CN105801602B - 5- tertiary Ding isoxazoles -3- bases -3- (4- substituted-phenyls) urea derivative and its preparation method and application - Google Patents
5- tertiary Ding isoxazoles -3- bases -3- (4- substituted-phenyls) urea derivative and its preparation method and application Download PDFInfo
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- OQILNVHKRLOZCB-UHFFFAOYSA-N CC(C)(C)Cc1cc(NC(Nc(cc2)ccc2-c2c[n](cc(C)cc3)c3n2)=O)n[o]1 Chemical compound CC(C)(C)Cc1cc(NC(Nc(cc2)ccc2-c2c[n](cc(C)cc3)c3n2)=O)n[o]1 OQILNVHKRLOZCB-UHFFFAOYSA-N 0.000 description 1
- WEOKOYXSFURICQ-UHFFFAOYSA-N CC(C)(C)c1cc(NC(CNC(C=CC2)=CC=C2c2c[n](ccc(OC)c3)c3n2)=O)n[o]1 Chemical compound CC(C)(C)c1cc(NC(CNC(C=CC2)=CC=C2c2c[n](ccc(OC)c3)c3n2)=O)n[o]1 WEOKOYXSFURICQ-UHFFFAOYSA-N 0.000 description 1
- INWAWOOMTQMSRP-UHFFFAOYSA-N CC(C)(C)c1cc(NC(Nc(cc2)ccc2-c(nc2[n]3cccc2)c3Cl)=O)n[o]1 Chemical compound CC(C)(C)c1cc(NC(Nc(cc2)ccc2-c(nc2[n]3cccc2)c3Cl)=O)n[o]1 INWAWOOMTQMSRP-UHFFFAOYSA-N 0.000 description 1
- GKIOEULGXKOJMK-GIDUJCDVSA-N CC(C)(C)c1cc(NC(Nc(cc2)ccc2-c2c[n](C)c(/C(/F)=C\C)n2)=O)n[o]1 Chemical compound CC(C)(C)c1cc(NC(Nc(cc2)ccc2-c2c[n](C)c(/C(/F)=C\C)n2)=O)n[o]1 GKIOEULGXKOJMK-GIDUJCDVSA-N 0.000 description 1
- ZONAZRSXCPAZRZ-UHFFFAOYSA-N CC(C)(C)c1cc(NC(Nc(cc2)ccc2-c2nc3ccccc3[o]2)=O)n[o]1 Chemical compound CC(C)(C)c1cc(NC(Nc(cc2)ccc2-c2nc3ccccc3[o]2)=O)n[o]1 ZONAZRSXCPAZRZ-UHFFFAOYSA-N 0.000 description 1
Abstract
The invention belongs to chemical medicine, and in particular to 5 tertiary 3 base 3 (4 substituted-phenyl) urea derivatives of Ding isoxazoles and its preparation method and application.The present invention provides a kind of 5 tertiary 3 base 3 (4 substituted-phenyl) urea derivatives of Ding isoxazoles, its structure is as shown in formula I.Present invention also offers the preparation method and purposes of 5 tertiary 3 base 3 (4 substituted-phenyl) urea derivatives of Ding isoxazoles.The present invention obtains tertiary 3 base 3 (4 substituted-phenyl) urea derivative of Ding isoxazoles in above-mentioned 5 on the basis of a large amount of screenings, with the activity for suppressing human muscle creatine kinase cell Proliferation, new selection is provided to treat the development and application of acute myeloid leukaemia medicine.
Description
Technical field
The invention belongs to chemical medicine, and in particular to 5- tertiary Ding isoxazoles -3- bases -3- (4- substituted-phenyls) urea spreads out
Biology and its preparation method and application.
Background technology
Receptor tyrosine kinase (RTKs) is a kind of important enzyme in human body, it is as growth factor receptors phosphorylation downstream
Albumen, plays central role in signal transduction pathway, so as to adjust the propagation of cell, growth, differentiation and death.In many diseases
Exception or overactivity and the activity imbalance of receptor tyrosine kinase (PTKs), such as cancer, inflammation can be observed in disease
Disease, autoimmune disease etc..
Vascular endothelial growth factor receptor (vascular endothelial growth factor receptor,
VEGFR) be RTKs member, it is closely related with Tumor Angiongesis.In most cancers (such as gastrointestinal cancer, pancreas gland, wing
Guang cancer, adenomyoma etc.) in can detect that VEGFR and its acceptor VEGFR1, VEGFR2 are overexpressed；Some intracranial tumors
VEGFR2 high is expressed in (such as glioblastoma, Von Hippel-Lindau syndromes).Have tens kinds of VEGFR2 at present
Inhibitor be in clinical investigation phase as antitumor drug.
Ret kinases is another class members of RTKs, is a proto-oncogene reset in conversion, its Codocyte
Membrane receptor tyrosine kinase, preliminary studies have shown that the signal transduction pathway that it is mediated is more unique.Ret gene mutations and the mankind 4
The generation of kind cancer is related：There are Ret genes and a variety of rearrangements of other genes for papillary adenocarcinoma of thyroid；Multiple Endocrine gland
There are 7 site point mutation for 2 type of knurl, familial inheritance medullary carcinoma of thyroid gland etc.；Congenital megacolon disease and Ret gene delection phases
Close.Some RET inhibitor have been demonstrated effectively to suppress tumour breed with nude mice model (Cancer Res.2003,
631,5559-5563)。
Platelet derived growth factor receptor (platelet-derived growth factor receptor, PDGFR)
And the member of RTKs, chemotactic, division and the propagation of cell can be promoted, in physiology mistakes such as body growth development, wound repairs
Play a part of positive important in journey, its activity imbalance, it has proved that related with a variety of proliferative diseases.Such as glioma and meat
It can detect that PDGFR raises (Oncogene.1992,7 in knurl patient:627-633)；Chronic myelocytic leukemia (CMML) is suffered from
Constitutively activated (the Blood.2002,100 of PDGFR is observed in person:1088-1091)；Gastroenteric tumor and idiopathic acidophilus
Property eosinophilic syndrome patient can detect PDGFR Functional mutations and fraction missing (Science.2003,299:
708-710)。
Macrophage colony-stimulating factor receptor (colony-stimulating factor-1Receptor, CSF-1R)
It is the 3rd member of PDGFR families, is a kind of cell factor with lineagespecific, is expressed by breast cancer, epithelioma etc..
The expression of CSF-1R can be detected in most of solid tumor and blood cancer cell, therefore, CSF-1R can be used as potential tumor
Therapy target carries out medicament research and development.
Stem cell factor receptor (stem cell factor receptor, Kit) is another member of PDGFR families,
Kit is mutated the examination criteria frequently as gastrointestinal stromal tumor canceration.Imatinib, is first RTPK inhibitor of FDA approvals,
For the treatment of the chronic myelocytic leukemia of c-Abl mediations, ratified to control as Kit inhibitor by FDA in 2002
Treat the gastrointestinal stromal tumor of Kit mediations.The Functional mutations of Kit and mast cell/granulocytic leukemia and seminoma/asexual
Related (the Nature.2001,411 (17) of cytoma:355-365).It has also been found that Kit is mutated in some melanomas, and conduct
Drug target is used for the treatment of melanoma.
FLT3 (tyrosine kinase 3 of Fms-like tyrosine kinase, FMS samples) be PDGFR families second into
Member, is expressed by Dendritic Cells (DC), prematurity candidate stem cell, ripe myeloid cell and lymphoid cell
(Leukemia.1995；9:1212-1218).In acute myeloid leukaemia (AML), the bone marrow cell of 90% patient detects
FLT3 high is expressed, and FLT3 plays an important role the propagation of bone marrow cell (Blood.2002,100,1532).
Hyperplasia, inflammation and the immunity disease of above-mentioned numerous receptor tyrosine kinase mediations, it would be highly desirable to research and develop new PTKs suppressions
Preparation is to treat the relevant diseases of PTKs.
The content of the invention
The present invention provides a kind of 5- tertiary Ding isoxazoles -3- bases -3- (4- substituted-phenyls) urea derivative, its structure such as formula
Shown in I：
Wherein, X1、X2、X3It independently is C, N, O or S；Y1、Y2、Y3It independently is C, N, O or S；
R1、R2For-H, halogen ,-CF3, C1~C6 alkyl, C1~C6 alkoxies, C2~C6 alkenyls, C2~C6 alkynyls, C3~
C8 cycloalkyl, C1~C6 acyloxy, heterocyclic radical, C6~C10 aryl or aromatic heterocyclic；The heterocyclic radical or aromatic heterocyclic be containing
There is 1~4 heteroatomic 5~10 yuan of rings, the hetero atom is N, O or S；
N=0~3.
As preferred embodiments of the present invention, X1、X2、X3It independently is C, N, O or S；Y1、Y2、Y3It independently is C, N or S；
R1、R2For H, halogen ,-CF3, C1~C6 alkyl, C1~C6 alkoxies, C2~C6 alkenyls, C2~C6 alkynyls, C3~C8 cycloalkyl,
C1~C6 acyloxy, heterocyclic radical, C6~C10 aryl or aromatic heterocyclic；The heterocyclic radical or aromatic heterocyclic are to contain 1~4
Heteroatomic 5~10 yuan of rings, the hetero atom are N, O or S；N=0~2.
Preferably, X1、X2、X3It independently is C, N, O or S；Y1、Y2、Y3It independently is C, N or S；R1、R2For-H, halogen ,-
CF3, C1~C4 alkyl, C1~C4 alkoxies, C2~C4 alkenyls, C2~C4 alkynyls, C3~C6 cycloalkyl, C1~C4 acyloxy,
Heterocyclic radical, phenyl, naphthyl or aromatic heterocyclic；The heterocyclic radical or aromatic heterocyclic are to contain 1~4 heteroatomic 5~10 yuan
Ring, the hetero atom are N, O or S；N=0~2.
It is further preferred that X1、X2、X3It independently is C, N, O or S；Y1、Y2、Y3It independently is C, N or S；R1、R2For-H,
Halogen ,-CF3, C1~C4 alkyl, C1~C4 alkoxies, C2~C4 alkenyls, C2~C4 alkynyls, C3~C6 cycloalkyl or C1~C4
Acyloxy；N=0~2.
Still more preferably, X1、X2、X3It independently is C, N, O or S；Y1、Y2、Y3It independently is C, N or S；R1For-H,
Halogen ,-CF3, C1~C4 alkyl, C1~C4 alkoxies, C2~C4 alkenyls, C2~C4 alkynyls, C3~C6 cycloalkyl or C2~C4
Acyloxy；R2For-H, halogen ,-CF3, C1~C4 alkyl；N=0~2.
Still further preferably, X1、X2、X3It independently is C, N, O or S；Y1、Y2、Y3It independently is C, N or S；R1For-H,
Halogen ,-CF3, C1~C4 alkyl, C1~C4 alkoxies, C2~C4 alkenyls, C2~C4 alkynyls, C3~C6 cycloalkyl or C2~C4
Acyloxy；R2For-H or halogen；N=0~2.
Optimal, X1、X2、X3It independently is C, N, O or S；Y1、Y2、Y3It independently is C, N or S；R1For-H, halogen ,-CF3、
C1~C4 alkyl, C1~C4 alkoxies, C2~C4 alkenyls, C2~C4 alkynyls, C3~C6 cycloalkyl or C2~C4 acyloxy；R2
For-H or halogen；N=0 or 1.
Above-mentioned 5- tertiary Ding isoxazoles -3- bases -3- (4- substituted-phenyls) urea derivative, works as X1、X3For N, X2For C when, its
Structure is as shown in formula II：
Wherein, Y1、Y2、Y3It independently is C, N, O or S；
R1、R2For-H, halogen ,-CF3, C1~C6 alkyl, C1~C6 alkoxies, C2~C6 alkenyls, C2~C6 alkynyls, C3~
C8 cycloalkyl, C1~C6 acyloxy, heterocyclic radical, C6~C10 aryl or aromatic heterocyclic；The heterocyclic radical or aromatic heterocyclic be containing
There is 1~4 heteroatomic 5~10 yuan of rings, the hetero atom is N, O or S；
N=0~3.
As preferred embodiments of the present invention, Y1、Y2、Y3It independently is C, N or S；R1、R2For H, halogen ,-CF3, C1~C6 alkane
Base, C1~C6 alkoxies, C2~C6 alkenyls, C2~C6 alkynyls, C3~C8 cycloalkyl, C1~C6 acyloxy, heterocyclic radical, C6~
C10 aryl or aromatic heterocyclic；The heterocyclic radical or aromatic heterocyclic be containing 1~4 heteroatomic 5~10 yuan of rings, it is described
Hetero atom is N, O or S；N=0~2.
Preferably, Y1、Y2、Y3It independently is C, N or S；R1、R2For-H, halogen ,-CF3, C1~C4 alkyl, C1~C4 alcoxyls
Base, C2~C4 alkenyls, C2~C4 alkynyls, C3~C6 cycloalkyl, C1~C4 acyloxy, heterocyclic radical, phenyl, naphthyl or heteroaromatic
Base；The heterocyclic radical or aromatic heterocyclic are that the hetero atom is N, O or S containing 1~4 heteroatomic 5~10 yuan of rings；n
=0~2.
It is further preferred that Y1、Y2、Y3It independently is C, N or S；R1、R2For-H, halogen ,-CF3, C1~C4 alkyl, C1~
C4 alkoxies, C2~C4 alkenyls, C2~C4 alkynyls, C3~C6 cycloalkyl or C1~C4 acyloxy；N=0~2.
Still more preferably, Y1、Y2、Y3It independently is C, N or S；R1For-H, halogen ,-CF3, C1~C4 alkyl, C1~
C4 alkoxies, C2~C4 alkenyls, C2~C4 alkynyls, C3~C6 cycloalkyl or C2~C4 acyloxy；R2For-H, halogen ,-CF3、C1
~C4 alkyl；N=0~2.
Still further preferably, Y1、Y2、Y3It independently is C, N or S；R1For-H, halogen ,-CF3, C1~C4 alkyl, C1~
C4 alkoxies, C2~C4 alkenyls, C2~C4 alkynyls, C3~C6 cycloalkyl or C2~C4 acyloxy；R2For-H or halogen；N=0~
2。
Optimal, Y1、Y2、Y3It independently is C, N or S；R1For-H, halogen ,-CF3, C1~C4 alkyl, C1~C4 alkoxies,
C2~C4 alkenyls, C2~C4 alkynyls, C3~C6 cycloalkyl or C2~C4 acyloxy；R2For-H or halogen；N=0 or 1.
Above-mentioned 5- tertiary Ding isoxazoles -3- bases -3- (4- substituted-phenyls) urea derivative, as n=1, its structure such as formula
Shown in III：
Wherein, Y1、Y2、Y3It independently is C, N, O or S；
R1、R2For-H, halogen ,-CF3, C1~C6 alkyl, C1~C6 alkoxies, C2~C6 alkenyls, C2~C6 alkynyls, C3~
C8 cycloalkyl, C1~C6 acyloxy, heterocyclic radical, C6~C10 aryl or aromatic heterocyclic；The heterocyclic radical or aromatic heterocyclic be containing
There is 1~4 heteroatomic 5~10 yuan of rings, the hetero atom is N, O or S.
As preferred embodiments of the present invention, Y1、Y2、Y3It independently is C, N or S；R1、R2For H, halogen ,-CF3, C1~C6 alkane
Base, C1~C6 alkoxies, C2~C6 alkenyls, C2~C6 alkynyls, C3~C8 cycloalkyl, C1~C6 acyloxy, heterocyclic radical, C6~
C10 aryl or aromatic heterocyclic；The heterocyclic radical or aromatic heterocyclic be containing 1~4 heteroatomic 5~10 yuan of rings, it is described
Hetero atom is N, O or S.
Preferably, Y1、Y2、Y3It independently is C, N or S；R1、R2For-H, halogen ,-CF3, C1~C4 alkyl, C1~C4 alcoxyls
Base, C2~C4 alkenyls, C2~C4 alkynyls, C3~C6 cycloalkyl, C1~C4 acyloxy, heterocyclic radical, phenyl, naphthyl or heteroaromatic
Base；The heterocyclic radical or aromatic heterocyclic are that the hetero atom is N, O or S containing 1~4 heteroatomic 5~10 yuan of rings.
It is further preferred that Y1、Y2、Y3It independently is C, N or S；R1、R2For-H, halogen ,-CF3, C1~C4 alkyl, C1
~C4 alkoxies, C2~C4 alkenyls, C2~C4 alkynyls, C3~C6 cycloalkyl or C1~C4 acyloxy.
Still more preferably, Y1、Y2、Y3It independently is C, N or S；R1For-H, halogen ,-CF3, C1~C4 alkyl, C1~
C4 alkoxies, C2~C4 alkenyls, C2~C4 alkynyls, C3~C6 cycloalkyl or C2~C4 acyloxy；R2For-H, halogen ,-CF3、C1
~C4 alkyl.
Still further preferably, Y1、Y2、Y3It independently is C, N or S；R1For-H, halogen ,-CF3, C1~C4 alkyl, C1~
C4 alkoxies, C2~C4 alkenyls, C2~C4 alkynyls, C3~C6 cycloalkyl or C2~C4 acyloxy；R2For-H or halogen.
Optimal, Y1、Y2、Y3It independently is C, N or S；R1For-H, halogen ,-CF3, C1~C4 alkyl, C1~C4 alkoxies,
C2~C4 alkenyls, C2~C4 alkynyls, C3~C6 cycloalkyl or C2~C4 acyloxy；R2For-H or halogen.
Above-mentioned 5- tertiary Ding isoxazoles -3- bases -3- (4- substituted-phenyls) urea derivative, works as Y1、Y2、Y3When being C, its
Structure is as shown in formula IV：
Wherein, R1、R2For-H, halogen ,-CF3, C1~C6 alkyl, C1~C6 alkoxies, C2~C6 alkenyls, C2~C6 alkynes
Base, C3~C8 cycloalkyl, C1~C6 acyloxy, heterocyclic radical, C6~C10 aryl or aromatic heterocyclic；The heterocyclic radical or virtue is miscellaneous
Ring group is that the hetero atom is N, O or S containing 1~4 heteroatomic 5~10 yuan of rings.
As preferred embodiments of the present invention, R1、R2For H, halogen ,-CF3, C1~C6 alkyl, C1~C6 alkoxies, C2~C6
Alkenyl, C2~C6 alkynyls, C3~C8 cycloalkyl, C1~C6 acyloxy, heterocyclic radical, C6~C10 aryl or aromatic heterocyclic；Described
Heterocyclic radical or aromatic heterocyclic are that the hetero atom is N, O or S containing 1~4 heteroatomic 5~10 yuan of rings.
Preferably, Y1、Y2、Y3It independently is C, N or S；R1、R2For-H, halogen ,-CF3, C1~C4 alkyl, C1~C4 alcoxyls
Base, C2~C4 alkenyls, C2~C4 alkynyls, C3~C6 cycloalkyl, C1~C4 acyloxy, heterocyclic radical, phenyl, naphthyl or heteroaromatic
Base；The heterocyclic radical or aromatic heterocyclic are that the hetero atom is N, O or S containing 1~4 heteroatomic 5~10 yuan of rings.
It is further preferred that Y1、Y2、Y3It independently is C, N or S；R1、R2For-H, halogen ,-CF3, C1~C4 alkyl, C1~
C4 alkoxies, C2~C4 alkenyls, C2~C4 alkynyls, C3~C6 cycloalkyl or C1~C4 acyloxy.
Still more preferably, Y1、Y2、Y3It independently is C, N or S；R1For-H, halogen ,-CF3, C1~C4 alkyl, C1~
C4 alkoxies, C2~C4 alkenyls, C2~C4 alkynyls, C3~C6 cycloalkyl or C2~C4 acyloxy；R2For-H, halogen ,-CF3、C1
~C4 alkyl.
Still further preferably, Y1、Y2、Y3It independently is C, N or S；R1For-H, halogen ,-CF3, C1~C4 alkyl, C1~
C4 alkoxies, C2~C4 alkenyls, C2~C4 alkynyls, C3~C6 cycloalkyl or C2~C4 acyloxy；R2For-H or halogen.
Optimal, Y1、Y2、Y3It independently is C, N or S；R1For-H, halogen ,-CF3, C1~C4 alkyl, C1~C4 alkoxies,
C2~C4 alkenyls, C2~C4 alkynyls, C3~C6 cycloalkyl or C2~C4 acyloxy；R2For-H or halogen.
The above-mentioned tertiary Ding isoxazoles -3- bases -3- of 5- (4- substituted-phenyls) urea derivative, its structural formula are：
Present invention also offers the preparation side of above-mentioned 5- tertiary Ding isoxazoles -3- bases -3- (4- substituted-phenyls) urea derivative
Method, synthetic route are：
Wherein, X1、X2、X3It independently is C, N, O or S；Y1、Y2、Y3It independently is C, N, O or S；R1、R2For-H, halogen ,-
CF3, C1~C6 alkyl, C1~C6 alkoxies, C2~C6 alkenyls, C2~C6 alkynyls, C3~C8 cycloalkyl, C1~C6 acyloxy,
Heterocyclic radical, C6~C10 aryl or aromatic heterocyclic；The heterocyclic radical or aromatic heterocyclic are to contain 1~4 heteroatomic 5~10
Yuan of rings, the hetero atom are N, O or S；N=0~3.
The preparation method of the above-mentioned tertiary Ding isoxazoles -3- bases -3- of 5- (4- substituted-phenyls) urea derivative, its reactions steps
For：
Triphosgene is placed in tetrahydrofuran, the tetrahydrofuran of the tertiary Ding isoxazoles of 3- amino -5- is slowly added dropwise thereto
Solution, then the tetrahydrofuran solution of triethylamine is slowly added dropwise, 15min is stirred at room temperature, decompression, which steams solvent, after the completion of reaction to produce
The thing 5- tert-butyl group -3- isoxazolyl isocyanates；The tertiary Ding isoxazoles of the triphosgene, 3- amino -5-, the molar ratio of triethylamine
For 1 ︰, 1 ︰ 3；
The 5- tert-butyl group -3- isoxazolyl isocyanates is dissolved in chloroform, then adds intermediate 1, heating reflux reaction
30min, room temperature is down to after the completion of reaction, and dichloromethane and water are added into reaction solution, 10min is vigorously stirred, gradually there is solid
Separate out, directly filter, wash solid with ethanol, obtain type I compound；The 5- tert-butyl groups -3- isoxazolyls the isocyanates is with
The molar ratio of mesosome 1 is 1 ︰ 1；The volume ratio of the dichloromethane and water is 1 ︰ 1.
Above-mentioned 5- tertiary Ding isoxazoles -3- bases -3- (4- substituted-phenyls) urea derivative of the present invention, includes the same of them
Position plain compound, racemic modification, optical active isomers, polymorphic forms or its mixture.
Present invention also offers the above-mentioned tertiary Ding isoxazoles -3- bases -3- of 5- (4- substituted-phenyls), urea derivative is pharmaceutically
Acceptable salt.
Present invention also offers the prodrug of the compounds of this invention, and according to the present invention, prodrug is the derivative of above-claimed cpd,
Their own may have weaker activity or even without activity, but upon administration, in physiological conditions (such as pass through generation
Thank, solvolysis or other mode) it is converted to corresponding biologically active form.
Present invention also offers the above-mentioned tertiary Ding isoxazoles -3- bases -3- of 5- (4- substituted-phenyls), urea derivative is pharmaceutically
Acceptable hydrate.
A kind of pharmaceutical composition, is as the 5- shown in formula I~IV tertiary Ding isoxazoles -3- bases -3- (4- substituted-phenyls) urea
Derivative and its salt or hydrate add what the pharmaceutically complementary component of acceptable was prepared.
Present invention also offers the above-mentioned tertiary Ding isoxazoles -3- bases -3- of 5- (4- substituted-phenyls) urea derivatives and its salt
Or hydrate prepare treat acute myeloid leukaemia medicine in purposes.
The present invention obtains the tertiary Ding isoxazoles -3- bases -3- of above-mentioned 5- (4- substituted-phenyls) on the basis of a large amount of screenings
Urea derivative, has the activity for suppressing human muscle creatine kinase cell Proliferation, for opening for treatment acute myeloid leukaemia medicine
Hair and application provide new selection.
Brief description of the drawings
Fig. 1 compounds 1- (the tertiary Ding isoxazoles -3- bases of 5-) -3- { 4- (imidazo [1,2-a] pyridine -2- bases) phenyl }
Urea (compound 2) suppresses MV4-11 cell Proliferation test results.
Fig. 2 compounds 1- (the tertiary Ding isoxazoles -3- bases of 5-) -3- { 4- (7- methylimidazoles simultaneously [1,2-a] pyridine -2- bases)
Phenyl } urea (compound 9) suppression MV4-11 cell Proliferation test results.
Fig. 3 compounds 1- (the tertiary Ding isoxazoles -3- bases of 5-) -3- { 4- (6- methylimidazoles simultaneously [1,2-a] pyridine -2- bases)
Phenyl } urea (compound 13) suppression MV4-11 cell Proliferation test results.
Fig. 4 compounds 1- (the tertiary Ding isoxazoles -3- bases of 5-) -3- { 4- (7- alkynyls imidazo [1,2-a] pyridine -2- bases)
Phenyl } urea (compound 26) suppression MV4-11 cell Proliferation test results.
Fig. 5 compounds 1- (the tertiary Ding isoxazoles -3- bases of 5-) -3- { 4- (6- alkynyls imidazo [1,2-a] pyridine -2- bases)
Phenyl } urea (compound 27) suppression MV4-11 cell Proliferation test results.
Fig. 6 compounds 1- (the tertiary Ding isoxazoles -3- bases of 5-) -3- { 4- (7- methoxyl groups imidazo [1,2-a] pyridine -2-
Base) phenyl } urea (compound 28) suppression MV4-11 cell Proliferation test results.
To the inhibitory activity of tumour in 28 body of Fig. 7 compounds.
Tumor staining figure after the processing of Fig. 8 compounds 28.
Effect of Fig. 9 compounds 28 to intracellular FLT3 phosphorylation levels.
Influence of Figure 10 compounds 28 to Apoptosis.
Embodiment
The synthesis of the 1 intermediate 5- tert-butyl group -3- isoxazolyl isocyanates of embodiment
Triphosgene 296.8mg (1mmol) is placed in 25mL round-bottomed flasks, and adds 3mL tetrahydrofurans and is dissolved, will
The tertiary Ding isoxazoles 140.2mg (1mmol) of 3- amino -5- are slowly added dropwise into above-mentioned flask after being dissolved in 2mL tetrahydrofurans, then
Measure triethylamine 0.416mL (3mmol) to be dissolved in 2mL tetrahydrofurans, be slowly added dropwise into above-mentioned flask, 15min be stirred at room temperature, instead
Decompression steams solvent and obtains the product 5- tert-butyl group -3- isoxazolyl isocyanates after the completion of answering.
2 1- of embodiment (the tertiary Ding isoxazoles -3- bases of 5-) -3- { 4- (imidazo [2,1-b] thiazole -6- bases) phenyl } urea
The synthesis of (compound 1)
Thiazolamine 500.65mg (5mmol) is dissolved in absolute ethyl alcohol (15mL), then adds the bromo- 4- nitrobenzenes of 2-
Benzoylformaldoxime 1.464g (6mmol), is heated to reflux 12h, there is solid precipitation after the completion of reaction, directly filters, washs to obtain 6- (4- nitre
Base phenyl) imidazo [2,1-B] thiazole.
6- (4- nitrobenzophenones) imidazo [2,1-b] thiazole 245mg (1mmol) are dissolved in absolute ethyl alcohol (9mL) and water
In (3mL), ammonium chloride 213.9mg (4mmol) and iron powder 558mg (10mmol) is then added, 30min is heated to reflux, has reacted
Unreacted iron powder is filtered to remove after, ethyl acetate (100mL), dilute hydrochloric acid (1N) washing three are dissolved in after filtrate decompression is concentrated
It is secondary, after adding anhydrous magnesium sulfate drying decompression steam solvent and obtain 6- (4- aminophenyls) imidazo [2,1-B] thiazole.
The 5- tert-butyl group -3- isoxazolyl isocyanates 132mg (0.8mmol) are dissolved in chloroform (15mL), are then added
6- (4- aminophenyls) imidazo [2,1-B] thiazole 172mg (0.8mmol), is heated to reflux 30min, is down to after the completion of reaction often
Temperature, dichloromethane (10mL) and water (10mL) are added into reaction solution, 10min is vigorously stirred, gradually there is solid precipitation, direct mistake
Filter, product 1- (the tertiary Ding isoxazoles -3- bases of 5-) -3- { 4- (imidazo [2,1-b] thiazole -6- bases) phenyl } is washed to obtain with ethanol
Urea.The Structural Identification data of product are as follows：
1H NMR (400MHz, DMSO) δ 9.53 (s, 1H), 8.87 (s, 1H), 8.14 (s, 1H), 7.92 (d, J=3.8Hz,
1H), 7.77 (d, J=8.1Hz, 2H), 7.49 (d, J=8.1Hz, 2H), 7.24 (d, J=3.8Hz, 1H), 6.53 (s, 1H),
1.30(s,9H)；MS(ES+)m/z：382.2(M+H+)。
3 1- of embodiment (the tertiary Ding isoxazoles -3- bases of 5-) -3- { 4- (imidazo [1,2-a] pyridine -2- bases) phenyl } urea
The synthesis of (compound 2)
By the preparation method of embodiment 2, raw material is replaced with into 2-aminopyridine, finally washs to obtain product 1- (5- with ethanol
Tertiary Ding isoxazoles -3- bases) -3- { 4- (imidazo [1,2-a] pyridine -2- bases) phenyl } urea.The Structural Identification data of product are such as
Under：
1H NMR (400MHz, DMSO) δ 9.56 (s, 1H), 8.92 (s, 1H), 8.50 (d, J=6.6Hz, 1H), 8.32 (s,
1H), 7.92 (d, J=7.8Hz, 2H), 7.56 (d, J=7.5Hz, 2H), 7.52 (d, J=6.68Hz, 1H), 7.22 (t, J=
7.8Hz, 1H), 6.87 (t, J=6.5Hz, 1H), 6.54 (s, 1H), 1.30 (s, 9H)；MS(ES+)m/z：376.2(M+H+)。
4 1- of embodiment (the tertiary Ding isoxazoles -3- bases of 5-) -3- { 4- (8- chlorine imidazo [1,2-a] pyridine -2- bases) benzene
Base } urea (compound 3) synthesis
By the preparation method of embodiment 2, raw material is replaced with into 2- amino -3- chloropyridines, finally washs to obtain product with ethanol
1- (the tertiary Ding isoxazoles -3- bases of 5-) -3- { 4- (8- chlorine imidazo [1,2-a] pyridine -2- bases) phenyl } urea.The structure mirror of product
Fixed number is according to as follows：
1H NMR(400MHz,DMSO)δ9.55(s,1H),8.91(s,1H),8.31(s,1H),8.18(s,1H),7.88
(d, J=8.0Hz, 2H), 7.53 (d, J=7.9Hz, 2H), 7.45 (d, J=9.2Hz, 1H), 6.96 (d, J=9.2Hz, 1H),
6.53(s,1H),1.30(s,9H)。MS(ES+)m/z：410.2(M+H+)。
5 1- of embodiment (the tertiary Ding isoxazoles -3- bases of 5-) -3- { 4- (8- methylimidazoles simultaneously [1,2-a] pyridine -2- bases) benzene
Base } urea (compound 4) synthesis
By the preparation method of embodiment 2, raw material is replaced with into 2- amino -3- picolines, finally being washed with ethanol to produce
Thing 1- (the tertiary Ding isoxazoles -3- bases of 5-) -3- { 4- (8- methylimidazoles simultaneously [1,2-a] pyridine -2- bases) phenyl } urea.The knot of product
Structure appraising datum is as follows：
1H NMR (400MHz, DMSO) δ 9.55 (s, 1H), 8.95 (s, 1H), 8.33 (d, J=6.6Hz, 1H), 8.29 (s,
1H), 7.92 (d, J=6.8Hz, 2H), 7.55 (d, J=6.9Hz, 2H), 7.01 (d, J=6.7Hz, 1H), 6.77 (t, J=
7.2Hz,1H),6.54(s,1H),2.53(s,3H),1.30(s,9H)。MS(ES+)m/z：390.2(M+H+)。
6 1- of embodiment (the tertiary Ding isoxazoles -3- bases of 5-) -3- { 4- (8- flumizoles simultaneously [1,2-a] pyridine -2- bases) benzene
Base } urea (compound 5) synthesis
By the preparation method of embodiment 2, raw material is replaced with into 2- amino -3- fluorine pyridines, finally washs to obtain product with ethanol
1- (the tertiary Ding isoxazoles -3- bases of 5-) -3- { 4- (8- flumizoles simultaneously [1,2-a] pyridine -2- bases) phenyl } urea.The structure mirror of product
Fixed number is according to as follows：
1H NMR(400MHz,DMSO)δ9.56(s,1H),8.95(s,1H),8.74(s,1H),8.32(s,1H),7.90
(d, J=8.2Hz, 2H), 7.62 (d, J=7.9,5.0Hz, 1H), 7.54 (d, J=8.2Hz, 2H), 7.31 (t, J=9.0Hz,
1H),6.53(s,1H),1.30(s,9H)。MS(ES+)m/z：394.2(M+H+)。
7 1- of embodiment (the tertiary Ding isoxazoles -3- bases of 5-) -3- { 4- (8- trifluoromethyl imidazoles simultaneously [1,2-a] pyridine -2-
Base) phenyl urea (compound 6) synthesis
By the preparation method of embodiment 2, raw material is replaced with into 2- amino -3- trifluoromethyl pyridines, is finally washed with ethanol
Product 1- (the tertiary Ding isoxazoles -3- bases of 5-) -3- { 4- (8- trifluoromethyl imidazoles simultaneously [1,2-a] pyridine -2- bases) phenyl } urea.
The Structural Identification data of product are as follows：
1H NMR (400MHz, DMSO) δ 9.55 (s, 1H), 8.97 (s, 1H), 8.79 (d, J=5.5Hz, 1H), 8.51 (s,
1H), 7.94 (d, J=7.4Hz, 2H), 7.69 (d, J=5.8Hz, 1H), 7.57 (d, J=7.5Hz, 2H), 7.02 (t, J=
7.8Hz,1H),6.54(s,1H),1.30(s,9H)。MS(ES+)m/z：444.2(M+H+)。
8 1- of embodiment (the tertiary Ding isoxazoles -3- bases of 5-) -3- { 4- (imidazo [1,2-a] pyrimidine -2-base) phenyl } urea
The synthesis of (compound 7)
By the preparation method of embodiment 2, raw material is replaced with into 2- aminopyrimidines, finally washs to obtain product 1- (5- with ethanol
Tertiary Ding isoxazoles -3- bases) -3- { 4- (imidazo [1,2-a] pyrimidine -2-base) phenyl } urea.The Structural Identification data of product are such as
Under：
1H NMR (400MHz, DMSO) δ 10.34 (s, 1H), 10.03 (s, 1H), 9.32 (d, J=6.1Hz, 1H), 8.96
(d, J=5.8Hz, 1H), 8.69 (s, 1H), 7.96 (d, J=7.9Hz, 2H), 7.66 (d, J=8.1Hz, 2H), 7.64 (t, J=
6.5Hz,1H),6.53(s,1H),1.30(s,9H)。MS(ES+)m/z：377.2(M+H+)。
9 1- of embodiment (the tertiary Ding isoxazoles -3- bases of 5-) -3- { 4- (7- chlorine imidazo [1,2-a] pyridine -2- bases) benzene
Base } urea (compound 8) synthesis
By the preparation method of embodiment 2, raw material is replaced with into 2- amino -4- chloropyridines, finally washs to obtain product with ethanol
1- (the tertiary Ding isoxazoles -3- bases of 5-) -3- { 4- (7- chlorine imidazo [1,2-a] pyridine -2- bases) phenyl } urea.The structure mirror of product
Fixed number is according to as follows：
1H NMR (400MHz, DMSO) δ 9.81 (s, 1H), 9.63 (s, 1H), 8.83 (d, J=7.1Hz, 1H), 8.65 (s,
1H), 8.01 (s, 1H), 7.92 (d, J=8.2Hz, 2H), 7.66 (d, J=8.2Hz, 2H), 7.48 (d, J=6.8Hz, 1H),
6.53(s,1H),1.30(s,9H)。MS(ES+)m/z：410.1(M+H+)。
10 1- of embodiment (the tertiary Ding isoxazoles -3- bases of 5-) -3- { 4- (7- methylimidazoles simultaneously [1,2-a] pyridine -2- bases)
Phenyl } urea (compound 9) synthesis
By the preparation method of embodiment 2, raw material is replaced with into 2-AMINO-4-PICOLINE, finally being washed with ethanol to produce
Thing 1- (the tertiary Ding isoxazoles -3- bases of 5-) -3- { 4- (7- methylimidazoles simultaneously [1,2-a] pyridine -2- bases) phenyl } urea.The knot of product
Structure appraising datum is as follows：
1H NMR (400MHz, DMSO) δ 9.57 (s, 1H), 8.97 (s, 1H), 8.37 (d, J=7.1Hz, 1H), 8.21 (s,
1H), 7.88 (d, J=8.2Hz, 2H), 7.53 (d, J=8.2Hz, 2H), 7.32 (s, 1H), 6.71 (d, J=6.8Hz, 1H),
6.53(s,1H),2.34(s,3H),1.29(s,9H)。MS(ES+)m/z：390.2(M+H+)。
11 1- of embodiment (the tertiary Ding isoxazoles -3- bases of 5-) -3- { 4- (7- bromines imidazo [1,2-a] pyridine -2- bases) benzene
Base } urea (compound 10) synthesis
By the preparation method of embodiment 2, raw material is replaced with into 2- amino -4- bromopyridines, finally washs to obtain product with ethanol
1- (the tertiary Ding isoxazoles -3- bases of 5-) -3- { 4- (7- bromines imidazo [1,2-a] pyridine -2- bases) phenyl } urea.The structure mirror of product
Fixed number is according to as follows：
1H NMR (400MHz, DMSO) δ 10.03 (s, 1H), 9.95 (s, 1H), 8.73 (s, 1H), 8.63 (d, J=
6.3Hz, 1H), 7.96 (d, J=8.1Hz, 2H), 7.66 (s), 7.64 (d, J=8.0Hz, 2H), 7.27 (d, J=5.8Hz,
1H),6.54(s,1H),1.30(s,9H)。MS(ES+)m/z：454.1(M+H+)。
12 1- of embodiment (the tertiary Ding isoxazoles -3- bases of 5-) -3- { 4- (imidazo [1,2-a] pyrazine -2- bases) phenyl } urea
The synthesis of (compound 11)
By the preparation method of embodiment 2, raw material is replaced with into 2- Aminopyrazines, finally washs to obtain product 1- (5- with ethanol
Tertiary Ding isoxazoles -3- bases) -3- { 4- (imidazo [1,2-a] pyrazine -2- bases) phenyl } urea.The Structural Identification data of product are such as
Under：
1H NMR (400MHz, DMSO) δ 9.58 (s, 1H), 9.05 (s, 1H), 8.99 (s, 1H), 8.58 (d, J=6.5Hz,
1H), 8.53 (s, 1H), 7.98 (d, J=7.9Hz, 2H), 7.89 (d, J=6.3Hz, 1H), 7.58 (d, J=8.2Hz, 2H),
6.53(s,1H),1.31(s,9H)。MS(ES+)m/z：377.2(M+H+)。
13 1- of embodiment (the tertiary Ding isoxazoles -3- bases of 5-) -3- { 4- (6- chlorine imidazo [1,2-a] pyridine -2- bases) benzene
Base } urea (compound 12) synthesis
By the preparation method of embodiment 2, raw material is replaced with into -5 chloropyridine of 2- amino, finally washs to obtain product 1- with ethanol
(the tertiary Ding isoxazoles -3- bases of 5-) -3- { 4- (6- chlorine imidazo [1,2-a] pyridine -2- bases) phenyl } urea.The Structural Identification of product
Data are as follows：
1H NMR (400MHz, DMSO) δ 8.96 (s, 1H), 8.80 (s, 1H), 8.30 (s, 1H), 7.91 (d, J=7.5Hz,
2H), 7.61 (d, J=9.3Hz, 1H), 7.55 (d, J=7.5Hz, 2H), 7.28 (d, J=9.3Hz, 1H), 6.54 (s, 1H),
1.31(s,9H)。MS(ES+)m/z：410.2(M+H+)。
14 1- of embodiment (the tertiary Ding isoxazoles -3- bases of 5-) -3- { 4- (6- methylimidazoles simultaneously [1,2-a] pyridine -2- bases)
Phenyl } urea (compound 13) synthesis
By the preparation method of embodiment 2, raw material is replaced with into 2- amino -5- picolines, finally being washed with ethanol to produce
Thing 1- (the tertiary Ding isoxazoles -3- bases of 5-) -3- { 4- (6- methylimidazoles simultaneously [1,2-a] pyridine -2- bases) phenyl } urea.The knot of product
Structure appraising datum is as follows：
1H NMR(400MHz,DMSO)δ9.88(s,1H),9.82(s,1H),8.68(s,1H),8.63(s,1H),7.93
(d, J=8.3Hz, 2H), 7.86 (d, J=9.1Hz, 1H), 7.79 (d, J=9.1Hz, 1H), 7.67 (d, J=8.2Hz, 2H),
6.54(s,1H),2.42(s,3H),1.31(s,9H)。MS(ES+)m/z：390.2(M+H+)。
15 1- of embodiment (the tertiary Ding isoxazoles -3- bases of 5-) -3- { 4- (6- flumizoles simultaneously [1,2-a] pyridine -2- bases) benzene
Base } urea (compound 14) synthesis
By the preparation method of embodiment 2, raw material is replaced with into 2- amino-5-fluorine pyridines, finally washs to obtain product with ethanol
1- (the tertiary Ding isoxazoles -3- bases of 5-) -3- { 4- (6- flumizoles simultaneously [1,2-a] pyridine -2- bases) phenyl } urea.The structure mirror of product
Fixed number is according to as follows：
1H NMR (400MHz, DMSO) δ 9.61 (s, 1H), 9.08 (s, 1H), 8.52 (d, J=6.3Hz, 1H), 8.45 (s,
1H), 7.93 (d, J=7.8Hz, 2H), 7.57 (d, J=7.8Hz, 2H), 7.41 (d, J=7.1Hz, 1H), 6.87 (t, J=
6.6Hz,1H),6.54(s,1H),1.30(s,9H)。MS(ES+)m/z：394.2(M+H+)。
16 1- of embodiment (the tertiary Ding isoxazoles -3- bases of 5-) -3- { 4- (6- bromines imidazo [1,2-a] pyridine -2- bases) benzene
Base } urea (compound 15) synthesis
By the preparation method of embodiment 2, raw material is replaced with into 2- amino -5- bromopyridines, finally washs to obtain product with ethanol
1- (the tertiary Ding isoxazoles -3- bases of 5-) -3- { 4- (6- bromines imidazo [1,2-a] pyridine -2- bases) phenyl } urea.The structure mirror of product
Fixed number is according to as follows：
1H NMR(400MHz,DMSO)δ9.60(s,1H),9.06(s,1H),8.86(s,1H),8.29(s,1H),7.90
(d, J=8.0Hz, 2H), 7.55 (d, J=8.5Hz, 2H), 7.50 (d, J=9.0Hz, 1H), 7.34 (d, J=9.5Hz, 1H),
6.53(s,1H),1.30(s,9H)。MS(ES+)m/z：454.1(M+H+)。
17 1- of embodiment (the tertiary Ding isoxazoles -3- bases of 5-) -3- { 4- (6- iodine imidazo [1,2-a] pyridine -2- bases) benzene
Base } urea (compound 16) synthesis
By the preparation method of embodiment 2, raw material is replaced with into 2- amino -5- iodine pyridines, finally washs to obtain product with ethanol
1- (the tertiary Ding isoxazoles -3- bases of 5-) -3- { 4- (6- iodine imidazo [1,2-a] pyridine -2- bases) phenyl } urea.The structure mirror of product
Fixed number is according to as follows：
1H NMR(400MHz,DMSO)δ9.92(s,1H),9.88(s,1H),9.21(s,1H),8.55(s,1H),8.04
(d, J=8.7Hz, 1H), 7.90 (d, J=7.4Hz, 2H), 7.73 (d, J=8.8Hz, 1H), 7.63 (d, J=7.7Hz, 2H),
6.52(s,1H),1.30(s,9H)。MS(ES+)m/z：502.1(M+H+)。
18 1- of embodiment (the tertiary Ding isoxazoles -3- bases of 5-) -3- { 4- (5- chlorine imidazo [1,2-a] pyridine -2- bases) benzene
Base } urea (compound 17) synthesis
By the preparation method of embodiment 2, raw material is replaced with into 2- amino -6- chloropyridines, finally washs to obtain product with ethanol
1- (the tertiary Ding isoxazoles -3- bases of 5-) -3- { 4- (5- chlorine imidazo [1,2-a] pyridine -2- bases) phenyl } urea.The structure mirror of product
Fixed number is according to as follows：
1H NMR (400MHz, DMSO) δ 9.99 (s, 1H), 9.93 (s, 1H), 8.89 (s, 1H), 8.07 (d, J=8.0Hz,
2H), 7.94 (d, J=8.5Hz, 1H), 7.86 (t, J=8.0Hz, 1H), 7.69 (d, J=7.2Hz, 1H), 7.63 (d, J=
8.0Hz,2H),6.55(s,1H),1.31(s,9H)。MS(ES+)m/z：410.2(M+H+)。
19 1- of embodiment (the tertiary Ding isoxazoles -3- bases of 5-) -3- { 4- (5- methylimidazoles simultaneously [1,2-a] pyridine -2- bases)
Phenyl } urea (compound 18) synthesis
By the preparation method of embodiment 2, raw material is replaced with into 2- amino -6- picolines, finally being washed with ethanol to produce
Thing 1- (the tertiary Ding isoxazoles -3- bases of 5-) -3- { 4- (5- methylimidazoles simultaneously [1,2-a] pyridine -2- bases) phenyl } urea.The knot of product
Structure appraising datum is as follows：
1H NMR (400MHz, DMSO) δ 9.57 (s, 1H), 8.93 (s, 1H), 8.28 (s, 1H), 7.98 (d, J=6.2Hz,
2H), 7.54 (d, J=6.4Hz, 2H), 7.46 (d, J=7.8Hz, 1H), 7.21 (t, J=7.6Hz, 1H), 6.77 (d, J=
6.8Hz,1H),6.54(s,1H),2.63(s,3H),1.30(s,9H)。MS(ES+)m/z：382.2(M+H+)。
20 1- of embodiment (the tertiary Ding isoxazoles -3- bases of 5-) -3- { 4- (imidazo [1,2-b] pyridazine -2- bases) phenyl } urea
The synthesis of (compound 19)
By the preparation method of embodiment 2, raw material is replaced with into 3- amino pyridazines, finally washs to obtain product 1- (5- with ethanol
Tertiary Ding isoxazoles -3- bases) -3- { 4- (imidazo [1,2-b] pyridazine -2- bases) phenyl } urea.The Structural Identification data of product are such as
Under：
1H NMR (400MHz, DMSO) δ 9.64 (s, 1H), 9.16 (s, 1H), 8.77 (s, 1H), 8.47 (d, J=8.3Hz,
1H), 8.09 (d, J=8.1Hz, 1H), 7.99 (d, J=7.1Hz, 2H), 7.57 (d, J=7.1Hz, 2H), 7.21 (t, d, J=
8.5Hz,1H),6.54(s,1H),1.29(s,9H)。MS(ES+)m/z：377.2(M+H+)。
21 1- of embodiment (the tertiary Ding isoxazoles -3- bases of 5-) -3- { 4- (6- cyclopropyl imidazo [1,2-a] pyridine -2-
Base) phenyl urea (compound 20) synthesis
The bromo- 2- of 6- (4- nitrobenzophenones) imidazo [1,2-a] pyridine 634mg (2mmol) are dissolved in toluene (25mL), according to
Secondary addition cyclopropylboronic acid 188.98mg (2.2mmol) and potassium phosphate 1.273g (6mmol), nitrogen protection is lower to add [1,1'- pair
(diphenylphosphine) ferrocene] palladium chloride dichloromethane complex 81.7mg (0.1mmol), it is heated to 90 DEG C of reaction 4h, reaction
After the completion of be filtered to remove unreacted [1,1'- double (diphenylphosphine) ferrocene] palladium chloride dichloromethane complex, by filtrate
It is concentrated under reduced pressure, 6- cyclopropyl -2- (4- nitrobenzophenones) imidazo [1,2-a] pyridine is obtained after column chromatography.
By the preparation method of embodiment 2, by 6- (4- nitrobenzophenones) imidazo [2,1-b] thiazole replace with 6- cyclopropyl-
2- (4- nitrobenzophenones) imidazo [1,2-a] pyridine, finally with ethanol wash product 1- (the tertiary Ding isoxazoles -3- bases of 5-) -
3- { 4- (6- cyclopropyl imidazo [1,2-a] pyridine -2- bases) phenyl } urea.The Structural Identification data of product are as follows：
1H NMR(400MHz,DMSO)δ9.52(s,1H),8.89(s,1H),8.32(s,1H),8.18(s,1H),7.87
(d, J=8.1Hz, 2H), 7.52 (d, J=8.1Hz, 2H), 7.45 (d, J=9.2Hz, 1H), 6.97 (d, J=9.3Hz, 1H),
6.52 (s, 1H), 2.04-1.88 (m, 1H), 1.30 (s, 9H), 0.94 (d, J=7.9Hz, 2H), 0.71 (d, J=4.4Hz,
2H)。MS(ES+)m/z：416.3(M+H+)。
22 1- of embodiment { 4- (benzo [d] oxazole -2- bases) phenyl } -3- ((change by 5- (tert-butyl group) isoxazole -3-bases) urea
Compound 21) synthesis
O-aminophenol 327mg (3mmol) is placed in 50mL round-bottomed flasks, adds 25mL glycerine and 5mL methanol, heating
To 90 DEG C of stirring 30min, paranitrobenzaldehyde 453.36mg (3mmol) is then added, 90 DEG C of reaction 2h, reaction is completed backward anti-
Addition 20mL ethyl acetate in system is answered, organic layer with water, saturated common salt water washing, depressurizes dense after anhydrous magnesium sulfate drying successively
Contracting removes solvent, and product is placed in mortar, adds [double (trifluoroacetyl epoxide) iodine] benzene 1.311g (3.05mmol), room temperature is ground
15min is ground, after the completion of reaction, reaction system is changed into black, there is liquid generation, and 50mL ethyl acetate is added into reaction system,
Organic layer with water, saturated common salt water washing, is concentrated under reduced pressure after anhydrous magnesium sulfate drying successively and removes solvent, and 2- is obtained after column chromatography
(4- nitrobenzophenones) benzo [d] oxazoles.
By the preparation method of embodiment 2,6- (4- nitrobenzophenones) imidazo [2,1-b] thiazole is replaced with into 2- (4- nitros
Phenyl) [d] oxazoles, finally wash to obtain product 1- { 4- (benzo [d] oxazole -2- bases) phenyl } -3- (5- (tertiary fourths to benzo with ethanol
Base) isoxazole -3-bases) urea.The Structural Identification data of product are as follows：
1H NMR (400MHz, DMSO) δ 9.67 (s, 1H), 9.21 (s, 1H), 8.15 (d, J=7.8Hz, 2H), 7.77 (s,
2H), 7.71 (d, J=7.8Hz, 2H), 7.40 (d, J=4.0Hz, 2H), 6.55 (s, 1H), 1.31 (s, 9H).MS(ES+)m/z：
377.2(M+H+)。
23 1- of embodiment { 4- (benzo [d] thiazol-2-yl) phenyl } -3- ((change by 5- (tert-butyl group) isoxazole -3-bases) urea
Compound 22) synthesis
By the preparation method of embodiment 22, raw material is replaced near amino thiophenols, finally washs 1- { 4- (benzene with ethanol
And [d] thiazol-2-yl) phenyl -3- (5- (tert-butyl group) isoxazole -3-bases) ureas.The Structural Identification data of product are as follows：
1H NMR (400MHz, DMSO) δ 9.65 (s, 1H), 9.19 (s, 1H), 8.12 (d, J=7.7Hz, 1H), 8.06 (d,
J=7.8Hz, 2H), 8.02 (d, J=7.5Hz, 1H), 7.67 (d, J=8.0Hz, 2H), 7.54 (t, J=7.2Hz, 1H), 7.44
(t, J=7.2Hz, 1H), 6.55 (s, 1H), 1.31 (s, 9H).MS(ES+)m/z：393.1(M+H+)。
24 1- of embodiment { 4- (benzo [d] thiophene -2- bases) phenyl } -3- ((change by 5- (tert-butyl group) isoxazole -3-bases) urea
Compound 23) synthesis
Benzothiophene -2- boric acid 783.2mg (4.4mmol) is dissolved in toluene (25mL), is sequentially added to Nitrobromobenzene
804mg (4mmol) and potassium phosphate 2.547g (12mmol), nitrogen protection is lower to add [1,1'- double (diphenylphosphine) ferrocene] two
Palladium bichloride dichloromethane complex 163.3mg (0.2mmol), is heated to 90 DEG C of reaction 4h, is filtered to remove after the completion of reaction not anti-
[1,1'- double (diphenylphosphine) ferrocene] palladium chloride dichloromethane complex answered, filtrate decompression is concentrated, after column chromatography
2- (4- nitrobenzophenones) benzo [d] thiophene.
By the preparation method of embodiment 2,6- (4- nitrobenzophenones) imidazo [2,1-b] thiazole is replaced with into 2- (4- nitros
Phenyl) benzo [d] thiophene, finally washs to obtain product 1- { 4- (benzo [d] thiophene -2- bases) phenyl } -3- (5- (tertiary fourths with ethanol
Base) isoxazole -3-bases) urea.The Structural Identification data of product are as follows：
1H NMR (400MHz, DMSO) δ 9.60 (s, 2H), 7.94 (d, J=7.7Hz, 1H), 7.81 (d, J=7.6Hz,
1H), 7.76 (s, 1H), 7.72 (d, J=8.0Hz, 2H), 7.60 (d, J=8.0Hz, 2H), 7.38 (d, J=7.8Hz, 1H),
7.33 (d, J=8.0Hz, 1H), 6.54 (s, 1H), 1.30 (s, 9H).MS(ES+)m/z：414.2(M+Na+)。
25 1- of embodiment { 4- (benzo [d] furans -2- bases) phenyl } -3- ((change by 5- (tert-butyl group) isoxazole -3-bases) urea
Compound 24) synthesis
Salicylaldhyde 610.6mg (5mmol) is dissolved in the DMF of 25mL, is then added to nitrobenzyl bromine 1.08g
(5mmol) and potassium tert-butoxide 1.68g (15mmol), is heated to reflux 4h, and after the completion of reaction, reaction solution is down to room temperature, adds
100mL ethyl acetate, organic layer with water, saturated common salt water washing, are concentrated under reduced pressure after anhydrous magnesium sulfate drying successively and remove solvent,
2- (4- nitrobenzophenones) benzo [d] furans is obtained after column chromatography.
By the preparation method of embodiment 2,6- (4- nitrobenzophenones) imidazo [2,1-b] thiazole is replaced with into 2- (4- nitros
Phenyl) benzo [d] furans, finally washs to obtain product 1- { 4- (benzo [d] furans -2- bases) phenyl } -3- (5- (tertiary fourths with ethanol
Base) isoxazole -3-bases) urea.The Structural Identification data of product are as follows：
1H NMR (400MHz, DMSO) δ 9.61 (s, 1H), 9.06 (s, 1H), 7.86 (d, J=6.0Hz, 2H), 7.62 (s,
4H), 7.29 (d, J=6.2Hz, 2H), 7.25 (s, 1H), 6.54 (s, 1H), 1.30 (s, 9H).MS(ES+)m/z：398.2(M+
Na+)。
26 1- of embodiment (the tertiary Ding isoxazoles -3- bases of 5-) -3- { 4- (3- chlorine imidazo [1,2-a] pyridine -2- bases) benzene
Base } urea (compound 25) synthesis
The chloro- 2- of 3- (4- nitrobenzophenones) imidazo [1,2-a] pyridine 478.14mg (2mmol) are dissolved in DMF (15mL),
N- chlorosuccinimides 280mg (2.1mmol) is added, stirring at normal temperature 6h, after the completion of reaction, 25mL is added into reaction system
Water, stir 15min, have solid precipitation, directly filter and be washed with water the chloro- 2- of product 3- (4- aminophenyls) imidazo [1,
2-a] pyridine.
By the preparation method of embodiment 2,6- (4- nitrobenzophenones) imidazo [2,1-b] thiazole is replaced with into the chloro- 2- (4- of 3-
Nitrobenzophenone) imidazo [1,2-a] pyridine, finally washs to obtain product 1- (the tertiary Ding isoxazoles -3- bases of 5-) -3- { 4- with ethanol
(3- chlorine imidazo [1,2-a] pyridine -2- bases) phenyl } urea.The Structural Identification data of product are as follows：
1H NMR (400MHz, DMSO) δ 9.88 (s, 1H), 9.81 (s, 1H), 8.81 (d, J=6.4Hz, 1H), 8.71 (s,
1H), 7.97 (s, 1H), 7.95 (d, J=8.2Hz, 2H), 7.67 (d, J=8.1Hz, 2H), 7.42 (d, J=6.5Hz, 1H),
6.54(s,1H),1.31(s,9H)。MS(ES+)m/z：410.2(M+H+)。
27 1- of embodiment (the tertiary Ding isoxazoles -3- bases of 5-) -3- { 4- (7- alkynyls imidazo [1,2-a] pyridine -2- bases)
Phenyl } urea (compound 26) synthesis
The bromo- 2- of 7- (4- nitrobenzophenones) imidazo [1,2-a] pyridine 634mg (2mmol) are dissolved in 20mL tetrahydrofurans,
Then bis-triphenylphosphipalladium palladium dichloride 140.4mg (0.2mmol), cuprous iodide 38.09mg (0.2mmol), triphen are sequentially added
Base phosphine 53mg (0.2mmol), is heated to 65 DEG C of reaction 30min, then adds trimethylsilyl acetylene 0.8mL (5.6mmol), three second
Amine 2.68mL (20mmol), 65 DEG C of the reaction was continued 12h, after the completion of reaction, column chromatography obtains 7- ((trimethyl silicon substrate) alkynyl) -2-
(4- nitrobenzophenones) imidazo [1,2-a] pyridine.
By 7- ((trimethyl silicon substrate) alkynyl) -2- (4- nitrobenzophenones) imidazo [1,2-a] pyridines 536.17mg
(1.6mmol) is dissolved in 15mL methanol, then adds Anhydrous potassium carbonate 442.27mg (3.2mmol), stirring at normal temperature 2h, has reacted
Cheng Hou, pressurization concentration remove solvent, add the dissolving of 50mL ethyl acetate, and organic layer is anhydrous successively with water, saturated common salt water washing
The removing solvent that is concentrated under reduced pressure after magnesium sulfate drying obtains 7- alkynyls -2- (4- aminophenyls) imidazo [1,2-a] pyridine.
By the preparation method of embodiment 2,6- (4- nitrobenzophenones) imidazo [2,1-b] thiazole is replaced with into 7- alkynyls -2-
(4- nitrobenzophenones) imidazo [1,2-a] pyridine, finally washs to obtain product 1- (the tertiary Ding isoxazoles -3- bases of 5-) -3- with ethanol
{ 4- (7- alkynyls imidazo [1,2-a] pyridine -2- bases) phenyl } urea.The Structural Identification data of product are as follows：
1H NMR (400MHz, DMSO) δ 9.83 (s, 1H), 9.68 (s, 1H), 8.74 (d, J=6.5Hz, 1H), 8.64 (s,
1H), 8.13 (s, 1H), 7.93 (d, J=7.8Hz, 2H), 7.66 (d, J=8.2Hz, 2H), 7.56 (d, J=6.1Hz, 1H),
6.53(s,1H),3.07(s,1H),1.30(s,9H)。MS(ES+)m/z：400.3(M+H+)。
28 1- of embodiment (the tertiary Ding isoxazoles -3- bases of 5-) -3- { 4- (6- alkynyls imidazo [1,2-a] pyridine -2- bases)
Phenyl } urea (compound 27) synthesis
By the preparation method of embodiment 27, it is bromo- to change the bromo- 2- of 7- (4- nitrobenzophenones) imidazo [1,2-a] pyridine into 6-
2- (4- nitrobenzophenones) imidazo [1,2-a] pyridine, finally with ethanol wash product 1- (the tertiary Ding isoxazoles -3- bases of 5-) -
3- { 4- (6- alkynyls imidazo [1,2-a] pyridine -2- bases) phenyl } urea.The Structural Identification data of product are as follows：
1H NMR (400MHz, DMSO) δ 9.33 (s, 1H), 8.74 (s, 1H), 8.56 (d, J=6.2Hz, 1H), 8.05 (d,
J=6.0Hz, 1H), 7.66 (d, J=8.2Hz, 2H), 7.31 (d, J=8.0Hz, 2H), 7.27 (s, 1H), 7.00 (s, 1H),
6.29(s,1H),4.02(s,1H),1.05(s,9H)。MS(ES+)m/z：400.3(M+H+)。
29 1- of embodiment (the tertiary Ding isoxazoles -3- bases of 5-) -3- { 4- (7- methoxyl groups imidazo [1,2-a] pyridine -2-
Base) phenyl urea (compound 28) synthesis
The bromo- 2- of 7- (4- nitrobenzophenones) imidazo [1,2-a] pyridine 1.902g (6mmol) are dissolved in the 1,4- dioxies of 20mL
In six rings, potassium acetate 1.766g (18mmol), connection boric acid pinacol ester 5.142g (13.5mmol) are sequentially added, under nitrogen protection
[1,1'- double (diphenylphosphine) ferrocene] palladium chloride dichloromethane complex 489mg (0.6mmol) is added, under nitrogen protection
90 DEG C of reaction 6h, reaction are completed to add glacial acetic acid 0.68mL (12mmol), 2mL water, 1.224mL in backward reaction system
The H of (12mmol) 30%2O2, 2h is reacted at room temperature, has solid generation after the completion of reaction, directly filters to obtain product 7- hydroxyl -2- (4-
Nitrobenzophenone) imidazo [1,2-a] pyridine.
7- hydroxyls -2- (4- nitrobenzophenones) imidazo [1,2-a] pyridine 510mg (2mmol) are dissolved in 15mL DMF (N, N-
Dimethylformamide) in, then sequentially add potassium carbonate 552.8mg (4mmol), 149.6 μ L (2.4mmol) of iodomethane, room temperature
2h is reacted, reaction is completed to add 30mL water in backward reaction solution, and stirring, has solid precipitation, directly filter to obtain 7- methoxyl groups -2-
(4- nitrobenzophenones) imidazo [1,2-a] pyridine.
By the preparation method of embodiment 2, by 6- (4- nitrobenzophenones) imidazo [2,1-b] thiazole replace with 7- methoxyl groups-
2- (4- nitrobenzophenones) imidazo [1,2-a] pyridine, finally with ethanol wash product 1- (the tertiary Ding isoxazoles -3- bases of 5-) -
3- { 4- (7- methoxyl groups imidazo [1,2-a] pyridine -2- bases) phenyl } urea.The Structural Identification data of product are as follows：
1H NMR (400MHz, DMSO) δ 9.54 (s, 1H), 8.91 (s, 1H), 8.36 (d, J=7.1Hz, 1H), 8.11 (s,
1H), 7.85 (d, J=7.9Hz, 2H), 7.52 (d, J=8.0Hz, 2H), 6.95 (s, 1H), 6.60 (d, J=7.0Hz, 1H),
6.53(s,1H),3.84(s,3H),1.30(s,9H)。MS(ES+)m/z：406.2(M+H+)。
30 1- of embodiment (the tertiary Ding isoxazoles -3- bases of 5-) -3- { 4- (7- ethyoxyls imidazo [1,2-a] pyridine -2-
Base) phenyl urea (compound 29) synthesis
By the preparation method of embodiment 29, iodomethane is replaced with into iodoethane, finally washs to obtain product 1- (uncles 5- with ethanol
Ding isoxazole -3- bases) -3- { 4- (7- ethyoxyls imidazo [1,2-a] pyridine -2- bases) phenyl } urea.The Structural Identification of product
Data are as follows：
1H NMR (400MHz, DMSO) δ 9.55 (s, 1H), 8.90 (s, 1H), 8.35 (d, J=7.2Hz, 1H), 8.11 (s,
1H), 7.84 (d, J=8.0Hz, 2H), 7.51 (d, J=8.1Hz, 2H), 6.92 (s, 1H), 6.59 (d, J=7.2Hz, 1H),
6.53 (s, 1H), 4.10 (dd, J=13.4,6.6Hz, 2H), 1.37 (t, J=6.4Hz, 3H), 1.30 (s, 9H).MS(ES+)m/
z：420.3 (M+H+)。
31 2- of embodiment (4- (3- (the tertiary Ding isoxazoles -3- bases of 5-) urea groups) phenylimidazole simultaneously [1,2-a] pyridine -6- bases
The synthesis of acetic acid esters (compound 30)
7- hydroxyls -2- (4- nitrobenzophenones) imidazo [1,2-a] pyridine 510mg (2mmol) are dissolved in 15mL dichloromethane
In, sequentially add chloroacetic chloride 284.4 μ L (4mmol), DMAP (4-dimethylaminopyridine) 24.5mg (0.2mmol), 30 DEG C of stirrings
React 15min, be concentrated under reduced pressure after the completion of reaction remove solvent, add 50mL ethyl acetate lysates, organic layer successively with water,
Saturated common salt water washing, the removing solvent that is concentrated under reduced pressure after anhydrous magnesium sulfate drying obtain 2- (4- nitrobenzophenones) imidazo [1,2-a]
Pyridin-7-yl acetic acid esters.
By the preparation method of embodiment 2,6- (4- nitrobenzophenones) imidazo [2,1-b] thiazole is replaced with into 2- (4- nitros
Phenyl) imidazo [1,2-a] pyridin-7-yl acetic acid esters, finally washs to obtain product 2- (4- (3- (the tertiary Ding Jiyi Evil of 5- with ethanol
Azoles -3- bases) urea groups) phenylimidazole simultaneously [1,2-a] pyridin-7-yl acetic acid esters.The Structural Identification data of product are as follows：
1H NMR (400MHz, DMSO) δ 9.56 (s, 1H), 8.96 (s, 1H), 8.55 (d, J=8.1Hz, 1H), 8.33 (s,
1H), 7.89 (d, J=7.3Hz, 2H), 7.54 (d, J=7.9Hz, 2H), 7.35 (s, 1H), 6.81 (d, J=7.4Hz, 1H),
6.53(s,1H),2.32(s,3H),1.31(s,9H)。MS(ES+)m/z：434.2(M+H+)。
Pharmacodynamic experiment part
Cell, kinases, the source of reagent used in the invention of this reality are：IMDM culture mediums, hyclone are purchased from Gibco
Company；Methyl thiazoly tetrazolium assay (MTT), lauryl sodium sulfate (SDS) are purchased from Sigma Co., USA；Pvdf membrane is purchased from the U.S.
Millipor companies；FLT3 antibody, the anti-rabbit IgG of horseradish peroxidase-labeled and anti-mouse are purchased from the limited public affairs of Beijing Zhong Shan Golden Bridge
Department；IgG, Annexin V-FICT/PI apoptosis detection kit, TUNEL (the transferase mediated notches of DNA end
End-labelling) detection kit, Ki67 detection kits be purchased from Roche companies of the U.S.；MV4-11 (human muscle creatine kinases
Cell line) from American Type Culture preservation center (ATCC)；NOD-SCID mouse, SPF (specific pathogen
Free) level, purchased from Beijing HFK Bio-Technology Co., Ltd.；FLT3 antibody, p-FLT3 antibody are purchased from U.S. Cell
Signaling companies；β-actin antibody is purchased from Chinese doctor's moral company.
32 Compound ira vitro MV4-11 cell inhibitory effects of embodiment are tested and the experiment of FLT3 kinase assays
MTT colorimetric methods are a kind of methods for detecting cell survival and growth, its action principle is that soluble tetrazole is blue
(Tetrazolium salt, MTT) is reduced to the bluish violet knot of slightly solubility in the presence of the mitochondrial dehydrogenase of living cells
Brilliant thing first a ceremonial jade-ladle, used in libation (formazan) is simultaneously deposited in cell, this effect is lost with the death of cell.The 20% water-soluble liquid energies of SDS are molten
Solve cell in first a ceremonial jade-ladle, used in libation, formed solution, by automatic microplate reader measure 570nm at optical density OD (Optical density,
OD) change of value or absorbance (A) value can directly reflect the survival rate of MV4-11 cells.In the range of certain cell quantity,
MTT crystallizes that the amount to be formed is directly proportional to living cells quantity, propagation that thus can be by OD values come pharmaceutical to MV4-11 cells
Inhibition level.
ADP-GloTMKinase assay kit is the kinase assay kit of a luminescence method, it detects institute in kinase reaction
The ADP of formation；ADP is converted to ATP, and then ATP is again by Ultra-GloTMLuciferase changes into light, luminous signal and kinases
Active positive correlation.After compound is combined with FLT3, ATP will be unable to be combined with FLT3, so that ADP can not be generated, cause to shine
Signal weakens, therefore the binding ability of luminous signal and compound and FLT3 is inversely proportional, and can be quantified by the power of luminous signal
The binding ability of compound and FLT3.
Table 1 is part of compounds to the in-vitro multiplication histamine result of MV4-11 (human muscle creatine kinase cell line) and right
FLT3 interaction in vitro results：
1 part of compounds of table suppresses and to FLT3 interaction in vitro results the in-vitro multiplication of MV4-11
Note：* literature value：IC50=0.56nM；* AC220 are the acute myeloid leukaemias in clinical II phase research
(AML) inhibitor, has an effect of preferable treatment AML, is in normal range (NR) in this, as positive control, the result during test
Within；Wherein, AC220, which is bought in Nanjing celestial worthy Ze Zhong Chemical Co., Ltd.s, its structural formula, is：
As can be seen from the above table, tester AC220 test results are in normal range (NR), from test data as can be seen that changing
Compound 2,9,13,26,27,28 is respectively provided with preferable external MV4-11 rejection abilities under low nanomolar concentration, also has to FLT3
Good inhibiting effect.
Compound 2, the inhibitory action of 9,13,26,27,28 couples of MV4-11 also have dose dependent, see Fig. 1~Fig. 6.
The activity in vivo of 33 compound 28 of embodiment and preliminary Mechanism Study result
1st, the activity in vivo of compound 28
Using the method for subcutaneous vaccination, MV4-11 neoplasm transplantations are established with NOD-SCID mouse, and by its point
For control group and medication therapy groups, medication therapy groups take orally give 10,30,60mg/kg compounds 28 respectively, and control group gives not
The blank formulation of drug containing.In experimentation, gross tumor volume, animation of nude mice etc. are recorded, according to control group and administration
The gross tumor volume of group carrys out the internal antitumor activity of preliminary judgement medicine, and when control group tumour grows to certain volume by mouse
Put to death, take tumour to carry out immunohistochemistry.
Experimental result shown in Fig. 7：60mg/kg/d dosage, which takes orally, to be given compound 28 tumour can be made to disappear at 27 days；
10mk/kg/d and 30mg/kg/d, which takes orally, gives compound 28, and tumour inhibiting rate is respectively 51% and 87%.During administration, mouse weight
There is not significant change.Prove it is oral give compound 28 and can effectively suppress the growth of MV4-11 tumours in Mice Body, and have
There is dose dependent.
Ki67 is a kind of relevant nuclear antigen of proliferative cell, its function and mitosis are closely related, in cell Proliferation
It is indispensable.Ki67 kit testing principles：Utilize complementation of the immunology antigen-antibody molecule due to structure and compatibility each other
Generation specifically binds principle, and makes the enzyme chromogenic reagent of labelled antibody by redox chemistry reaction to determine tissue
Intracellular antigen, positions it, is qualitative.It is to be coupled the anti-human Ki67 immunohistochemistry monoclonal antibody of mouse and structural first
Ki67 antigens；Second is enzyme mark sheep anti-Mouse/rabbit igg polymer identification connected Ki67 antibody；3rd, add bottom
Thing, the horseradish peroxidase part on polymer can be catalyzed the H in DAB nitrite ions2O2Decompose, become benzidine oxidation
Biphenyl imines, makes occur yellow or brownish discoloration in histotomy on antigen site；Finally sample is redyed and mounting.
By microscopic develop the color situation, infer histotomy on Ki67 there are site and situation.
TUNEL cell apoptosis detection kits are the crack conditions for detecting cell nucleus DNA in apoptotic process,
Its principle is effects of the dUTP in deoxyribonucleotide terminal enzyme (DNA) (TdT Enzyme) of biotin (biotin) mark
Under, it may be connected to the 3'-OH ends for the DNA being broken in apoptotic cell, and with being connected horseradish peroxidase (HRP, horse-
Radish peroxidase) anti-fluorescein antibody specific binding, the latter again with HRP substrates diaminobenzidine (DAB) react
Very strong color reaction (being in dark-brown) is produced, is specifically positioned exactly just in the cell of apoptosis, thus under an optical microscope
Can observing apoptosis cell；It is opposite, the normal or cell bred almost without DNA fracture, because without 3'-OH
Formed, so being less able to be colored.
Experimental result shown in Fig. 8：Tumour after the processing of compound 28 is found after being dyed with Ki67, with solvent control group phase
Than when, the cell of propagation significantly reduces；And apoptosis in the tumor tissues after the processing of compound 28 is found after being dyed with TUNEL
Cell showed increased.These as shown by data compounds 28 can suppress the propagation of tumour cell and promote tumour cell in vivo
Apoptosis.
2nd, effect of the compound 28 to intracellular FLT3 phosphorylation levels
Experimental principle：For Western Blot using polyacrylamide gel electrophoresis, detected material is protein, " is visited
Pin " is antibody, and " colour developing " uses the secondary antibody of mark.By the separated protein examples of PAGE, solid phase carrier (pvdf membrane) is transferred to
On, solid phase carrier adsorbed proteins in the form of non-covalent bond, and the polypeptide forms and its biological activity of electrophoretic separation can be kept
It is constant.Using the protein on solid phase carrier or polypeptide as antigen, immune response is played with corresponding antibody, then with enzyme or isotope
The secondary antibody of mark reacts, by substrate colour developing or autoradiograph to detect the specific target gene table of electrophoretic separation
The protein ingredient reached.
After handling 2 hours of MV4-11 cells with the compound 28 of various concentrations, compound 28 is shown to FLT3 tables
Up to amount and the dose-dependent inhibitory action of phosphorylation level, as shown in Figure 9.
3rd, influence of the compound 28 to Apoptosis
Annexin V-FITC apoptosis detection kit testing principles：In normal living cells, phosphatidylserine
(phosphotidylserine, PS) is located at the inner side of cell membrane, but in the cell of early apoptosis, and PS is from the inner side of cell membrane
The surface of cell membrane is turned to, in extracellular environment.Annexin-V (annexin-V) is that a kind of molecular weight is 35-
The Ca of 36KD2+Dependence cardiolipin binding protein, can be combined with PS high-affinities.With fluorescein (FITC, Alexa Fluor488
Deng) mark Annexin-V as probe, the generation of Apoptosis is can detect using flow cytometer or fluorescence microscope.Separately
Outside, propidium iodide (propidine iodide, PI) is a kind of nucleic acid dye, it cannot pass through complete cell membrane, but wither
The cell and dead cell of middle and advanced stage are died, PI can pass through cell membrane and make the red dye of nucleus.Therefore Annexin-V is matched with PI
Use, it is possible to detect the ratio of apoptosis early stage, late period and dead cell.
The compound 28 of various concentrations acts on MV4-11,48 it is small when after detect Apoptosis situation, as shown in Figure 10.
The results show compound 28 can induce MV4-11 Apoptosis, during 100nM concentration, apoptotic cell with dose dependent
Ratio is 29.8%.
In conclusion compound provided by the invention has MV4-11 stronger in-vitro multiplication inhibitory action, and dividing
Sub- level also has good inhibiting effect to FLT3.In addition, (4- substitutes the tertiary Ding isoxazoles -3- bases -3- of 5- of the present invention
Phenyl) preparation method of urea derivative has that reaction condition is gentle, abundant raw material is easy to get, operate and post-process simply, gross production rate
Higher, the advantages that environmental pollution is smaller.
Claims (25)
1.5- tertiary Ding isoxazoles -3- bases -3- (4- substituted-phenyls) urea derivative, its structure is as shown in formula I：
Wherein, X1、X2、X3It independently is C, N, O or S；Y1、Y2、Y3It independently is C, N, O or S；
R1For-H, halogen ,-CF3, C1~C6 alkyl, C1~C6 alkoxies, C2~C6 alkenyls, C2~C6 alkynyls, C3~C8 cycloalkanes
Base or C1~C6 acyloxy；R2For-H, halogen ,-CF3Or C1~C6 alkyl；N=0~3.
2. the tertiary Ding isoxazoles -3- bases -3- of 5- (4- substituted-phenyls) urea derivative according to claim 1, its feature exist
In：X1、X2、X3It independently is C, N, O or S；Y1、Y2、Y3It independently is C, N or S；R1For H, halogen ,-CF3, C1~C6 alkyl, C1
~C6 alkoxies, C2~C6 alkenyls, C2~C6 alkynyls, C3~C8 cycloalkyl or C1~C6 acyloxy；R2For-H, halogen ,-CF3Or
C1~C6 alkyl；N=0~2.
3. the tertiary Ding isoxazoles -3- bases -3- of 5- (4- substituted-phenyls) urea derivative according to claim 2, its feature exist
In：X1、X2、X3It independently is C, N, O or S；Y1、Y2、Y3It independently is C, N or S；R1For-H, halogen ,-CF3, C1~C4 alkyl,
C1~C4 alkoxies, C2~C4 alkenyls, C2~C4 alkynyls, C3~C6 cycloalkyl or C1~C4 acyloxy；R2For-H, halogen ,-CF3
Or C1~C4 alkyl；N=0~2.
4. the tertiary Ding isoxazoles -3- bases -3- of 5- (4- substituted-phenyls) urea derivative according to claim 3, its feature exist
In：X1、X2、X3It independently is C, N, O or S；Y1、Y2、Y3It independently is C, N or S；R1For-H, halogen ,-CF3, C1~C4 alkyl,
C1~C4 alkoxies, C2~C4 alkenyls, C2~C4 alkynyls, C3~C6 cycloalkyl or C2~C4 acyloxy；R2For-H, halogen ,-
CF3, C1~C4 alkyl；N=0~2.
5. the tertiary Ding isoxazoles -3- bases -3- of 5- (4- substituted-phenyls) urea derivative according to claim 4, its feature exist
In：X1、X2、X3It independently is C, N, O or S；Y1、Y2、Y3It independently is C, N or S；R1For-H, halogen ,-CF3, C1~C4 alkyl,
C1~C4 alkoxies, C2~C4 alkenyls, C2~C4 alkynyls, C3~C6 cycloalkyl or C2~C4 acyloxy；R2For-H or halogen；n
=0~2.
6. the tertiary Ding isoxazoles -3- bases -3- of 5- (4- substituted-phenyls) urea derivative according to claim 5, its feature exist
In：X1、X2、X3It independently is C, N, O or S；Y1、Y2、Y3It independently is C, N or S；R1For-H, halogen ,-CF3, C1~C4 alkyl,
C1~C4 alkoxies, C2~C4 alkenyls, C2~C4 alkynyls, C3~C6 cycloalkyl or C2~C4 acyloxy；R2For-H or halogen；n
=0 or 1.
7. the tertiary Ding isoxazoles -3- bases -3- of 5- (4- substituted-phenyls) urea derivative according to claim 1, its feature exist
In：Work as X1、X3For N, X2For C when, its structure is as shown in formula II：
Wherein, Y1、Y2、Y3It independently is C, N, O or S；
R1For-H, halogen ,-CF3, C1~C6 alkyl, C1~C6 alkoxies, C2~C6 alkenyls, C2~C6 alkynyls, C3~C8 cycloalkanes
Base or C1~C6 acyloxy；R2For-H, halogen ,-CF3Or C1~C6 alkyl；N=0~3.
8. the tertiary Ding isoxazoles -3- bases -3- of 5- (4- substituted-phenyls) urea derivative according to claim 7, its feature exist
In：Y1、Y2、Y3It independently is C, N or S；R1For H, halogen ,-CF3, C1~C6 alkyl, C1~C6 alkoxies, C2~C6 alkenyls, C2
~C6 alkynyls, C3~C8 cycloalkyl or C1~C6 acyloxy；R2For-H, halogen ,-CF3Or C1~C6 alkyl；N=0~2.
9. the tertiary Ding isoxazoles -3- bases -3- of 5- (4- substituted-phenyls) urea derivative according to claim 8, its feature exist
In：Y1、Y2、Y3It independently is C, N or S；R1For-H, halogen ,-CF3, C1~C4 alkyl, C1~C4 alkoxies, C2~C4 alkenyls,
C2~C4 alkynyls, C3~C6 cycloalkyl or C1~C4 acyloxy；R2For-H, halogen ,-CF3Or C1~C4 alkyl；N=0~2.
10. the tertiary Ding isoxazoles -3- bases -3- of 5- (4- substituted-phenyls) urea derivative according to claim 9, its feature exist
In：Y1、Y2、Y3It independently is C, N or S；R1For-H, halogen ,-CF3, C1~C4 alkyl, C1~C4 alkoxies, C2~C4 alkenyls,
C2~C4 alkynyls, C3~C6 cycloalkyl or C2~C4 acyloxy；R2For-H, halogen ,-CF3Or C1~C4 alkyl；N=0~2.
11. the tertiary Ding isoxazoles -3- bases -3- of 5- (4- substituted-phenyls) urea derivative according to claim 10, its feature
It is：Y1、Y2、Y3It independently is C, N or S；R1For-H, halogen ,-CF3, C1~C4 alkyl, C1~C4 alkoxies, C2~C4 alkene
Base, C2~C4 alkynyls, C3~C6 cycloalkyl or C2~C4 acyloxy；R2For-H or halogen；N=0~2.
12. the tertiary Ding isoxazoles -3- bases -3- of 5- (4- substituted-phenyls) urea derivative according to claim 11, its feature
It is：Y1、Y2、Y3It independently is C, N or S；R1For-H, halogen ,-CF3, C1~C4 alkyl, C1~C4 alkoxies, C2~C4 alkene
Base, C2~C4 alkynyls, C3~C6 cycloalkyl or C2~C4 acyloxy；R2For-H or halogen；N=0 or 1.
13. the tertiary Ding isoxazoles -3- bases -3- of 5- (4- substituted-phenyls) urea derivative according to claim 7, its feature exist
In：As n=1, its structure is as shown in formula III：
Wherein, Y1、Y2、Y3It independently is C, N, O or S；
R1For-H, halogen ,-CF3, C1~C6 alkyl, C1~C6 alkoxies, C2~C6 alkenyls, C2~C6 alkynyls, C3~C8 cycloalkanes
Base or C1~C6 acyloxy；R2For-H, halogen ,-CF3Or C1~C6 alkyl.
14. the tertiary Ding isoxazoles -3- bases -3- of 5- (4- substituted-phenyls) urea derivative according to claim 13, its feature
It is：Y1、Y2、Y3It independently is C, N or S；R1For-H, halogen ,-CF3, C1~C6 alkyl, C1~C6 alkoxies, C2~C6 alkene
Base, C2~C6 alkynyls, C3~C8 cycloalkyl or C1~C6 acyloxy；R2For-H, halogen ,-CF3Or C1~C6 alkyl.
15. the tertiary Ding isoxazoles -3- bases -3- of 5- (4- substituted-phenyls) urea derivative according to claim 14, its feature
It is：Y1、Y2、Y3It independently is C, N or S；R1For-H, halogen ,-CF3, C1~C4 alkyl, C1~C4 alkoxies, C2~C4 alkene
Base, C2~C4 alkynyls, C3~C6 cycloalkyl or C1~C4 acyloxy；R2For-H, halogen ,-CF3Or C1~C4 alkyl.
16. the tertiary Ding isoxazoles -3- bases -3- of 5- (4- substituted-phenyls) urea derivative according to claim 15, its feature
It is：Y1、Y2、Y3It independently is C, N or S；R1For-H, halogen ,-CF3, C1~C4 alkyl, C1~C4 alkoxies, C2~C4 alkene
Base, C2~C4 alkynyls, C3~C6 cycloalkyl or C2~C4 acyloxy；R2For-H, halogen ,-CF3Or C1~C4 alkyl.
17. the tertiary Ding isoxazoles -3- bases -3- of 5- (4- substituted-phenyls) urea derivative according to claim 16, its feature
It is：Y1、Y2、Y3It independently is C, N or S；R1For-H, halogen ,-CF3, C1~C4 alkyl, C1~C4 alkoxies, C2~C4 alkene
Base, C2~C4 alkynyls, C3~C6 cycloalkyl or C2~C4 acyloxy；R2For-H or halogen.
18. the tertiary Ding isoxazoles -3- bases -3- of 5- (4- substituted-phenyls) urea derivative according to claim 13, its feature
It is：Work as Y1、Y2、Y3When being C, its structure is as shown in formula IV：
Wherein, R1For-H, halogen ,-CF3, C1~C6 alkyl, C1~C6 alkoxies, C2~C6 alkenyls, C2~C6 alkynyls, C3~C8
Cycloalkyl or C1~C6 acyloxy；R2For-H, halogen ,-CF3Or C1~C6 alkyl.
19. the tertiary Ding isoxazoles -3- bases -3- of 5- (4- substituted-phenyls) urea derivative according to claim 18, its feature
It is：R1For-H, halogen ,-CF3, C1~C4 alkyl, C1~C4 alkoxies, C2~C4 alkenyls, C2~C4 alkynyls, C3~C6 cycloalkanes
Base or C1~C4 acyloxy；R2For-H, halogen ,-CF3Or C1~C4 alkyl.
20. the tertiary Ding isoxazoles -3- bases -3- of 5- (4- substituted-phenyls) urea derivative according to claim 19, its feature
It is：R1For-H, halogen ,-CF3, C1~C4 alkyl, C1~C4 alkoxies, C2~C4 alkenyls, C2~C4 alkynyls, C3~C6 cycloalkanes
Base or C2~C4 acyloxy；R2For-H, halogen ,-CF3Or C1~C4 alkyl.
21. the tertiary Ding isoxazoles -3- bases -3- of 5- (4- substituted-phenyls) urea derivative according to claim 20, its feature
It is：R1For-H, halogen ,-CF3, C1~C4 alkyl, C1~C4 alkoxies, C2~C4 alkenyls, C2~C4 alkynyls, C3~C6 cycloalkanes
Base or C2~C4 acyloxy；R2For-H or halogen.
22.5- tertiary Ding isoxazoles -3- bases -3- (4- substituted-phenyls) urea derivative, its structural formula are：
Claim 1~22 any one of them 5- 23. tertiary Ding isoxazoles -3- bases -3- (4- substituted-phenyls) urea derivative medicine
Acceptable salt on.
24. a kind of pharmaceutical composition, is by the tertiary Ding isoxazoles -3- bases -3- (4- of claim 1~22 any one of them 5-
Substituted-phenyl) pharmaceutically the complementary component of acceptable is prepared for urea derivative or salt addition described in claim 23
's.
25. the tertiary Ding isoxazoles -3- bases -3- of claim 1~22 any one of them 5- (4- substituted-phenyls) urea derivatives or
Salt described in claim 23 is preparing the purposes in treating acute myeloid leukaemia medicine.
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