₁-C ₁₀Alkyl, R ²¹-replace or assorted alkyl, the R of unsubstituted 2-10 unit ²¹-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ²¹-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ²²-replace or unsubstituted aryl or R ²²-replace or unsubstituted heteroaryl, wherein R ⁴¹¹And R ⁴¹²Randomly the nitrogen that is connected with them lumps together and forms R ²¹-replace or the Heterocyclylalkyl or the R of unsubstituted 3-7 unit ²²-replace or unsubstituted heteroaryl;

(e) R ⁶Be hydrogen, R ²¹-replace or unsubstituted C ₁-C ₁₀Alkyl, R ²¹-replace or assorted alkyl, the R of unsubstituted 2-10 unit ²¹-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ²¹-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ²²-replacement or unsubstituted aryl, R ²²-replace or unsubstituted heteroaryl or-NR ⁶¹R ⁶², wherein

(i) R ⁶¹And R ⁶²Be hydrogen, R ²¹-replace or unsubstituted C ₁-C ₁₀Alkyl, R ²¹-replace or assorted alkyl, the R of unsubstituted 2-10 unit ²¹-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ²¹-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ²²-replace or unsubstituted aryl or R ²²-replace or unsubstituted heteroaryl, wherein R ⁶¹And R ⁶²Randomly the nitrogen that is connected with them lumps together and forms R ²¹-replace or the Heterocyclylalkyl or the R of unsubstituted 3-7 unit ²²-replace or unsubstituted heteroaryl;

(f) L ²²Be key, unsubstituted C ₁-C ₁₀Alkylidene group or unsubstituted assorted alkylidene group;

(g) R ²¹Be oxo ,-OH ,-COOH ,-CF ₃,-OCF ₃,-CN, amino, halogen, R ²³-replace or alkyl, the R of unsubstituted 2-10 unit ²³-replace or assorted alkyl, the R of unsubstituted 2-10 unit ²³-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ²³-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ²⁴-replace or unsubstituted aryl or R ²⁴-replace or unsubstituted heteroaryl;

(h) R ²²Be-OH ,-COOH, amino, halogen ,-CF ₃,-OCF ₃,-CN, R ²³-alkyl, R that replace or unsubstituted 2-10 unit ²³-replace or assorted alkyl, the R of unsubstituted 2-10 unit ²³-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ²³-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ²⁴-replace or unsubstituted aryl or R ²⁴-replace or unsubstituted heteroaryl;

(i) R ²³Be oxo ,-OH ,-COOH, amino, halogen ,-CF ₃,-OCF ₃,-CN, unsubstituted C ₁-C ₁₀Assorted alkyl, the unsubstituted C of alkyl, unsubstituted 2-10 unit ₃-C ₇Heterocyclylalkyl, unsubstituted aryl, the unsubstituted heteroaryl of cycloalkyl, unsubstituted 3-7 unit; And

(j) R ²⁴Be-OH ,-COOH, amino, halogen ,-CF ₃,-OCF ₃,-CN, unsubstituted C ₁-C ₁₀Assorted alkyl, the unsubstituted C of alkyl, unsubstituted 2-10 unit ₃-C ₇Heterocyclylalkyl, unsubstituted aryl, the unsubstituted heteroaryl of cycloalkyl, unsubstituted 3-7 unit.

15, the described compound of claim 14, wherein L ²It is key.

16, the described compound of claim 14, wherein R ²Be (3), (4), (7) or (8).

17, the described compound of claim 15, wherein R ²Be (7) or (8).

18, the described compound of claim 17, wherein (7) and (8) quilt-L ²²-C (X ³) R ³,-L ²²-OR ⁴,-L ²²-NR ⁵¹R ⁵²,-L ²²-C (NH)-NR ³²R ³³Or-L ²²-S (O) _qR ⁶Replace.

19, the described compound of claim 18, wherein

R ³Be-NR ³²R ³³

X ³Be=O or=NR ¹⁷

R ⁶Be-NR ⁶¹R ⁶²

R ⁵¹Be-C (O) R ⁴¹Or-S (O) _vR ⁴¹

20, the described compound of claim 19, wherein R ⁴¹Be-NR ⁴¹¹R ⁴¹²

21, the described compound of claim 15, wherein R ²Be (7) or (8), wherein (7) and (8) quilt-OH ,-CF ₃Assorted alkyl, the unsubstituted C of ,-COOH, amino, halogen, unsubstituted 2-10 unit ₃-C ₇The Heterocyclylalkyl of cycloalkyl, unsubstituted 3-7 unit, unsubstituted aryl, unsubstituted heteroaryl or-L ²²-C (X ³) R ³Replace, wherein

X ³Be=O;

R ³Be unsubstituted C ₁-C ₁₀Assorted alkyl, the unsubstituted C of alkyl, unsubstituted 2-10 unit ₃-C ₇The Heterocyclylalkyl of cycloalkyl, unsubstituted 3-7 unit, unsubstituted aryl, unsubstituted heteroaryl or-NR ³²R ³³, wherein

R ³²And R ³³Be hydrogen, R independently ²¹-replace or unsubstituted C ₁-C ₁₀Alkyl, R ²¹-replace or assorted alkyl, the R of unsubstituted 2-10 unit ²¹-replace or unsubstituted C ₃-C ₇Cycloalkyl, R ²¹-replace or Heterocyclylalkyl, the R of unsubstituted 3-7 unit ²²-replace or unsubstituted aryl or R ²²-replace or unsubstituted heteroaryl,

R wherein ³²And R ³³Randomly the nitrogen that is connected with them lumps together and forms R ²¹-replace or the Heterocyclylalkyl or the R of unsubstituted 3-7 unit ²²-replace or unsubstituted heteroaryl.

22, the described compound of claim 15, wherein R ²Be (7) or (8), wherein (7) and (8) by the assorted alkyl of unsubstituted 2-10 unit or-L ²²-C (O) R ³Replace,

Wherein

L ²²It is key; And

R ³Be-NR ³²R ³³, wherein

23, the described compound of claim 1, wherein R ¹Be to replace or unsubstituted fused ring aryl or replacement or unsubstituted condensed ring heteroaryl.

24, the described compound of claim 1, wherein R ²Be to replace or unsubstituted indyl, replacement or unsubstituted quinolines base or replacement or unsubstituted benzo dioxolyl.

25, the described compound of claim 1, wherein R ²Be to replace or unsubstituted fused ring aryl or replacement or unsubstituted condensed ring heteroaryl.

26, the described compound of claim 1, wherein R ¹Be to replace or unsubstituted indyl, replacement or unsubstituted quinolines base or replacement or unsubstituted benzo dioxolyl.

27, the described compound of claim 14, wherein R ¹And R ²Be to replace or unsubstituted glycolylurea base independently, replace or unsubstituted dioxolanyl, replace or unsubstituted alkyl dioxin, replace or unsubstituted trioxa alkyl, replace or unsubstituted tetrahydro-thienyl, replace or unsubstituted tetrahydrofuran base, replace or unsubstituted tetrahydro-thienyl, replace or unsubstituted THP trtrahydropyranyl, replace or unsubstituted tetrahydro thiapyran base, replace or unsubstituted pyrrolidyl, replace or unsubstituted morpholinyl, replace or unsubstituted piperidyl, replace or unsubstituted pyrazolyl, replace or unsubstituted furyl, replace or unsubstituted imidazolyl, replace or unsubstituted isoxazolyl, replace or do not replace De oxadiazole base, replace or do not replace the De oxazolyl, replace or the unsubstituted pyridine base, replace or unsubstituted pyrazinyl, replace or unsubstituted pyrimidyl, replace or unsubstituted pyridazinyl, replace or unsubstituted thiazolyl, replace or unsubstituted isothiazolyl, replace or unsubstituted triazolyl, replace or unsubstituted thienyl, replace or unsubstituted triazinyl, replace or unsubstituted thiadiazolyl group or replacement or unsubstituted tetrazyl.

28, following formula: compound:

Wherein

R ¹And R ²Be to replace or unsubstituted cycloalkyl, replacement or unsubstituted Heterocyclylalkyl, replacement or unsubstituted heteroaryl or replacement or unsubstituted aryl independently.

29, following formula: compound:

L wherein ¹And L ²Be independently key ,-S (O) _n-,-O-,-NH-, unsubstituted C ₁-C ₅The assorted alkylidene group of alkylidene group or unsubstituted 2-5 unit, wherein n is the integer of 0-2, and

30, regulate the method for protein kinase activity, this method comprises described protein kinase is contacted with a described compound in the claim 1,28 or 29.

31, in the individuality of the described treatment of needs, treat cancer, allergy, asthma, inflammation, obstructive airway diseases, autoimmune disease, metabolic trouble, infection, CNS disease, cerebral tumor, obesity, asthma, blood disease, sex change neuropathy, cardiovascular disorder or with the method for vasculogenesis, neovascularization or blood vessel generation diseases associated, described method comprises a described compound in the claim 1 of described individual administering therapeutic significant quantity, 28 or 29.

32, regulate the method for protein kinase activity, this method comprises described protein kinase is contacted with following formula: compound:

Wherein

33, the described method of claim 32, wherein said protein kinase are Abelson Tyrosylprotein kinase, Ron receptor tyrosine kinase, Met receptor tyrosine kinase, Fms sample Tyrosylprotein kinase-3, Aurora kinases, p21-activatory kinases-4 or 3-phosphoinositide dependent kinases-1.

34, the described method of claim 32, wherein said protein kinase are to have the Bcr-Abl kinases that is selected from following sudden change: M244V, L248V, G250E, G250A, Q252H, Q252R, Y253F, Y253H, E255K, E255V, D276G, F311L, T315I, T315N, T315A, F317V, F317L, M343T, M351T, E355G, F359A, F359V, V379I, F382L, L387M, H396P, H396R, S417Y, E459K and F486S.

35, the described method of claim 34, wherein said protein kinase have the T315I sudden change.

36, in the individuality of the described treatment of needs, treat cancer, allergy, asthma, inflammation, obstructive airway diseases, autoimmune disease, metabolic trouble, infection, CNS disease, cerebral tumor, obesity, asthma, blood disease, sex change neuropathy, cardiovascular disorder or with the method for vasculogenesis, neovascularization or blood vessel generation diseases associated, described method comprises the following formula: compound to individual administering therapeutic significant quantity:

Wherein

37, the described method of claim 36, wherein said cancer is selected from leukemia or myeloproliferative disease.

38, comprise the described compound in pharmaceutically acceptable vehicle and the claim 1,28 or 29 or the pharmaceutical composition of following formula: compound:

Wherein

L ¹And L ²Be independently key ,-S (O) n-,-O-,-NH-, unsubstituted C ₁-C ₅The assorted alkylidene group of alkylidene group or unsubstituted 2-5 unit, wherein n is the integer of 0-2, and