CN102020638B

CN102020638B - 2-indolinone derivative with protein kinase inhibition activity and histone deacetylase inhibition activity and preparation method and application thereof

Info

Publication number: CN102020638B
Application number: CN 200910176473
Authority: CN
Inventors: 鲁先平; 李志斌; 余金迪; 山松; 吴仲闻; 王祥辉; 马保顺; 宁志强
Original assignee: SHENZHEN WEIXIN BIOLOGICAL SCIENCE AND TECHNOLOGY Co Ltd
Current assignee: Shenzhen microbiology Polytron Technologies Inc
Priority date: 2009-09-16
Filing date: 2009-09-16
Publication date: 2013-05-08
Anticipated expiration: 2029-09-16
Also published as: CN102020638A

Abstract

The invention discloses a 2-indolinone derivative with protein kinase inhibition activity and histone deacetylase inhibition activity and a preparation method and application thereof. The structure of the 2-indolinone derivative is shown as a general formula (I), wherein the definitions of X, R1, R2, R3, R4, R5, R6, R7, R8 and n are shown in the specifications. The compound simultaneously has the protein kinase inhibition activity and the histone deacetylase inhibition activity and can be used for treating protein kinase activity abnormality or histone deacetylase activity abnormality-related diseases which comprise inflammation, autoimmune diseases, cancers, nerve system diseases, neurodegenerative disorders, cardiovascular diseases, allergy, asthma and hormone-related diseases.

Description

Have protein kinase inhibiting activity and histon deacetylase (HDAC) and suppress active 2-dihydroindole ketone derivate, its preparation method and application

Technical field

The present invention relates to have simultaneously that protein kinase inhibiting activity and histon deacetylase (HDAC) suppress active 2-dihydroindole ketone derivate, it is synthetic and the clinical application aspect the active extremely relevant disease of and histon deacetylase (HDAC) abnormal to protein kinase activity in treatment.

Background technology

Protein kinase is the phosphorylation of the hydroxyl on specific tyrosine, Serine and threonine residues in the class of enzymes of catalytic protein phosphorylation, particularly catalytic protein.Protein kinase all plays a key effect in regulating many cell physiological processes, comprises metabolism, cell proliferation, cytodifferentiation, cell survival, environment-main body reaction, immunne response and vasculogenesis.Numerous disease all should be relevant with the cell of being regulated the abnormality that causes by protein kinase.These diseases comprise inflammation, autoimmune disorder, cancer, nervous system disorders and neurodegenerative disorders, cardiovascular disorder, allergy, asthma and with hormone relative disease (Tan, S-L., 2006, J.Immunol., 176:2872-2879; Healy, A.ea al., 2006, J.Immunol., 177:1886-1893; Salek-Ardakani, S.et al., 2005, J.Immunol., 175:7635-7641; Kim, J.et al., 2004, J.Clin.Invest., 114:823-827).Therefore, people are devoted to seek effectively to treat the kinases inhibitor of these diseases always.

Protein kinase is divided into two classes usually, i.e. protein tyrosine kinase (PTKs) and serine-threonine kinase (STKs).

Protein tyrosine kinase (PTKs) can be divided into two classes, i.e. non-cross-film Tyrosylprotein kinase and transmembrane growth factor receptor Tyrosylprotein kinase (RTKs).At present, at least 19 kinds of different subtribes of RTKs have been determined, as EGF-R ELISA (EGFR), vascular endothelial growth factor receptor (VEGFR), platelet-derived growth factor receptor (PDGFR) and fibroblast growth factor receptor (FGFR).

EGF-R ELISA (EGFR) family has comprised four kinds of cross-film Tyrosylprotein kinase growth factor receptorses, i.e. HER1, HER2, HER3 and HER4.After the combination of one group of specific part and acceptor, promoted the dimerization of EGFR, and caused the autophosphorylation (Arteaga, C-L., 2001, Curr.Opin.Oncol., 6:491-498) of tyrosine residues in acceptor.In case after the acceptor autophosphorylation, the downstream of several signal transduction pathways of EGFR just has been activated.The process that the signal transduction pathway of EGFR and tumour generate is closely related, comprises motion, invasion and the transfer of cell cycle progression, apoptotic inhibition, tumour cell.The activation of EGFR has also stimulated vascular endothelial growth factor (VEGF), and it is the main inductor (Petit, A-M.et al., 1997, Am.J.Pathol., 151:1523-1530) of vasculogenesis.In experimental model, the downward of the signal transduction pathway of EGFR mediation and the generation closely related (Wikstrand, C-J.et al., 1998, J Natl Cancer Inst., 90:799-800) of tumour.Can observe continuous amplification activation and the overexpression of the EGFR albumen that causes of sudden change in many human tumors, comprising the tumour of chest, lung, ovary and kidney.These sudden changes are determinatives (Wikstrand, C-J.etal., 1998, J Natl Cancer Inst., 90:799-800) of neoplasm invasiveness.Right and wrong are usually seen in nonsmall-cell lung cancer (NSCLC) in the EGFR overexpression.The activity of EGFR can be by blocking the outer ligand binding domain of born of the same parents or suppressing by suppressing the EGFR Tyrosylprotein kinase with small molecules with anti-EGFR-antibodies, suppress thereby reach the purpose (Mendelsohn that EGFR passage downstream signal transmits, J., 1997, Clin.Can.Res., 3:2707-2707).

Nearly all solid tumor and mesenchymoma all can be secreted vascular endothelial growth factor (VEGF) in the hypoxemia situation.It is highly single-minded to blood vessel endothelium, and can play regulating effect to vascular proliferation and infiltration.The overexpression of VEGF level and the increase of total microvessel density, cancer return and survival rate reduce all closely related (Parikh, A-A., 2004; , Hematol.Oncol.Clin.N.Am., 18:951-971 Parikh, A-A., 2004; , Hematol.Oncol.Clin.M Am., 18:951-971).6 kinds of different parts: VEGF-A are arranged to E and placenta growth factor concerning vegf receptor.Part combines with special receptor (being mainly VEGFR-2) on endotheliocyte.The combination of VEGF-A and VEGFR-1 causes the migration of endotheliocyte, causes endothelial cell proliferation, infiltration and survival with the combination of VEGFR-2, and VEGFR-3 is considered to regulate lymph vessels and generates.The combination of VEGF and VEGFR-2 acceptor has caused activation and the autophosphorylation in intracellular tyrosine kinases zone, and generation (Parikh, A-A., 2004 of further having triggered a succession of signal in cell, Hematol.Oncol.Clin.N.Am., 18:951-971).

Serine-threonine kinase (STKs) mainly is present in cell.Also there is the receptor kinase of a few STKs type.STKs is the modal form of cytosol kinases, and they are in their function of tenuigenin position execution, rather than the organoid in tenuigenin and cytoskeleton.

GSK-3 (GSK-3) is a kind of serine-threonine protein kinase enzyme, has comprised two kinds of isomeric form of α and β, and these two kinds of isomeric form are separately with the genes encoding of uniqueness.It is found that GSK-3 can make many modulin phosphorylations, and the activity that can regulate them.GSK-3 is relevant to various diseases, comprises diabetes, senile dementia, central nervous system disorder such as manic depressive disorder and neurodegenerative disease, myocardial hypertrophy etc. (Haq, et al., 2000, J.Cell Biol., 151:117).

Aurora-2 is a kind of serine-threonine protein kinase enzyme, and it and human body cancer are closely related, as colorectal carcinoma, mastocarcinoma and other solid tumor.It is believed that it is relevant with the protein phosphorylation of regulating the cell cycle.Especially, in the mitotic division process, Aurora-2 has played effect in the chromosomal accurate separation of control.The abnormal adjusting of cell cycle can cause cell proliferation and other abnormal.In the human colon cancerous tissue, it is found that Aurora-2 albumen is by overexpression (Schumacher, et al., 1998, J.CellBiol., 143:1635-1646; Kimura et al., 1997, J.Biol.Chem., 272:13766-13771).

Cell cycle dependant kinase (CDKs) is a kind of serine-threonine protein kinase enzyme, and it regulates mammiferous cell fission.At present, people have determined nine kinase subunits (CDK 1-9).Each kinases and a specific controlled plant combine, and have consisted of together the active catalytic site.Proliferation out of control is characteristics of cancer cells, in many important solid tumors, the abnormal adjusting of CDK function usually occurs.People cherish a special interest to CDK2 and CDK4, because in many human tumors, their activity is usually regulated extremely.

The Raf kinases is a kind of downstream effect albumen of ras oncoprotein.It is by the crucial setter of cell surface to nuclear signal transduction pathway.Suppress the Raf kinases with in vivo with the growth of the many human tumors of vitro inhibition closely related (Moniaet al., 1996, Nat.Med., 2:668-675).

Other serine-threonine protein kinase enzyme comprises protein kinase A, B and C.These kinases (being PKA, PKB and PKC) play keying action in signal transduction pathway.

People are devoted to seek to have protein kinase inhibiting activity always and can treat micromolecular compound to the extremely moving relevant disease of protein kinase activity.the compound of bibliographical information has ring compound (US Patent No. 7, 151, 096), dicyclic compound (US Patent No. 7, 189, 721), tricyclic compound (US Patent No. 7, 132, 533), (2-oxyindole base-3-methylene radical) acetogenin (US Patent No. 7, 214, 700), 3-(4-acid amides pyrryl-2-methylene)-2-dihydroindole ketone derivate (US Patent No. 7, 179, 910), fused pyrazole derivatives (US Patent No. 7, 166, 597), amido furazan compound (US Patent No. 7, 157, 476), the pyrroles replace 2-indolinone compound (US Patent No. 7, 125, 905), triazole compounds (US Patent No. 7, 115, 739), quinazoline compound (the US Patent No. 7 that the pyrazolyl amido replaces, 098, 330) and indazole compound (US Patent No. 7, 041, 687) etc.There are several kinases inhibitors to be used for cancer therapy by the FDA approval, as Glivec, Sutent and Sorafenib.Clinical use shows, compares with traditional chemotherapy, and these medicines are with the obvious advantage.Excite thus people based on mechanism, methods for the treatment of to be improved, optimize the compound molecule skeleton, to find to have better oral administration biaavailability, higher antitumour activity and more hypotoxic new compound.

Summary of the invention

One of the object of the invention is to disclose a class to be had the protein kinase selective inhibitory activity and has simultaneously the 2-dihydroindole ketone derivate that histon deacetylase (HDAC) suppresses activity;

Two of the object of the invention is to disclose the preparation method of the described compound of this class;

Three of the object of the invention is to disclose the clinical application of the described compound of this class active extremely relevant disease of and histon deacetylase (HDAC) abnormal to protein kinase activity aspect as treatment.

Play keying action aspect the genetic expression of histon deacetylase (HDAC) (HDAC) albumen in the regulation and control body, it changes the accessibility that transcription factor is attached to genome DNA.Especially HDAC can remove the ethanoyl of acetylize lysine residue in histone, thereby causes nucleosome reconstruct (Grunstein, M., 1997, Nature, 389:349-352).Because HDAC albumen plays keying action in genetic expression, so they and many cell functions are closely related, comprise that (Ruijter occurs for cell cycle regulating, cell proliferation, differentiation, gene program expression and cancer, A-J-M., 2003, Biochem.J., 370:737-749; Grignani, F., 1998, Nature, 391:815-818; Lin, R-J., 1998,391:811-814; Marks, P-A., 2001, NatureReviews Cancer, 1:194).Closely related by the abnormal deacetylation due to the histon deacetylase (HDAC) mistuning and many clinical diseases, as Lu-Tai (Rubinstein-Taybi) two Cotards, fragile X chromosome syndromes, leukemia and other various cancers (Langley B et al., 2005, Current Drug Targets-CNS﹠amp; Neurological Disorders, 4:41-50).Test shows, hdac inhibitor can suppress the growth of the tumour in the mankind and animal body, comprises (Dokmanovic, M., 2005, J.Cell Biochenm., 96:293-304) such as lung cancer, cancer of the stomach, mammary cancer, prostate cancer and lymphomas.

Histon deacetylase (HDAC) is active abnormal closely related with various nervous system disorderss and neurodegenerative disease, comprises apoplexy, Huntington chorea (Huntington) disease, amyotrophic lateral sclerosis (Amyotrophic Lateral Sclerosis) and alzheimer's disease (Alzheimer ' s disease).Inhibition of histone deacetylation endonuclease capable is induced the expression of antimitotic and anti-apoptotic genes expression, as p21 and HSP-70, is conducive to Normocellular survival.Hdac inhibitor also can act on other neurocyte of central nervous system, as reactive astrocytes and microgliacyte, is reduced in inflammation and secondary injury in nerve injury or disease.Increase acetylize by hdac inhibitor, can induce Dendritic Sprouting (increase of cynapse quantity), the Reconstruction of Learning behavior enters long-term memory.Hdac inhibitor can reverse the gene silencing of friedreich's ataxia (Friedreich ' s ataxia).These data show, it is a kind of method (Langley B et al., 2005, Current Drug Targets-CNS that has the many central nervous system diseases for the treatment of of hope that HDAC suppresses; Neurological Disorders, 4:41-50; FischerA., et al, 2007, Nature 447 (10): 178-183; Herman D., et al., 2006, Nature Chemical Biology 2 (10): 551-558).

According to sequence homology, mammiferous HDACs can be divided three classes.The first kind forms (HDAC 1,2,3,8 and 11) by yeast Rpd3-proteinoid.Equations of The Second Kind forms (HDAC 4,5,6,7,9 and 10) by yeast HDA1-proteinoid.The 3rd class forms (SIRT 1,2,3,4,5,6 and 7) by yeast SIR2-proteinoid.

The activity of HDAC1 and cell proliferation (sign of cancer) are relevant.Mammalian cell reduces HDAC1 by siRNA expresses, and has anti proliferative (Glaser, K-B., 2003, Biochem.Biophys.Res.Comm., 310:529-536).The deratization of HDAC1 clpp gene is embryonic death, and all the other cells show different Growth of Cells (Lagger, G., 2002, EMBO J., 21:2672-2681).The mouse cell of HDAC1 overexpression shows G2 and the M phase extends and the speed of growth reduces (Bartl.S., 1997, Mol.Cell Biol., 17:5033-5043).Therefore, testing data shows, HDAC1 and cell cycle regulating and cell proliferation are closely related.

The expression of the many fetal rhythm myoprotein isomer of HDAC2 regulation and control.HDAC2 lacks or can stop by chemical process inhibition of histone deacetylase the expression again of embryonic gene, reduces ventricular hypertrophy.Anti-hypertrophy is with the inositol polyphosphate hydrochlorate-expression of 5-Phosphoric acid esterase f (Inpp5f) encoding gene increases relevant, this increase causes the proto-oncogene (Akt) of thymoma filtrable virus and protein kinase-1 inactivation of 3-phosphoinositide-dependence, has activated glycogen synthase kinase 3 (Gsk3).On the contrary, HDAC2 transgenic mouse ventricular hypertrophy increases, and this Gsk3 β with inactivation is relevant.The Gsk3 β that suppresses activation by chemical process makes adult that HDAC2-lacks become responsive to the stimulation of ventricular hypertrophy.These results show, HDAC2 is an important molecular target of hdac inhibitor in heart.HDAC2 and Gsk3 β are the integral parts of regulatory pathway, and this is treatment ventricular hypertrophy and the treatment target (Trivedi, C-M., 2007, Nat.Med .13:324-331) that very attractive is provided in heart failure.

HDAC3 expresses at most in the propagation pit cell of normal small intestine.The HDAC3 expression silencing causes cell growth inhibition, cell survival to reduce and the apoptosis increase in colon carcinoma cell line.Reticent HDAC2 expresses and can observe similar effect, and for HDAC1, effect is so not remarkable.The HDAC3 gene silencing also optionally causes the expression (sign of colon cell maturation) of alkaline phosphatase.The overexpression inhibition basal transcription of HDAC3 and the P21 that butyric ester is induced transcribe, and reticent HDAC3 stimulates the active of P21 gene promoter and expresses.These discoveries show that HDAC3 is the gene of lowering in the human colon cancer, be a kind of novel conditioning agent (Wilson, A-J., 2006, J.Biol.Chem., 281:13548-13558) that colon cell is ripe and P21 expresses.

HDAC6 is a hypotype of HDAC family, it sloughs-and the ethanoyl of tubulin, increase cell mobility.Clone and upper quantitative real-time RT-PCR and the Western Blots technical Analysis used of normal oral cavity keratinocyte (NOKs) in nine groups of oral squamous cell carcinoma (OSCC), compare with NOKs, HDAC6mRNA and protein expression level have all raise in all cancer cells.By the immunofluorescence technique analysis, HDAC6 albumen detected in the tenuigenin of OSCC clone.Similar with OSCC clone, in human body OSCC tumour in early days, HDAC6 raises obviously, and mRNA reaches 74%, and albumen reaches 51%.By the analysis to clinical variable, it is found that the expression status of the developmental stage of clinical tumor and HDAC6 is relevant.The analysis showed that, there is significant difference (P=0.014) in the expression level of HDAC6 in tumour early stage (I and II phase) and late period (III and IV phase).These results show that the expression of HDAC6 may be relevant with the grade malignancy of tumour, and this also provides clue (Sakuma, T., 2006, Int.J.Oncol., 29:117-124) for designing new methods for the treatment of.

IIa class HDAC comprises HDAC4,5 and 7.In the past few years, the researchist has determined IIa class HDAC some important physiological function in live body.It is shocking, these seem does not have associated physiological process that common feature is arranged, and they all depend on IIa class HDAC MEF2 is transcribed movable strict control.The crucial physiological processs such as formation, megalocardia, bone development, T-cytodifferentiation and neuronal survival such as skeletal muscle are all controlled by IIa class HDAC, this fact means that the pathological Intervention Therapy of many mankind is possible, comprise blood vessel kind disease such as arteriosclerosis, apoplexy and aneurysma, tumor-blood-vessel growth and transfer, nanism, skeleton deformity, autoimmunization and lymphoproliferative syndrome, neurodegenerative disorders and myocardial hypertrophy (Martin M.et al, 2007, Oncogene 26:5450-5467).

It is one of main mechanism of many pathologic processes that HDAC makes Functional genomics apparent gene silence.Wherein, the function genes involved is suppressed by HDAC or resets, and causes phenotype disappearance in last differentiation eventually, maturation and growth control, and loses function of organization.For example, by reticent, hdac inhibitor can be induced the expression of these tumor suppressor genes to tumor suppressor gene in the evolution of the cancer of being everlasting, thus inhibition tumor cell growth and differentiation (Glaros S et al., 2007, Oncogene June 4Epub aheadof print; Mai, A, et al., 2007, Int J.Biochem Cell Bio., April 4, Epub ahead of print; Vincent A.et al., 2007, Oncogene, April 30, Epub ahead of print; Our unpublished results).The inhibition of structure gene (as the FXN gene relevant to Friedreich ' s ataxia and the SMN gene relevant with spinal muscular atrophy) can be reversed by HDAC, make FXN and SMN gene again express, and recovery organization function (Herman D et al., 2006, NatureChemical Biology, 2 (10): 551-8; Avila AM et al., 2007, J Clinic Investigation, 117 (3) 659-71; DeBore J, 2006, Tissue Eng.12 (10): 2927-37).Hdac inhibitor is in karyomit(e) 6p21-22 readjustment HDAC " focus " program, induce whole MHC II family gene to express, further extended apparent gene regulation and control (Gialitakis M et al., 2007 of immune identification and immune response, Nucleic Acids Res., 34 (1); 765-72).

Determined at present a few class hdac inhibitors, having comprised: 1) short chain fatty acid, as butyric acid and benzenebutanoic acid; 2) organic hydroximic acid is as suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA); 3) contain 2-amido-8-oxygen-9, the cyclic tetrapeptide of 10-epoxy decanoyl (AOE) is as trapoxin and HC-toxin; 4) do not contain 2-amino-8-oxygen-9, the cyclic tetrapeptide of 10-epoxy decanoyl is as apicidin and FK228; 5) benzamides is as MS-275 (European patent EP 0847992A1, United States Patent (USP) 2002/0103192A1, world patent 02/26696A1, world patent 01/70675A2, world patent 01/18171A2).Although HDAC is a drug targets that has prospect, the SAHA of at present Merck company development only is confined to the treatment to cutaneous T cell lymphoma, and to solid tumor curative effect and not obvious.Therefore, be necessary to continue the development novel compound, make it have stronger HDAC and suppress active, stronger antitumour activity, better HDAC subtype-selective and lower toxicity.

Targeted therapy is praised highly by the cancer drug development personnel always.People wish to design medicine, and it both can accurately arrive certain particular target and the kill tumor cell of tumour cell, simultaneously not only to normal cell but also not injury.Yet tumour cell can use multiple biological initiator and path grow and propagate.At a target spot, tumour cell is hit, they can and redeploy along new growth path restructuring.People have been developed the combination targeted therapy thus, and are becoming the new example of cancer therapy.Several many target spots kinase inhibitor are are now researched and developed, and wherein Sorafenib and Sutent are in U.S.'s listing that gets the Green Light.Sorafenib (Bayer company exploitation) is first medicine take RAF/MEK/ERK path (relevant with cell proliferation) and VEGFR2/PDGFR β cascade signal path (with associated angiogenesis) as target spot simultaneously, and this medicine gets the Green Light in December, 2005 and is used for the treatment of advanced renal cell cancer.Although these target therapeutic agents are effectively during some solid tumors in treatment, when treating other solid tumor, curative effect is unsatisfactory and have a toxic side effect.

Compound of the present invention, combine the angiogenesis inhibitor of RTK inhibitor and antiproliferative activity and hdac inhibitor have induce differentiation, immunity modulation, block cell cycle, impel apoptotic activity, be intended to that solid tumor is had better curative effect, overcome simultaneously the toxic side effect of commercially available RTK inhibitor, as hypertension, QT interval prolongation, Tiroidina degeneration, fash and parachromatosis, pain etc.

Compound of the present invention, its chemical structure is as shown in general formula (I):

Wherein,

X is=CH-or=N-N=CH-;

R ¹, R ², R ³And R ⁴Be respectively hydrogen, halogen, alkyl, alkoxyl group, nitro or trifluoromethyl;

R ⁵, R ⁶, R ⁷And R ⁸Be respectively hydrogen, halogen, alkyl, alkoxyl group or trifluoromethyl;

N is 2 to 6 integer;

The form, enantiomer, diastereomer or the hydrate that comprise its free form, salt.More preferably, the structure of described compound as shown in general formula (I), wherein,

X is=CH-or=N-N=CH-;

R ¹, R ², R ³And R ⁴Be respectively hydrogen or halogen;

R ⁵, R ⁶, R ⁷And R ⁸Be respectively hydrogen or halogen;

N is 2 to 6 integer.

More preferably, the structure of described compound as shown in general formula (I), wherein,

X is=CH-or=N-N=CH-;

R ¹, R ², R ³And R ⁴Be respectively hydrogen or fluorine;

R ⁵, R ⁶, R ⁷And R ⁸Be respectively hydrogen or fluorine;

N is 2 to 4 integer.

" halogen " of the present invention is fluorine, chlorine, bromine, iodine;

" alkyl " of the present invention, comprise straight chain, side chain or cyclic alkyl, as methyl, ethyl, n-propyl, sec.-propyl, normal-butyl, isobutyl-, tertiary butyl, n-pentyl, isopentyl, n-hexyl, isohexyl, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl etc.;

" alkoxyl group " of the present invention, refer to the alkyl formed group that is connected with Sauerstoffatom, wherein, Sauerstoffatom has free bonding power, as methoxyl group, oxyethyl group, propoxy-, butoxy, pentyloxy, isopropoxy, tert-butoxy, ring propoxy-, cyclohexyl oxygen base etc.;

The synthetic route of compound of the present invention is as follows:

(a) 6-chlorine apellagrin and compound 1 are carried out condensation reaction and obtain compound 2;

(b) compound 2 and compound 3 are carried out condensation reaction and obtain compound 4;

(c) compound 4 and compound 5 are carried out condensation reaction and obtain compound 6.

Above-mentioned condensation reaction (a) and (c) take the peptide condensing agent as catalyzer, as 1-ethyl-3-(3-dimethylamine propyl) carbodiimide (EDC), N, N '-dicyclohexylcarbodiimide (DCC), N, N '-phosphinylidyne diimidazole (CDI) etc.Temperature of reaction is 0～80 ℃, and the reaction times is 4～72 hours.The reaction solvent for use is common solvent, as benzene, toluene, tetrahydrofuran (THF), dioxane, methylene dichloride, chloroform, DMF etc.In case of necessity, also can add alkali such as sodium hydroxide, triethylamine or pyridine.

Above-mentioned condensation reaction (b), temperature of reaction are 40～120 ℃, and the reaction times is 1～24 hour.The reaction solvent for use is common solvent, as benzene, toluene, tetrahydrofuran (THF), dioxane, methylene dichloride, chloroform, DMF etc.In case of necessity, also can add alkali such as sodium hydroxide, triethylamine or pyridine.

The compound 5 that above-mentioned condensation reaction (c) is used, when X be=during CH-, its preparation method is:

Compound (7), compound (8) and piperidines are added in dehydrated alcohol, and back flow reaction 6 hours is neutralized to pH=4 with 2M hydrochloric acid, obtains compound (5).

The compound 5 that above-mentioned condensation reaction (c) is used, when X be=during N-N=CH-, its preparation method is:

Compound (9) and 50% hydrazine hydrate were obtained compound (10) in 3 hours in 80～90 ℃ of reactions.Compound (10), compound (8) and piperidines are added in dehydrated alcohol, back flow reaction 6 hours is neutralized to pH=4 with 2M hydrochloric acid again, obtains compound (5).

The described compound of general formula (I) and intermediate (2) and intermediate (4) can adopt common separation method to carry out purifying, as extraction, recrystallization, column chromatography etc.

Compound of the present invention; have simultaneously protein kinase inhibiting activity and histon deacetylase (HDAC) and suppress active; can be used for the treatment of abnormal to protein kinase activity and the active abnormal relevant disease of histon deacetylase (HDAC), especially leukemia and solid tumor be had excellent curative effect.

Compound of the present invention, the medicinal preparations that is processed to commonly use is as tablet, capsule, pulvis, syrup, liquor, suspension agent, injection.Said preparation contains the compound of the general formula (I) as active ingredient, and pharmaceutical carrier, auxiliary material or thinner.Usually contain 0.5～70% activeconstituents in said preparation, better content is 1～20%.The formulation specification of this medicinal preparations is 0.0001～200mg.

The said pharmaceutical carrier of the present invention, auxiliary material or thinner, comprise " pharmaceutical excipient handbook (American Pharmaceutical Association, in October, 1986) or " pharmaceutical excipient handbook (the Handbook of Pharmaceutical Excipients that publishes of Chemical Industry Press, original work the 4th edition) listed carrier filler, but be not limited to these carrier fillers.

Compound of the present invention can carry out medication to Mammals (comprising the people) by oral or injection system clinically, and is wherein especially best with oral way.Dosage is 0.0001～200mg/kg body weight every day, and better dosage is 0.01～100mg/kg body weight every day, and best dosage is 0.1～50mg/kg body weight every day, simultaneously, optimal dose depending on individuality, when usually beginning, dosage is less, then increases gradually consumption.

Representative compound of the present invention is as shown in table 1.Compound number is consistent with " embodiment numbering " in the embodiment part, and namely in table 1, synthesizing in " embodiment 5 " of compound 5 described, and in table 1, synthesizing in " embodiment 53 " of compound 53 described.

Table 1 representative compound of the present invention

Embodiment

Further illustrate content of the present invention below in conjunction with example, but protection scope of the present invention is not limited only to these examples.Per-cent of the present invention all is weight percentage except indicating especially.Numerical range described in specification sheets as measure unit, reaction conditions, compound physical state or per-cent, is all for undoubted desk reference is provided.Those skilled in the art use outside this scope or are different from the temperature, concentration, quantity, carbonatoms etc. of single numerical value when putting into practice this patent, still can obtain expected result.

Embodiment 1

5-(5-fluoro-2-oxygen-1,2-dihydro-indoles-(3Z)-ylidenylmethyl)-2, the preparation of 4-dimethyl-1H-pyrroles-compounds such as 3-carboxylic acid

(US 7 to add 1.51g (10mmol) 5-fluoro-2-indolone in reaction flask; 179; 910B2, Example1), 1.67g (10mmol) 5-formyl radical-2, (US 7 for 4-dimethyl-1H-pyrroles-3-carboxylic acid; 179; 910B2, Example1), 1.28g (15mmol) piperidines and 25ml dehydrated alcohol, heating reflux reaction 6 hours; be chilled to room temperature, filter.Filter cake is changed in beaker, add 100ml water, be neutralized to pH=4 with 2M hydrochloric acid, solid collected by filtration, solid washes with water, and vacuum-drying gets yellow solid 5-(5-fluoro-2-oxygen-1,2-dihydro-indoles-(3Z)-ylidenylmethyl)-2,4-dimethyl-1H-pyrroles-3-carboxylic acid (1.56g, productive rate 52%).LC-MS(m/z)301(M+1)。

Take 2-indolone (be purchased from Aldrich company) as raw material, adopt similar approach to make 5-(2-oxygen-1,2-dihydro-indoles-(3Z)-ylidenylmethyl)-2,4-dimethyl-1H-pyrroles-3-carboxylic acid, LC-MS (m/z) 283 (M+1).

Take 5-chloro-2-indolone (be purchased from Aldrich company) as raw material, adopt similar approach to make 5-(5-chloro-2-oxygen-1,2-dihydro-indoles-(3Z)-ylidenylmethyl)-2,4-dimethyl-1H-pyrroles-3-carboxylic acid, LC-MS (m/z) 317 (M+1).

With 4-methyl-2-indolone (US 2004/0063773 A1, Examples of Oxindole Syntheses) be raw material, adopt similar approach to make 5-(4-methyl-2-oxygen-1,2-dihydro-indoles-(3Z)-ylidenylmethyl)-2,4-dimethyl-1H-pyrroles-3-carboxylic acid, LC-MS (m/z) 297 (M+1).

With 5-nitro-2-indolone (US 2004/0063773A1, Examples of Oxindole Syntheses) be raw material, adopt similar approach to make 5-(5-nitro-2-oxygen-1,2-dihydro-indoles-(3Z)-ylidenylmethyl)-2,4-dimethyl-1H-pyrroles-3-carboxylic acid, LC-MS (m/z) 328 (M+1).

Take 6-methoxyl group-2-indolone (be purchased from Austin chemical company) as raw material, adopt similar approach to make 5-(6-methoxyl group-2-oxygen-1,2-dihydro-indoles-(3Z)-ylidenylmethyl)-2,4-dimethyl-1H-pyrroles-3-carboxylic acid, LC-MS (m/z) 313 (M+1).

Take 6-trifluoromethyl-2-indolone (be purchased from DSL chemical company) as raw material, adopt similar approach to make 5-(6-trifluoromethyl-2-oxygen-1,2-dihydro-indoles-(3Z)-ylidenylmethyl)-2,4-dimethyl-1H-pyrroles-3-carboxylic acid, LC-MS (m/z) 351 (M+1).

Embodiment 2

5-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2, the preparation of 4-dimethyl-1H-pyrroles-compounds such as 3-carboxylic acid

Add 8.25g (50mmol) 5-fluoro indigo red (be purchased from Aldrich company) and 150ml 50% hydrazine hydrate in reaction flask, in 80～90 ℃ of reactions 3 hours, be chilled to room temperature, filter, solid is collected in washing, and vacuum-drying gets yellow solid 5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit hydrazine (6.0g, productive rate 67%).LC-MS(m/z)180(M+1)。

Add 1.79g (10mmol) 5-fluoro-2-oxygen-1 in reaction flask; 2-dihydro-indoles-3-subunit hydrazine, 1.67g (10mmol) 5-formyl radical-2; (US 7 for 4-dimethyl-1H-pyrroles-3-carboxylic acid; 179; 910B2, Example1), 1.28g (15mmol) piperidines and 25ml dehydrated alcohol, heating reflux reaction 6 hours; be chilled to room temperature, filter.Filter cake is changed in beaker, add 100ml water, be neutralized to pH=4 with 2M hydrochloric acid, solid collected by filtration, solid washes with water, and vacuum-drying gets red solid 5-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl-1H-pyrroles-3-carboxylic acid (1.18g, productive rate 36%).LC-MS(m/z)329(M+1)。

Take isatin (be purchased from Aldrich company) as raw material, adopt similar approach to make 5-(((2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl-1H-pyrroles-3-carboxylic acid, LC-MS (m/z) 311 (M+1).

Take 5-chlorisatide (be purchased from Aldrich company) as raw material, adopt similar approach to make 5-(((5-chloro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl-1H-pyrroles-3-carboxylic acid, LC-MS (m/z) 345 (M+1).

With 4-methylisatin (Sadler P.W., 1956, J.Org.Chem, Vol.21:69) be raw material, adopt similar approach to make 5-(((4-methyl-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl-1H-pyrroles-3-carboxylic acid, LC-MS (m/z) 325 (M+1).

Take 5-nitro isatin (be purchased from Aldrich company) as raw material, adopt similar approach to make 5-(((5-nitro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl-1H-pyrroles-3-carboxylic acid, LC-MS (m/z) 356 (M+1).

With 6-methoxyl group isatin (Sadler P.W., 1956, J.Org.Chem, Vol.21:69) be raw material, adopt similar approach to make 5-(((6-methoxyl group-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl-1H-pyrroles-3-carboxylic acid, LC-MS (m/z) 341 (M+1).

With 6-trifluoromethyl isatin (Simet L., 1963, J.Org.Chem, Vol.28:3580-3581) be raw material, adopt similar approach to make 5-(((6-trifluoromethyl-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl-1H-pyrroles-3-carboxylic acid, LC-MS (m/z) 398 (M+1).

Embodiment 3

The preparation of N-(2-aminophenyl)-6-chloro-nicotinamide

157.5mg (1mmol) 6-chlorine apellagrin (be purchased from Aldrich company) is dissolved in 8ml DMF, then adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 216mg (2mmol) O-Phenylene Diamine.Mixture at room temperature stirred 20 hours, then with the dilution of 400ml salt solution, then used the 200ml ethyl acetate extraction.Vacuum is removed ethyl acetate, adds the 5ml dehydrated alcohol in residue.Vacuum filtration is collected solid.The a small amount of absolute ethanol washing of solid, vacuum-drying get brown solid (138mg, productive rate 56%).LC-MS(m/z)248(M+1)。

Embodiment 4

The preparation of N-(2-aminophenyl)-6-(2-aminoethylamino) niacinamide

248mg (1mmol) N-(2-aminophenyl)-6-chloro-nicotinamide and 5ml quadrol are mixed, be heated to 80 ℃, reacted 3 hours.Vacuum is removed excessive quadrol.Add the NaOH solution of 5ml 0.20M in the residue, then use the 100ml ethyl acetate extraction.Decompression is removed ethyl acetate and is got brown solid (150mg, productive rate 55%).LC-MS(m/z)272(M+1)。

Embodiment 5

The preparation of (Z)-N-(2-aminophenyl)-6-(2-(2-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl)-3,5-dimethyl-1H-pyrroles-4-amide group) ethylamino)-niacinamide

With 300mg (1mmol) 5-(5-fluoro-2-oxygen-1,2-dihydro-indoles-(3Z)-ylidenylmethyl)-2,4-dimethyl-1H-pyrroles-3-carboxylic acid is dissolved in 8ml DMF, then adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 284mg (1.05mmol) N-(2-aminophenyl)-6-(2-aminoethylamino) niacinamide.Mixture at room temperature stirred 20 hours, and then with the dilution of 400ml salt solution, vacuum filtration is collected solid.Solid washes with water, and vacuum-drying gets yellow solid (493mg, productive rate 89%). ¹HNMR(DMSO-d ₆)δ2.41(s，3H，pyrrole-CH ₃)，2.43(s，3H，pyrrole-CH ₃)，3.43(m，2H，CH ₂)，3.48(m，2H，CH ₂)，4.86(s，2H，benzene-NH ₂)，6.56(m，2H)，6.76(d，J＝8.0Hz，1H)，6.84(m，1H)，6.92(m，2H)，7.12(d，J＝8.0Hz，1H)，7.26(s?1H)，7.71～7.77(m，3H)，7.94(d，J＝8.0Hz，1H)，8.65(s，1H)，9.38(s，1H，benzene-NH)，10.90(s，1H，indolinone-NH)，13.69(s，1H，pyrrole-NH)。LC-MS(m/z)554(M+1)。

Embodiment 6

The preparation of N-(2-aminophenyl)-6-(2-(2-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-3,5-dimethyl-1H-pyrroles-4-amide group) ethylamino)-niacinamide

With 328mg (1mmol) 5-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl-1H-pyrroles-3-carboxylic acid is dissolved in 8ml DMF, then adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 284mg (1.05mmol) N-(2-aminophenyl)-6-(2-aminoethylamino) niacinamide.Mixture at room temperature stirred 20 hours, and then with the dilution of 400ml salt solution, vacuum filtration is collected solid.Solid washes with water, and vacuum-drying gets yellow solid (425mg, productive rate 73%). ¹H?NMR(DMSO-d ₆)δ2.35(s，3H，pyrrole-CH ₃)，2.44(s，3H，pyrrole-CH ₃)，3.42(m，2H，CH ₂)，3.48(m，2H，CH ₂)，4.85(s，2H，benzene-NH ₂)，6.56(m，2H)，6.76(d，J＝8.0Hz，1H)，6.85(m，1H)，6.92(m，1H)，7.12(d，J＝8.0Hz，1H)，7.20～7.25(m，2H)，7.71(s，1H)，7.93(d，J＝8.0Hz，1H)，8.33(d，J＝8.0Hz，1H)，8.64(s，2H)，9.38(s，1H，benzene-NH)，10.73(s，1H，indolinone-NH)，11.84(s，1H，pyrrole-NH)。LC-MS(m/z)582(M+1)。

Embodiment 7

The preparation of N-(2-aminophenyl)-6-(3-amino propyl amino) niacinamide

With 248mg (1mmol) N-(2-aminophenyl)-6-chloro-nicotinamide and 6ml 1, the 3-propylene diamine mixes, and is heated to 80 ℃, reacts 3 hours.It is excessive 1 that vacuum is removed, the 3-propylene diamine.Add the NaOH solution of 5ml 0.20M in the residue, then use the 100ml ethyl acetate extraction.Decompression is removed ethyl acetate and is got brown solid (168mg, productive rate 59%).LC-MS(m/z)286(M+1)。

Embodiment 8

The preparation of (Z)-N-(2-aminophenyl)-6-(3-(2-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl)-3,5-dimethyl-1H-pyrroles-4-amide group) propyl group is amino)-niacinamide

With 300mg (1mmol) 5-(5-fluoro-2-oxygen-1,2-dihydro-indoles-(3Z)-ylidenylmethyl)-2,4-dimethyl-1H-pyrroles-3-carboxylic acid is dissolved in 8ml DMF, then adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 299mg (1.05mmol) N-(2-aminophenyl)-6-(3-amino propyl amino) niacinamide.Mixture at room temperature stirred 20 hours, and then with the dilution of 400ml salt solution, vacuum filtration is collected solid.Solid washes with water, and vacuum-drying gets yellow solid (465mg, productive rate 82%). ¹HNMR(DMSO-d ₆)δ1.79(m，2H，CH2)，2.42(s，3H，pyrrole-CH ₃)，2.44(s，3H，pyrrole-CH ₃)，3.30(m，2H，CH2)，3.38(m，2H，CH2)，4.85(s，2H，benzene-NH ₂)，6.51(m，1H)，6.58(m，1H)，6.75(d，J＝8.0Hz，1H)，6.83(t，J＝8.0Hz，1H)，6.92(m，2H)，7.12(d，J＝8.0Hz，1H)，7.19(s，1H)，7.71～7.77(m，3H)，7.91(d，J＝8.0Hz，1H)，8.64(s，1H)，9.37(s，1H，benzene-NH)，10.90(s，1H，indolinone-NH)，13.68(s，1H，pyrrole-NH)。LC-MS(m/z)568(M+1)。

Embodiment 9

The preparation of N-(2-aminophenyl)-6-(3-(2-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-3,5-dimethyl-1H-pyrroles-4-amide group) propyl group is amino)-niacinamide

With 328mg (1mmol) 5-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl-1H-pyrroles-3-carboxylic acid is dissolved in 8ml DMF, then adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 299mg (1.05mmol) N-(2-aminophenyl)-6-(3-amino propyl amino) niacinamide.Mixture at room temperature stirred 20 hours, and then with the dilution of 400ml salt solution, vacuum filtration is collected solid.Solid washes with water, and vacuum-drying gets yellow solid (452mg, productive rate 76%). ¹H?NMR(DMSO-d ₆)δ1.78(m，2H，CH ₂)，2.36(s，3H，pyrrole-CH ₃)，2.45(s，3H，pyrrole-CH ₃)，3.30(m，2H，CH ₂)，3.38(m，2H，CH ₂)，4.85(s，2H，benzene-NH ₂)，6.51(m，1H)，6.57(m，1H)，6.75(d，J＝8.0Hz，1H)，6.85(m，1H)，6.93(m，1H)，7.12(d，J＝8.0Hz，1H)，7.20(m，2H)，7.71(s，1H)，7.92(d，J＝8.0Hz，1H)，8.34(d，J＝8.0Hz，1H)，8.64(s，2H)，9.37(s，1H，benzene-NH)，10.74(s，1H，indolinone-NH)，11.85(s，1H，pyrrole-NH)。LC-MS(m/z)596(M+1)。

Embodiment 10

The preparation of N-(2-aminophenyl)-6-(the 4-aminobutyl is amino) niacinamide

248mg (1mmol) N-(2-aminophenyl)-6-chloro-nicotinamide and 7ml Putriscine are mixed, be heated to 80 ℃, reacted 3 hours.Vacuum is removed excessive Putriscine.Add the NaOH solution of 5ml 0.20M in the residue, then use the 100ml ethyl acetate extraction.Decompression is removed ethyl acetate and is got brown solid (158mg, productive rate 53%).LC-MS(m/z)300(M+1)。

Embodiment 11

The preparation of (Z)-N-(2-aminophenyl)-6-(4-(2-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl)-3,5-dimethyl-1H-pyrroles-4-amide group) butyl is amino)-niacinamide

With 300mg (1mmol) 5-(5-fluoro-2-oxygen-1,2-dihydro-indoles-(3Z)-ylidenylmethyl)-2,4-dimethyl-1H-pyrroles-3-carboxylic acid is dissolved in 8ml DMF, then adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 314mg (1.05mmol) N-(2-aminophenyl)-6-(the 4-aminobutyl is amino) niacinamide.Mixture at room temperature stirred 20 hours, and then with the dilution of 400ml salt solution, vacuum filtration is collected solid.Solid washes with water, and vacuum-drying gets yellow solid (447mg, productive rate 77%). ¹HNMR(DMSO-d ₆)δ1.59(m，4H，CH ₂CH ₂)，2.39(s，3H，pyrrole-CH ₃)，2.41(s，3H，pyrrole-CH ₃)，3.25(m，4H，2×CH ₂)，4.85(s，2H，benzene-NH ₂)，6.49(m，1H)，6.57(m，1H)，6.75(d，J＝8.0Hz，1H)，6.83(m，1H)，6.91(m，2H)，7.12(d，J＝8.0Hz，1H)，7.18(s，1H)，7.67～7.76(m，3H)，7.90(d，J＝8.0Hz，1H)，8.63(s，1H)，9.35(s，1H，benzene-NH)，10.88(s，1H，indolinone-NH)，13.66(s，1H，pyrrole-NH)。LC-MS(m/z)582(M+1)。

Embodiment 12

The preparation of N-(2-aminophenyl)-6-(4-(2-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-3,5-dimethyl-1H-pyrroles-4-amide group) butyl is amino)-niacinamide

With 328mg (1mmol) 5-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl-1H-pyrroles-3-carboxylic acid is dissolved in 8ml DMF, then adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 314mg (1.05mmol) N-(2-aminophenyl)-6-(the 4-aminobutyl is amino) niacinamide.Mixture at room temperature stirred 20 hours, and then with the dilution of 400ml salt solution, vacuum filtration is collected solid.Solid washes with water, and vacuum-drying gets yellow solid (444mg, productive rate 73%). ¹H?NMR(DMSO-d ₆)δ1.59(m，4H，CH ₂CH ₂)，2.32(s，3H，pyrrole-CH ₃)，2.43(s，3H，pyrrole-CH ₃)，3.24(m，4H，2×CH ₂)，4.85(s，2H，benzene-NH ₂)，6.49(m，1H)，6.57(m，1H)，6.76(d，J＝8.0Hz，1H)，6.85(m，1H)，6.93(m，1H)，7.12(d，J＝8.0Hz，1H)，7.20(m，2H)，7.67(s，1H)，7.89(d，J＝8.0Hz，1H)，8.33(d，J＝8.0Hz，1H)，8.63(s，2H)，9.35(s，1H，benzene-NH)，10.70(s，1H，indolinone-NH)，11.82(s，1H，pyrrole-NH)。LC-MS(m/z)610(M+1)。

Embodiment 13

The preparation of N-(2-amino-4-fluorophenyl)-6-chloro-nicotinamide

157.5mg (1mmol) 6-chlorine apellagrin is dissolved in 8ml DMF, then adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 151mg (1.2mmol) 4-fluorine O-Phenylene Diamine (be purchased from Alfa Aesar company).Mixture at room temperature stirred 20 hours, then with the dilution of 400ml salt solution, then used the 200ml ethyl acetate extraction.Vacuum is removed ethyl acetate, adds the 5ml dehydrated alcohol in residue.Vacuum filtration is collected solid.The a small amount of absolute ethanol washing of solid, vacuum-drying get brown solid (193mg, productive rate 73%).LC-MS(m/z)266(M+1)。

Embodiment 14

The preparation of N-(the amino 4-fluorophenyl of 2-)-6-(2-aminoethylamino) niacinamide

266mg (1mmol) N-(2-amino-4-fluorophenyl)-6-chloro-nicotinamide and 5ml quadrol are mixed, be heated to 80 ℃, reacted 3 hours.Vacuum is removed excessive quadrol.Add the NaOH solution of 5ml 0.20M in the residue, then use the 100ml ethyl acetate extraction.Decompression is removed ethyl acetate and is got brown solid (176mg, productive rate 61%).LC-MS(m/z)290(M+1)。

Embodiment 15

The preparation of (Z)-N-(2-amino-4-fluorophenyl)-6-(2-(2-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl)-3,5-dimethyl-1H-pyrroles-4-amide group) ethylamino)-niacinamide

With 300mg (1mmol) 5-(5-fluoro-2-oxygen-1,2-dihydro-indoles-(3Z)-ylidenylmethyl)-2,4-dimethyl-1H-pyrroles-3-carboxylic acid is dissolved in 8ml DMF, then adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 303mg (1.05mmol) N-(2-amino-4-fluorophenyl)-6-(2-aminoethylamino) niacinamide.Mixture at room temperature stirred 20 hours, and then with the dilution of 400ml salt solution, vacuum filtration is collected solid.Solid washes with water, and vacuum-drying gets yellow solid (457mg, productive rate 80%). ¹H?NMR(DMSO-d ₆)δ2.41(s，3H，pyrrole-CH ₃)，2.43(s，3H，pyrrole-CH ₃)，3.43(m，2H，CH ₂)，3.48(m，2H，CH ₂)，5.18(s，2H，benzene-NH ₂)，6.33(m，1H)，6.53(m，2H)，6.84(m，1H)，6.91(m，1H)，7.07(m，1H)，7.25(s，1H)，7.71(m，3H)，7.92(d，J＝8.0Hz，1H)，8.64(s，1H)，9.31(s，1H，benzene-NH)，10.89(s，1H，indolinone-NH)，13.68(s，1H，pyrrole-NH)。LC-MS(m/z)572(M+1)。

Embodiment 16

The preparation of N-(2-amino-4-fluorophenyl)-6-(2-(2-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-3,5-dimethyl-1H-pyrroles-4-amide group) ethylamino)-niacinamide

With 328mg (1mmol) 5-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl-1H-pyrroles-3-carboxylic acid is dissolved in 8ml DMF, then adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 303mg (1.05mmol) N-(2-amino-4-fluorophenyl)-6-(2-aminoethylamino) niacinamide.Mixture at room temperature stirred 20 hours, and then with the dilution of 400ml salt solution, vacuum filtration is collected solid.Solid washes with water, and vacuum-drying gets yellow solid (407mg, productive rate 68%). ¹H?NMR(DMSO-d ₆)δ2.35(s，3H，pyrrole-CH ₃)，2.44(s，3H，pyrrole-CH ₃)，3.42(m，2H，CH ₂)，3.47(m，2H，CH ₂)，5.18(s，2H，benzene-NH ₂)，6.33(m，1H)，6.53(m，2H)，6.85(m，1H)，7.06(m，1H)，7.21～7.25(m，2H)，7.71(s，1H)，7.93(d，J＝8.0Hz，1H)，8.33(d，J＝8.0Hz，1H)，8.64(s，2H)9.31(s，1H，benzene-NH)，10.73(s，1H，indolinone-NH)，11.84(s，1H，pyrrole-NH)。LC-MS(m/z)600(M+1)。

Embodiment 17

The preparation of N-(2-amino-4-fluorophenyl)-6-(3-amino propyl amino) niacinamide

With 266mg (1mmol) N-(2-amino-4-fluorophenyl)-6-chloro-nicotinamide and 6ml 1, the 3-propylene diamine mixes, and is heated to 80 ℃, reacts 3 hours.It is excessive 1 that vacuum is removed, the 3-propylene diamine.Add the NaOH solution of 5ml 0.20M in the residue, then use the 100ml ethyl acetate extraction.Decompression is removed ethyl acetate and is got brown solid (158mg, productive rate 52%).LC-MS(m/z)304(M+1)。

Embodiment 18

The preparation of (Z)-N-(2-amino-4-fluorophenyl)-6-(3-(2-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl)-3,5-dimethyl-1H-pyrroles-4-amide group) propyl group is amino)-niacinamide

With 300mg (1mmol) 5-(5-fluoro-2-oxygen-1,2-dihydro-indoles-(3Z)-ylidenylmethyl)-2,4-dimethyl-1H-pyrroles-3-carboxylic acid is dissolved in 8ml DMF, then adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 318mg (1.05mmol) N-(2-amino-4-fluorophenyl)-6-(3-amino propyl amino) niacinamide.Mixture at room temperature stirred 20 hours, and then with the dilution of 400ml salt solution, vacuum filtration is collected solid.Solid washes with water, and vacuum-drying gets yellow solid (456mg, productive rate 78%). ¹H?NMR(DMSO-d ₆)δ1.78(m，2H，CH ₂)，2.42(s，3H，pyrrole-CH ₃)，2.44(s，3H，pyrrole-CH ₃)，3.30(m，2H，CH ₂)，3.38(m，2H，CH ₂)，5.18(s，2H，benzene-NH ₂)，6.33(m，1H)，6.51(m，2H)，6.84(m，1H)，6.90(m，1H)，7.06(t，J＝8.0Hz，1H)，7.20(s，1H)，7.71～7.76(m，3H)，7.91(d，J＝8.0Hz，1H)，8.63(s，1H)，9.30(s，1H，benzene-NH)，10.90(s，1H，indolinone-NH)，13.68(s，1H，pyrrole-NH)。LC-MS(m/z)586(M+1)。

Embodiment 19

The preparation of N-(2-amino-4-fluorophenyl)-6-(3-(2-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit)-hydrazono-) methyl)-3,5-dimethyl-1H-pyrroles-4-amide group) propyl group is amino)-niacinamide

With 328mg (1mmol) 5-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl-1H-pyrroles-3-carboxylic acid is dissolved in 8ml DMF, then adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 318mg (1.05mmol) N-(2-amino-4-fluorophenyl)-6-(3-amino propyl amino) niacinamide.Mixture at room temperature stirred 20 hours, and then with the dilution of 400ml salt solution, vacuum filtration is collected solid.Solid washes with water, and vacuum-drying gets yellow solid (441mg, productive rate 72%). ¹H?NMR(DMSO-d ₆)δ1.77(m，2H，CH ₂)，2.36(s，3H，pyrrole-CH ₃)，2.45(s，3H，pyrrole-CH ₃)，3.29(m，2H，CH ₂)，3.38(m，2H，CH ₂)，5.18(s，2H，benzene-NH ₂)，6.32(m，1H)，6.51(m，2H)，6.85(m，1H)，7.06(m，1H)，7.20(m，2H)，7.71(s，1H)，7.90(d，J＝8.0Hz，1H)，8.33(d，J＝8.0Hz，1H)，8.64(s，2H)，9.29(s，1H，benzene-NH)，10.73(s，1H，indolinone-NH)，11.84(s，1H，pyrrole-NH)。LC-MS(m/z)614(M+1)。

Embodiment 20

The preparation of N-(2-amino-4-fluorophenyl)-6-(the 4-aminobutyl is amino) niacinamide

266mg (1mmol) N-(2-amino-4-fluorophenyl)-6-chloro-nicotinamide and 7ml Putriscine are mixed, be heated to 80 ℃, reacted 3 hours.Vacuum is removed excessive Putriscine.Add the NaOH solution of 5ml 0.20M in the residue, then use the 100ml ethyl acetate extraction.Decompression is removed ethyl acetate and is got brown solid (149mg, productive rate 47%).LC-MS(m/z)318(M+1)。

Embodiment 21

The preparation of (Z)-N-(2-amino-4-fluorophenyl)-6-(4-(2-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl)-3,5-dimethyl-1H-pyrroles-4-amide group) butyl is amino)-niacinamide

With 300mg (1mmol) 5-(5-fluoro-2-oxygen-1,2-dihydro-indoles-(3Z)-ylidenylmethyl)-2,4-dimethyl-1H-pyrroles-3-carboxylic acid is dissolved in 8ml DMF, then adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162 milligrams of (1.2mmol) I-hydroxybenzotriazoles, 404mg (4mmol) triethylamine and 333mg (1.05mmol) N-(2-amino-4-fluorophenyl)-6-(the 4-aminobutyl is amino) niacinamide.Mixture at room temperature stirred 20 hours, and then with the dilution of 400ml salt solution, vacuum filtration is collected solid.Solid washes with water, and vacuum-drying gets yellow solid (485mg, productive rate 81%). ¹H?NMR(DMSO-d ₆)δ1.59(m，4H，CH ₂CH ₂)，2.39(s，3H，pyrrole-CH ₃)，2.41(s，3H，pyrrole-CH ₃)，3.24(m，2H，CH ₂)，3.34(m，2H，CH ₂)，5.17(s，2H，benzene-NH ₂)，6.33(m，1H)，6.50(m，2H)，6.83(m，1H)，6.91(m，1H)，7.06(t，J＝8.0Hz，1H)，7.18(s，1H)，7.67～7.76(m，3H)，7.89(d，J＝8.0Hz，1H)，8.63(s，1H)，9.28(s，1H，benzene-NH)，10.89(s，1H，indolinone-NH)，13.67(s，1H，pyrrole-NH)。LC-MS(m/z)600(M+1)。

Embodiment 22

The preparation of N-(2-amino-4-fluorophenyl)-6-(4-(2-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit)-hydrazono-) methyl)-3,5-dimethyl-1H-pyrroles-4-amide group) butyl is amino)-niacinamide

With 328mg (1mmol) 5-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl-1H-pyrroles-3-carboxylic acid is dissolved in 8ml DMF, then adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 333mg (1.05mmol) N-(2-amino-4-fluorophenyl)-6-(the 4-aminobutyl is amino) niacinamide.Mixture at room temperature stirred 20 hours, and then with the dilution of 400ml salt solution, vacuum filtration is collected solid.Solid washes with water, and vacuum-drying gets yellow solid (433mg, productive rate 69%). ¹H?NMR(DMSO-d ₆)δ1.58(m，4H，CH ₂CH ₂)，2.32(s，3H，pyrrole-CH ₃)，2.42(s，3H，pyrrole-CH ₃)，3.24(m，2H，CH ₂)，3.35(m，2H，CH ₂)，5.18(s，2H，benzene-NH ₂)，6.33(m，1H)，6.50(m，2H)，6.85(m，1H)，7.06(m，1H)，7.20(m，2H)，7.67(s，1H)，7.89(d，J＝8.0Hz，1H)，8.33(d，J＝8.0Hz，1H)，8.63(s，2H)，9.29(s，1H，benzene-NH)，10.74(s，1H，indolinone-NH)，11.83(s，1H，pyrrole-NH)。LC-MS(m/z)628(M+1)。

Embodiment 23

The preparation of N-(2-amino-4-chloro-phenyl-)-6-chloro-nicotinamide

157.5mg (1mmol) 6-chlorine apellagrin is dissolved in 8ml DMF, then adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 171mg (1.2mmol) 4-chlorine O-Phenylene Diamine (be purchased from Alfa Aesar company).Mixture at room temperature stirred 20 hours, then with the dilution of 400ml salt solution, then used the 200ml ethyl acetate extraction.Vacuum is removed ethyl acetate, adds the 5ml dehydrated alcohol in residue.Vacuum filtration is collected solid.The a small amount of absolute ethanol washing of solid, vacuum-drying get brown solid (135mg, productive rate 48%).LC-MS(m/z)282(M+1)。

Embodiment 24

The preparation of N-(the amino 4-chloro-phenyl-of 2-)-6-(2-aminoethylamino) niacinamide

282mg (1mmol) N-(2-amino-4-chloro-phenyl-)-6-chloro-nicotinamide and 5ml quadrol are mixed, be heated to 80 ℃, reacted 3 hours.Vacuum is removed excessive quadrol.Add the NaOH solution of 5ml 0.20M in the residue, then use the 100ml ethyl acetate extraction.Decompression is removed ethyl acetate and is got brown solid (180mg, productive rate 59%).LC-MS(m/z)306(M+1)。

Embodiment 25

The preparation of (Z)-N-(2-amino-4-chloro-phenyl-)-6-(2-(2-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl)-3,5-dimethyl-1H-pyrroles-4-amide group) ethylamino)-niacinamide

With 300mg (1mmol) 5-(5-fluoro-2-oxygen-1,2-dihydro-indoles-(3Z)-ylidenylmethyl)-2,4-dimethyl-1H-pyrroles-3-carboxylic acid is dissolved in 8ml DMF, then adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 321mg (1.05mmol) N-(2-amino-4-chloro-phenyl-)-6-(2-aminoethylamino) niacinamide.Mixture at room temperature stirred 20 hours, and then with the dilution of 400ml salt solution, vacuum filtration is collected solid.Solid washes with water, and vacuum-drying gets yellow solid (446mg, productive rate 76%).LC-MS(m/z)588(M+1)。

Embodiment 26

The preparation of N-(2-amino-4-chloro-phenyl-)-6-(2-(2-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit)-hydrazono-) methyl)-3,5-dimethyl-1H-pyrroles-4-amide group) ethylamino)-niacinamide

With 328mg (1mmol) 5-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl-1H-pyrroles-3-carboxylic acid is dissolved in 8ml DMF, then adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 321mg (1.05mmol) N-(2-amino-4-chloro-phenyl-)-6-(2-aminoethylamino) niacinamide.Mixture at room temperature stirred 20 hours, and then with the dilution of 400ml salt solution, vacuum filtration is collected solid.Solid washes with water, and vacuum-drying gets yellow solid (406mg, productive rate 66%).LC-MS(m/z)616(M+1)。

Embodiment 27

The preparation of N-(2-amino-4-aminomethyl phenyl)-6-chloro-nicotinamide

157.5mg (1mmol) 6-chlorine apellagrin is dissolved in 8ml DMF, then adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 146mg (1.2mmol) 4-methyl-o-phenylenediamine (be purchased from Alfa Aesar company).Mixture at room temperature stirred 20 hours, then with the dilution of 400ml salt solution, then used the 200ml ethyl acetate extraction.Vacuum is removed ethyl acetate, adds the 5ml dehydrated alcohol in residue.Vacuum filtration is collected solid.The a small amount of absolute ethanol washing of solid, vacuum-drying get brown solid (164mg, productive rate 63%).LC-MS(m/z)262(M+1)。

Embodiment 28

The preparation of N-(2-amino-4-aminomethyl phenyl)-6-(2-aminoethylamino) niacinamide

261mg (1mmol) N-(2-amino-4-aminomethyl phenyl)-6-chloro-nicotinamide and 5ml quadrol are mixed, be heated to 80 ℃, reacted 3 hours.Vacuum is removed excessive quadrol.Add the NaOH solution of 5ml 0.20M in the residue, then use the 100ml ethyl acetate extraction.Decompression is removed ethyl acetate and is got brown solid (145mg, productive rate 51%).LC-MS(m/z)286(M+1)。

Embodiment 29

The preparation of (Z)-N-(2-amino-4-aminomethyl phenyl)-6-(2-(2-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl)-3,5-dimethyl-1H-pyrroles-4-amide group) ethylamino)-niacinamide

With 300mg (1mmol) 5-(5-fluoro-2-oxygen-1,2-dihydro-indoles-(3Z)-ylidenylmethyl)-2,4-dimethyl-1H-pyrroles-3-carboxylic acid is dissolved in 8ml DMF, then adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 299mg (1.05mmol) N-(2-amino-4-aminomethyl phenyl)-6-(2-aminoethylamino) niacinamide.Mixture at room temperature stirred 20 hours, and then with the dilution of 400ml salt solution, vacuum filtration is collected solid.Solid washes with water, and vacuum-drying gets yellow solid (420mg, productive rate 74%).LC-MS(m/z)568(M+1)。

Embodiment 30

The preparation of N-(2-amino-4-aminomethyl phenyl)-6-(2-(2-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit)-hydrazono-) methyl)-3,5-dimethyl-1H-pyrroles-4-amide group) ethylamino)-niacinamide

With 328mg (1mmol) 5-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl-1H-pyrroles-3-carboxylic acid is dissolved in 8ml DMF, then adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 299mg (1.05mmol) N-(2-amino-4-aminomethyl phenyl)-6-(2-aminoethylamino) niacinamide.Mixture at room temperature stirred 20 hours, and then with the dilution of 400ml salt solution, vacuum filtration is collected solid.Solid washes with water, and vacuum-drying gets yellow solid (363mg, productive rate 61%).LC-MS(m/z)596(M+1)。

Embodiment 31

The preparation of N-(2-amino-4-methoxyl phenyl)-6-chloro-nicotinamide

157.5mg (1mmol) 6-chlorine apellagrin is dissolved in 8ml DMF, then adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 166mg (1.2mmol) 4-methoxyl group O-Phenylene Diamine (be purchased from AlfaAesar company).Mixture at room temperature stirred 20 hours, then with the dilution of 400ml salt solution, then used the 200ml ethyl acetate extraction.Vacuum is removed ethyl acetate, adds the 5ml dehydrated alcohol in residue.Vacuum filtration is collected solid.The a small amount of absolute ethanol washing of solid, vacuum-drying get brown solid (144mg, productive rate 52%).LC-MS(m/z)278(M+1)。

Embodiment 32

The preparation of N-(the amino 4-p-methoxy-phenyl of 2-)-6-(2-aminoethylamino) niacinamide

277mg (1mmol) N-(2-amino-4-methoxyl phenyl)-6-chloro-nicotinamide and 5ml quadrol are mixed, be heated to 80 ℃, reacted 3 hours.Vacuum is removed excessive quadrol.Add the NaOH solution of 5ml 0.20M in the residue, then use the 100ml ethyl acetate extraction.Decompression is removed ethyl acetate and is got brown solid (144mg, productive rate 48%).LC-MS(m/z)302(M+1)。

Embodiment 33

The preparation of (Z)-N-(2-amino-4-methoxyl phenyl)-6-(2-(2-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl)-3,5-dimethyl-1H-pyrroles-4-amide group) ethylamino)-niacinamide

With 300mg (1mmol) 5-(5-fluoro-2-oxygen-1,2-dihydro-indoles-(3Z)-ylidenylmethyl)-2,4-dimethyl-1H-pyrroles-3-carboxylic acid is dissolved in 8ml DMF, then adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 316mg (1.05mmol) N-(2-amino-4-methoxyl phenyl)-6-(2-aminoethylamino) niacinamide.Mixture at room temperature stirred 20 hours, and then with the dilution of 400ml salt solution, vacuum filtration is collected solid.Solid washes with water, and vacuum-drying gets yellow solid (478mg, productive rate 82%).LC-MS(m/z)584(M+1)。

Embodiment 34

The preparation of N-(2-amino-4-methoxyl phenyl)-6-(2-(2-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-3,5-dimethyl-1H-pyrroles-4-amide group) ethylamino)-niacinamide

With 328mg (1mmol) 5-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl-1H-pyrroles-3-carboxylic acid is dissolved in 8ml DMF, then adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 316mg (1.05mmol) N-(2-amino-4-methoxyl phenyl)-6-(2-aminoethylamino) niacinamide.Mixture at room temperature stirred 20 hours, and then with the dilution of 400ml salt solution, vacuum filtration is collected solid.Solid washes with water, and vacuum-drying gets yellow solid (397mg, productive rate 65%).LC-MS(m/z)612(M+1)。

Embodiment 35

The preparation of N-(2-amino-4-trifluoromethyl)-6-chloro-nicotinamide

157.5mg (1mmol) 6-chlorine apellagrin is dissolved in 8ml DMF, then adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 211mg (1.2mmol) 4-trifluoromethyl O-Phenylene Diamine (be purchased from Alfa Aesar company).Mixture at room temperature stirred 20 hours, then with the dilution of 400ml salt solution, then used the 200ml ethyl acetate extraction.Vacuum is removed ethyl acetate, adds the 5ml dehydrated alcohol in residue.Vacuum filtration is collected solid.The a small amount of absolute ethanol washing of solid, vacuum-drying get brown solid (132mg, productive rate 42%).LC-MS(m/z)316(M+1)。

Embodiment 36

The preparation of N-(the amino 4-trifluoromethyl of 2-)-6-(2-aminoethylamino) niacinamide

266mg (1mmol) N-(2-amino-4-trifluoromethyl)-6-chloro-nicotinamide and 5ml quadrol are mixed, be heated to 80 ℃, reacted 3 hours.Vacuum is removed excessive quadrol.Add the NaOH solution of 5ml 0.20M in the residue, then use the 100ml ethyl acetate extraction.Decompression is removed ethyl acetate and is got brown solid (159mg, productive rate 47%).LC-MS(m/z)340(M+1)。

Embodiment 37

The preparation of (Z)-N-(2-amino-4-trifluoromethyl)-6-(2-(2-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl)-3,5-dimethyl-1H-pyrroles-4-amide group) ethylamino)-niacinamide

With 300mg (1mmol) 5-(5-fluoro-2-oxygen-1,2-dihydro-indoles-(3Z)-ylidenylmethyl)-2,4-dimethyl-1H-pyrroles-3-carboxylic acid is dissolved in 8ml DMF, then adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 356mg (1.05mmol) N-(2-amino-4-trifluoromethyl)-6-(2-aminoethylamino) niacinamide.Mixture at room temperature stirred 20 hours, and then with the dilution of 400ml salt solution, vacuum filtration is collected solid.Solid washes with water, and vacuum-drying gets yellow solid (422mg, productive rate 68%).LC-MS(m/z)622(M+1)。

Embodiment 38

N-(2-amino-4-trifluoromethyl)-6-(2-(2-(((5-fluoro-2-oxygen-1, the preparation of 2-dihydro-indoles-3-subunit) hydrazono-) methyl)-3,5-dimethyl-1H-pyrroles-4-amide group) ethylamino)-niacinamide

With 328mg (1mmol) 5-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl-1H-pyrroles-3-carboxylic acid is dissolved in 8ml DMF, then adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 356mg (1.05mmol) N-(2-amino-4-trifluoromethyl)-6-(2-aminoethylamino) niacinamide.Mixture at room temperature stirred 20 hours, and then with the dilution of 400ml salt solution, vacuum filtration is collected solid.Solid washes with water, and vacuum-drying gets yellow solid (350mg, productive rate 54%).LC-MS(m/z)650(M+1)。

Embodiment 39

The preparation of (Z)-N-(2-aminophenyl)-6-(2-(2-((2-oxygen-1,2-dihydro-indoles-3-subunit) methyl)-3,5-dimethyl-1H-pyrroles-4-amide group) ethylamino)-niacinamide

With 282mg (1mmol) 5-(2-oxygen-1,2-dihydro-indoles-(3Z)-ylidenylmethyl)-2,4-dimethyl-1H-pyrroles-3-carboxylic acid is dissolved in 8ml DMF, then adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 284mg (1.05mmol) N-(2-aminophenyl)-6-(2-aminoethylamino) niacinamide.Mixture at room temperature stirred 20 hours, and then with the dilution of 400ml salt solution, vacuum filtration is collected solid.Solid washes with water, and vacuum-drying gets yellow solid (440mg, productive rate 86%).LC-MS(m/z)536(M+1)。

Embodiment 40

The preparation of N-(2-aminophenyl)-6-(2-(2-(((2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-3,5-dimethyl-1H-pyrroles-4-amide group) ethylamino)-niacinamide

With 310mg (1mmol) 5-(((2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl-1H-pyrroles-3-carboxylic acid is dissolved in 8ml DMF, then adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 284mg (1.05mmol) N-(2-aminophenyl)-6-(2-aminoethylamino) niacinamide.Mixture at room temperature stirred 20 hours, and then with the dilution of 400ml salt solution, vacuum filtration is collected solid.Solid washes with water, and vacuum-drying gets yellow solid (394mg, productive rate 70%).LC-MS(m/z)564(M+1)。

Embodiment 41

The preparation of (Z)-N-(2-aminophenyl)-6-(2-(2-((5-chloro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl)-3,5-dimethyl-1H-pyrroles-4-amide group) ethylamino)-niacinamide

With 316mg (1mmol) 5-(5-chloro-2-oxygen-1,2-dihydro-indoles-(3Z)-ylidenylmethyl)-2,4-dimethyl-1H-pyrroles-3-carboxylic acid is dissolved in 8ml DMF, then adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 284mg (1.05mmol) N-(2-aminophenyl)-6-(2-aminoethylamino) niacinamide.Mixture at room temperature stirred 20 hours, and then with the dilution of 400ml salt solution, vacuum filtration is collected solid.Solid washes with water, and vacuum-drying gets yellow solid (444mg, productive rate 77%).LC-MS(m/z)570(M+1)。

Embodiment 42

The preparation of N-(2-aminophenyl)-6-(2-(2-(((5-chloro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-3,5-dimethyl-1H-pyrroles-4-amide group) ethylamino)-niacinamide

With 344mg (1mmol) 5-(((5-chloro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl-1H-pyrroles-3-carboxylic acid is dissolved in 8ml DMF, then adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 284mg (1.05mmol) N-(2-aminophenyl)-6-(2-aminoethylamino) niacinamide.Mixture at room temperature stirred 20 hours, and then with the dilution of 400ml salt solution, vacuum filtration is collected solid.Solid washes with water, and vacuum-drying gets yellow solid (376mg, productive rate 63%).LC-MS(m/z)598(M+1)。

Embodiment 43

The preparation of (Z)-N-(2-aminophenyl)-6-(2-(2-((4-methyl-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl)-3,5-dimethyl-1H-pyrroles-4-amide group) ethylamino)-niacinamide

With 296mg (1mmol) 5-(4-methyl-2-oxygen-1,2-dihydro-indoles-(3Z)-ylidenylmethyl)-2,4-dimethyl-1H-pyrroles-3-carboxylic acid is dissolved in 8ml DMF, then adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 284mg (1.05mmol) N-(2-aminophenyl)-6-(2-aminoethylamino) niacinamide.Mixture at room temperature stirred 20 hours, and then with the dilution of 400ml salt solution, vacuum filtration is collected solid.Solid washes with water, and vacuum-drying gets yellow solid (445mg, productive rate 81%).LC-MS(m/z)550(M+1)。

Embodiment 44

The preparation of N-(2-aminophenyl)-6-(2-(2-(((4-methyl-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-3,5-dimethyl-1H-pyrroles-4-amide group) ethylamino)-niacinamide

With 324mg (1mmol) 5-(((4-methyl-2-oxygen-1,2-dihydro-hydrogen indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl-1H-pyrroles-3-carboxylic acid is dissolved in 8ml DMF, then adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 284mg (1.05mmol) N-(2-aminophenyl)-6-(2-aminoethylamino) niacinamide.Mixture at room temperature stirred 20 hours, and then with the dilution of 400ml salt solution, vacuum filtration is collected solid.Solid washes with water, and vacuum-drying gets yellow solid (438mg, productive rate 76%).LC-MS(m/z)578(M+1)。

Embodiment 45

The preparation of (Z)-N-(2-aminophenyl)-6-(2-(2-((5-nitro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl)-3,5-dimethyl-1H-pyrroles-4-amide group) ethylamino)-niacinamide

With 327mg (1mmol) 5-(5-nitro-2-oxygen-1,2-dihydro-indoles-(3Z)-ylidenylmethyl)-2,4-dimethyl-1H-pyrroles-3-carboxylic acid is dissolved in 8ml DMF, then adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 284mg (1.05mmol) N-(2-aminophenyl)-6-(2-aminoethylamino) niacinamide.Mixture at room temperature stirred 20 hours, and then with the dilution of 400ml salt solution, vacuum filtration is collected solid.Solid washes with water, and vacuum-drying gets yellow solid (383mg, productive rate 66%).LC-MS(m/z)581(M+1)。

Embodiment 46

The preparation of N-(2-aminophenyl)-6-(2-(2-(((5-nitro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-3,5-dimethyl-1H-pyrroles-4-amide group) ethylamino)-niacinamide

With 355mg (1mmol) 5-(((5-nitro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl-1H-pyrroles-3-carboxylic acid is dissolved in 8ml DMF, then adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 284mg (1.05mmol) N-(2-aminophenyl)-6-(2-aminoethylamino) niacinamide.Mixture at room temperature stirred 20 hours, and then with the dilution of 400ml salt solution, vacuum filtration is collected solid.Solid washes with water, and vacuum-drying gets yellow solid (450mg, productive rate 74%).LC-MS(m/z)609(M+1)。

Embodiment 47

The preparation of (Z)-N-(2-aminophenyl)-6-(2-(2-((6-methoxyl group-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl)-3,5-dimethyl-1H-pyrroles-4-amide group) ethylamino)-niacinamide

With 312mg (1mmol) 5-(6-methoxyl group-2-oxygen-1,2-dihydro-indoles-(3Z)-ylidenylmethyl)-2,4-dimethyl-1H-pyrroles-3-carboxylic acid is dissolved in 8ml DMF, then adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 284mg (1.05mmol) N-(2-aminophenyl)-6-(2-aminoethylamino) niacinamide.Mixture at room temperature stirred 20 hours, and then with the dilution of 400ml salt solution, vacuum filtration is collected solid.Solid washes with water, and vacuum-drying gets yellow solid (463mg, productive rate 82%).LC-MS(m/z)566(M+1)。

Embodiment 48

The preparation of N-(2-aminophenyl)-6-(2-(2-(((6-methoxyl group-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-3,5-dimethyl-1H-pyrroles-4-amide group) ethylamino)-niacinamide

With 340mg (1mmol) 5-(((6-methoxyl group-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl-1H-pyrroles-3-carboxylic acid is dissolved in 8ml DMF, then adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 284mg (1.05mmol) N-(2-aminophenyl)-6-(2-aminoethylamino) niacinamide.Mixture at room temperature stirred 20 hours, and then with the dilution of 400ml salt solution, vacuum filtration is collected solid.Solid washes with water, and vacuum-drying gets yellow solid (397mg, productive rate 67%).LC-MS(m/z)594(M+1)。

Embodiment 49

The preparation of (Z)-N-(2-aminophenyl)-6-(2-(2-((6-trifluoromethyl-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl)-3,5-dimethyl-1H-pyrroles-4-amide group) ethylamino)-niacinamide

With 350mg (1mmol) 5-(6-trifluoromethyl-2-oxygen-1,2-dihydro-indoles-(3Z)-ylidenylmethyl)-2,4-dimethyl-1H-pyrroles-3-carboxylic acid is dissolved in 8ml DMF, then adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 284mg (1.05mmol) N-(2-aminophenyl)-6-(2-aminoethylamino) niacinamide.Mixture at room temperature stirred 20 hours, and then with the dilution of 400ml salt solution, vacuum filtration is collected solid.Solid washes with water, and vacuum-drying gets yellow solid (356mg, productive rate 59%).LC-MS(m/z)604(M+1)。

Embodiment 50

The preparation of N-(2-aminophenyl)-6-(2-(2-(((6-trifluoromethyl-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-3,5-dimethyl-1H-pyrroles-4-amide group) ethylamino)-niacinamide

With 378mg (1mmol) 5-(((6-trifluoromethyl-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl-1H-pyrroles-3-carboxylic acid is dissolved in 8ml DMF, then adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 284mg (1.05mmol) N-(2-aminophenyl)-6-(2-aminoethylamino) niacinamide.Mixture at room temperature stirred 20 hours, and then with the dilution of 400ml salt solution, vacuum filtration is collected solid.Solid washes with water, and vacuum-drying gets yellow solid (341mg, productive rate 54%).LC-MS(m/z)632(M+1)。

Embodiment 51

The preparation of N-(2-aminophenyl)-6-(6-amino cyclohexyl amino) niacinamide

With 248mg (1mmol) N-(2-aminophenyl)-6-chloro-nicotinamide and 5.8g (50mmol) 1, the 6-hexanediamine mixes, and is heated to 80 ℃, reacts 3 hours.It is excessive 1 that vacuum is removed, the 6-hexanediamine.Add the NaOH solution of 5ml 0.20M in the residue, then use the 100ml ethyl acetate extraction.Decompression is removed ethyl acetate and is got brown solid (219mg, productive rate 67%).

LC-MS(m/z)328(M+1)。

Embodiment 52

The preparation of (Z)-N-(2-aminophenyl)-6-(6-(2-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl)-3,5-dimethyl-1H-pyrroles-4-amide group) hexyl is amino)-niacinamide

With 300mg (1mmol) 5-(5-fluoro-2-oxygen-1,2-dihydro-indoles-(3Z)-ylidenylmethyl)-2,4-dimethyl-1H-pyrroles-3-carboxylic acid is dissolved in 8ml DMF, then adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 343mg (1.05mmol) N-(2-aminophenyl)-6-(6-amino cyclohexyl amino) niacinamide.Mixture at room temperature stirred 20 hours, and then with the dilution of 400ml salt solution, vacuum filtration is collected solid.Solid washes with water, and vacuum-drying gets yellow solid (487mg, productive rate 80%).LC-MS(m/z)610(M+1)。

Embodiment 53

The preparation of N-(2-aminophenyl)-6-(6-(2-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-3,5-dimethyl-1H-pyrroles-4-amide group) hexyl is amino)-niacinamide

With 328mg (1mmol) 5-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl-1H-pyrroles-3-carboxylic acid is dissolved in 8ml DMF, then adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 343mg (1.05mmol) N-(2-aminophenyl)-6-(6-amino cyclohexyl amino) niacinamide.Mixture at room temperature stirred 20 hours, and then with the dilution of 400ml salt solution, vacuum filtration is collected solid.Solid washes with water, and vacuum-drying gets yellow solid (427mg, productive rate 67%).LC-MS(m/z)638(M+1)。

The preparation of embodiment 54 tablets

Prescription (1000):

Compound 5:5g

Microcrystalline Cellulose: 90g

Sodium starch glycolate: 5g

4% PVP K30 ethanol solution: 50g

Talcum powder: 0.5g

Preparation technology: compound 5 is crossed 100 mesh sieves, Microcrystalline Cellulose, sodium starch glycolate and talcum powder are crossed 80 mesh sieves, take Microcrystalline Cellulose and the sodium starch glycolate of recipe quantity, mix, then with compound 5 with it by the equivalent method mixing that progressively increases, add 4% PVP K30 ethanol solution to granulate in right amount, add recipe quantity talcum powder mixing after drying, compressing tablet and get final product.

The preparation of embodiment 55 capsules

Prescription (1000):

Compound 5:5g

Microcrystalline Cellulose: 55g

Lactose: 35g

Sodium starch glycolate: 5g

Magnesium Stearate: 0.5g

Preparation technology: compound 5 is crossed 100 mesh sieves, Microcrystalline Cellulose, lactose, sodium starch glycolate and Magnesium Stearate are crossed 80 mesh sieves, take Microcrystalline Cellulose, lactose and the sodium starch glycolate of recipe quantity, mix, then with compound 5 with it by the equivalent method mixing that progressively increases, add recipe quantity Magnesium Stearate mixing, can capsule and get final product.

The preparation of embodiment 56 injection liquids

Prescription:

Compound 5:1.00mg

Medicinal DMSO:0.10ml

Medicinal alcohol: 1.00ml

Preparation technology: compound 5 is dissolved in medicinal DMSO, adds medicinal alcohol and get final product.

Embodiment 57

Compound 5-22 suppresses tyrosine kinase receptor c-Kit in cell, the ligand dependent cell proliferation experiment of PDGFR β and VEGFR2 enzymic activity

(1) PDGF dependent cell propagation

The mouse NIH of employing stably express people PDGFR β-3T3 becomes the fiber engineering cell strain to estimate the cell proliferation that PDGF relies on.PDGFR β/NIH-3T3 cell divides with the density in 5000 every holes plants in 96 hole microwell plates, at CO ₂37 ℃ of cultivations of incubator changed the serum free medium overnight incubation into after 24 hours.Add testing compound and PDGF BB (50ng/ml), continue to cultivate 72 hours in serum free medium.The cell proliferation situation detects with MTS method (Promega) according to explanation.At CO ₂37 ℃ of cultivations of incubator 2 hours, the then absorbance value of measurement 490nm on the ELISA microplate reader.

(2) VEGF dependent cell propagation

The HUVEC cell divides with the density in 6000 every holes plants in 96 hole microwell plates, at CO ₂37 ℃ of cultivations of incubator changed in serum free medium after 24 hours cultivated 2 hours.Add testing compound and VEGF 165 (50ng/ml), continue to cultivate 72 hours in serum free medium.The cell proliferation situation detects with MTS method (Promega) according to explanation.At CO ₂37 ℃ of cultivations of incubator 2 hours, the then absorbance value of measurement 490nm on the ELISA microplate reader.

(3) SCF dependent cell propagation

The Mo7e cell divided kind in 96 hole microwell plates with the density in 15000 every holes, with 1640 culture medium culturing that contains 10%FBS and SCF (50ng/ml) 24 hours.Add testing compound and solvent control, continue to cultivate 72 hours.The cell proliferation situation detects with MTS method (Promega) according to explanation.At CO ₂37 ℃ of cultivations of incubator 2 hours, the then absorbance value of measurement 490nm on the ELISA microplate reader.

Experimental result sees Table 2.

Table 2

Embodiment 58

Compound 5-22 vitro inhibition tyrosine kinase receptor c-Kit, PDGFR β, the enzymic activity experiment of VEGFR2 and Flt3

(1) the PDGFR 'beta ' activity is measured

Use the ELISA method to measure the kinase activity of external PDGFR.

Experimental procedure:

1. add the ATP of 10 μ l 10mM in the peptide substrate of 1.25ml 6 μ M.With mixed solution dH ₂O is diluted to 2.5ml, obtains 2XATP/ substrate mixture ([ATP]=40 μ M, [substrate]=3 μ m).

2. enzyme is transferred on ice from-80 ℃ of refrigerators, thaws on ice.

3.4 ℃ centrifugal in short-term bottom that makes enzyme be deposited in bottle moves on ice at once.

4. 10 μ l DTT (1.25M) are added 2.5ml 4X HTScan ^TMTyrosylprotein kinase damping fluid (240mMHEPES pH 7.5,20mM MgCl ₂, 20mM MnCl ₂, 12 μ M Na ₃VO ₄) obtain DTT/ kinase buffer liquid mixture.

5. 1.25ml DTT/ kinase buffer liquid mixture is mixed to get 4X reaction mixture (enzyme in the 4X reaction mixture=4ng/ μ L) with kinases again.

6. the testing compound of the beforehand dilution in the 4X reaction mixture of 12.5 μ l and 12.5 μ l/ holes is put together in incubated at room 5 minutes.

7. 25 μ l 2X ATP/ substrate mixture are added 25 μ l/ holes in advance in cultured reaction mixture/compound.The final condition of 50 μ l reaction solutions is:

60mM?HEPES?pH?7.5

5mM?MgCl ₂

5mM?MnCl ₂

3μM?Na ₃VO ₄

1.25mM?DTT

20μM?ATP

1.5 μ M peptide substrate

The 50ng pdgf receptor kinase

8. with microwell plate placing response 30 minutes at room temperature.

9. add 50 μ l/ hole stop buffer (50mM EDTA, pH 8) termination reactions.

10. get each reaction solution 25 μ l and 75 μ l dH ₂Then at room temperature hatched 60 minutes to 96 coated plates of Streptavidin in the O/ hole.

11. with 200 μ l/ hole PBS/T washing three times.

12. in the PBS/T that contains 1%BSA, every hole added 100 μ l to the monoclonal antibody (P-Tyr-100) that will resist tyrosine phosphorylation with the dilution proportion of 1: 1000.

13. at room temperature hatched 60 minutes.

14. with 200 μ l/ hole PBS/T washing three times.

15. with the anti-mouse lgG of HRP-mark with the dilution proportion of 1: 500 in the PBS/T that contains 1%BSA, every hole adds 100 μ l.

16. at room temperature hatched 30 minutes.

17. with 200 μ l/ hole PBS/T washing five times.

18. add 100 μ l/ hole ECL solution.

19. detect with luminous microplate reader.

(2) VEGFR2 determination of activity

Use the ELISA method to measure the kinase activity of external VEGFR2.

Experimental procedure:

1. add the ATP of 10 μ l 10mM in the peptide substrate of 1.25ml 6 μ M.With mixed solution dH ₂O is diluted to 2.5ml, obtains 2X ATP/ substrate mixture ([ATP]=40 μ M, [substrate]=3 μ m).

2. enzyme is transferred on ice from-80 ℃ of refrigerators, thaws on ice.

60mM?HEPES?pH?7.5

5mM?MgCl ₂

5mM?MnCl ₂

3μM?Na ₃VO ₄

1.25mM?DTT

20μM?ATP

1.5 μ M peptide substrate

100ng VEGFR2 kinases

8. will react microwell plate at room temperature hatched 30 minutes.

9. add 50 μ l/ hole stop buffers (50mM EDTA, pH 8), termination reaction.

11. with 200 μ l/ hole PBS/T washing three times.

13. at room temperature hatched 60 minutes.

14. with 200 μ l/ hole PBS/T washing three times.

16. at room temperature hatched 30 minutes.

17. with 200 μ l/ hole PBS/T washing five times.

18. add 100 μ l/ hole ECL solution.

19. detect with luminous microplate reader.

(3) c-KIT determination of activity

Use the ELISA method to measure the kinase activity of external c-KIT.

Experimental procedure:

1. add the ATP of 10 μ l 10mM in the peptide substrate of 1.25ml 6 μ M.With mixed solution dH ₂O is diluted to 2.5ml,

Obtain 2X ATP/ substrate mixture ([ATP]=40 μ M, [substrate]=3 μ m).

2. enzyme is transferred on ice from-80 ℃ of refrigerators, thaws on ice.

60mM?HEPES?pH?7.5

5mM?MgCl ₂

5mM?MnCl ₂

3μM?Na ₃VO ₄

1.25mM?DTT

20μM?ATP

1.5 μ M peptide substrate

100ng c-KIT kinases

8. will react microwell plate at room temperature hatched 30 minutes.

9. add 50 μ l/ hole stop buffers (50mM EDTA, pH 8), termination reaction.

11. with 200 μ l/ hole PBS/T washing three times.

13. at room temperature hatched 60 minutes.

14. with 200 μ l/ hole PBS/T washing three times.

16. at room temperature hatched 30 minutes.

17. with 200 μ l/ hole PBS/T washing five times.

18. add 100 μ l/ hole ECL solution.

19. detect with luminous microplate reader.

(4) Flt3 determination of activity

Use the ELISA method to measure the kinase activity of external Flt3.

Experimental procedure:

1. add the ATP of 100 μ l 10mM in the peptide substrate of 1.25ml 6 μ M.With mixed solution dH ₂O is diluted to 2.5ml, obtains 2X ATP/ substrate mixture ([ATP]=400 μ M, [substrate]=3 μ m).

2. enzyme is transferred on ice from-80 ℃ of refrigerators, thaws on ice.

60mM?HEPES?pH?7.5

5mM?MgCl ₂

5mM?MnCl ₂

3μM?Na ₃VO ₄

1.25mM?DTT

20μM?ATP

1.5 μ M peptide substrate

10 Flt3 of unit kinases

8. will react microwell plate at room temperature hatched 30 minutes.

9. add 50 μ l/ hole stop buffers (50mM EDTA, pH 8), termination reaction.

11. with 200 μ l/ hole PBS/T washing three times.

13. at room temperature hatched 60 minutes.

14. with 200 μ l/ hole PBS/T washing three times.

16. at room temperature hatched 30 minutes.

17. with 200 μ l/ hole PBS/T washing five times.

18. add 100 μ l/ hole ECL solution.

19. detect with luminous microplate reader.

Experimental result sees Table 3.

Table 3

Embodiment 59

Compound 5-22 vitro inhibition HDAC total enzyme activity, the test of vitro inhibition HDAC hypotype activity

(1) mensuration of external HDAC total enzyme activity

The mensuration of external HDAC total enzyme activity is that we adopt the HDAC Fluorimetric Assay/DrugDiscovery Kit of BIOMOL company that the vitro inhibition activity of inhibitor is tested.The principle of experiment is as follows: a kind of special substrate Fluor deLys (adopts the nuclear extract of HeLa cell at histon deacetylase (HDAC) in experiment; be rich in the HDAC of multiple hypotype) effect under can remove an ethanoyl; expose free amino, can produce a kind of derivable fluorescence after adding Developer.The excitation wavelength of this fluorescence is 360nm, and emission wavelength is 460nm.The substrate deacetylation is more abundant, and the fluorescent value of inducing is just higher, and the fluorescent value in unrestraint agent situation is contrast; And when having inhibitor to exist, derivative fluorescent value can reduce, and during without enzyme the fluorescent value of (being equivalent to enzymic activity fully suppressed) for blank.And the fluorescent value after general the inhibition can be between contrast and blank.When analyzing we as 0, contrast is calculated as 1 with blank, be worth less explanation inhibition activity higher.

(2) mensuration of reporter gene to inhibitor HDAC subtype-selective

The HDAC different subtype can from different transcription factor combinations, participate in heterogeneic expression regulation.Select the controlling element of suitable transcription factor to be built into reporter gene, can be used for assessing inhibitor to the selective inhibitory of HDAC hypotype.Density reaches the fusion of 50-80% when transfection the day before yesterday enters HeLa cell kind 96 orifice plates and makes transfection.Inserted the p21-promotor in the luciferase gene upstream, the gdf11-promotor, the MEF-binding member, the reporter plasmid of the regulating and controlling sequences such as Nur77-promotor uses respectively FuGene6 (Roche) transfection reagent, carries out transfection according to operation instructions.Simultaneously in order to proofread and correct transfection efficiency, cotransfection the expression plasmid of green fluorescent protein (GFP).Add compound or solvent control (DMSO) after 24 hours in transfection.Collected and lysing cell after 24 hours, use luciferase (Promega) detection kit that the amount of luciferase is assessed according to operation instructions.Further measure simultaneously the content of GFP, transfection efficiency is proofreaied and correct.

Experimental result sees Table 4.

Table 4

Nd: undetermined

CS055: chidamide is the antitumor hdac inhibitor of little core bio tech ltd in the clinical study stage

Embodiment 60

The compound 5-22 MTS test that increment suppresses to tumour cell

With tumour cell with trysinization after, divide kind in 96 hole microwell plates with the density in 3000 every holes, cultivated 24 hours containing in the perfect medium of 10%FBS.Add testing compound and solvent control, final compound concentration is that 100nmol/L is to 100 μ mol/L.Then cultivated 72 hours in perfect medium.Add MTS reagent (Promega) according to the method for specification sheets, at 37 ℃ of CO ₂Cultivated 2 hours in incubator, then read the absorption value of 490nm on the ELISA microplate reader.

Experimental result sees Table 5.

Table 5

CS055: chidamide is the antitumor hdac inhibitor of little core bio tech ltd in the clinical study stage; Sorafinib and Sutent are the how active tyrosine kinase inhibitors that goes on the market.

Embodiment 61

The inhibition experiment of compound 5,11 couples of Human Colonic Tumor in Nude Mice transplanted tumor HCT-8

Select nude mice, female, 14-16g, through normal breeding observing 3 days as without abnormal, with breeding observing after 50 conventional oxter inoculation Human Colonic Tumor in Nude Mice HCT-8, random packet when confirming oncocyte implantation propagation and knurl piece diameter greater than 6mm, totally 5 groups of 8/groups, wherein two administration concentration of 2 kinds of tested materials are totally 4 groups, one group of solvent control.This day administration, 1 times/day of oral administration.Administration 21 is put to death animal rear next day and is stripped tumour and claim knurl heavy, by formula: and [(the average knurl of the average knurl weight-experimental group of control group is heavy)/average knurl of control group is heavy] * 100%, calculate and respectively organize tumor control rate, and the knurl tuple is checked according to carrying out t.Experimental result sees Table 6.

Table 6

^*The whole story course for the treatment of

Claims

1. 2-dihydroindole ketone derivate with general formula (I):

Wherein,

X is=CH-or=N-N=CH-;

R ¹, R ², R ³And R ⁴Be respectively hydrogen, chlorine, fluorine, methyl, methoxyl group, nitro or trifluoromethyl;

R ⁵, R ⁶, R ⁷And R ⁸Be respectively hydrogen, chlorine, fluorine, methyl, methoxyl group or trifluoromethyl;

N is 2 to 6 integer.

2. compound as claimed in claim 1 is characterized in that described compound is:

X is=CH-or=N-N=CH-;

R ¹, R ², R ³And R ⁴Be respectively hydrogen, chlorine or fluorine;

R ⁵, R ⁶, R ⁷And R ⁸Be respectively hydrogen, chlorine or fluorine;

N is 2 to 6 integer.

3. compound as claimed in claim 1 is characterized in that described compound is:

X is=CH-or=N-N=CH-

R ¹, R ², R ³And R ⁴Be respectively hydrogen or fluorine;

R ⁵, R ⁶, R ⁷And R ⁸Be respectively hydrogen or fluorine;

N is 2 to 4 integer.

4. the preparation method of the compound of general formula (I), the method comprises the steps:

(a) 6-chlorine apellagrin and compound (1) are carried out condensation reaction and obtain compound (2);

(b) compound (2) and compound (3) are carried out condensation reaction and obtain compound (4);

(c) compound (4) and compound (5) are carried out the compound that condensation reaction obtains general formula (I);

Wherein,

X is=CH-or=N-N=CH-;

N is 2 to 6 integer.

5. medicinal preparations that is used for the treatment of the active abnormal relevant disease of or histon deacetylase (HDAC) abnormal to protein kinase activity, it is characterized in that: said preparation comprises as the compound of the described general formula of the claim 1 of active ingredient (I) and pharmaceutical carrier, auxiliary material or thinner.

6. medicinal preparations as claimed in claim 5, it is characterized in that: the specification of described medicinal preparations is 0.0001～200mg.

7. medicinal preparations as claimed in claim 5, it is characterized in that: described medicinal preparations is tablet, capsule, pulvis, syrup, suspension agent or injection.

8. compound as claimed in claim 1 is for the preparation of the application in the medicine for the treatment of inflammation, autoimmune disorder, cancer, nervous system disorders and neurodegenerative disorders, allergy, asthma, cardiovascular disorder or the disease relevant to hormone.