CN100447143C - Compounds and compositions as inhibitors of receptor tyrosine kinase activity - Google Patents

Abstract

The invention provides a novel class of compounds, pharmaceutical compositions comprising such compounds and methods of using such compounds to treat or prevent diseases or disorders associated with cSRC, Lck, FGFR3, Flt3, TrkB, Bmx, and/or PFGFRalpha kinase activity.

Description

The anilino purine that replaces as the 6-of RTK inhibitor

The cross reference of related application

The application requires the benefit of priority of following U.S. Provisional Patent Application: 60/495,406 of submission on August 15th, 2003; 60/524,357 of submission on November 21st, 2003; With 60/565,367 of submission on April 26th, 2004.Whole disclosure integral body of these applications are incorporated herein by reference widely.

Background of invention

Invention field

The pharmaceutical composition that the invention provides a class new compound, comprises this compounds with use the active relevant disease of this compounds for treating or prevention and cSRC, Lck, FGFR3, Flt3, TrkB, Bmx and/or PFGFR alpha kinase or the method for illness.

Background technology

Protein kinase is represented big nation's protein, and they are being regulated various cell processes and are keeping and play central role aspect the control of cellular function.The non-limiting catalogue of these kinase whose parts comprises: receptor tyrosine kinase, as the receptor kinase of Fms-sample Tyrosylprotein kinase 3 (Fit3), platelet-derived growth factor receptor kinase (PDGF-R), c-kit, trk C, trkB and fibroblast growth factor acceptor (FGFR3) and STEM CELL FACTOR; Non--receptor tyrosine kinase, as Abl and fusion kinase b CR-Abl, Fes, Lck and Syk; And serine/threonine kinase, as b-RAF, map kinase (for example MKK6) and SAPK2 β.In the numerous disease state, observed the aberrant kinase activity, the disease that comprises optimum and neoplasm venereal disease disease and cause because of immunity and the inappropriate activation of neural system.

New compound of the present invention suppresses one or more protein kinase activities, estimates to can be used for treating the disease relevant with kinases thus.

Summary of the invention

On the one hand, the invention provides formula I compound and N-oxide derivative thereof, prodrug derivatives, protected derivative, independent isomer and the pharmacy acceptable salt and the solvate (for example hydrate) of isomer mixture and this compounds:

Wherein:

R ₁Be selected from hydrogen, halogen, C _1-6Alkyl, halogen-replacement-C _1-6Alkyl, C _1-6Alkoxyl group, halogen-replacement-C _1-6Alkoxyl group ,-OXOR ⁵,-OXR ⁶,-OXNR ₅R ₆,-OXONR ₅R ₆,-XR ₆,-XNR ₅R ₆With-XNR ₇XNR ₇R ₇Wherein X is selected from key, C _1-6Alkylidene group, C _2-6Alkylene group and C _2-6Alkynylene; R wherein ₇Be independently selected from hydrogen or C _1-6Alkyl;

R ₅Be selected from hydrogen, C _1-6Alkyl and-XOR ₇Wherein X is selected from key, C _1-6Alkylidene group, C _2-6Alkylene group and C _2-6Alkynylene; And R ₇Be independently selected from hydrogen or C _1-6Alkyl;

R ₆Be selected from hydrogen, C _1-6Alkyl, C _3-12Cycloalkyl C _0-4Alkyl, C _3-8Heterocyclylalkyl C _0-4Alkyl, C _6-10Aryl C _0-4Alkyl and C _5-10Heteroaryl C _0-4Alkyl; Or

R ₅And R ₆With R ₅And R ₆The nitrogen-atoms that is connected forms C together _3-8Heterocyclylalkyl or C _5-8Heteroaryl; R wherein ₅And R ₆Methylene radical in formed any Heterocyclylalkyl can choose wantonly by-C (O)-or-S (O) ₂-substitute;

R wherein ₆Any aryl, heteroaryl, cycloalkyl or Heterocyclylalkyl or R ₅And R ₆Combination can choose wantonly by 1-3 group and replace, described group is independently selected from-XNR ₇R ₇,-XOR ₇,-XNR ₇R ₇,-XC (O) NR ₇R ₇,-XNR ₇C (O) R ₇,-XOR ₇,-XC (O) OR ₇,-XC (O) R ₇, C _1-6Alkyl, C _3-8Heterocyclylalkyl, C _5-10Heteroaryl, C _3-12Cycloalkyl and C _6-10Aryl C _0-4Alkyl; R wherein ₁Any alkyl or alkylidene group can choose wantonly and have by divalent group alternate methylene radical, described divalent group is selected from-NR ₇C (O)-,-C (O) NR ₇-,-NR ₇-,-C (O)-,-O-,-S-,-S (O)-and-S (O) ₂-; And R wherein ₆Any alkyl or alkylidene group can choose wantonly by 1-3 group and replace, described group is independently selected from C _5-8Heteroaryl ,-NR ₇R ₇,-C (O) NR ₇R ₇,-NR ₇C (O) R ₇, halogen and hydroxyl; R wherein ₇Be independently selected from hydrogen or C _1-6Alkyl;

R ₂Be selected from hydrogen, C _6-10Aryl and C _5-10Heteroaryl; R wherein ₂Any aryl or heteroaryl optional by 1-3 group replacement, described group is independently selected from-XNR ₇R ₇,-XOR ₇,-XOR ₈,-XC (O) OR ₇,-XC (O) R ₇, C _1-6Alkyl, C _1-6Alkoxyl group, nitro, cyano group, hydroxyl, halogen and halogen-replacement-C _1-6Alkyl; Wherein X and R ₇As mentioned above; And R ₈Be C _6-10Aryl C _0-4Alkyl;

R ₃Be selected from hydrogen and C _1-6Alkyl;

R ₄Be selected from C _3-12Cycloalkyl C _0-4Alkyl, C _3-8Heterocyclylalkyl C _0-4Alkyl, C _6-10Aryl C _0-4Alkyl and C _5-10Heteroaryl C _0-4Alkyl; R wherein ₄Any alkylidene group can choose wantonly and have by divalent group alternate methylene radical, described divalent group is selected from-C (O)-,-S-,-S (O)-and-S (O) ₂-; R wherein ₄Described aryl, heteroaryl, cycloalkyl or Heterocyclylalkyl optional by 1-3 group replacement, described group is selected from halogen, C _1-6Alkyl, C _1-6Alkoxyl group, halogen-replacement-C _1-6Alkyl, halogen-replacement-C _1-6Alkoxyl group ,-XR ₉,-XOR ₉,-XS (O) _0-2R ₇,-XS (O) _0-2R ₉,-XC (O) R ₇,-XC (O) OR ₇,-XP (O) R ₇R ₇,-XC (O) R ₉,-XC (O) NR ₇XNR ₇R ₇,-XC (O) NR ₇R ₇,-XC (O) NR ₇R ₉With-XC (O) NR ₇XOR ₇Wherein X and R ₇As mentioned above; R ₉Be selected from C _3-12Cycloalkyl C _0-4Alkyl, C _3-8Heterocyclylalkyl C _0-4Alkyl, C _6-10Aryl and C _5-10Heteroaryl; R wherein ₉Any aryl, heteroaryl, cycloalkyl or Heterocyclylalkyl optional be selected from C by 1-3 _1-6Alkyl ,-XC (O) R ₇With-XC (O) NR ₇R ₇Group replace; Wherein X and R ₇As mentioned above.

Second aspect the invention provides pharmaceutical composition, and it contains formula I compound or its N-oxide derivative, independent isomer and isomer mixture or its pharmacy acceptable salt and one or more suitable vehicle.

The third aspect, the invention provides the method for treatment Animal diseases, wherein suppress cSRC, Lck, FGFR3, Flt3, TrkB, PDGFR α and/or Bmx activity and can prevent, suppress or improve the pathology and/or the symptom of described disease, this method comprises treats the formula I compound of significant quantity or its N-oxide derivative, independent isomer and isomer mixture or its pharmacy acceptable salt to described animal.

Fourth aspect the invention provides formula I compound and is used for the treatment of purposes in the medicine of Animal diseases in preparation, and wherein cSRC, Lck, FGFR3, Flt3, TrkB, PDGFR α and/or Bmx activity work to the pathology and/or the symptom of described disease.

The 5th aspect the invention provides preparation I compound and N-oxide derivative thereof, prodrug derivatives, independent isomer and the method for isomer mixture and pharmacy acceptable salt thereof.

Detailed Description Of The Invention

Definition

" alkyl " as group and as other group for example halogen-replacement-structural unit of alkyl and alkoxyl group, can be for straight or branched.C _1-4Alkoxyl group comprises methoxyl group, oxyethyl group etc.The alkyl of halogen-replacement comprises trifluoromethyl, pentafluoroethyl group etc.

" aryl " refers to monocycle or the condensed-bicyclic aromatic ring system that contains 6-10 ring carbon atom.For example, aryl can be phenyl or naphthyl, preferred phenyl.

" arylidene " refers to the divalent group derived from aryl." heteroaryl " as defining aryl, and wherein one or more in the annular atoms are heteroatoms.For example, heteroaryl comprises pyridyl, indyl, indazolyl, quinoxalinyl, quinolyl, benzofuryl, benzopyranyl, benzo thiapyran base, benzo [1,3] dioxole, imidazolyl, benzo-imidazolyl, pyrimidyl, furyl, oxazolyl, isoxazolyl, triazolyl, tetrazyl, pyrazolyl, thienyl etc.

" cycloalkyl " refer to contain shown in the undersaturated monocycle of saturated or part, condensed-bicyclic or the bridge joint multi-loop system of annular atoms number.For example, C _3-10Cycloalkyl comprises cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl etc." Heterocyclylalkyl " refers to as defined cycloalkyl among the application, condition be shown in the one or more be selected from-O-of ring in the carbon ,-N=,-NR-,-C (O)-,-S-,-S (O)-or-S (O) ₂-part substitute, wherein R is hydrogen, C _1-4Alkyl or nitrogen-protecting group.For example, as being the employed C of description The compounds of this invention among the application _3-8Heterocyclylalkyl comprises morpholino, pyrrolidyl, piperazinyl, piperidyl, piperidyl ketone, 1,4-two oxa-s-8-azepine-spiral shell [4.5] last of the ten Heavenly stems-8-base etc.

" halogen " (or halo) preferably represented chlorine or fluorine, but also can be bromine or iodine.

" treatment " refers to and alleviates or palliate a disease and/or the method for its subsidiary symptom.In this manual, term " treatment " comprises the treatment of preventative (prophylactic) or preventing property (preventative) and cures or suppress treatment of diseases, comprises that treatment place danger catches or suspects the patient that caught and ill patient.This term further comprises the treatment that is used to delay disease progression.

Term used herein " healing " refers to the effect for the treatment of in the PD incident that relates to the imbalance of Flt3 receptor tyrosine kinase activity.

Term " preventative " refers to prevention or prevents to relate to the outbreak or the recurrence of the disease of Flt3 receptor tyrosine kinase activity imbalance.

The patient that term used herein " delay of progression " refers to preposition stage (pre-stage) for the treatment of disease to being in or early stage (early phase) gives active compound, described patient for example is diagnosed as the prefixed form of corresponding disease, or be under the condition in the medical procedure for example or under the unexpected condition that causes, may develop corresponding disease with this understanding.

Term used herein " disease that relates to the imbalance of Flt3 receptor tyrosine kinase activity " includes but not limited to leukemia, and comprising acute myeloid leukaemia (AML), having three is AML (AML/TMDS), acute lymphoblastic leukemia (ALL) and the myelodysplastic syndrome (MDS) of myelodysplasia.The disease that this term also causes particularly including the Flt3 receptor mutation.

Preferred embodiment is described

The invention provides a class new compound, comprise the pharmaceutical composition of this compounds disease relevant with cSRC, Lck, FGFR3, Flt3, TrkB, PFGFR α and/or Bmx kinase activity or the method for illness with using this compounds for treating or prevention.Especially, described compound shows the height effect to F1t3 and FGFR3 receptor kinase.

Relate to formula I compound in one embodiment:

R ₁Be selected from hydrogen, halogen, C _1-6Alkoxyl group ,-OXOR ⁵,-OXR ⁶,-OXNR ₅R ₆,-OXONR ₅R ₆,-XR ₆,-XNR ₇XNR ₇R ₇With-XNR ₅R ₆Wherein X is selected from key, C _1-6Alkylidene group, C _2-6Alkylene group and C _2-6Alkynylene;

R ₅Be selected from hydrogen, C _1-6Alkyl and-XOR ₇Wherein X is selected from key, C _1-6Alkylidene group, C _2-6Alkylene group and C _2-6Alkynylene; And R ₇Be independently selected from hydrogen or C _1-6Alkyl;

R ₆Be selected from hydrogen, C _1-6Alkyl, C _3-12Cycloalkyl C _0-4Alkyl, C _3-8Heterocyclylalkyl C _0-4Alkyl, C _6-10Aryl C _0-4Alkyl and C _5-10Heteroaryl C _0-4Alkyl; R ₆Be hydrogen or C _1-6Alkyl; Or

R ₅And R ₆With R ₅And R ₆The nitrogen-atoms that is connected forms C together _3-8Heterocyclylalkyl or C _5-8Heteroaryl; R wherein ₅And R ₆Methylene radical in formed any Heterocyclylalkyl can choose wantonly by-C (O)-or-S (O) ₂-substitute;

R wherein ₆Any aryl, heteroaryl, cycloalkyl or Heterocyclylalkyl or R ₅And R ₆Combination can choose wantonly by 1-3 group and replace, described group is independently selected from-XNR ₇R ₇,-XC (O) NR ₇R ₇,-XOR ₇,-XNR ₇R ₇,-XNR ₇C (O) R ₇,-XOR ₇,-XC (O) R ₇, C _1-6Alkyl, C _3-8Heterocyclylalkyl and C _6-10Aryl C _0-4Alkyl; R wherein ₁Any alkyl or alkylidene group can choose wantonly and have by divalent group alternate methylene radical, described divalent group is selected from-NR ₇C (O)-,-C (O) NR ₇-,-NR ₇-,-O-; And R wherein ₁Any alkyl or alkylidene group can choose wantonly by 1-3 group and replace, described group is independently selected from C _5-8Heteroaryl ,-NR ₇R ₇,-C (O) NR ₇R ₇,-NR ₇C (O) R ₇, halogen and hydroxyl; R wherein ₇Be independently selected from hydrogen or C _1-6Alkyl;

R ₂Be selected from hydrogen, C _6-10Aryl and C _5-10Heteroaryl; R wherein ₂Any aryl or heteroaryl optional by 1-3 group replacement, described group is independently selected from-XNR ₇R ₇,-XOR ₇,-XOR ₈,-XC (O) OR ₇, C _1-6Alkyl, C _1-6Alkoxyl group, nitro, cyano group, halogen, halogen-replacement-C _1-6Alkoxyl group and halogen-replacement-C _1-6Alkyl; Wherein X and R ₇As mentioned above; And R ₈Be C _6-10Aryl C _0-4Alkyl;

R ₃Be hydrogen; And

R ₄Be selected from C _6-10Aryl C _0-4Alkyl and C _5-10Heteroaryl C _0-4Alkyl; R wherein ₄Described aryl or heteroaryl replaced by 1-3 group, described group be selected from halogen ,-XR ₉,-XOR ₉,-XS (O) ₂R ₇,-XS (O) ₂R ₉,-XC (O) R ₇,-XC (O) OR ₇,-XP (O) R ₇R ₇,-XC (O) R ₉,-XC (O) NR ₇XNR ₇R ₇,-XC (O) NR ₇R ₇,-XC (O) NR ₇R ₉With-XC (O) NR ₇XOR ₇Wherein X and R ₇As mentioned above; R ₉Be C _3-8Heterocyclylalkyl C _0-4Alkyl; R wherein ₉Optional by the individual C that is selected from of 1-3 _1-6Alkyl ,-XC (O) R ₇With-XC (O) NR ₇R ₇Group replace; Wherein X and R ₇As mentioned above.

In another embodiment, R ₁Be selected from hydrogen, halogen, C _1-6Alkoxyl group ,-OXOR ⁵,-OXR ⁶,-OXNR ₅R ₆,-OXONR ₅R ₆,-XR ₆With-XNR ₅R ₆Wherein X is selected from key, C _1-6Alkylidene group, C _2-6Alkylene group and C _2-6Alkynylene; R ₅Be selected from hydrogen, methyl, hydroxyl-ethyl and methoxyl group-ethyl; R ₆Be selected from hydrogen, phenyl, benzyl, cyclopentyl, cyclobutyl, dimethylamino-propenyl, cyclohexyl, 2,3-dihydroxyl-propyl group, piperidyl, amino-carbonyl-ethyl, methyl-carbonyl-amino-ethyl, methyl-amino-ethyl, amino-propyl group, methyl-amino-propyl group, 1-hydroxymethyl-butyl, amyl group, butyl, propyl group, methoxyl group-ethynyl, methoxyl group-vinyl, dimethyl-amino-butyl, dimethyl-amino-ethyl, dimethyl-amino-propyl group, THP trtrahydropyranyl, tetrahydrofuran base-methyl, pyridyl-methyl, azepine ring-1-in heptan base, [1,4] oxygen azepine ring-4-in heptan base, piperidyl-ethyl, diethyl-amino-ethyl, amino-butyl, amino-sec.-propyl, amino-ethyl, hydroxyl-ethyl, 2-acetylamino-ethyl, formamyl-ethyl, 4-methyl-[1,4] diaza ring-1-in heptan base, 2-hydroxyl-propyl group, hydroxyl-propyl group, 2-hydroxy-2-methyl-propyl group, methoxyl group-ethyl, amino-propyl group, methyl-amino-propyl group, 2-hydroxyl-2-phenyl-ethyl, pyridyl-ethyl, morpholino-propyl group, morpholino-ethyl, pyrrolidyl, pyrrolidyl-methyl, pyrrolidyl-ethyl, pyrrolidyl-propyl group, pyrazinyl, quinoline-3-base, quinoline-5-base, imidazolyl-ethyl, pyridyl-methyl, styroyl, tetrahydrochysene-pyrans-4-base, pyrimidyl, furyl isoxazolyl-methyl, pyridyl, benzo [1,3] dioxole-5-base, thiazolyl-ethyl and thiazolyl-methyl; Or R ₅And R ₆With R ₅And R ₆The nitrogen-atoms that is connected forms pyrrolidyl, piperazinyl, piperidyl, imidazolyl, 3-oxo-piperazine-1-base, [1,4] diaza ring-1-in heptan base, morpholino, 3-oxo-piperazine-1-base, 1,1-dioxo-l λ together ⁶-thiomorpholine-4-base or pyrazolyl;

R wherein ₆Any aryl, heteroaryl, cycloalkyl or Heterocyclylalkyl or R ₅And R ₆Combination can choose wantonly by 1-3 group and replace, described group is independently selected from methyl-carbonyl, amino-methyl, amino-carbonyl, methyl-alkylsulfonyl, methoxyl group, methoxyl group-methyl, formyl radical, fluoro-ethyl, hydroxyl-ethyl, amino, dimethylamino, hydroxyl, methyl, ethyl, ethanoyl, sec.-propyl, pyrrolidyl, pyrimidyl, morpholino, pyridyl and benzyl; R wherein ₆Any alkyl or alkylidene group can choose wantonly have be selected from-NHC (O)-or-the divalent group alternate methylene radical of C (O) NH-; And R wherein ₆Any alkyl or alkylidene group can choose wantonly by 1 to 2 group that is independently selected from amino, halogen, piperidyl and hydroxyl and replace.

In another embodiment, R ₂Be selected from hydrogen, phenyl, thienyl, pyridyl, pyrazolyl, thiazolyl, pyrazinyl, naphthyl, furyl, benzo [1,3] dioxole-5-base, isothiazolyl, imidazolyl and pyrimidyl; R wherein ₂Any aryl or heteroaryl optional by 1-3 group replacement, described group is independently selected from methyl, sec.-propyl, halogen, ethanoyl, trifluoromethyl, nitro, 1-hydroxyl-ethyl, 1-hydroxyl-1-methyl-ethyl, hydroxyl-ethyl, hydroxyl-methyl, formamyl, methoxyl group, benzyloxy, carboxyl, amino, cyano group, amino-carbonyl, amino-methyl and oxyethyl group.

In another embodiment, R ₄Be selected from phenyl, benzyl, pyridyl and 1-oxo-indane-5-base; Wherein said phenyl; benzyl; indanyl or pyridyl are optional by following replacement: halogen; ethanoyl; trifluoromethyl; cyclopropyl-amino-carbonyl; azetidine-1-carbonyl; piperidyl-carbonyl; morpholino; methyl-carbonyl; piperazinyl; methyl-alkylsulfonyl; piperidyl-alkylsulfonyl; 4-methyl-piperazinyl-carbonyl; dimethyl-amino-ethyl-amino-carbonyl; morpholino-carbonyl; morpholino-methyl; amino-carbonyl; propyl group-amino-carbonyl; hydroxyl-ethyl-amino-carbonyl; morpholino-ethyl-amino-carbonyl; 4-ethanoyl-piperazine-1-carbonyl; 4-amino-carbonyl-piperazine-1-carbonyl; phenyl-carbonyl; pyrrolidyl-l-carbonyl; propyl group-carbonyl; butyl; sec.-propyl-oxygen base-carbonyl; cyclohexyl-carbonyl; cyclopropyl-carbonyl; methyl-alkylsulfonyl; dimethyl-inferior phosphono (phosphinoyl); 4-methyl-piperazinyl-alkylsulfonyl; 1-oxo-indane-5-base; trimethylene oxide-3-alkylsulfonyl; amino-alkylsulfonyl and tetrahydrochysene-pyrans-4-alkylsulfonyl.

Describe preferred formula I compound among embodiment hereinafter and the table 1,2 and 3 in detail.Other preferred embodiment is selected from: N ⁶-(4-methanesulfinyl-phenyl)-N ²-methyl-N ²-(tetrahydrochysene-pyrans-4-yl)-9-thiazole-4-base-9H-purine-2, the 6-diamines; (4-methylsulfonyl-phenyl)-[2-(2-methyl-morpholine-4-yl)-9-thiazole-4-base-9H-purine-6-yl]-amine; 1-{4-[2-(2-methyl-morpholine-4-yl)-9-thiazole-4-base-9H-purine-6-base is amino]-phenyl }-ethyl ketone; [4-(dimethyl-inferior phosphono)-phenyl]-[2-(2-methyl-morpholine-4-yl)-9-thiazole-4-base-9H-purine-6-yl]-amine; Azetidine-1-base-4-[2-(4-morpholine-4-base-piperidines-1-yl)-9-thiazole-4-base-9H-purine-6-base is amino]-phenyl }-ketone; 1-(4-{2-[methyl-(1-methyl-piperidin-4-yl)-amino]-9-thiazole-4-base-9H-purine-6-base is amino }-phenyl)-ethyl ketone; 1-{4-[2-(2-methyl-morpholine-4-yl)-9-thiene-3-yl--9H-purine-6-base is amino]-phenyl }-ethyl ketone; (4-methylsulfonyl-phenyl)-[2-(4-morpholine-4-base-piperidines-1-yl)-9-thiazole-4-base-9H-purine-6-yl]-amine; N ⁶-(4-methylsulfonyl-phenyl)-N ²-methyl-N ²-(1-methyl-piperidin-4-yl)-9-thiazole-4-base-9H-purine-2, the 6-diamines; [2-(2-methyl-morpholine-4-yl)-9-thiazole-4-base-9H-purine-6-yl]-(4-morpholine-4-base-phenyl)-amine; N ²-methyl-N ²-(1-methyl-piperidin-4-yl)-N ⁶-(4-morpholine-4-base-phenyl)-9-thiazole-4-base-9H-purine-2, the 6-diamines; N ²-methyl-N ²-(1-methyl-piperidin-4-yl)-N ⁶-(4-morpholine-4-base-phenyl)-9-thiene-3-yl--9H-purine-2, the 6-diamines; [2-(2,2-dimethyl-morpholine-4-yl)-9-thiazole-4-base-9H-purine-6-yl]-(4-methylsulfonyl-phenyl)-amine; [2-(2,6-dimethyl-morpholine-4-yl)-9-thiazole-4-base-9H-purine-6-yl]-(4-methylsulfonyl-phenyl)-amine; [4-(dimethyl-inferior phosphono)-phenyl]-[2-(2-ethyl-morpholine-4-yl)-9-thiene-3-yl--9H-purine-6-yl]-amine; [4-(dimethyl-inferior phosphono)-phenyl]-[2-(2-methyl fluoride-morpholine-4-yl)-9-thiene-3-yl--9H-purine-6-yl]-amine; [2-(2,6-dimethyl-morpholine-4-yl)-9-thiazole-4-base-9H-purine-6-yl]-[4-(dimethyl-inferior phosphono)-phenyl]-amine; [2-(2,6-dimethyl-morpholine-4-yl)-9-thiene-3-yl--9H-purine-6-yl]-[4-(dimethyl-inferior phosphono)-phenyl]-amine; [4-(dimethyl-inferior phosphono)-phenyl]-[2-(2-methyl-morpholine-4-yl)-9-thiene-3-yl--9H-purine-6-yl]-amine; [4-(dimethyl-inferior phosphono)-phenyl]-[2-(3-methyl-piperidines-1-yl)-9-thiazole-4-base-9H-purine-6-yl]-amine; N ⁶-(4-methylsulfonyl-phenyl)-N ²-methyl-N ²-pyridine-2-ylmethyl-9-thiene-3-yl--9H-purine-2, the 6-diamines; N ²-methyl-N ⁶-(4-morpholine-4-base-phenyl)-N ²-pyridine-2-ylmethyl-9-thiene-3-yl--9H-purine-2, the 6-diamines; (2-azepine ring-1-in heptan base-9-thiazole-4-base-9H-purine-6-yl)-[4-(dimethyl-inferior phosphono)-phenyl]-amine; N ²-cyclohexyl-N ⁶-[4-(dimethyl-inferior phosphono)-phenyl]-N ²-methyl-9-thiazole-4-base-9H-purine-2, the 6-diamines; N ⁶-(4-methylsulfonyl-phenyl)-N ²-methyl-N ²-(tetrahydrochysene-pyrans-4-yl)-9-thiazole-4-base-9H-purine-2, the 6-diamines; N ⁶-(4-methylsulfonyl-phenyl)-N ²-pyridine-2-ylmethyl-9-thiazole-4-base-9H-purine-2, the 6-diamines; N ²-cyclohexyl-N ⁶-(4-methanesulfinyl-phenyl)-N ²-methyl-9-thiazole-4-base-9H-purine-2, the 6-diamines; R-(4-methanesulfinyl-phenyl)-[2-(2-methyl-morpholine-4-yl)-9-thiazole-4-base-9H-purine-6-yl]-amine; N ⁶-(4-methylsulfonyl-phenyl)-N ²-methyl-N ²-pyridine-2-ylmethyl-9-thiazole-4-base-9H-purine-2, the 6-diamines; 4-[6-(4-methylsulfonyl-phenyl amino)-2-(methyl-pyridine-2-ylmethyl-amino)-purine-9-yl]-phenyl }-methyl alcohol; R-(4-methylsulfonyl-phenyl)-[2-(2-methyl-morpholine-4-yl)-9-thiazole-4-base-9H-purine-6-yl]-amine; R-4-[2-(2-methyl-morpholine-4-yl)-9-thiazole-4-base-9H-purine-6-base is amino]-benzsulfamide; 4-[6-(4-methylsulfonyl-phenyl amino)-2-(2-methyl-morpholine-4-yl)-purine-9-yl]-phenyl }-methyl alcohol.

Pharmacology and application

Compound of the present invention suppresses the active of Flt3 receptor tyrosine kinase and can be used for treating disease or the illness that FLT3 activity wherein works to the pathology and/or the symptom of disease at this point.

Flt3 is the member of III receptor Tyrosylprotein kinase (RTK) family.Flt3 (fms-sample Tyrosylprotein kinase) is also referred to as FLk-2 (fetal livers kinases 2).It is unusual to be documented in grownup and the leukemia of children Flt3 expression of gene, and comprising acute myeloid leukaemia (AML), having three is AML (AML/TMDS), acute lymphoblastic leukemia (ALL) and the myelodysplastic syndrome (MDS) of myelodysplasia.In about 35% the patient who suffers from acute myeloblastic leukemia (AML), observed the sudden change of Flt3 receptor activation, and relevant with poor prognosis.Modal sudden change comprises in the frame in the nearly membrane structure territory duplicates, and also has the patient of 5-10% to have point mutation on 835 l-asparagines.These sudden changes all composing type activation with the tyrosine kinase activity of FIt3 are relevant, and do not having in the presence of the part generation breed and the viability signal.Verified, the healing chance of expressing the patient of mutant acceptor reduces.Therefore, the cumulative evidence that exists overactive (sudden change) Flt3 kinase activity in human leukemia and myelodysplastic syndrome, to act on.This impels the applicant to seek new Flt3 acceptor inhibitor, as the patient that operability almost can't be provided these present pharmacotherapys with before to the patient's that can utilize the failure of pharmacotherapy and/or stem cell transplantation therapy at present possible treatment means.

Leukemia is generally by due to acquired (nongenetic) gene damage of prematurity hematopoietic cell DNA in marrow, lymphoglandula, spleen or other blood and the immunity system organ.Its effect is: accelerating growth in the cell maturation and retardance, cause being known as the cellular accumulation of " leukemia meiocyte (leukemic blast) ", and it does not play the normal plasma cell effect; With can not produce normal myelocyte, cause red corpuscle (anaemia), thrombocyte and normal white corpuscle to lack.Parent cell is generally produced by marrow and develops into mature blood cell usually, account for all myelocytic 1%.In leukemia, parent cell is can't be suitably ripe and accumulate in marrow.In acute myeloid leukaemia (AML), they are called myeloblast, and in acute lymphoblastic leukemia (ALL), they are called lymphocytoblast.Another kind of leukemia is for mixing the leukemia (MLL) of pedigree (mixed-lineage).

Term " having three is the AML (AML/TMDS) of myelodysplasia " relates to a kind of uncommon leukemia form, it is characterized in that acute leukemia follow DH, to the Low Response of inducing chemotherapy and with the tendency of simple myelodysplastic syndrome recurrence.

Term " myelodysplastic syndrome (MDS) " relates to one group of blood disorder, and wherein marrow stops to work orderly, and causes healthy blood cell quantity to lack.Comparing with a large amount of leukemia that produce of one type hemocyte, is arbitrarily and sometimes that all types of hemocytes all are affected in MDS.Have at least every year 10,000 new cases to occur in the U.S..Be diagnosed as among the patient of MDS and acute myeloid leukaemias take place up to 1/3rd.Owing to this reason, the preleukemia that this disease being called sometimes.Myelodysplastic syndrome is also referred to as the myelodysplasia myelocyte sometimes and generates unusual or original oligodendroglia leukemia.When the parent cell of high quantity was retained in the marrow, MDS was also referred to as insidiousness (smoldering) leukemia.

Myelodysplastic syndrome such as leukemia are due to the gene damage of unicellular DNA in the marrow.Some that occurs among the MDS patient in the karyomit(e) is unusual.These are called transposition unusually, and it takes place when being connected a chromosomal part fracture and with the breaking portion of coloured differently body.In acute myeloid leukaemia, find identical defective usually.Yet MDS is different with leukemia, because all hemocytes of patient are all unusual, and all derives from the stem cell of identical infringement.In the leukaemic, marrow contains ill and mixture healthy blood cell.

At present, use the cytotoxicity chemotherapeutic of high dosage such as cytosine arabinoside and daunorubicin treatment AML and late period myelodysplastic syndrome.Such therapy makes about 70% patient enter the hematology remission.Yet,, in entering the patient of alleviation, surpass half and recurred afterwards although give chemotherapy for a long time.Nearly all patient who fails to enter alleviation at first or recur after obtaining alleviation finally dies from leukemia.Bone marrow transplantation can be cured the patient who accepts this operation who reaches 50-60%, 1/3rd suitable accepts graft but only have an appointment in suffering from all patients of AML or MDS.Press for new and effectively medicine treat and use standard treatment to fail to enter the patient of the patient of alleviation, later stage recurrence and the unfavorable patient of stem cell transplantation.In addition, effective novel drugs can be joined in the standard treatment, and rational expectation its chemotherapy of inducing of all patients will be improved.

FGFR3 is the integral part by the tyrosine kinase receptor family of the structurally associated of 4 kinds of different genes codings.Specificity point mutation in the different structure territory of FGFR3 gene causes acceptor composing type activation, and with the bladder relevant with cervical cancer (23 roll up for Cappellen etc., Nature) of autosomal dominant bone disorders, multiple myeloma and larger proportion.The FGFR3 target growth plate cartilage that activation suddenlys change and mouse is activated that is arranged in mouse FGFR3 gene can cause nanism.Similar with our viewpoint, the target of FGFR3 breaks and causes long bone and vertebra overgrowth in the mouse.In addition, the multiple myeloma cells of 20-25% contains t (4; 14) (p16.3; Q32.3) chromosome translocation, breaking point are positioned at the 50-100kb place in FGFR3 kinetochore at 4p16.In rare case of multiple myeloma, had been found that observed FGFR3 activation sudden change in bone disorders in the past, and followed this chromosome translocation all the time.Recently, in the transitional cell bladder carcinoma cell line of vast scale and some cervical cancer cell, identified FGFR3 missense somatic mutation (R248C, S249C, G372C and K652E), and in fact they be that the neonatal period reproduction activation sudden change of fatefulue nanism form is identical with causing the lethality skeleton development bad.Compound of the present invention can be by more effective and can multiple myeloma be had treatment effectiveness, by avoiding changing the cystectomy of life bladder cancer be had treatment effectiveness than present therapy, and those are wished that the cervical cancer among the patient of the following fertility of protection has treatment effectiveness.

Compound of the present invention not only can be as the material that suppresses tumour; for example in small cell lung cancer; and can be used as promoting agent and be used for the treatment of non-malignant proliferation venereal disease disease such as atherosclerosis, thrombosis, psoriatic, scleroderma and fibrosis; and be used to protect stem cell; for example resist the hemotoxin effect of chemotherapeutics such as 5 FU 5 fluorouracil, and be used for asthma.Compound of the present invention especially can be used for the treatment of the disease that inhibition is replied to pdgf receptor kinase.

Compound of the present invention shows useful effect in treatment in by the illness of transplanting due to for example allograft, and described illness is tissue rejection especially, as bronchiolitis obliterans (OB) especially, i.e. and the chronic rejection of allos lung transplantation thing.Opposite with the patient who does not suffer from OB, those patients that suffer from OB show PDGF concentration rising in bronchoalveolar lavage fluid usually.

Compound of the present invention is also to the disease effective (wherein PDGF and PDGF-R usually also work) relevant with propagation with vascular smooth muscle cells migration, as restenosis and atherosclerosis.These to the effect of vascular smooth muscle cell proliferation in the external and body or migration and result can by give compound of the present invention, can also by study it to mechanical injuries in the body after vascellum tunica interna incrassation be used for confirm.

The trk family of neurotrophin acceptor (trkA, trkB, trkC) promotes survival, growth and the differentiation of neurone and non-neuron tissue.Express in the monocyte of α cell, lymphoglandula and the spleen of the neuroendocrine type cell of TrkB albumen in small intestine and colon, pancreas and scavenger cell and the epidermal granular layer (Shibayama and Koizumi, 1996).The proteic expression of TrkB is relevant with the unfavorable development of wilms' tumor and neuroblastoma.In addition, TkrB expresses in carcinous prostatic cell, and does not express in normal cell.The signal transduction path downstream of Trk acceptor relates to the activation cascade (Sugimoto etc., 2001) of MAPK by Shc, activatory Ras, ERK-1 and ERK-2 gene and PLC-gammal transduction pathway.

Kinases c-Src conducts the carcinogenic signal of many acceptors.For example, EGFR or the HER2/neu overexpression in tumour causes the composing type activation of c-src, and it is the feature of malignant cell, but does not exist in normal cell.On the other hand, the mouse that c-src expresses defective shows the Osteopetrosis phenotype, has shown that the key of c-src in the osteoclast function participates in possible related with in associated conditions.

Fibroblast growth factor receptor3 has shown to be brought into play negative regulating effect and suppresses chondrocyte proliferation osteogenesis.Thanatophoric dysplasia is to be caused by the sudden change of the difference in the fibroblast growth factor receptor3, and a kind of sudden change is the composing type tyrosine kinase activity that TDII FGFR3 has transcriptional factors Statl, cause cell cycle inhibitor expression, growth-inhibiting and osteodysplasty (Su etc., Nature, 1997,386,288-292).FGFR3 also expresses in multiple myeloma type cancer usually.

Lck works in the T-cell signaling.The ability that lacks the mice develop thymocyte of Lck gene.Lck is as the function prompt of T-cell signaling positivity activator: the Lck inhibitor can be used for the treatment of autoimmune disease, as rheumatoid arthritis.

According to above-mentioned, the present invention further provides the method for in the object of needs treatment, preventing or treating above-mentioned disease or illness, this method comprises formula I compound or its pharmacy acceptable salt of described object being treated significant quantity.With regard to above-mentioned any purposes, required dosage will change according to administering mode, the disease specific of being treated and required effect.

Administration and pharmaceutical composition

Generally speaking, by arbitrarily commonly used and acceptable manner as known in the art, separately or with one or more therapeutical agent couplings, give compound of the present invention with the treatment significant quantity.The treatment significant quantity can broadly change according to the different of the effect of disease severity, age and relevant health condition, the compound used therefor of object and other factors.Generally speaking, dosage every day of about 0.03-2.5mg/kg body weight shows the effect that obtains general satisfaction.In than large mammals, for example people, be fit to every day dosage in the scope of the about 100mg of about 0.5mg-, for example divide 4 every day at the most with divided dose or with the administration expediently of slowly-releasing form.The suitable unit dosage forms that is used for oral administration comprises about 1-50mg active ingredient.

Compound of the present invention can be used as pharmaceutical composition by any conventional route administration, and is for example oral particularly through enteral administration, for example with tablet or capsule form; Or by parenteral admin, for example with Injectable solution or suspension form; By local mode, for example with lotion, gel, ointment or cream, or with nose usefulness or suppository form.The The compounds of this invention that comprises free form or pharmacy acceptable salt form and the pharmaceutical composition of at least a pharmaceutically acceptable carrier or thinner can be in a usual manner, by mix, granulation or coating method preparation.For example, oral compositions can be tablet or gelatine capsule, they comprise active ingredient and: a) thinner, for example lactose, glucose, sucrose, mannitol, sorbyl alcohol, Mierocrystalline cellulose and/or glycine; B) lubricant, for example silicon-dioxide, talcum, stearic acid, its magnesium or calcium salt and/or polyoxyethylene glycol; The c that also has that is used for tablet) tackiness agent, for example neusilin, starch paste, gelatin, tragakanta, methylcellulose gum, Xylo-Mucine and/or polyvinylpyrrolidone; If desired, also have d) disintegrating agent, for example starch, agar, alginic acid or its sodium salt or effervescent mixture; And/or e) absorption agent, tinting material, correctives and sweeting agent.Injectable composition can be moisture isotonic solution or suspension, and suppository can be by lipomul or suspension preparation.Composition can be sterilization and/or contain adjuvant, as sanitas, stablizer, wetting agent or emulsifying agent, dissolution accelerator, be used to regulate the salt and/or the buffer reagent of osmotic pressure.In addition, they can also contain upward valuable material of other treatment.The appropriate formulation that is used for transdermal administration comprises the The compounds of this invention and the carrier of significant quantity.Carrier can comprise that absorbable pharmaceutically acceptable solvent is to help the skin by the host.For example, transdermal device is a form of bandage, comprise backing part (backing member), contain the storage of the optional and carrier of compound, optional rate-controlling barrier to be being delivered to host skin with described compound with controlled and set rate in prolonging period, and make device and skin fixed means.Can also use the matrix preparation capable of permeating skin.The appropriate formulation that the part is used for skin for example and eye is preferably the aqueous solution well-known in the art, ointment, creme or gel.This class preparation can contain solubilizing agent, stablizer, tension-elevating agent, buffer reagent and sanitas.

Compound of the present invention can be with one or more therapeutical agents, comprise radiotherapy and bone marrow transplantation associating (drug regimen), give with the treatment significant quantity.Can be with the limiting examples of the compound of The compounds of this invention coupling: the cytotoxicity chemotherapeutic, as cytosine arabinoside, daunorubicin, endoxan, VP-16, mitoxantrone, daunorubicin, cytosine arabinoside, methotrexate, vincristine(VCR), 6-Tioguanine, Ismipur, taxol etc.; The angiogenesis inhibitor medicine is such as but not limited to cyclooxygenase inhibitors, as celecoxib; Immunomodulatory or anti-inflammatory substance, for example ciclosporin, rapamycin or ascosin, or its immunosuppressor analogue, for example Ciclosporin A (CsA), ciclosporin G, FK-506, rapamycin or suitable compound; Reflunomide; Endoxan; Azathioprine; Methotrexate; Bai Ruikuaer; Take fluorine Lip river rice; Mizoribine; The wheat phenolic acid; The Mycophenolic Acid morpholine ethyl ester; The 15-Gusperimus; The monoclonal antibody of immunosuppressor antibody, especially leukocyte receptors, for example MHC, CD2, CD3, CD4, CD7, CD25, CD28, B7, CD45, CD58 or its part, or other immunomodulatory compounds are as CTLA41g.In addition, compound of the present invention can with the drug combination of other signal transduction inhibitor or other target oncogene to obtain collaborative significantly therapy.

When compound of the present invention and other therapies being united when giving, the dosage of common administered compound changes according to the type of the common medicine that uses, used concrete medicine, the disease of being treated etc. certainly.

The present invention also provides drug regimen, and medicine box for example comprises: a) first kind of promoting agent is the free form that discloses of this paper or the The compounds of this invention of pharmacy acceptable salt form; And b) at least a coagent.This medicine box can comprise the administration specification sheets.

Term used herein " co-administered " or " Combined Preparation " etc. give selected therapeutical agent in order to comprise to single patient, and in order to comprise that promoting agent is not necessarily by identical route of administration or the treatment plan that gives at the same time.

" drug regimen " used herein refers to and mixes or merge more than one active ingredients and the product that obtains, and comprises fixing and unfixed active ingredient combination.Term " fixed combination " refers to active ingredient, for example formula I compound and coagent with the form of single entities or dosage simultaneously to patient's administration.Term " unfixed combination " refer to active ingredient, for example formula I compound and coagent as the entity that separates simultaneously, common or do not have the specified time restriction successively to patient's administration, wherein this class administration provides 2 kinds of compounds of treatment level of significance in patient's body.The latter also is applicable to drug cocktail therapy (treatment) (cocktail therapy), for example gives active ingredient more than 3 kinds or 3 kinds.

The method for preparing The compounds of this invention

The present invention also comprises the method for preparing The compounds of this invention.In described reaction, when in end product, needing them, have necessary protective reaction functional group, for example hydroxyl, amino, imino-, sulfo-or carboxyl unnecessarily participate in reaction to avoid them.Can use protecting group commonly used according to standard practices, for example referring to T.W.Greene and P.G.M.Wuts: " protecting group in the organic chemistry ", John Wiley and Sons, 1991.

R wherein ₅For the formula I compound of hydrogen can prepare by carrying out following reaction scheme I:

Reaction scheme I

R wherein ₁, R ₂, R ₃And R ₄As in the summary of the invention formula I being defined, PG represents that nitrogen-protecting group (for example tetrahydrochysene-pyrans-2-base etc.) and Z represent halogen, for example iodine or chlorine, preferred chlorine.

Formula 3 compounds can be by making formula 2 compounds and NHR ₃R ₄In the presence of suitable solvent (for example ethanol, butanols, THF etc.), use suitable alkali (for example DIEA, Na ₂CO ₃Deng) reaction and prepare.Formula 4 compounds can be by making formula 3 compounds and R ₁H in the presence of suitable solvent (for example DME, ethanol, butanols, THF etc.), optional appropriate catalyst (for example palladium catalyst etc.), the suitable alkali of use (for example DIEA, Na ₂CO ₃Deng) reaction and prepare.Formula I compound can by at first in the presence of the appropriate catalyst (for example p-TSA etc.), in suitable solvent (for example MeOH etc.), remove protecting group (PG) and prepare.Reaction is proceeded, and makes formula 4 compounds of deprotection and Y wherein represent the R of halogen, for example iodine, bromine and chlorine ₂The Y reaction.Be reflected under the about 110 ℃ temperature of about 70-, use suitable alkali (for example potassiumphosphate etc.), in the presence of suitable solvent (for example DMF, diox etc.), carry out, and can consuming timely reach 24 hours and finish.

Formula I compound can prepare by carrying out following scheme II:

Scheme II

R wherein ₁, R ₂, R ₃And R ₄Define as mutual-through type I in the summary of the invention, PG represents that nitrogen-protecting group (for example tetrahydrochysene-pyrans-2-base etc.) and Z represent halogen, for example iodine or chlorine, preferred chlorine.

Formula 3 compounds can be by making formula 2 compounds and NHR ₃R ₄In the presence of suitable solvent (for example ethanol, butanols, THF etc.), use suitable alkali (for example DIEA, Na ₂CO ₃Deng) reaction and prepare.Formula 5 compounds can by at first in the presence of the appropriate catalyst (for example p-TSA etc.), in suitable solvent (for example MeOH etc.), remove protecting group (PG) and prepare.Reaction is proceeded, and makes formula 3 compounds and the R of deprotection ₂B (OH) ₂In the presence of suitable solvent (for example diox, methylene dichloride etc.) and appropriate catalyst (for example venus crystals etc.), use suitable alkali (for example pyridine, TEA etc.) reaction.Be reflected under the about 80 ℃ temperature of about 20-and carry out, and can consuming timely reach 168 hours and finish.Formula I compound can be by making formula 5 compounds and R ₁H in the presence of suitable solvent (for example butanols, ethanol etc.), use suitable alkali (for example DIEA, Na ₂CO ₃Deng) reaction and prepare.

Formula I compound can prepare by carrying out following scheme II I:

Scheme II I

R wherein ₁, R ₂, R ₃And R ₄Define and Z represents halogen for example iodine or chlorine, preferred chlorine as mutual-through type I in the summary of the invention.

Formula 7 compounds can be by making formula 6 compounds and R ₂B (OH) ₂In the presence of suitable solvent (for example diox, methylene dichloride etc.) and appropriate catalyst (for example venus crystals etc.), use suitable alkali (for example pyridine, TEA etc.) reaction and prepare.Be reflected under the about 80 ℃ temperature of about 20-and carry out, and can consuming timely reach 168 hours and finish.Formula 5 compounds can be by making formula 7 compounds and NHR ₃R ₄In the presence of suitable solvent (for example DME, ethanol, butanols, THF etc.), optional use appropriate catalyst (for example palladium catalyst etc.) and use suitable alkali (for example DIEA, Na ₂CO ₃Deng) reaction and prepare.Formula I compound can by make formula 5 compounds and R1H in the presence of suitable solvent (for example ethanol, butanols, THF etc.), use suitable alkali (for example DIEA, Na ₂CO ₃Deng) reaction and prepare.

Other method of preparation The compounds of this invention

Compound and pharmaceutically acceptable mineral acid or organic acid reaction by making free alkali form can become pharmaceutically-acceptable acid addition with compound of the present invention.Perhaps, by compound and pharmaceutically acceptable mineral alkali or the organic bases reaction that makes free acid form, can prepare the pharmaceutically acceptable base addition salt of The compounds of this invention.Perhaps, can use the salt of raw material or intermediate to prepare the salt form of The compounds of this invention.

The free acid of The compounds of this invention or free alkali form can be respectively by corresponding base addition salt or the preparations of acid salt form.For example, by handling, the The compounds of this invention of acid salt form can be changed into corresponding free alkali with suitable alkali (for example solution of ammonium hydroxide, sodium hydroxide etc.).By handling, the The compounds of this invention of base addition salt form can be changed into corresponding free acid with suitable acid (for example hydrochloric acid etc.).

Not the The compounds of this invention of oxidised form can be by the N-oxide compound of The compounds of this invention, in 0-80 ℃ and suitable inert organic solvents (for example acetonitrile, the ethanol, diox aqueous solution etc.), handle and prepare with reductive agent (for example sulphur, sulfurous gas, triphenyl phosphine, lithium borohydride, sodium borohydride, phosphorus trichloride, phosphorus tribromide etc.).

The prodrug derivatives of The compounds of this invention can (for example further be described in detail referring to Saulnier etc. by well known to a person skilled in the art method preparation, (1994), Bioorganic andMedicinal Chemistry Letters, the 4th volume, p.1985).For example, by making deutero-The compounds of this invention not and suitable carbamyl reagent (for example 1,1-acyloxyalkylcarbanochloridate, carbonic acid be right-nitrophenyl ester etc.) reaction, can prepare the suitable precursor medicine.

The protected derivative of The compounds of this invention can prepare by well known to a person skilled in the art mode.The detailed description that is applicable to the technology that generates protecting group and be removed is found in T.W.Greene, " protecting group in the organic chemistry " the 3rd edition, John Wiley and Sons, Inc., 1999.

In the process of the inventive method, can expediently compound of the present invention be made or make it to form solvate (for example hydrate).As dioxin, tetrahydrofuran (THF) or methyl alcohol recrystallization from water/ORGANIC SOLVENT MIXTURES, can prepare the hydrate of The compounds of this invention by with an organic solvent expediently.

Compound of the present invention can be prepared as follows into their independent steric isomer: make the racemic mixture and the reaction of optically active resolving agent of compound, it is right to generate diastereomeric compound, separates diastereomer and reclaims optically pure enantiomorph.Split preferred dissociable mixture (for example crystallization diastereo-isomerism salt) although can use the covalency diastereo-isomerism derivative of The compounds of this invention to carry out enantiomorph.Diastereomer has different physical property (for example fusing point, boiling point, solubleness, reactivity etc.) and can be easily by utilizing these differences to separate.Diastereomer can be by chromatography or preferred by the separation/disassemble technique separation based on dissolubility difference.Then by not causing the practical way of racemization to reclaim optically pure enantiomorph and resolving agent arbitrarily.The more detailed description that is applicable to the technology of the steric isomer that splits compound from racemic mixture is found in Jean Jacques, Andre Collet, Samuel H.Wilen, " enantiomorph, racemoid and fractionation ", John Tiley and Sons, Inc., 1981.

Overview, formula I compound can prepare by the method that comprises the following steps:

(a) those methods of reaction scheme I, II and III for example make formula 5 compounds and R according to scheme II or III ₁The H coupling; With

(b) optional compound of the present invention is changed into pharmacy acceptable salt;

(c) optional salt form with The compounds of this invention changes into salt-independent shape;

(d) optional not oxidised form with The compounds of this invention changes into the acceptable N-oxide compound of pharmacy;

(e) optional N-oxide form with The compounds of this invention changes into its not oxidised form;

(f) the optional independent isomer that from isomer mixture, splits The compounds of this invention;

(g) optional underivatized compound of the present invention is changed into pharmaceutically acceptable prodrug derivatives; With

(h) optional prodrug derivatives with The compounds of this invention changes into its not derivative form.

With regard to the production of raw material is not described especially, these compounds be known or can according to similar mode of method as known in the art or as the method preparation that hereinafter discloses among the embodiment.

Those skilled in the art will appreciate that above-mentioned conversion only is the representative of preparation The compounds of this invention method and can uses other well-known method similarly.

Embodiment

The following example provides the detailed description of representative compounds preparation, but it is provided with explaining and non-limiting the present invention.

Embodiment 1

4-[2-(4-amino-cyclohexyl amino)-9-phenyl-9H-purine-6-base is amino]-phenyl }-piperidines-1-base -ketone

To 0 ℃ piperidines (18.0g, 211.8mmol) divide in the solution in methylene dichloride (360mL) the careful adding of several parts 4-nitrobenzoyl chloride (18.6g, 100mmol).This reaction mixture was at room temperature stirred 10 minutes, and (1%, 2 * 200mL) solution and water (300mL) wash and use Na after this to use HCl ₂SO ₄Dry.Behind evaporating solvent, obtain (4-nitro-phenyl)-piperidines-1-base-ketone (23.2g, 99%) and be directly used in hydrogenation (the 400mL ethanol that contains 1.0g 10%Pd/C).After filtering catalyst and ethanol evaporation, obtain (4-amino-phenyl)-piperidines-1-base-ketone (19.6g, 96%).

With 2, the 6-dichloropurine (18.80g, 100mmol), 3,4-dihydro-2H-pyrans (12.62g, 150mmol), the tosic acid monohydrate (1.90g, 10mmol) and the mixture of anhydrous methylene chloride (200mL) at room temperature stirred 4 hours.After the filtration, it is used Na ₂CO ₃(10% aqueous solution, 100mL), water (100mL) washs and use Na ₂SO ₄Dry.Evaporating solvent grinds in ethyl acetate (5mL) and hexane (60mL) subsequently, brings out precipitation, after filtration, obtains 2,6-two chloro-9-(tetrahydrochysene-pyrans-2-yl)-9H-purine (24.01g, 88%).

With 2,6-two chloro-9-(tetrahydrochysene-pyrans-2-yl)-9H-purine (5.44g, 20mmol), (4-amino-phenyl)-piperidines-1-base-ketone (4.08g, 20mmol), the mixture of diisopropylethylamine (24mmol) and ethanol (100mL) refluxed 24 hours.Add anti-form-1 then, (6.84g's 4-cyclohexane diamine again 60mmol) and diisopropylethylamine (24mmol), and refluxed this mixture 24 hours.Handle the oily resistates that obtains after the ethanol evaporation with ethyl acetate (250mL) and water (200mL).(2 * 100mL) aqueous phase extracted are used Na with ethyl acetate ₂SO ₄The dry organic phase that merges.After evaporation, (3.80g 20mmol) handles the oily resistates that obtains 4 hours and monitoring reaction, up to finishing deprotection to be used in tosic acid monohydrate in the methyl alcohol (100mL) at 55 ℃.

Add diisopropylethylamine with this mixture that neutralizes.To the oily resistates carry out column chromatography (EtOAc: MeOH=9: 1, CH then ₂Cl ₂: MeOH (containing～7N ammonia)=9: 1), obtain 2-(4-amino-cyclohexyl amino)-6-[4-(piperidines-1-carbonyl)-phenyl amino]-9H-purine (6.50g, 75%).

To contain as above 2-(4-amino-cyclohexyl amino)-6-[4-(piperidines-1-the carbonyl)-phenyl amino of preparation]-9H-purine (86.8mg, 0.2mmol), cupric iodide (I) (38.2mg, 0.2mmol) and potassiumphosphate (170mg, the degassing of the reaction flask of mixture 0.8mmol) also refills exsiccant nitrogen.Be added in N ' N '-dimethyl-ethylenediamine among the DMF (700 μ L) (35.3mg, 43 μ L, 0.4mmol) and iodobenzene (40.8mg, 0.2mmol) and this mixture is spent the night 88 ℃ of stirrings.Adding AcOH-MeOH (1: 10,1.5mL) with this mixture that neutralizes, filter by syringe filter subsequently.Carry out column chromatography (EtOAc: MeOH=9: 1. CH then ₂Cl ₂: MeOH (containing～7N ammonia)=9: 1), obtain { 4-[2-(4-amino-ring Hexyl amino)-9-phenyl-9H-purine-6-base is amino]-phenyl }-piperidines-1-base-ketone, be solid; ¹HNMR 400MHz (CD ₃OD) d 8.03 (s, 1H), 7.90-7.95 (m, 2H), 7.75-7.65 (m, 2H), 7.50-7.42 (m, 2H), 7.38-7.30 (m, 3H), 3.80-3.50 (m, 5H), 2.83-2.73 (m, 1H), 2.15-2.05 (m, 2H), 1.95-1.90 (m, 2H), 1.70-1.40 (m, 6H), and 1.40-1.20 (m, 4H); MSm/z 511.3 (M+1).

Embodiment 2

[4-(2-chloro-9-phenyl-9H-purine-6-base is amino)-phenyl]-piperidines-1-base-ketone

With 2,6-two chloro-9-(tetrahydrochysene-pyrans-2-yl)-9H-purine (10g, 36.6mmol), (4-amino-phenyl)-piperidines-1-base-ketone (7.48g, 36.6mmol) and diisopropylethylamine (9.5g, 73.5mmol) mixture in ethanol (110mL) refluxes and to spend the night.This mixture is cooled to room temperature and concentrated in a vacuum, obtains [4-(2-chloro-9H-purine-6-base is amino)-phenyl]-piperidines-1-base-ketone (14.7g, 91%), be the dark yellow solid.

Will [4-(2-chloro-9H-purine-6-base amino)-phenyl]-piperidines-1-base-ketone (10g, 22.7mmol) and right-toluenesulphonic acids monohydrate (0.86g, 4.5mmol) mixture in methyl alcohol (100mL) stirred 2 hours at 50 ℃.This mixture is cooled to room temperature and is suspended in the methyl alcohol.Collecting precipitation and with ethyl acetate washing obtains [4-(2-chloro-9H-purine-6-base is amino)-phenyl]-piperidines-1-base-ketone (7.69g, 95%), is light yellow solid.

(add [4-(2-chloro-9H-purine-6-base is amino)-phenyl]-piperidines-1-base-ketone (4g in the suspension in the 4.2g) Zai diox (35mL) to the activatory molecular sieve, 11.2mmol), phenyl is for boric acid (2.73g, 22.4mmol), venus crystals (3.05g, 16.8mmol) and pyridine (3.54g, 44.8mmol).With this mixture at room temperature stir spend the night and then 40 ℃ the heating 5 hours.This mixture is cooled to room temperature, with THF (50mL) dilution, by diatomite filtration and use methanol wash.Under reduced pressure concentrated filtrate also by flash column chromatography purifying resistates (MeOH/ methylene dichloride=1/50), obtains [4-(2-chloro-9-benzene Base-9H-purine-6-base is amino)-phenyl]-piperidines-1-base-ketone(3.89g, 80%) is yellow solid; ¹HNMR 400MHz (CDCl ₃) d 8.17 (s, 1H), 8.06 (s, 1H), 7.93 (d, 2H, J=8.8Hz), 7.69 (d, 2H, J=8.8Hz), 7.58 (d, 2H, J=8Hz), 7.49 (t, 3H, J=7.2Hz), 7.41 (d, 1H, J=7.2Hz), 2.93-2.90 (m, 4H), 2.18-1.96 (m, 2H), 1.58-1.53 (m, 4H), 1.35-1.29 (m, 2H); MS m/z 433.2 (M+1).

Embodiment 3

4-[2-(3-dimethylamino-tetramethyleneimine-1-yl)-9-phenyl-9H-purine-6-base is amino]-phenyl }-piperazine Pyridine-1-base-ketone

Will [4-(2-chloro-9-phenyl-9H-purine-6-base amino)-phenyl]-piperidines-1-base ketone (129mg, 0.3mmol) and 3-(dimethylamino)-tetramethyleneimine (103mg, 0.9mmol) mixture in 1-butanols (0.6mL) was 120 ℃ of stirrings 12 hours.This mixture is cooled to room temperature and under reduced pressure concentrated.By flash column chromatography purifying resistates (MeOH/ methylene dichloride=1/50), obtain { 4-[2-(3-dimethylamino Base-tetramethyleneimine-1-yl)-9-phenyl-9H-purine-6-base is amino]-phenyl }-piperidines-1-base-ketone(73.3mg, 49%) is dark pink solid; ¹H NMR 400MHz (MeOH-d ₄) d 8.22 (s, 1H), 7.95 (d, 2H, J=8.4Hz), 7.83 (d, 2H, J=7.6Hz), 7.53 (t, 2H, J=7.6Hz), 7.43 (d, 1H, J=7.6Hz), 7.40 (d, 2H, J=8.8Hz), and 4.04-3.96 (m, 1H), 3.94-3.83 (m, 1H), 3.70-3.36 (m, 6H), 2.95 (s, 6H), 2.51-2.46 (m, 1H), and 2.25-2.19 (m, 1H), 1.78-1.47 (m, 6H); MS m/z 511.3 (M+1).

Embodiment 4

4-(2-imidazoles-1-base-9-phenyl-9H-purine-6-base is amino)-phenyl] piperidines-1-base-ketone

In quartz reaction container (2mL), be added in [4-(2-chloro-9-phenyl-9H-purine-6-base-amino)-phenyl]-piperidines among the NMP (0.3mL)-1-base ketone (43mg, 0.1mmol) and imidazoles (20.4mg, 0.3mmol).Then this reaction vessel is put into microwave reactor chamber (Emrys optimizer) and 200 ℃ the irradiation 30 minutes.By preparation HPLC purifying crude product mixture, obtain 4-(2-imidazoles -1-base-9-phenyl-9H-purine-6-base is amino)-phenyl] piperidines-1-base-ketoneTrifluoroacetate (18.7mg), be light yellow solid; ¹H NMR 400MHz (MeOH-d ₄) d9.52 (s, 1H), 8.58 (s, 1H), 8.26 (s, 1H), 7.91 (d, 2H, J=6.8Hz), 7.86 (d, 2H, J=8.8Hz), 7.65 (m, 3H), 7.56 (d, 1H, J=7.6Hz), 7.51 (d, 2H, J=8.8Hz), 3.70-3.49 (m, 4H), 1.77-1.60 (m, 6H); MS m/z 465.3 (M+1).

Embodiment 5

4-[9-phenyl-2-(quinoline-3-base is amino)-9H-purine-6-base is amino]-phenyl }-piperidines-1-base-ketone

To in vitro adding [4-(2-chloro-9-phenyl-9H-purine-6-base is amino)-phenyl]-piperidines-1-base ketone (43mg, 0.1mmol), 3-quinolylamine (21.6mg, 0.15mmol), three (dibenzalacetones), two palladiums (0) (7mg, 0.008mmol), 2-(two-tert-butyl phosphino-) biphenyl (8.9mg, 0.03mmol), potassiumphosphate (100mg, 0.47mmol), vacuumize and anti-inflated with nitrogen.Under nitrogen, add DME (0.7mL).This reaction mixture was stirred 16 hours at 85 ℃.Gained light brown suspension is cooled to room temperature and passes through the preparation HPLC purifying, obtain { 4-[9-phenyl-2-(quinoline-3-base is amino)-9H-purine-6-base Amino]-phenyl }-trifluoroacetate of piperidines-1-base-ketone(24.5mg), be yellow solid; ¹H NMR400MHz (MeOH-d ₄) d 9.29 (d, 1H, J=2.4Hz), 9.13 (d, 1H, J=2.0Hz), 8.18 (s, 1H), 7.92 (d, 1H, J=8.4Hz), and 7.81-7.70 (m, 7H), 7.58 (t, 2H, J=8.0Hz), 7.48 (t, 1H, J=7.2Hz), 7.30 (d, 2H, J=8.4Hz), 3.87-3.35 (m, 4H), 1.80-1.43 (m, 6H); MS m/z 541.3 (M+1).

Embodiment 6

N ² -(4-amino-cyclohexyl)-N ⁶ -(4-morpholine-4-base-phenyl)-9-phenyl-9H-purine-2, the 6-diamines

Under 150 ℃ and vacuum with molecular sieve (4A, 12.0g) dried overnight and be cooled to room temperature.Add then 2-fluoro-6-chloro-purine (6.0g, 35mmol), phenyl for boric acid (8.3g, 70mmol), venus crystals (9.0g, 52mmol) and triethylamine (19mL mixes 140mmol) and in dry diox (100mL).With the drying tube that connects this reaction mixture was at room temperature stirred 2 days.After reaction is finished, this reaction mixture is diluted in methylene dichloride (200mL), filter and wash by Celite pad with methylene dichloride (200mL).Merge organic phase and remove and desolvate by rotary evaporation.By silica gel flash column chromatography purifying crude product, as eluent, obtain 2-fluoro-6-chloro-9-phenyl-9H-purine (2.1g, 24%) with hexane/ethyl acetate (2: 1), be faint yellow solid, MS m/z 249.1 (M+1).

With 2-fluoro-6-chloro-9-phenyl-9H-purine (50mg, 0.20mmol), 4-morpholine-4-base-phenyl amine (39mg, 0.22mmol) and diisopropylethylamine (35 μ L 0.2mmol) mix in 1-butanols (0.4mL).This is reflected at 80 ℃ stirred 2 hours, after this add trans 1, the 4-cyclohexane diamine (68mg, 0.6mmol) and diisopropylethylamine (70 μ L, 0.4mmol).This reaction mixture is spent the night 110 ℃ of stirrings.Remove by rotary evaporation and to desolvate.Crude mixture is dissolved in DMSO again and passes through the HPLC purifying, obtain N ² -(4-amino-cyclohexyl)-N ⁶ -(4-morpholine-4-base-phenyl)-9-phenyl-9H-purine-2,6- DiaminesTrifluoroacetate, be white powder; ¹H NMR 400MHz (DMSO-d ₆) δ 9.29 (s, 1H), 8.23 (1 1H), 7.84 (t, 4H, J=9.4Hz), 7.51 (t, 2H, J=8.0Hz), 7.35 (t, 1H, J=7.2Hz), 6.84 (d, 2H, J=9.2Hz), 6.48 (d, 1H, J=7.2Hz), 3.71 (t, 4H, J=4.8Hz), 3.57 (s, 1H), 3.01 (t, 4H, J=4.8Hz), 1.93 (d, 2H, J=12Hz), 1.77 (d, 2H, J=11.2Hz), 1.24 (m, 4H), 0.90 (t, 1H, J=7.2Hz); MS m/z 485.3 (M+1).

Embodiment 7

N ² -(4-amino-cyclohexyl)-N ⁶ -[3-(4-methyl-piperazine-1-yl)-phenyl]-9-phenyl-9H-purine -2, the 6-diamines

(1.0g 7mmol) mixes with 1-methyl-piperazine (2.0mL) and this reaction is added a cover, and stirs 2 hours at 190 ℃ with 1-chloro-3-nitro-benzene.After the reaction, remove excessive 1-methyl-piperazine, obtain crude product, be yellow oil by rotary evaporation.By the quick column purification crude product of silica gel, obtain 1.2g 1-methyl-4-(3-nitro-phenyl)-piperazine (productive rate 78%).

With 1-methyl-4-(3-nitro-phenyl)-piperazine (1.2g, 5.4mmol) be dissolved in methyl alcohol (50mL) and in this solution, add Pd/C (5%, 120mg).The hydrogen capsule is connected with flask.This solution at room temperature stirred spend the night.After reaction is finished, filter Pd/C, collect filtrate and concentrated by rotary evaporation, obtain 3-(4-methyl-piperazine-1-yl)-phenyl amine.

With 2-fluoro-6-chloro-9-phenyl-9H-purine (50mg, 0.20mmol), 3-(4-methyl-piperazine-1-yl)-phenyl amine (42mg, 0.22mmol) and diisopropylethylamine (35 μ L 0.2mmol) mix in 1-butanols (0.4mL).This is reflected at 80 ℃ stirred 2 hours, after this add trans 1, the 4-cyclohexane diamine (68mg, 0.6mmol) and diisopropylethylamine (70 μ L, 0.4mmol).This reaction mixture is spent the night 110 ℃ of stirrings., except that desolvating crude product is dissolved in DMSO again and passes through the HPLC purifying by rotary evaporation, obtain N ² -(4-amino-cyclohexyl)-N ⁶ -[3-(4-methyl-piperazine-1-yl)-phenyl]-9- Phenyl-9H-purine-2, the 6-diamines, be white powder; ¹H NMR 400MHz (DMSO-d ₆) δ 9.12 (s, 1H), 8.16 (s, 1H), 7.78 (d, 2H, J=6.0Hz), 7.58 (d, 1H, J=7.6Hz), 7.42 (m, 2H), 7.24 (m, 2H), 7.00 (t, 1H, J=8.0Hz), 6.48 (m, 2H), 3.53 (s, 1H), 3.25 (m, 4H), 3.01 (t, 4H, J=4.8Hz), 2.09 (s, 3H), 1.74 (m, 2H), 1.66 (s, 2H), 0.92 (m, 4H), 0.79 (t, 1H, J=7.2Hz); MS m/z 498.3 (M+1).

Embodiment 8

1-{4-[2-(2-methyl-morpholine-4-yl)-9-thiazole-4-base-9H-purine-6-base is amino]-phenyl }-ethyl ketone

With 1-(4-amino-phenyl)-ethyl ketone (1.0g, 7.4mmol) with 2-fluoro-6-chloro-9-(tetrahydrochysene-pyrans-2-yl)-9H-purine (1.90g, 7.4mmol), diisopropylethylamine (1.54mL, 8.9mmol) and propyl carbinol 50mL mix.This is reflected at 95 ℃ stirred 14 hours.Be cooled to room temperature and,, obtaining 1-{4-[2-fluoro-9-(tetrahydrochysene-pyrans-2-yl)-9H-purine-6-base amino by using the purified by flash chromatography crude product of MeOH/DCM (5%: 95%) except that after desolvating]-phenyl }-ethyl ketone, be white solid 2.49g.

1-{4-[2-fluoro-9-(tetrahydrochysene-pyrans-2-yl)-9H-purine-6-base is amino]-phenyl }-ethyl ketone (100mg, 0.28mmol) and 2-methyl-morpholine HCl salt (58mg, 0.45mmol), diisopropylethylamine (121 μ L, 0.70mmol) and the 5mL propyl carbinol mix.This is reflected at 100 ℃ stirred 14 hours.In cooling and except that after desolvating, by using the purified by flash chromatography crude product of EA/ hexane (1: 1), it is amino to obtain 1-{4-[2-(2-methyl-morpholine-4-yl)-9-(tetrahydrochysene-pyrans-2-yl)-9H-purine-6-base]-phenyl }-ethyl ketone, be yellow solid 115mg.

1-{4-[2-(2-methyl-morpholine-4-yl)-9-(tetrahydrochysene-pyrans-2-yl)-9H-purine-6-base is amino]-phenyl }-(115mg 0.26mmol) is dissolved in 10mL ethanol and mix with 200 μ L TFA to ethyl ketone.This is reflected at 60 ℃ stirred 2 hours.Be cooled to room temperature and remove fully desolvate and TFA after, with crude product and cupric iodide (I) (50mg, 0.26mmol) and potassiumphosphate (220mg, 0.8mmol) mixing and outgasing refills exsiccant nitrogen.Be added in the N ' among the DMF (4mL), N '-dimethyl-ethylenediamine (46mg, 0.52mmol) and the iodo-thiazole (53mg 0.26mmol), and stirs this mixture 14 hours at 90 ℃.After being cooled to room temperature, and adding AcOH-MeOH (1: 10,1.6mL) with this mixture that neutralizes, filter by syringe filter subsequently.Except that after desolvating, crude product is dissolved in DMSO and, obtains light solid 1-{4-[2-(2-methyl-morpholine-4-yl)-9-thiazole-4-base-9H-purine-6-base amino by the preparation HPLC purifying]-phenyl }-ethyl ketone 71mg. ¹H?NMR?600MHz(DMSO-d ₆)δ10.21(s，1H)，9.26(d，1H，J＝2.2)，8.60(s，1H)，8.27(d，1H，J＝2.0Hz)，8.07(d，2H，J＝8.8Hz)，7.95(d，2H，J＝8.8Hz)，4.50(dd，2H，J＝3.0Hz)，3.95(dd，1H，J＝2.6Hz)，3.59(m，2H)，3.04(m，1H)，2.72(m，1H)，2.54(s，3H)，1.22(d，3H，J＝6.2Hz)；MS?m/z?436.2(M+1)。

Embodiment 9

(4-methylsulfonyl-phenyl)-[2-(4-morpholine-4-base-piperidines-1-yl)-9-thiazole-4-base-9H-purine-6- Base]-amine

With 4-methylsulfonyl-phenyl amine (1.27g, 7.4mmol) with 2-fluoro-6-chloro-9-(tetrahydrochysene-pyrans-2-yl)-9H-purine (1.90g, 7.4mmol), diisopropylethylamine (1.54mL, 8.9mmol) and propyl carbinol 50mL mix.This is reflected at 95 ℃ stirred 14 hours.Being cooled to room temperature and,, obtaining [2-fluoro-9-(tetrahydrochysene-pyrans-2-yl)-9H-purine-6-yl]-(4-methylsulfonyl-phenyl)-amine, be white solid 2.75g by using the purified by flash chromatography crude product of MeOH/DCM (7%: 93%) except that after desolvating.

With [2-fluoro-9-(tetrahydrochysene-pyrans-2-yl)-9H-purine-6-yl]-(4-methylsulfonyl-phenyl)-amine (110mg; 0.28mmol) and 4-piperidin-4-yl-morpholine (76mg; 0.45mmol), diisopropylethylamine (121 μ L, 0.70mmol) and the 5mL propyl carbinol mix.This is reflected at 100 ℃ stirred 14 hours.In cooling and except that after desolvating; by using the purified by flash chromatography crude product of EA/ hexane (6: 4); obtain (4-methylsulfonyl-phenyl)-[2-(4-morpholine-4-base-piperidines-1-yl)-9-(tetrahydrochysene-pyrans-2-yl)-9H-purine-6-yl]-amine, be yellow solid 145mg.

(145mg 0.26mmol) is dissolved in 10mL ethanol and mix with 200 μ L TFA with (4-methylsulfonyl-phenyl)-[2-(4-morpholine-4-base-piperidines-1-yl)-9-(tetrahydrochysene-pyrans-2-yl)-9H-purine-6-yl]-amine.This is reflected at 60 ℃ stirred 2 hours.Be cooled to room temperature and remove fully desolvate and TFA after, with crude product and cupric iodide (I) (50mg, 0.26mmol) and potassiumphosphate (220mg 0.8mmol) mixes, outgases also to fill again and adds exsiccant nitrogen.Be added in the N ' among the DMF (4mL), (46mg, 0.52mmol) (53mg 0.26mmol) and with this mixture stirred 14 hours at 90 ℃ N '-dimethyl-ethylenediamine with the iodo-thiazole.After being cooled to room temperature, and adding AcOH-MeOH (1: 10,1.6mL) with this mixture that neutralizes, filter by syringe filter subsequently.Except that after desolvating, crude product is dissolved in DMSO and by the preparation HPLC purifying, obtains white solid (4-methylsulfonyl-phenyl)-[2-(4-morpholine-4-base-piperidines-1-yl)-9-thiazole-4-base-9H-purine-6-yl]-amine 95mg. ¹H?NMR?400MHz(DMSO-d ₆)δ10.44(s，1H)，9.41(s，1H)，8.72(s，1H)，8.40(d，1H，J＝2.4Hz)，8.31(d，2H，J＝8.8Hz)，8.01(d，2H，J＝8.0Hz)，4.86(d，2H，J＝12.8Hz)，3.71(s，4H)，3.52(m，4H)，3.33(s，3H)，3.15(t，2H，J＝12.0Hz)，2.06(d，2H，J＝11.2Hz)，1.55(m，2H)；MS?m/z?541.3(M+1)。

Embodiment 10

N ⁶ -(4-methylsulfonyl-phenyl)-N ² -pyridine-2-ylmethyl-9-thiazole-4-base-9H-purine-2, the 6-diamines

With 2-fluoro-6-chloropurine (17.26g, 100mmol), 3,4-dihydro-2H-pyrans (12.62g, 150mmol) and the tosic acid monohydrate (1.90g, mixture 10mmol) are dissolved in anhydrous methylene chloride (200mL) and at room temperature stirred 4 hours.Filter this reaction mixture, use Na ₂CO ₃(10% aqueous solution, 100mL) and water (100mL) wash and use Na ₂SO ₄Dry organic layer.Evaporating solvent obtains oily matter, it is ground to bring out precipitation with ethyl acetate (10mL) and hexane (60mL) form.Collect product 2-fluoro-6-chloro-9-(tetrahydrochysene-pyrans-2-yl)-9H-purine by filtering.

With 2-fluoro-6-chloro-9-(tetrahydrochysene-pyrans-2-yl)-9H-purine (2.56g, 10mmol), 4-(methylthio group) aniline (1.39g, 10mmol) and DIEA (1.93g, 15mmol) mixture in ethanol (20mL) spends the night 78 ℃ of stirrings.This mixture is cooled to room temperature.Evaporating solvent carries out column chromatography (EtOAc/DCM 10% to 30%) subsequently, obtains [2-fluoro-9-(tetrahydrochysene-pyrans-2-yl)-9H-purine-6-yl]-(4-methylthio group-phenyl)-amine, is white solid.

To the compound of above-mentioned acquisition (3.33g, 9.25mmol) progressively add lentamente in the solution in DCM (10mL) the 3-chloroperoxybenzoic acid (6.22g, maximum 77%, 27.8mmol) (in ice bath).After interpolation, with this mixture restir 2 hours at room temperature.Dilute this mixture and use saturated Na with DCM (50ml) ₂S ₂O ₃(50ml) with saturated NaHCO ₃(50mL * 2) washing suspension is clarified up to organic phase.Water (50ml) and salt solution (50ml) further wash organic layer and use MgSO ₄Dry.Evaporating solvent carries out column chromatography (EtOAc/DCM 30% to 70%) subsequently, obtains [2-fluoro-9-(tetrahydrochysene-pyrans-2-yl)-9H-purine-6-yl]-(4-methyl sulphonyl-phenyl)-amine, is light yellow solid.

With 2-fluoropurine substrate (4.6g, 11.8mmol) and the mixture of 2-(amino methyl) pyridine (15.0g) heated overnight in 84 ℃ oil bath.This mixture is distributed between ethyl acetate (200mL) and the water (200mL).Use NH ₄Cl (2 * 150mL, saturated aqueous solution) and water (200mL) washing organic phase are also used Na ₂SO ₄Dry.Evaporating solvent obtains crude product, and it is used for next step reaction without being further purified.

(1.93g, 4.02mmol) (950mg 5.0mmol) is stirred in methyl alcohol (20mL) and no longer detects raw material (by TLC or LC-MS monitoring) with the tosic acid monohydrate at 60 ℃ of compounds with above-mentioned acquisition.Add triethylamine (1.0mL).Be cooled to room temperature with this reaction mixture, precipitation forms, and collects by filtering, and obtains the product of deprotection.

With 2 of deprotection, the dibasic purine of 6-(1.98g, 5.0mmol), CuI (475mg, 2.50mmol) and K ₃PO ₄(3.18g 15mmol) merges (anti-inflated with nitrogen) in flask.Be added in the trans-N among the DMF (9.0mL), N '-dimethyl cyclohexane-1, (355mg, 2.50mmol) (932mg, 88% is pure, 5.0mmol) also this mixture spent the night 88 ℃ of stirrings with the 4-bromo thiazole for the 2-diamines.After this mixture is cooled to room temperature, adds acetate (1.0mL) and this mixture is filtered (washing with DMF) by syringe filter.By anti-phase preparation type LC-MS purifying filtrate (acetonitrile/water/TFA gradient 10-90%CH ₃CN, in 7.5 minutes, Ultro 1205 μ M C18Q, 75 * 30mmID).With the water/MeCN solution evaporation of collected product to remove acetonitrile.Add NaHCO ₃(saturated aqueous solution) is so that pH rises to 9.With the DCM extraction product and use Na ₂SO ₄Dry organic phase.Evaporating solvent obtains product, is free alkali N ⁶-(4-methylsulfonyl-phenyl)-N ²-pyridine-2-ylmethyl-9-thiazole-4-base-9H-purine-2, the 6-diamines is white powder; ¹H NMR 400MHz (d-DMSO) δ 10.21 (s, 1H), 9.26 (s, 1H), 8.53-7.70 (m, 9H), 7.42 (d, 1H, J=8.0Hz), 7.24 (t, 1H, J=6.0Hz), 4.67 (d, 2H, J=5.6Hz), 3.17 (s, 3H), MS m/z 479.3 (M+1).

Embodiment 11

R-(4-methylsulfonyl-phenyl)-[2-(2-methyl-morpholine-4-yl)-9-thiazole-4-base-9H-purine-6-yl]-amine

With N-benzyl ethyl alcohol amine (9.06g, 60mmol) with (R)-(+)-propylene oxide (6.96g, 99%, 120mmol) the sealing in vitro spend the night in 45 ℃ of stirrings.Evaporate excessive propylene oxide in a vacuum, obtain the glycol resistates, it is directly used in next step.

Described glycol is dissolved in diox (60mL, anhydrous).Add KOH (10.08g, 180mmol) and three (3, the 6-dioxaheptyl) amine (200mg 0.62mmol), and is cooled to 0 ℃ with this mixture, after this drips toluene sulfonyl chloride (12.58g, 66mmol is in the no Shui diox of 60mL).This reaction mixture was stirred 45 minutes at 0 ℃, after this with its temperature to room temperature and restir 4 hours.Filter this reaction mixture and evaporated filtrate in a vacuum.(2N 200mL), and with ethyl acetate (150mL * 2) washing gained acidic aqueous solution, is cooled to 0 ℃ also by adding the NaOH neutralization with this solution to add HCl in product.Use the ethyl acetate extraction product then.Use Na ₂SO ₄Dry organic phase and evaporating then.Resistates is carried out chromatography (DCM that contains 5～20% ethyl acetate), obtain cyclisation product (6.66g).

Free alkali is changed into HCl salt and following recrystallization: (the 2M diethyl ether solution 50mL) is handled the free alkali of above-mentioned acquisition and evaporating, and obtains HCl salt with HCl.This salt (6.0 gram) mixed with ethyl acetate (120mL) and be heated to backflow.Careful dropping EtOH dissolves up to all solids.Then this system is cooled to room temperature and remains in the refrigerator and spend the night.Filter the precipitation that obtains, obtain pure products (2.8g).

Under pressure (55psi) and room temperature, use salt (1.35g, 5.94mmol) solution in ethanol (30mL) of 10%Pd/C (0.20g) hydrogenation recrystallization.This mixture by diatomite filtration (washing with EtOH) and evaporated filtrate, is obtained oily matter.Add ether and evaporation subsequently, obtain the R-2-methyl morpholine hydrochloride, be solid.

With 2-fluoropurine substrate (4.6g, 11.8mmol), the R-2-methyl morpholine hydrochloride (1.78g, 12.9mmol) and DIEA (3.78g, 29.4mmol) mixture in ethanol (20ml) refluxes and to spend the night.Ethanol evaporation also is dissolved in DCM (100ml) again with resistates.It is used saturated NaHCO ₃(50ml), water (50ml), salt solution (50ml) wash and use MgSO ₄Dry.Evaporating solvent carries out column chromatography (EtOAc/DCM 30% to 50%) subsequently, obtains R-4-methylsulfonyl-phenyl)-(2-(2-methyl-morpholine-4-yl)-9-(tetrahydrochysene-pyrans-2-yl)-9H-purine-6-yl)-amine, be the light brown solid.

(1.90g, 4.02mmol) (380mg 2.0mmol) is stirred in methyl alcohol (20mL) and no longer detects raw material (by TLC or LC-MS monitoring) with the tosic acid monohydrate at 60 ℃ of compounds with above-mentioned acquisition.Add triethylamine (0.5mL) and ethanol evaporation.Carry out column chromatography (MeOH/DCM0 to 5%), obtain the product of deprotection.

With 2, (5.00g 20.7mmol) puts into the anti-flask that fills three argon gas to 4-two bromo thiazoles.Add anhydrous diethyl ether (82mL) and this solution is cooled to-78 ℃.Just add-(the 2.5M cyclohexane solution 10.0mL) and with this reaction mixture stirred 90 minutes at-78 ℃ butyllithium, after this used HCl/ diethyl ether solution (2.0m * 15mL) quencher.With this reaction mixture temperature to room temperature.Use NaHCO ₃(saturated aqueous solution 60ml) washs this mixture and use Na ₂SO ₄Dry organic phase.After the evaporation, obtain the 4-bromo thiazole, be crude product.

With 2 of deprotection, the dibasic purine of 6-(1.44g, 3.71mmol), CuI (352mg, 1.86mmol) and CS ₂CO ₃(3.62g 3.0eq) merges (filling with argon gas is counter in advance) in flask.Be added in the trans-N among the DMF (8.0mL), N-dimethyl cyclohexane-1, (264mg, 1.86mmol) (691mg, 88% is pure, 3.71mmol) also this mixture spent the night 88 ℃ of stirrings with the 4-bromo thiazole for the 2-diamines.After this mixture is cooled to room temperature, adds acetate (1.0mL) and filter this mixture by syringe filter (washing) with DMF.By anti-phase preparation type LC-MS (acetonitrile/water/TFA gradient 10-90 10%CH ₃CN, in 7.5 minutes, Ultro 120 5uM CI8Q, 75 * 30mmID) purifying filtrates.With the water/MeCN solution evaporation of collected product to remove acetonitrile.Add NaHCO ₃(saturated aqueous solution) is so that pH rises to 9.With the DCM extraction product and use Na ₂SO ₄Dry organic phase.Evaporating solvent obtains R-(4-methylsulfonyl-phenyl)-[2-(2-methyl-morpholine-4-yl)-9-thiazole-4-base-9H-purine-6-yl]- Amine, be free alkali/white powder; ¹H NMR 400MHz (CDCl ₃) δ 9.69 (s, 1H), 8.87 (d, 1H, J=2.4Hz), 8.83 (s, 1H), 8.26 (d, 1H, J=2.4Hz), 8.07 (d, 2H, J=8.8Hz), 7.95 (d, 2H, J=8.8Hz), 4.53 (t, 2H, J=10.8Hz), 4.10-4.07 (m, 1H), 3.74-3.65 (m, 2H), 3.25-3.10 (m, 1H), 3.08 (s, 3H), 2.90-2.84 (m, 1H), 1.33 (d, 3H, J=6.4Hz); MS m/z 472.3 (M+1).

Embodiment 12

1-(4-{2-[methyl-(1-methyl-piperidin-4-yl)-amino]-9-thiazole-4-base-9H-purine-6-base ammonia Base }-phenyl)-ethyl ketone

With 1-(4-amino-phenyl)-ethyl ketone (1.0g, 7.4mmol) with 2-fluoro-6-chloro-9-(tetrahydrochysene-pyrans-2-yl)-9H-purine (1.90g, 7.4mmol), diisopropylethylamine (1.54mL, 8.9mmol) and propyl carbinol 50mL mix.This is reflected at 95 ℃ stirred 14 hours.Be cooled to room temperature and,, obtaining 1-{4-[2-fluoro-9-(tetrahydrochysene-pyrans-2-yl)-9H-purine-6-base amino by using the purified by flash chromatography crude product of MeOH/DCM (5%: 95%) except that after desolvating]-phenyl }-ethyl ketone, be white solid 2.49g.

1-{4-[2-fluoro-9-(tetrahydrochysene-pyrans-2-yl)-9H-purine-6-base is amino]-phenyl }-ethyl ketone (100mg, 0.28mmol) and methyl-(1-methyl-piperidin-4-yl)-amine (58mg, 0.45mmol), diisopropylethylamine (121 μ L, 0.70mmol) and the 5mL propyl carbinol mix.This is reflected at 100 ℃ stirred 14 hours.In cooling and except that after desolvating, by using the purified by flash chromatography crude product of EA/ hexane (1: 1), obtain 1-{4-[2-[methyl-(1-methyl-piperidin-4-yl)-amino]-9-(tetrahydrochysene-pyrans-2-yl)-9H-purine-6-base is amino]-phenyl }-ethyl ketone, be yellow solid 115mg.

With 1-{4-[2-[methyl-(1-methyl-piperidin-4-yl)-amino]-9-(tetrahydrochysene-pyrans-2-yl)-9H-purine-6-base is amino]-phenyl }-(115mg 0.26mmol) is dissolved in 10mL ethanol and mix with 200 μ L TFA to ethyl ketone.This is reflected at 60 ℃ stirred 2 hours.Be cooled to room temperature and remove fully desolvate and TFA after, with crude product and cupric iodide (I) (50mg, 0.26mmol) and potassiumphosphate (220mg 0.8mmol) mixes, outgases and refills exsiccant nitrogen.Be added in the N ' among the DMF (4mL), (46mg, 0.52mmol) (53mg 0.26mmol) and with this mixture stirred 14 hours at 90 ℃ N '-dimethyl-ethylenediamine with the iodo-thiazole.After being cooled to room temperature, and adding AcOH-MeOH (1: 10,1.6mL) with this mixture that neutralizes, filter by syringe filter subsequently.Remove desolvate after, crude product is dissolved in DMSO and by the preparation HPLC purifying, obtain light yellow solid 1-(4-{2-[methyl-(1-methyl-piperidin-4-yl)-amino]-9-thiazole-4-base-9H-purine-6-base is amino-phenyl)-ethyl ketone: ¹H NMR 400MHz (DMSO-d ₆) δ 10.22 (s, 1H), 9.28 (d, 1H, J=2.3), 8.61 (s, 1H), 8.25 (d, 1H, J=2.1Hz), 8.12 (d, 2H, J=8.7Hz), 7.98 (d, 2H, J=8.7Hz), 3.57 (m, 4H), 3.21 (t, 1H, J=4.6Hz), 3.10 (s, 3H), 2.79 (d, 3H, J=4.6Hz), 2.55 (s, 3H), 2.00 (m, 4H) (MS m/z 463.3 (M+1).

By repeating step described in the foregoing description, using proper raw material to obtain the following formula I compound of determining as in the table 1,2 and 3.

Table 1

Composition in the table 1 merges accepted way of doing sth I compound, and for example, the composition of compound 13 is merged into N2-(1-benzyl-piperidin-4-yl)-9-phenyl-N6-[4-(piperidines-1-the alkylsulfonyl)-phenyl with following structure]-9H-purine-2, the 6-diamines:

Similarly, composition in the table 2 merges accepted way of doing sth I compound, for example, the composition of compound 425 be merged into have following structure (4-{2-[2-(4-methyl-thiazole-5-yl)-oxyethyl group]-9-thiene-3-yl--9H-purine-6-base is amino-phenyl)-piperidines-1-base-ketone:

Table 2

Table 3

Composition in the table 3 merges accepted way of doing sth I compound, and for example, the composition of compound 605 is merged into [2-(2-methyl-morpholine-4-yl)-9-thiazole-4-base-9H-purine-6-yl]-[4-(tetrahydrochysene-pyrans-4-alkylsulfonyl)-the phenyl]-amine with following structure:

Test

Result (embodiment 10-13) explaination The compounds of this invention by following pharmacological testing relates to effect in the disease of Flt3 and/or the imbalance of FGFR3 receptor tyrosine kinase activity in treatment.These embodiment are used to explain the present invention, and limit scope of the present invention never in any form.

Embodiment 13

Flt-3: active generation and mensuration

Be with or without in the presence of the different concns inhibitor, by measuring ³³P from ³³P-ATP mixes detection of active to suitable substrate.

Use the tyrosine protein kinase test of the GST-Flt-3 of purifying to carry out in 40 μ L final volume, described final volume contains 500ng enzyme (30mM Tris-HCl (pH7.5), 3mMMnCl in kinase buffer liquid ₂, 15mM MgCl ₂, 1.5mM DTT, 15 μ M Na ₃VO ₄, 7.5mg/ml PEG, 0.25 μ M be poly--EY (Glu, Tyr), 1%DMSO (under the maximum concentration compound), 10 μ M ATP and γ- ³³P-ATP (0.1 μ Ci)).Prepare two kinds of solution: first kind of 10 μ l solution contains Flt-3 enzyme and inhibitor.Second kind of solution contain substrate in 30 μ l kinase buffer liquid (poly--EY), ATP and γ- ³³P-ATP.With two kinds of solution in advance with 70% ethanol moistening and with the 96-hole PVDF filter plate of 1M Tris (7.4) flushing (MA USA) goes up and mixes for Millipore, Bedford.Should react and at room temperature hatch 20 minutes, stop, pass through flat panel filter with vacuum manifold then, substrate is combined with film with 0.1% phosphoric acid.With 0.1% phosphoric acid flat board is washed 5 times then, be fixed on PackardTopCount 96-hole and connect dull and stereotyped and adding 50 μ L Microscint TM (Packard), after this counting in each hole.

Suppress to carry out linear regression analysis by the per-cent that every kind of compound (in duplicate) (is diluted according to 1: 3 from 1 μ M-0.0005 μ M) under 8 kinds of concentration, calculate IC ₅₀Value.In this test, the IC of The compounds of this invention ₅₀Value is in 0.1nM-2 μ M scope.

Embodiment 14

General technology comprise with possible inhibitor to the effect that depends on mutant Flt3 and carry out proliferating cells system with do not compare relying on the effect that mutant Flt3 carries out proliferating cells system.Be chosen in the compound that has different activities (sensitivity differences is more than or equal to 10 times) between Flt3+ clone and the Flt3-clone and be used for further research.

The cell that is used for preliminary screening is the subbreed of Ba/F3 cell, its be designed in case behind the retrovirus of infect expressing suitable Flt3 cDNA overexpression mutant or wild-type (not sudden change) Flt3.Parent cell is that Ba/F3 depends on interleukin 3 and breeds, and when forfeiture IL-3, cell stops propagation and dead rapidly.The viral LTR of the reflexive record of retrovirus expresses Flt3 and expresses the neo gene from the IRES site.In G418, select the Ba/F3 cell and pass through the expression that fluorescence amplifying cell separator (FACS) is analyzed Flt3.Use has the clone of two kinds of different Flt3 sudden changes.A kind of mutant is expressed in the nearly membrane structure territory by exons 11 coding has 14 amino acid multiple Flt-3, concrete repeats to be ... VDFREYEYDLKWEF... (being called Ba/F3-Flt3-ITD).Second kind of sudden change has the point mutation (being called Ba/F3-Flt3-D835Y) that the l-asparagine on 835 is changed into tyrosine.Two kinds of sudden changes all cause the Flt-3 kinase activation and make it not rely on IL-3, and the cell of expression is not having growth in the presence of the IL-3.Produce to express the Ba/F3 cell of wild-type Flt3 similarly and used as " contrast " clone.Parental generation (not infecting) clone and wild-type " contrast " cell keep depending on IL-3 and breed.

With the Ba/F3 cell (contrast ,-Flt3-ITD or-Flt3-D835Y) be cultured to and in the 30mL culture, reach 500,000 cell/mL, with containing the RPMI 1640 of 10% foetal calf serum as substratum.The substratum that is used for control cells (but not being mutant-Flt3 cell) contains 10% conditioned medium from WEHI-3B clone as the IL-3 source.Prepare 10mM " deposit " solution of each compound in methyl-sulphoxide (DMSO).In the RPMI 1640 that contains 10% foetal calf serum, prepare diluent then, make final drug level typically be 1nM-10 μ M.Make similar diluent as vehicle Control by DMSO.After adding compound 48 hours, detect the proliferation rate and the cytotoxicity of cell.

Add Yo-Pro-l iodide (molecular probe) in cell, the final concentration in NaCl/ citric acid Na damping fluid is 2.5 μ M.Cell and Yo-Pro were at room temperature hatched 10 minutes, and reading on photofluorometer then is to measure cytotoxicity.Next use the NP40/EDTA/EGTA damping fluid with lysis 90 minutes, at room temperature hatched 90 minutes and reading, to measure propagation.

Optionally Ba/F3-Flt3-ITD cell comparison wild-type contrast Ba/F3 cell is had more toxic compound with the further test of the cell of expressing Flt3-D835Y.

In addition, be exposed to different concns active compound front and back, using α-Flt3 antibody mediated immunity precipitation Flt3 albumen.By the albumen of sodium dodecyl sulfate polyacrylamide gel separation immunoprecipitation, go to pvdf membrane by the electrophoresis mode, and with α-phosphorus- ⁵⁹¹Y-Flt3 antibody carries out immunoblotting.Whether this test determination compound can reduce the distinctive Flt3 of mutant form acceptor " autophosphorylation " level.

Compound of the present invention generally shows the antiproliferative activity to Flt3-ITD in the nmole scope, and contrast-Flt3 is not had toxicity reaching under the 10 μ M yet.Compound of the present invention also reduces the autophosphorylation activity of cell Flt-3 in the nmole scope.

The formula I compound of free form or pharmacy acceptable salt form shows the valuable pharmacological characteristic, for example, and shown in the in vitro tests described in the application.For example, in above-mentioned test, the IC that formula I compound shows Flt3 ₅₀Preferably 1 * 10 ^-10-2 * 10 ^-6In the M scope, preferably be lower than 100nM.For example, { 4-[2-(4-amino-cyclohexyl amino)-9-thiene-3-yl--9H-purine-6-base Amino]-phenyl }-piperidines-1-base-ketoneIC in embodiment 14 described tests ₅₀Be 5nM, and the IC that in embodiment 13 described tests, shows ₅₀Be 7nM.

Embodiment 15

FGFR3: active mensuration

Be with or without in the presence of the different concns inhibitor, coming detection of active by the phosphorylation of using HTRF to measure peptide substrates.

Use the tyrosine protein kinase test of the FGFR3 (Upstate) of purifying to carry out in 10 μ L final volume, described final volume contains kinase buffer liquid (30mM Tris-HCl pH7.5,15mMMgCl ₂, 4.5mM MnCl ₂, 15 μ M Na ₃VO ₄With 50 μ M/mL BSA) in 0.25 μ g/mL enzyme and substrate (5 μ g/mL vitamin Hs-poly--EY (Glu, Tyr) (CIS-US, Inc.) and 3 μ MATP).Prepare two kinds of solution: first kind of 5 μ l solution contains the FGFR3 enzyme in kinase buffer liquid, at first it is allocated into 384-lattice Proxiplate

(Perkin-Elmer), add the compound that 50nL is dissolved in DMSO subsequently, in each hole, add second kind of solution of 5 μ l then, its contain substrate in kinase buffer liquid (poly--EY) and ATP.To react and at room temperature hatch 1 hour, detect the mixture termination by adding 10 μ LHTRF, described detection mixture contains 30mM Tris-HCl pH7.5,0.5MKF, 50mM ETDA, 0.2mg/mL BSA, 15 μ g/mL streptavidin-XL665 (CIS-US, Inc.) and anti--phosphotyrosine antibody of puting together of 150ng/mL kryptofix 222 (CIS-US, Inc.).At room temperature hatched 1 hour so that after streptavidin-vitamin H interaction, go up time for reading at Analyst GT (Molecular Devices Corp.) and differentiate fluorescent signal.

By the per-cent of every kind of compound (in duplicate) under 12 kinds of concentration (diluting according to 1: 3 from 10 μ M-0.05nM) is suppressed to carry out linear regression analysis, calculate IC ₅₀Value.In this test, the IC of The compounds of this invention ₅₀Value is in 0.1nM-2 μ M scope.

Embodiment 16

General technology comprise with possible inhibitor to the effect that depends on FGFR3 and carry out proliferating cells system with do not compare relying on the effect that FGFR3 carries out proliferating cells system.Be chosen in the compound that has different activities (sensitivity differences is more than or equal to 10 times) between FGFR3+ clone and the FGFR3-clone and be used for further research.

The cell that is used for preliminary screening is the subbreed of Ba/F3 cell, and it is designed to overexpression TEL-FGFR3 syzygy after infecting the retrovirus of expressing TEL-FGFR3 cDNA.Parent cell is that Ba/F3 depends on interleukin 3 (IL-3) and breeds, and when forfeiture IL-3, cell stops propagation and dead rapidly.On the contrary, in the Ba/F3 of FGFR3 overexpression cell, the TEL-FGFR3 syzygy causes part-independence FGFR3 dimerization and FGFR3 kinase activation subsequently, makes the Ba/F3 cell of overexpression not have growth in the presence of the IL-3.

(TEL-FGFR3) cell is cultured to 800,000 cell/mL in suspension, uses and has replenished the RPMI 1640 of 10% foetal calf serum as substratum with the Ba/F3 of wild-type Ba/F3 and conversion.The substratum that is used for control cells contains 10ng/ml reorganization IL-3 (R﹠amp; D Research).Prepare 10mM " deposit " solution of each compound in methyl-sulphoxide (DMSO).Dilution is gone among the DMSO then, makes final drug level typically be 0.05nM-10 μ M.After adding compound 48 hours, detect the proliferation rate of cell.With AlamarBlue

(TREK Diagnostic Systems) joins in the cell, and the final concentration in cell culture medium is 10%.With cell and AlamarBlue

Hatched 4 hours in 37 ℃ of incubator for tissue cultures, reading in the fluorescence reader is bred to measure then.

In addition, in Western trace, detect the phosphorylation TEL-FGFR3 protein level in the lysate that is exposed to overexpression Ba/F3 behind the different concns active compound with anti--phosphorylation-FGFR3 antibody mediated immunity trace.Whether this test determination compound can reduce " autophosphorylation " level of the distinctive FGFR3 of mutant acceptor.

Compound of the present invention generally shows the antiproliferative activity to TEL-FGFR3 in the nmole scope, and wild-type Ba/F3 is not had toxicity reaching under the 10 μ M yet.Compound of the present invention also reduces the autophosphorylation activity of cell TEL-FGFR3 in the nmole scope.

Embodiment 17

Upstate KinaseProfiler ^TM-radiation enzyme filter-binding assay

The compounds of this invention being suppressed the ability of each member in one group of kinases (the non-limiting kinases catalogue of part comprises: cSRC, Lck, FGFR3, F1t3, TrkB and PFGFR α) estimates.According to this generality scheme in duplicate with final concentration 10 μ M test compounds.Notice that kinase buffer liquid composition and substrate are because of " Upstate KinaseProfiler ^TM" the different kinases that comprise in the group and changing.According to this generality scheme in duplicate with final concentration 10 μ M test compounds.Notice that kinase buffer liquid composition and substrate are because of " Upstate KinaseProfiler ^TM" the different kinases that comprise in the group and changing.(2.5 μ L, 10x-contains MnCl if desired with kinase buffer liquid ₂), active kinases (0.001-0.01 unit; 2.5 μ L), the specificity in kinase buffer liquid or poly-(Glu4-Tyr) peptide (5-500 μ M or .01mg/ml) and kinase buffer liquid (50 μ M; 5 μ L) mix in the eppendorf pipe on ice.Add Mg/ATP mixture (10 μ L; (67.5 or 33.75) mM MgCl ₂, 450 (or 225) μ MATP and 1 μ Ci/ μ l[γ- ³²P]-ATP (3000Ci/mmol)), and this is reflected at about 30 ℃ hatched about 10 minutes.With this reaction mixture point sample (20 μ L) at 2cm * 2cm P81 (phosphorylated cotton is used for positively charged peptide substrates) or Whatman No.1 (being used to gather (Glu4-Tyr) peptide substrates) square of paper piece.To test square of paper piece washing 4 times with 0.75% phosphoric acid, each 5 minutes, and with washing with acetone one time 5 minutes.To test the square of paper piece and change scintillation vial over to, add 5mL flicker mixture, and use the Beckman scintillometer mixing in the peptide substrates ³²P (cpm) carries out quantitatively.Inhibition per-cent is calculated in each reaction.

Preferably, the inhibition per-cent that formula I compound shows cSRC, Lck, FGFR3, Flt3, TrkB and PFGFR alpha kinase under 10 μ M concentration is preferably greater than 60%, more preferably greater than 70% greater than 50%.For example:

(i) compound 539: N ² -methyl-N ² -(1-methyl-piperidin-4-yl)-N ⁶ -(4 morpholines-4-base-phenyl)-9- Thiazole-4-base-9H-purine-2, the 6-diaminesShow following rejection characteristic: Bmx (90%); C-Src (97%); Lck (99%); FIt3 (100%); Rsk1 (82%); And TrkB (99%);

(ii) compound 554 (embodiment 10): N ⁶ -(4-methylsulfonyl-phenyl)-N ² -pyridine-2-ylmethyl-9- Thiazole-4-base-9H-purine-2, the 6-diaminesShow following rejection characteristic: Abl (98%); Bmx (86%); C-Src (99%); Lck (95%); FIt3 (100%); FGFR3 (98%); And TrkB (99%); With

(iii) compound 503: (4-methylsulfonyl-phenyl)-(2-morpholine-4-base-9-thiazole-4-base-9H-purine -6-yl)-amineShow following rejection characteristic: Abl (81%); Bmx (71%); C-Src (98%); Lck (99%); FIt3 (99%); TrkB (99%).

Should be understood that embodiment described herein and embodiment only are used for task of explanation, and prompting those skilled in the art can carry out various modifications and change according to them, these modifications and change should be included in the application and the claim that awaits the reply essence and scope in.All open source literatures, patent and the patent application of this paper citation are hereby incorporated by and are used for all purposes.

Claims (7)

1. formula I compound and pharmacy acceptable salt thereof and isomer:

Wherein:

R ₁Be selected from hydrogen, halogen, C _1-6Alkoxyl group ,-OXOR ⁵,-OXR ⁶,-OXNR ₅R ₆,-OXONR ₅R ₆,-XR ₆,-XNR ₇XNR ₇R ₇With-XNR ₅R ₆Wherein X is selected from key, C _1-6Alkylidene group, C _2-6Alkylene group and C _2-6Alkynylene;

R ₅Be selected from hydrogen, C _1-6Alkyl and-XOR ₇Wherein X is selected from key, C _1-6Alkylidene group, C _2-6Alkylene group and C _2-6Alkynylene; And R ₇Be independently selected from hydrogen or C _1-6Alkyl;

R ₆Be selected from hydrogen, C _1-6Alkyl, C _3-12Cycloalkyl C _0-4Alkyl, C _3-8Heterocyclylalkyl C _0-4Alkyl, C _6-10Aryl C _0-4Alkyl and C _5-10Heteroaryl C _0-4Alkyl; Or

R ₅And R ₆With R ₅And R ₆The nitrogen-atoms that is connected forms C together _3-8Heterocyclylalkyl or C _5-8Heteroaryl; R wherein ₅And R ₆Methylene radical in formed any Heterocyclylalkyl can choose wantonly by-C (O)-or-S (O) ₂-substitute;

R wherein ₆Any aryl, heteroaryl, cycloalkyl or Heterocyclylalkyl or R ₅And R ₆Combination can choose wantonly by 1-3 group and replace, described group is independently selected from-XNR ₇R ₇,-XC (O) NR ₇R ₇,-XOR ₇,-XNR ₇R ₇,-XNR ₇C (O) R ₇,-XOR ₇,-XC (O) R ₇, C _1-6Alkyl, C _3-8Heterocyclylalkyl and C _6-10Aryl C _0-4Alkyl; R wherein ₁Any alkyl or alkylidene group can choose wantonly and have by divalent group alternate methylene radical, described divalent group is selected from-NR ₇C (O)-,-C (O) NR ₇-,-NR ₇-,-O-; And R wherein ₁Any alkyl or alkylidene group can choose wantonly by 1-3 group and replace, described group is independently selected from C _5-8Heteroaryl ,-NR ₇R ₇,-C (O) NR ₇R ₇,-NR ₇C (O) R ₇, halogen and hydroxyl; R wherein ₇Be independently selected from hydrogen or C _1-6Alkyl;

R ₂Be selected from hydrogen, C _6-10Aryl and C _5-10Heteroaryl; R wherein ₂Any aryl or heteroaryl optional by 1-3 group replacement, described group is independently selected from-XNR ₇R ₇,-XOR ₇,-XOR ₈,-XC (O) OR ₇, C _1-6Alkyl, C _1-6Alkoxyl group, nitro, cyano group, halogen, halogen-replacement-C _1-6Alkoxyl group and halogen-replacement-C _1-6Alkyl; Wherein X and R ₇As mentioned above; And R ₈Be C _6-10Aryl C _0-4Alkyl;

R ₃Be hydrogen; And

R ₄Be selected from C _6-10Aryl C _0-4Alkyl and C _5-10Heteroaryl C _0-4Alkyl; R wherein ₄Described aryl or heteroaryl replaced by 1-3 group, described group is selected from-XR ₉,-XOR ₉,-XS (O) ₂R ₇,-XS (O) ₂R ₉,-XC (O) R ₇,-XC (O) OR ₇,-XP (O) R ₇R ₇,-XC (O) R ₉,-XC (O) NR ₇XNR ₇R ₇,-XC (O) NR ₇R ₇,-XC (O) NR ₇R ₉With-XC (O) NR ₇XOR ₇Wherein X and R ₇As mentioned above; R ₉Be C _3-8Heterocyclylalkyl C _0-4Alkyl; R wherein ₉Optional by the individual C that is selected from of 1-3 _1-6Alkyl ,-XC (O) R ₇With-XC (O) NR ₇R ₇Group replace; Wherein X and R ₇As mentioned above.

2. the compound of claim 1, wherein R ₁Be selected from hydrogen, halogen, C _1-6Alkoxyl group ,-OXOR ⁵,-OXR ⁶,-OXNR ₅R ₆,-OXONR ₅R ₆,-XR ₆With-XNR ₅R ₆Wherein X is selected from key, C _1-6Alkylidene group, C _2-6Alkylene group and C _2-6Alkynylene; R ₅Be selected from hydrogen, methyl, hydroxyl-ethyl and methoxyl group-ethyl; R ₆Be selected from hydrogen, phenyl, benzyl, cyclopentyl, cyclobutyl, dimethylamino-propenyl, cyclohexyl, 2,3-dihydroxyl-propyl group, piperidyl, amino-carbonyl-ethyl, methyl-carbonyl-amino-ethyl, methyl-amino-ethyl, amino-propyl group, methyl-amino-propyl group, 1-hydroxymethyl-butyl, amyl group, butyl, propyl group, methoxyl group-ethynyl, methoxyl group-vinyl, dimethyl-amino-butyl, dimethyl-amino-ethyl, dimethyl-amino-propyl group, THP trtrahydropyranyl, tetrahydrofuran base-methyl, pyridyl-methyl, azepine ring-1-in heptan base, [1,4] oxygen azepine ring-4-in heptan base, piperidyl-ethyl, diethyl-amino-ethyl, amino-butyl, amino-sec.-propyl, amino-ethyl, hydroxyl-ethyl, 2-acetylamino-ethyl, formamyl-ethyl, 4-methyl-[1,4] diaza ring-1-in heptan base, 2-hydroxyl-propyl group, hydroxyl-propyl group, 2-hydroxy-2-methyl-propyl group, methoxyl group-ethyl, amino-propyl group, methyl-amino-propyl group, 2-hydroxyl-2-phenyl-ethyl, pyridyl-ethyl, morpholino-propyl group, morpholino-ethyl, pyrrolidyl, pyrrolidyl-methyl, pyrrolidyl-ethyl, pyrrolidyl-propyl group, pyrazinyl, quinoline-3-base, quinoline-5-base, imidazolyl-ethyl, pyridyl-methyl, styroyl, tetrahydrochysene-pyrans-4-base, pyrimidyl, furyl isoxazolyl-methyl, pyridyl, benzo [1,3] dioxole-5-base, thiazolyl-ethyl and thiazolyl-methyl; Or R ₅And R ₆With R ₅And R ₆The nitrogen-atoms that is connected forms pyrrolidyl, piperazinyl, piperidyl, imidazolyl, 3-oxo-piperazine-1-base, [1,4] diaza ring-1-in heptan base, morpholino, 3-oxo-piperazine-1-base, 1,1-dioxo-l λ together ⁶-thiomorpholine-4-base or pyrazolyl;

R wherein ₆Any aryl, heteroaryl, cycloalkyl or Heterocyclylalkyl or R ₅And R ₆Combination can choose wantonly by 1-3 group and replace, described group is independently selected from methyl-carbonyl, amino-methyl, amino-carbonyl, methyl-alkylsulfonyl, methoxyl group, methoxyl group-methyl, formyl radical, fluoro-ethyl, hydroxyl-ethyl, amino, dimethyl-amino, hydroxyl, methyl, ethyl, ethanoyl, sec.-propyl, pyrrolidyl, pyrimidyl, morpholino, pyridyl and benzyl; R wherein ₆Any alkyl or alkylidene group can choose wantonly have be selected from-NHC (O)-or-the divalent group alternate methylene radical of C (O) NH-; And R wherein ₆Any alkyl or alkylidene group can choose a group that is independently selected from amino, halogen, piperidyl and hydroxyl by 1-2 wantonly and replace.

3. the compound of claim 1, wherein R ₂Be selected from hydrogen, phenyl, thienyl, pyridyl, pyrazolyl, thiazolyl, pyrazinyl, naphthyl, furyl, benzo [1,3] dioxole-5-base, isothiazolyl, imidazolyl and pyrimidyl; R wherein ₂Any aryl or heteroaryl optional by 1-3 group replacement, described group is independently selected from methyl, sec.-propyl, halogen, ethanoyl, trifluoromethyl, nitro, 1-hydroxyl-ethyl, 1-hydroxyl-1-methyl-ethyl, hydroxyl-ethyl, hydroxyl-methyl, formamyl, methoxyl group, benzyloxy, carboxyl, amino, cyano group, amino-carbonyl, amino-methyl and oxyethyl group.

4. the compound of claim 1, wherein R ₄Be selected from phenyl, benzyl, pyridyl and 1-oxo-indane-5-base; Wherein said phenyl; benzyl; indanyl or pyridyl are by following replacement: ethanoyl; cyclopropyl-amino-carbonyl; azetidine-1-carbonyl; piperidyl-carbonyl; morpholino; methyl-carbonyl; piperazinyl; methyl-alkylsulfonyl; piperidyl-alkylsulfonyl; 4-methyl-piperazinyl-carbonyl; dimethyl-amino-ethyl-amino-carbonyl; morpholino-carbonyl; morpholino-methyl; amino-carbonyl; propyl group-amino-carbonyl; hydroxyl-ethyl-amino-carbonyl; morpholino-ethyl-amino-carbonyl; 4-ethanoyl-piperazine-1-carbonyl; 4-amino-carbonyl-piperazine-1-carbonyl; phenyl-carbonyl; pyrrolidyl-1-carbonyl; propyl group-carbonyl; sec.-propyl-oxygen base-carbonyl; cyclohexyl-carbonyl; cyclopropyl-carbonyl; methyl-alkylsulfonyl; dimethyl-inferior phosphono; 4-methyl-piperazinyl-alkylsulfonyl; 1-oxo-indane-5-base; trimethylene oxide-3-alkylsulfonyl; amino-alkylsulfonyl and tetrahydrochysene-pyrans-4-alkylsulfonyl.

5. the compound of claim 1 is selected from: N ⁶-(4-methanesulfinyl-phenyl)-N ²-methyl-N ²-(tetrahydrochysene-pyrans-4-yl)-9-thiazole-4-base-9H-purine-2, the 6-diamines; (4-methylsulfonyl-phenyl)-[2-(2-methyl-morpholine-4-yl)-9-thiazole-4-base-9H-purine-6-yl]-amine; 1-{4-[2-(2-methyl-morpholine-4-yl)-9-thiazole-4-base-9H-purine-6-base is amino]-phenyl }-ethyl ketone; [4-(dimethyl-inferior phosphono)-phenyl]-[2-(2-methyl-morpholine-4-yl)-9-thiazole-4-base-9H-purine-6-yl]-amine; Azetidine-1-base-4-[2-(4-morpholine-4-base-piperidines-1-yl)-9-thiazole-4-base-9H-purine-6-base is amino]-phenyl }-ketone; 1-(4-{2-[methyl-(1-methyl-piperidin-4-yl)-amino]-9-thiazole-4-base-9H-purine-6-base is amino }-phenyl)-ethyl ketone; 1-{4-[2-(2-methyl-morpholine-4-yl)-9-thiene-3-yl--9H-purine-6-base is amino]-phenyl }-ethyl ketone; (4-methylsulfonyl-phenyl)-[2-(4-morpholine-4-base-piperidines-1-yl)-9-thiazole-4-base-9H-purine-6-yl]-amine; N ⁶-(4-methylsulfonyl-phenyl)-N ²-methyl-N ²-(1-methyl-piperidin-4-yl)-9-thiazole-4-base-9H-purine-2, the 6-diamines; [2-(2-methyl-morpholine-4-yl)-9-thiazole-4-base-9H-purine-6-yl]-(4-morpholine-4-base-phenyl)-amine; N ²-methyl-N ²-(1-methyl-piperidin-4-yl)-N ⁶-(4-morpholine-4-base-phenyl)-9-thiazole-4-base-9H-purine-2, the 6-diamines; N ²-methyl-N ²-(1-methyl-piperidin-4-yl)-N ⁶-(4-morpholine-4-base-phenyl)-9-thiene-3-yl--9H-purine-2, the 6-diamines; [2-(2,2-dimethyl-morpholine-4-yl)-9-thiazole-4-base-9H-purine-6-yl]-(4-methylsulfonyl-phenyl)-amine; [2-(2,6-dimethyl-morpholine-4-yl)-9-thiazole-4-base-9H-purine-6-yl]-(4-methylsulfonyl-phenyl)-amine; [4-(dimethyl-inferior phosphono)-phenyl]-[2-(2-ethyl-morpholine-4-yl)-9-thiene-3-yl--9H-purine-6-yl]-amine; [4-(dimethyl-inferior phosphono)-phenyl]-[2-(2-methyl fluoride-morpholine-4-yl)-9-thiene-3-yl--9H-purine-6-yl]-amine; [2-(2,6-dimethyl-morpholine-4-yl)-9-thiazole-4-base-9H-purine-6-yl]-[4-(dimethyl-inferior phosphono)-phenyl]-amine; [2-(2,6-dimethyl-morpholine-4-yl)-9-thiene-3-yl--9H-purine-6-yl]-[4-(dimethyl-inferior phosphono)-phenyl]-amine; [4-(dimethyl-inferior phosphono)-phenyl]-[2-(2-methyl-morpholine-4-yl)-9-thiene-3-yl--9H-purine-6-yl]-amine; [4-(dimethyl-inferior phosphono)-phenyl]-[2-(3-methyl-piperidines-1-yl)-9-thiazole-4-base-9H-purine-6-yl]-amine; N ⁶-(4-methylsulfonyl-phenyl)-N ²-methyl-N ²-pyridine-2-ylmethyl-9-thiene-3-yl--9H-purine-2, the 6-diamines; N ²-methyl-N ⁶-(4-morpholine-4-base-phenyl)-N ²-pyridine-2-ylmethyl-9-thiene-3-yl--9H-purine-2, the 6-diamines; (2-azepine ring-1-in heptan base-9-thiazole-4-base-9H-purine-6-yl)-[4-(dimethyl-inferior phosphono)-phenyl]-amine; N ²-cyclohexyl-N ⁶-[4-(dimethyl-inferior phosphono)-phenyl]-N ²-methyl-9-thiazole-4-base-9H-purine-2, the 6-diamines; N ⁶-(4-methylsulfonyl-phenyl)-N ²-methyl-N ²-(tetrahydrochysene-pyrans-4-yl)-9-thiazole-4-base-9H-purine-2, the 6-diamines; N ⁶-(4-methylsulfonyl-phenyl)-N ²-pyridine-2-ylmethyl-9-thiazole-4-base-9H-purine-2, the 6-diamines; N ²-cyclohexyl-N ⁶-(4-methanesulfinyl-phenyl)-N ²-methyl-9-thiazole-4-base-9H-purine-2, the 6-diamines; R-(4-methanesulfinyl-phenyl)-[2-(2-methyl-morpholine-4-yl)-9-thiazole-4-base-9H-purine-6-yl]-amine; N ⁶-(4-methylsulfonyl-phenyl)-N ²-methyl-N ²-pyridine-2-ylmethyl-9-thiazole-4-base-9H-purine-2, the 6-diamines; 4-[6-(4-methylsulfonyl-phenyl amino)-2-(methyl-pyridine-2-ylmethyl-amino)-purine-9-yl]-phenyl }-methyl alcohol; R-(4-methylsulfonyl-phenyl)-[2-(2-methyl-morpholine-4-yl)-9-thiazole-4-base-9H-purine-6-yl]-amine; R-4-[2-(2-methyl-morpholine-4-yl)-9-thiazole-4-base-9H-purine-6-base is amino]-benzsulfamide; 4-[6-(4-methylsulfonyl-phenyl amino)-2-(2-methyl-morpholine-4-yl)-purine-9-yl]-phenyl }-methyl alcohol.

6. pharmaceutical composition comprises the compound and the pharmaceutically acceptable vehicle of the claim 1 for the treatment of significant quantity.

7. the compound of claim 1 is used for the treatment of purposes in the medicine of Animal diseases in preparation, and wherein the kinase activity of cSRC, Lck, FGFR3, F1t3, TrkB and/or Bmx works to the pathology and/or the symptom of described disease.