CN101397295B

CN101397295B - 2-dihydroindolemanone derivates as histone deacetylase inhibitor, preparation method and use thereof

Info

Publication number: CN101397295B
Application number: CN2008101721613A
Authority: CN
Inventors: 鲁先平; 李志斌; 余金迪; 山松; 马保顺; 吴仲闻; 王祥辉; 宁志强
Original assignee: SHENZHEN WEIXIN BIOLOGICAL SCIENCE AND TECHNOLOGY Co Ltd
Current assignee: Shenzhen microbiology Polytron Technologies Inc
Priority date: 2008-11-12
Filing date: 2008-11-12
Publication date: 2012-04-25
Anticipated expiration: 2028-11-12
Also published as: CN101397295A

Abstract

The invention discloses 2-isatin derivatives, the preparation method and the application. The structure of the invention is shown as the formula (I), wherein, the definitions of R<1>, R<2>, R<3>, R<4>, R<5>, X, Ar and n are shown in the description of instruction. The compound is provided with the restraining activity against histone deacetylase so as to treat the diseases relevant to the abnormal condition of the histone deacetylase, including Rubinstein-Taybi II-syndrome, fragile x syndrome, leukemia, cancer, immunological diseases, cardiac hypertrophy, bone diseases, nervous system diseases and neurodegenerative diseases.

Description

2-dihydroindole ketone derivate, its method for making and purposes as NSC 630176

Technical field

The present invention relates to have histon deacetylase (HDAC) and suppress the synthetic of active 2-dihydroindole ketone derivate and the clinical application aspect the active relevant unusually disease of treatment and histon deacetylase (HDAC) thereof.

Background technology

Histon deacetylase (HDAC) (HDAC) albumen is playing keying action aspect the intravital genetic expression of regulation and control, and it changes the accessibility of transcription factor to genome DNA.Especially HDAC removes the ethanoyl of acetylize lysine residue in the histone, thus cause nucleosome reconstruct (Grunstein, M., 1997, Nature, 389:349-352).Because HDAC albumen plays keying action in genetic expression,, comprise cell cycle regulating, cell proliferation, differentiation, gene program expression and cancer generation (Ruijter so they are closely related with many cell functions; 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, Nature Reviews Cancer, 1:194).Closely related by the unusual deacetylation due to the histon deacetylase (HDAC) mistuning and many clinical diseases; Like Lu-Tai (Rubinstein-Taybi) two Cotards, fragile X chromosome syndromes, white blood disease and other various cancers (Langley B etal.; 2005; Current Drug Targets-CNS & Neurological Disorders, 4:41-50).Test shows that hdac inhibitor can suppress the mankind and the intravital growth of tumor of animal, comprise lung cancer, cancer of the stomach, mammary cancer, prostate cancer and lymphoma etc. (Dokmanovic, M., 2005, J.Cell Biochenm., 96:293-304).

Histon deacetylase (HDAC) is active unusual 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, like p21 and HSP-70, helps Normocellular survival.Hdac inhibitor also can act on other neurocyte of cns, like reactive astrocytes and microgliacyte, is reduced in inflammation and secondary injury in nerve injury or the disease.Increase acetylize through hdac inhibitor, can induce dendron to germinate (increase of cynapse quantity), rebuild learning behavior, get into long-term memory.Hdac inhibitor can reverse the gene silencing of friedreich's ataxia (Friedreich ' s ataxia).These data show that it is a kind of method (LangleyB et al., 2005, Current Drug Targets-CNS Neurological Disorders, 4:41-50 that has the many central nervous system diseases of treatment of hope that HDAC suppresses; Fischer A., et al, 2007, Nature447 (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 is formed (HDAC1,2,3,8 and 11) by yeast Rpd3-proteinoid.Form (HDAC4,5,6,7,9 and 10) by yeast HDA1-proteinoid for second type.Form (SIRT1,2,3,4,5,6 and 7) by yeast SIR2-proteinoid for the 3rd type.

The activity of HDAC1 is relevant with cell proliferation (sign of cancer).Mammalian cell reduces HDAC1 through siRNA expresses, have anti proliferative (Glaser, K-B., 2003, Biochem.Biophys.Res.Comm., 310:529-536).The deratization of HDAC1 clpp gene is an embryonic death, all the other cells show the growth of different cell (Lagger, G., 2002, EMBO J., 21:2672-2681).The mouse cell of HDAC1 over-expresses shows G ₂Prolong with the M phase and the speed of growth reduce (Bartl.S., 1997, Mol.Cell Biol., 17:5033-5043).Therefore, testing data shows that HDAC1 and cell cycle regulating and cell proliferation are closely related.

HDAC2 regulates and control the expression of many fetal rhythm myoprotein isomer.HDAC2 lacks or can stop the expression again of embryonic gene through chemical process inhibition of histone deacetylase, reduces ventricular hypertrophy.Anti-loose with the inositol polyphosphate hydrochlorate-expression of 5-Phosphoric acid esterase f (Inpp5f) encoding sox 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 β (Gsk3 β).On the contrary, HDAC2 transgenic mouse ventricular hypertrophy increases, and this Gsk3 β with inactivation is relevant.Suppress adult that activatory Gsk3 β makes that HDAC2-lacks through chemical process the stimulation of ventricular hypertrophy is become responsive.These results show that HDAC2 is an important molecular target of hdac inhibitor in heart.HDAC2 and Gsk3 β are the integral parts of regulatory pathway, this for treatment ventricular hypertrophy and the treatment target that very attractive is provided in heart failure (Trivedi, C-M., 2007, Nat.Med .13:324-331).

HDAC3 expresses at most in the propagation pit cell of normal small intestine.The HDAC3 expression silencing causes cell growth-inhibiting, cell survival to reduce and the apoptosis increase in colon carcinoma cell line.Reticent HDAC2 expresses and can observe similar effects, and for HDAC1, effect is then so not remarkable.The HDAC3 gene silencing also optionally causes the expression (the sophisticated sign of colon cell) of Ostase.The over-expresses of HDAC3 suppresses basal transcription and butyric ester inductive P21 transcribes, and reticent HDAC3 then stimulates the active of P21 gene promoter and expresses.These discoveries show that HDAC3 is the gene of in the human colon cancer, reducing, be colon cell ripe with the P21 expression a kind of novel regulator (Wilson, A-J., 2006, J.Biol.Chem., 281:13548-13558).

HDAC6 is a hypotype of HDAC family, and it sloughs the ethanoyl of alpha-tubulin, increases cell mobility.Clone at nine groups of OSCCs (OSCC) goes up quantitative real-time RT-PCR of utilization and Western Blots technical Analysis with normal oral cavity keratinocyte (NOKs); Compare with NOKs, HDAC6mRNA and protein expression level have all raise in all cancer cells.Through the immunofluorescence technique analysis, in the tenuigenin of OSCC clone, detected HDAC6 albumen.Similar with OSCC clone, HDAC6 raises obviously in the human body OSCC tumour in early days, and mRNA reaches 74%, and albumen reaches 51%.Through analysis, it is found that the expression status of developmental stage and HDAC6 of clinical tumor is relevant to clinical variable.Analysis revealed, 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 maybe be relevant with the grade malignancy of tumour, this also for the design new treat-ment provide clue (Sakuma, T., 2006, Int.J.Oncol., 29:117-124).

IIa class HDAC comprises HDAC4,5 and 7.In the past few years, the researchist has confirmed IIa class HDAC some important physiological function in live body.It is shocking that these seem does not have the physiological process of contact that the common characteristic is arranged, they all depend on IIa class HDAC MEF2 are transcribed the active strict control.Crucial physiological processs such as formation, megalocardia, bone development, T-cytodifferentiation and neuronal survival such as Skelettmuskel 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 (MartinM.et al; 2007, Oncogene 26:5450-5467).

It is one of main mechanism of many pathologic processes that HDAC makes function karyomit(e) 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 forfeiture 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, thereby suppresses growth of tumour cell and differentiation (Glaros S et al.; 2007, Oncogene June 4 Epub ahead ofprint; Mai, A, et al., 2007, IntJ.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 (like FXN gene relevant with Friedreich ' s ataxy and the SMN gene relevant with spinal muscular atrophy) can be reversed by HDAC; Make FXN and SMN gene express again; 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; De BoreJ, 2006, Tissue Eng.12 (10): 2927-37).Hdac inhibitor is in karyomit(e) 6p21-22 readjustment HDAC " focus " program; Induce whole M HC 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).

Confirmed several types of hdac inhibitors at present, having comprised: 1) short chain fatty acid, like butyric acid and benzenebutanoic acid; 2) organic hydroximic acid is like suberoylanilide hydroxamic acid (SAHA) and trichostatin A (TSA); 3) contain 2-amido-8-oxygen-9, the cyclic tetrapeptide of 10-epoxy decanoyl (AOE) is like trapoxin and HC-toxin; 4) do not contain 2-amino-8-oxygen-9, the cyclic tetrapeptide of 10-epoxy decanoyl is like apicidin and FK228; 5) benzamides is like MS-275 (European patent EP 0847992A1, 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 Merck company development at present 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 to develop new compound, make it have stronger HDAC and suppress active, stronger antitumour activity, better HDAC subtype-selective and lower toxicity.

Summary of the invention

The object of the present invention is to provide and a kind ofly have histon deacetylase (HDAC) and suppress active 2-dihydroindole ketone derivate;

Another object of the present invention is to provide a kind of method for preparing above-claimed cpd;

Further purpose of the present invention is to provide a kind of medicinal prepns of treating rubinstein-Taybi syndrome, fragile X chromosome syndromes, white blood disease, cancer, amynologic disease, myocardial hypertrophy, skeletal diseases, nervous system disorders and neurodegenerative disorders.

Another object of the present invention is to provide above-claimed cpd and medicinal prepns; Inhibition of histone deacetylase selectively; Being used to treat and the active unusual relevant disease of histon deacetylase (HDAC), like the application in the medicine of Lu-Tai (Rubinstein-Taybi) two Cotards, fragile X chromosome syndromes, white blood disease, cancer, amynologic disease, myocardial hypertrophy, skeletal diseases and various nervous system disorders and neurodegenerative disorders.

Compound according to the invention, its chemical structure is shown in general formula (I):

Wherein,

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

R ⁵For-NHOH or

R ⁶, R ⁷, R ⁸And R ⁹Be respectively hydrogen, halogen, alkyl, alkoxyl group or trifluoromethyl;

Ar is phenyl ring or 2,4-dimethyl--1H-pyrroles;

When Ar is phenyl ring, X is=CH-or=N-N=CH-; When Ar is 2, during 4-dimethyl--1H-pyrroles, X is=N-N=CH-;

N is 1 to 7 integer;

The form, enantiomer, diastereomer or the hydrate that comprise its free form, salt.

Said halogen is a kind of in fluorine, chlorine, bromine, the iodine, is preferably fluorine or chlorine, most preferably is fluorine.

Said alkyl comprises straight chain, side chain or cyclic alkyl, is preferably a kind of in methyl, ethyl, n-propyl, sec.-propyl, normal-butyl, isobutyl-, tertiary butyl, n-pentyl, isopentyl, n-hexyl, isohexyl, cyclopropyl, cyclobutyl, cyclopentyl, the cyclohexyl; A kind of in methyl, ethyl, n-propyl, the sec.-propyl more preferably; Most preferably be methyl or ethyl.

Said alkoxyl group refers to the alkyl formed group that links to each other with Sauerstoffatom; Wherein, Sauerstoffatom has free bonding power, is preferably a kind of in methoxyl group, oxyethyl group, propoxy-, butoxy, pentyloxy, isopropoxy, tert-butoxy, ring propoxy-, the cyclohexyl oxygen base; A kind of in methoxyl group, oxyethyl group, the propoxy-more preferably; Most preferably be methoxyl group, oxyethyl group.

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

(a) compound 1 and compound 2 are carried out condensation reaction and obtain compound 3;

(b) compound 3 hydrolysis are obtained compound 4;

(c) compound 4 and azanol are carried out condensation reaction and obtain compound 5a;

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

Above-mentioned condensation reaction (a) and (d) be catalyzer with the peptide condensing agent, like 1-ethyl-3-(3-dimethylamine propyl) carbodiimide (EDC), N, N '-NSC 57182 (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 a common solvent, like benzene, toluene, THF, dioxane, methylene dichloride, chloroform, N, dinethylformamide etc.In case of necessity, also can add alkali such as sodium hydroxide, triethylamine or pyridine.

Above-mentioned condensation reaction (c) is condensing agent with ClCOOEt, and temperature of reaction is 0～80 ℃, and the reaction times is 1～24 hour.The reaction solvent for use is a common solvent, like benzene, toluene, THF, dioxane, methylene dichloride, chloroform, N, dinethylformamide etc.In case of necessity, also can add alkali such as sodium hydroxide, triethylamine or pyridine.

Said hydrolyzed reaction (b) is with Lithium Hydroxide MonoHydrate, and sodium hydroxide or Pottasium Hydroxide are hydrolysing agent, and temperature of reaction is 0～80 ℃, and the reaction times is 2～72 hours.The reaction solvent for use is a common solvent, like water, methyl alcohol, ethanol, THF, 1,4-dioxane, N, dinethylformamide etc.

The employed compound 1 of above-mentioned condensation reaction (a), when X be=during CH-, its preparation method is:

The temperature of reaction of this reaction is 70～80 ℃, and the reaction times is 4～72 hours, and the reaction solvent for use is an ethanol.

The employed compound 1 of above-mentioned condensation reaction (a), when X be=during N-N=CH-, its preparation method is:

Step 1:

Step 2:

Wherein, the temperature of reaction of step 1 is 70～100 ℃, and the reaction times is 1～10 hour, and the reaction solvent for use is a water; The temperature of reaction of step 2 is 70～80 ℃, and the reaction times is 2～72 hours, and the reaction solvent for use is an ethanol.

Described compound of general formula (I) and midbody (3) and midbody (4) can adopt common separation method to carry out purifying, like extraction, recrystallization, column chromatography etc.

Compound of the present invention and medicinal prepns have histon deacetylase (HDAC) and suppress active; Can be used for preparing treatment and the active medicine of relevant disease unusually of histon deacetylase (HDAC), especially Lu-Tai (Rubinstein-Taybi) two Cotards, fragile X chromosome syndromes, white blood disease, cancer, amynologic disease, myocardial hypertrophy, skeletal diseases and various nervous system disorders and neurodegenerative disorders are had excellent curative effect.

Compound of the present invention can be processed into medicinal prepns commonly used according to the conventional working method of pharmaceutical field, like tablet, capsule, pulvis, syrup, liquor, suspension agent, injection.Said preparation comprises the compound of the general formula (I) as active ingredient, and pharmaceutical carrier, auxiliary material or thinner.Usually contain 0.5～70% activeconstituents in the said preparation, preferable content is 1～20%.

The formulation specification of this medicinal prepns is 0.0001～200mg.

The said pharmaceutical carrier of the present invention, auxiliary material or thinner; Comprise " the listed carrier filler of pharmaceutical excipient handbook (this handbook original work " HandbookofPharmaceutical Excipients " by name is published by U.S. Pharmaceutical Press), but be not limited to these carrier fillers.

Compound of the present invention, clinically can administered through oral or injection system Mammals (comprising the people) is carried out medication, wherein best with oral way especially.Dosage is 0.0001～200mg/kg body weight every day, and preferable 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 is looked individuality and is decided, and dosage is less when beginning usually, increases consumption then gradually.

Representative compound of the present invention is as shown in table 1." embodiment numbering " in compound number and the embodiment part is consistent, and promptly synthesizing of compound 5 obtains describing in " embodiment 5 " in the table 1, and synthesizing of compound 58 obtains describing in " embodiment 58 " in the table 1.

Table 1 representative compound of the present invention

Further illustrate content of the present invention below in conjunction with instance, but protection scope of the present invention is not limited only to these instances.Per-cent of the present invention all is weight percentage except that indicating especially.Numerical range described in the specification sheets like measure unit, reaction conditions, compound physical state or per-cent, all is 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

4-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl) phenylformic acid reaches

The benzoic preparation of 3-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl)

In reaction flask, add 1.51g (10mmol) 5-fluoro-2-indolone (US7,179,910B2; Example1), 1.50g (10mmol) 4-carboxyl benzaldehyde (purchasing company), 1.28g (15mmol) piperidines and 25ml absolute ethyl alcohol in AlfaAesar; Heating reflux reaction 6 hours is chilled to room temperature, filters.Filter cake is changed in the beaker, add 100ml water, be neutralized to pH=4 with 2M hydrochloric acid, solid collected by filtration, solid is used water washing, and vacuum-drying gets brown solid 4-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl) phenylformic acid (1.33g, productive rate 47%).LC-MS(m/z)284(M+1)。

With 5-fluoro-2-indolone and 3-carboxyl benzaldehyde (purchasing the company in AlfaAesar) is raw material, adopts similar approach to make 3-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl) phenylformic acid, LC-MS (m/z) 284 (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

In reaction flask, add 8.25g (50mmol) 5-fluoro indigo red (purchasing company) and 150ml50% Hydrazine Hydrate 80,, be chilled to room temperature in 80～90 ℃ of reactions 3 hours in Aldrich; 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)。

In reaction flask, add 1.79g (10mmol) 5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit hydrazine, 1.67g (10mmol) 5-formyl radical-2,4-dimethyl--1H-pyrroles-3-carboxylic acid (US7; 179; 910B2, Examplel), 1.28g (15mmol) piperidines and 25ml absolute ethyl alcohol, heating reflux reaction 6 hours; Be chilled to room temperature, filter.Filter cake is changed in the beaker, add 100ml water, be neutralized to pH=4 with 2M hydrochloric acid; Solid collected by filtration; Solid is used water washing, 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)。

With isatin (purchasing the company in Aldrich) is raw material, adopts 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).

With 5-chlorisatide (purchasing the company in Aldrich) is raw material, adopts 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-methyl isatin (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).

With 5-nitro isatin (purchasing the company in Aldrich) is raw material, adopts 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) is raw material; Adopt similar approach to make 5-(((6-trifluoromethyl-2-oxygen-1; 2-dihydro-indoles-3-subunit) methyl hydrazono-))-2,4-dimethyl--1H-pyrroles-3-carboxylic acid, LC-MS (m/z) 398 (M+1).

With 5-fluoro indigo red (purchasing the company in Aldrich) and 4-carboxyl benzaldehyde (purchasing the company in Alfa Aesar) is raw material, adopts similar approach to make 4-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl) phenylformic acid, LC-MS (m/z) 312 (M+1).

Embodiment 3

2-(5-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2, the 4-dimethyl--

1H-pyrroles-3-carboxamido-group) preparation of methyl acetate

With 328mg (1mmol) 5-(((5-fluoro-2-oxygen-1; 2-dihydro-indoles-3-subunit) methyl hydrazono-))-2; 4-dimethyl--1H-pyrroles-3-carboxylic acid is dissolved among 8 milliliters of DMF, adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 151.8mg (1.2mmol) glycine methyl ester hydrochloride then.Mixture at room temperature stirred 24 hours.Dilute with 400ml salt solution then.Vacuum filtration is collected solid.Solid is used water washing.Vacuum-drying gets red solid 2-(5-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl--1H-pyrroles-3-carboxamido-group) methyl acetate (244mg, productive rate 61%).LC-MS(m/z)400(M+1)。

Embodiment 4

1H-pyrroles-3-carboxamido-group) preparation of acetate

399mg (1mmol) 2-(5-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl--1H-pyrroles-3-carboxamido-group) methyl acetate is dissolved in the 300ml methyl alcohol, adds the lithium hydroxide aqueous solution of 25ml4M then.Mixture at room temperature stirred 24 hours.Using hydrochloric acid to be neutralized to the pH value in mixture is 7, and methyl alcohol is removed in decompression.Residue is with salt acid for adjusting pH value to 3.Vacuum filtration is collected solid, washing, and vacuum-drying gets red solid 2-(5-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl--1H-pyrroles-3-carboxamido-group) acetate (128mg, productive rate 33%).LC-MS(m/z)386(M+1)。

Embodiment 5

1H-pyrroles-3-carboxamido-group)-preparation of N-hydroxyl acetamide

With 385mg (1mmol) 2-(5-(((5-fluoro-2-oxygen-1; 2-dihydro-indoles-3-subunit) methyl hydrazono-))-2; 4-dimethyl--1H-pyrroles-3-carboxamido-group) acetate, 151mg (1.5mmol) triethylamine are dissolved among the 20ml DMF, add 163mg (1.5mmol) Vinyl chloroformate in 0 ℃.Mixture adds the aqueous hydroxylamine of 1.32g (20mmol) 50% after stirring 2 hours under 0 ℃.Mixture at room temperature continued to stir 3 hours, diluted with 1000ml salt solution then.Vacuum filtration is collected solid, washing, and vacuum-drying must get red solid 2-(5-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl--1H-pyrroles-3-carboxamido-group)-N-hydroxyl acetamide (140mg, productive rate 35%).LC-MS(m/z)401(M+1)。

Embodiment 6

1H-pyrroles-3-carboxamido-group)-preparation of N-(2-aminophenyl) ethanamide

With 385mg (1mmol) 2-(5-(((5-fluoro-2-oxygen-1; 2-dihydro-indoles-3-subunit) methyl hydrazono-))-2; 4-dimethyl--1H-pyrroles-3-carboxamido-group) acetate is dissolved among the 8ml DMF, adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 432mg (4mmol) O-Phenylene Diamine then.Mixture at room temperature stirred 24 hours.Dilute with 400ml salt solution then.Vacuum filtration is collected solid.Solid is used water washing.Vacuum-drying gets red solid 2-(5-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl--1H-pyrroles-3-carboxamido-group)-N-(2-aminophenyl) ethanamide (342mg, productive rate 72%).LC-MS(m/z)476(M+1)。

Embodiment 7

5-(5-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2, the 4-dimethyl--

1H-pyrroles-3-carboxamido-group) preparation of methyl valerate

With 328mg (1mmol) 5-(((5-fluoro-2-oxygen-1; 2-dihydro-indoles-3-subunit) methyl hydrazono-))-2; 4-dimethyl--1H-pyrroles-3-carboxylic acid is dissolved among 8 milliliters of DMF, adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 202mg (1.2mmol) 5-aminovaleric acid methyl ester hydrochloride then.Mixture at room temperature stirred 24 hours.Dilute with 400ml salt solution then.Vacuum filtration is collected solid.Solid is used water washing.Vacuum-drying gets red solid 5-(5-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl--1H-pyrroles-3-carboxamido-group) methyl valerate (300mg, productive rate 68%).LC-MS(m/z)442(M+1)。

Embodiment 8

1H-pyrroles-3-carboxamido-group) preparation of valeric acid

441mg (1mmol) 5-(5-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl--1H-pyrroles-3-carboxamido-group) methyl valerate is dissolved in the 300ml methyl alcohol, adds the lithium hydroxide aqueous solution of 25ml4M then.Mixture at room temperature stirred 24 hours.Using hydrochloric acid to be neutralized to the pH value in mixture is 7, and methyl alcohol is removed in decompression.Residue is with salt acid for adjusting pH value to 3.Vacuum filtration is collected solid, washing, and vacuum-drying gets red solid 5-(5-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl--1H-pyrroles-3-carboxamido-group) valeric acid (167mg, productive rate 39%).LC-MS(m/z)428(M+1)。

Embodiment 9

1H-pyrroles-3-carboxamido-group)-preparation of N-(2-aminophenyl) valeramide

With 427mg (1mmol) 5-(5-(((5-fluoro-2-oxygen-1; 2-dihydro-indoles-3-subunit) methyl hydrazono-))-2; 4-dimethyl--1H-pyrroles-3-carboxamido-group) valeric acid is dissolved among the 8ml DMF, adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 432mg (4mmol) O-Phenylene Diamine then.Mixture at room temperature stirred 24 hours.Dilute with 400ml salt solution then.Vacuum filtration is collected solid.Solid is used water washing.Vacuum-drying gets red solid 5-(5-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl--1H-pyrroles-3-carboxamido-group)-N-(2-aminophenyl) valeramide (388mg, productive rate 75%).LC-MS(m/z)518(M+1)。

Embodiment 10

1H-pyrroles-3-carboxamido-group)-preparation of N-(2-amino-4-fluorophenyl)-valeramide

With 427mg (1mmol) 5-(5-(((5-fluoro-2-oxygen-1; 2-dihydro-indoles-3-subunit) methyl hydrazono-))-2; 4-dimethyl--1H-pyrroles-3-carboxamido-group) valeric acid is dissolved among the 8ml DMF, 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 (purchasing the company in AlfaAesar) then.Mixture at room temperature stirred 24 hours.Dilute with 400ml salt solution then.Vacuum filtration is collected solid.Solid is used water washing.Vacuum-drying gets red solid 5-(5-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl--1H-pyrroles-3-carboxamido-group)-N-(the fluorine-based phenyl of 2-amino-4-) valeramide (439mg, productive rate 82%).LC-MS(m/z)536(M+1)。

Embodiment 11

6-(5-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2, the 4-dimethyl--

1H-pyrroles-3-carboxamido-group) preparation of compound such as methyl caproate

With 328mg (1mmol) 5-(((5-fluoro-2-oxygen-1; 2-dihydro-indoles-3-subunit) methyl hydrazono-))-2; 4-dimethyl--1H-pyrroles-3-carboxylic acid is dissolved among 8 milliliters of DMF, adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 219mg (1.2mmol) 6-aminocaprolc acid methyl ester hydrochloride then.Mixture at room temperature stirred 24 hours.Dilute with 400ml salt solution then.Vacuum filtration is collected solid.Solid is used water washing.Vacuum-drying gets red solid 6-(5-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl--1H-pyrroles-3-carboxamido-group) methyl caproate (287mg, productive rate 63%).LC-MS(m/z)456(M+1)。

With 5-(((2-oxygen-1; 2-dihydro-indoles-3-subunit) methyl hydrazono-))-2; 4-dimethyl--1H-pyrroles-3-carboxylic acid is a raw material, adopts similar approach to make 6-(5-(((2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2; 4-dimethyl--1H-pyrroles-3-carboxamido-group) methyl caproate, LC-MS (m/z) 438 (M+1).

With 5-(((5-chloro-2-oxygen-1; 2-dihydro-indoles-3-subunit) methyl hydrazono-))-2; 4-dimethyl--1H-pyrroles-3-carboxylic acid is a raw material, adopts similar approach to make 6-(5-(((5-chloro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2; 4-dimethyl--1H-pyrroles-3-carboxamido-group) methyl caproate, LC-MS (m/z) 472 (M+1).

With 5-(((4-methyl-2-oxygen-1; 2-dihydro-indoles-3-subunit) methyl hydrazono-))-2; 4-dimethyl--1H-pyrroles-3-carboxylic acid is a raw material, adopts similar approach to make 6-(5-(((4-methyl-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2; 4-dimethyl--1H-pyrroles-3-carboxamido-group) methyl caproate, LC-MS (m/z) 452 (M+1).

With 5-(((5-nitro-2-oxygen-1; 2-dihydro-indoles-3-subunit) methyl hydrazono-))-2; 4-dimethyl--1H-pyrroles-3-carboxylic acid is a raw material, adopts similar approach to make 6-(5-(((5-nitro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2; 4-dimethyl--1H-pyrroles-3-carboxamido-group) methyl caproate, LC-MS (m/z) 483 (M+1).

With 5-(((6-methoxyl group-2-oxygen-1; 2-dihydro-indoles-3-subunit) methyl hydrazono-))-2; 4-dimethyl--1H-pyrroles-3-carboxylic acid is a raw material, adopts similar approach to make 6-(5-(((6-methoxyl group-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2; 4-dimethyl--1H-pyrroles-3-carboxamido-group) methyl caproate, LC-MS (m/z) 468 (M+1).

With 5-(((6-trifluoromethyl-2-oxygen-1; 2-dihydro-indoles-3-subunit) methyl hydrazono-))-2; 4-dimethyl--1H-pyrroles-3-carboxylic acid is a raw material, adopts similar approach to make 6-(5-(((6-trifluoromethyl-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2; 4-dimethyl--1H-pyrroles-3-carboxamido-group) methyl caproate, LC-MS (m/z) 506 (M+1).

Embodiment 12

1H-pyrroles-3-carboxamido-group) preparation of compound such as caproic acid

455mg (1mmol) 6-(5-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl--1H-pyrroles-3-carboxamido-group) methyl caproate is dissolved in the 300ml methyl alcohol, adds the lithium hydroxide aqueous solution of 25ml4M then.Mixture at room temperature stirred 24 hours.Using hydrochloric acid to be neutralized to the pH value in mixture is 7, and methyl alcohol is removed in decompression.Residue is with salt acid for adjusting pH value to 3.Vacuum filtration is collected solid, washing, and vacuum-drying gets red solid 6-(5-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl--1H-pyrroles-3-carboxamido-group) caproic acid (163mg, productive rate 37%).LC-MS(m/z)442(M+1)。

With 6-(5-(((2-oxygen-1; 2-dihydro-indoles-3-subunit) methyl hydrazono-))-2; 4-dimethyl--1H-pyrroles-3-carboxamido-group) methyl caproate is a raw material, adopts similar approach to make 6-(5-(((2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2; 4-dimethyl--1H-pyrroles-3-carboxamido-group) caproic acid, LC-MS (m/z) 424 (M+1).

With 6-(5-(((5-chloro-2-oxygen-1; 2-dihydro-indoles-3-subunit) methyl hydrazono-))-2; 4-dimethyl--1H-pyrroles-3-carboxamido-group) methyl caproate is a raw material, adopts similar approach to make 6-(5-(((5-chloro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2; 4-dimethyl--1H-pyrroles-3-carboxamido-group) caproic acid, LC-MS (m/z) 458 (M+1).

With 6-(5-(((4-methyl-2-oxygen-1; 2-dihydro-indoles-3-subunit) methyl hydrazono-))-2; 4-dimethyl--1H-pyrroles-3-carboxamido-group) methyl caproate is a raw material, adopts similar approach to make 6-(5-(((4-methyl-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2; 4-dimethyl--1H-pyrroles-3-carboxamido-group) caproic acid, LC-MS (m/z) 438 (M+1).

With 6-(5-(((5-nitro-2-oxygen-1; 2-dihydro-indoles-3-subunit) methyl hydrazono-))-2; 4-dimethyl--1H-pyrroles-3-carboxamido-group) methyl caproate is a raw material, adopts similar approach to make 6-(5-(((5-nitro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2; 4-dimethyl--1H-pyrroles-3-carboxamido-group) caproic acid, LC-MS (m/z) 469 (M+1).

With 6-(5-(((6-methoxyl group-2-oxygen-1; 2-dihydro-indoles-3-subunit) methyl hydrazono-))-2; 4-dimethyl--1H-pyrroles-3-carboxamido-group) methyl caproate is a raw material, adopts similar approach to make 6-(5-(((6-methoxyl group-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2; 4-dimethyl--1H-pyrroles-3-carboxamido-group) caproic acid, LC-MS (m/z) 454 (M+1).

With 6-(5-(((6-trifluoromethyl-2-oxygen-1; 2-dihydro-indoles-3-subunit) methyl hydrazono-))-2; 4-dimethyl--1H-pyrroles-3-carboxamido-group) methyl caproate is a raw material, adopts similar approach to make 6-(5-(((6-trifluoromethyl-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2; 4-dimethyl--1H-pyrroles-3-carboxamido-group) caproic acid, LC-MS (m/z) 492 (M+1).

Embodiment 13

1H-pyrroles-3-carboxamido-group)-preparation of N-hydroxyl hexanamide

With 441mg (1mmol) 6-(5-(((5-fluoro-2-oxygen-1; 2-dihydro-indoles-3-subunit) methyl hydrazono-))-2; 4-dimethyl--1H-pyrroles-3-carboxamido-group) caproic acid, 151mg (1.5mmol) triethylamine are dissolved among the 20ml DMF, add 163mg (1.5mmol) Vinyl chloroformate in 0 ℃.Mixture adds the aqueous hydroxylamine of 1.32g (20mmol) 50% after stirring 2 hours under 0 ℃.Mixture at room temperature continued to stir 3 hours, diluted with 1000ml salt solution then.Vacuum filtration is collected solid, washing, and vacuum-drying must get red solid 6-(5-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl--1H-pyrroles-3-carboxamido-group)-N-hydroxyl hexanamide (201mg, productive rate 44%).LC-MS(m/z)457(M+1)。

Embodiment 14

1H-pyrroles-3-carboxamido-group)-preparation of N-(2-amino-4-fluorophenyl) hexanamide

With 441mg (1mmol) 6-(5-(((5-fluoro-2-oxygen-1; 2-dihydro-indoles-3-subunit) methyl hydrazono-))-2; 4-dimethyl--1H-pyrroles-3-carboxamido-group) caproic acid is dissolved among the 8ml DMF, 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 then.Mixture at room temperature stirred 24 hours.Dilute with 400ml salt solution then.Vacuum filtration is collected solid.Solid is used water washing.Vacuum-drying gets red solid 6-(5-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl--1H-pyrroles-3-carboxamido-group)-N-(2-amino-4-fluorophenyl) hexanamide (417mg, productive rate 76%). ¹HNMR(DMSO-d ₆)δ?1.35(m，2H，CH ₂)，1.53(m，2H，CH ₂)，1.62(m，2H，CH ₂)，2.30(t，J＝8.0Hz，2H，CH ₂CO)，2.33(s，3H，pyrrole-CH ₃)，2.43(s，3H，pyrrole-CH ₃)，3.21(m，2H，NCH ₂)，5.12(s，2H，benzene-NH ₂)，6.28(m，1H，Ar-H)，6.47(dd，J＝4.0and8.0Hz，1H，Ar-H)，6.85(dd，J＝4.0and8.0Hz，1H，Ar-H)，7.07(td，J＝4.0and8.0Hz，1H，Ar-H)，7.20(td，J＝4.0and8.0Hz，1H，Ar-H)，7.64(t，J＝4.0Hz，CONH)，8.33(dd，J＝4.0and8.0Hz，1H，Ar-H)，8.63(s，1H，vinyl-H)，9.02(s，1H，benzene-NH)，10.72(s，1H，indolinone-NH)，11.82(s，1H，pyrrole-NH)。LC-MS(m/z)550(M+1)。

Embodiment 15

1H-pyrroles-3-carboxamido-group)-preparation of N-(2-aminophenyl) hexanamide

With 441mg (1mmol) 6-(5-(((5-fluoro-2-oxygen-1; 2-dihydro-indoles-3-subunit) methyl hydrazono-))-2; 4-dimethyl--1H-pyrroles-3-carboxamido-group) caproic acid is dissolved among the 8ml DMF, adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 432mg (4mmol) O-Phenylene Diamine then.Mixture at room temperature stirred 24 hours.Dilute with 400ml salt solution then.Vacuum filtration is collected solid.Solid is used water washing.Vacuum-drying gets red solid 6-(5-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl--1H-pyrroles-3-carboxamido-group)-N-(2-aminophenyl) hexanamide (430mg, productive rate 81%). ¹H?NMR(DMSO-d ₆)δ?1.38(m，2H，CH ₂)，1.54(m，2H，CH ₂)，1.62(m，2H，CH ₂)，2.30(s，3H，pyrrole-CH ₃)，2.32(t，J＝8.0Hz，2H，CH ₂CO)，2.41(s，3H，pyrrole-CH ₃)，3.22(m，2H，NCH ₂)，4.82(s，2H，benzene-NH ₂)，6.51(t，J＝8.0Hz，1H，Ar-H)，6.69(d，J＝8.0Hz，1H，Ar-H)，6.87(m，2H，Ar-H)，7.14(d，J＝8.0Hz，1H，Ar-H)，7.20(m，1H，Ar-H)，7.65(t，J＝4.0Hz，1H，CONH)，8.33(dd，J＝4.0and8.0Hz，1H，Ar-H)，8.63(s，1H，vinyl-H)，9.11(s，1H，benzene-NH)，10.73(s，1H，indolinone-NH)，11.82(s，1H，pyrrole-NH)。LC-MS(m/z)532(M+1)。

Embodiment 16

1H-pyrroles-3-carboxamido-group)-preparation of N-(2-amino-4-chloro-phenyl-) hexanamide

With 441mg (1mmol) 6-(5-(((5-fluoro-2-oxygen-1; 2-dihydro-indoles-3-subunit) methyl hydrazono-))-2; 4-dimethyl--1H-pyrroles-3-carboxamido-group) caproic acid is dissolved among the 8ml DMF, 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 (purchasing the company in AlfaAesar) then.Mixture at room temperature stirred 24 hours.Dilute with 400ml salt solution then.Vacuum filtration is collected solid.Solid is used water washing.Vacuum-drying gets red solid 6-(5-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl--1H-pyrroles-3-carboxamido-group)-N-(2-amino-4-chloro-phenyl-) hexanamide (402mg, productive rate 71%).LC-MS(m/z)566(M+1)。

Embodiment 17

1H-pyrroles-3-carboxamido-group)-preparation of N-(2-amino-4-aminomethyl phenyl) hexanamide

With 441mg (1mmol) 6-(5-(((5-fluoro-2-oxygen-1; 2-dihydro-indoles-3-subunit) methyl hydrazono-))-2; 4-dimethyl--1H-pyrroles-3-carboxamido-group) caproic acid is dissolved among the 8ml DMF, 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 (purchasing the company in Alfa Aesar) then.Mixture at room temperature stirred 24 hours.Dilute with 400ml salt solution then.Vacuum filtration is collected solid.Solid is used water washing.Vacuum-drying gets red solid 6-(5-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl--1H-pyrroles-3-carboxamido-group)-N-(2-amino-4-aminomethyl phenyl) hexanamide (376mg, productive rate 69%).LC-MS(m/z)546(M+1)。

Embodiment 18

1H-pyrroles-3-carboxamido-group)-preparation of N-(2-amino-4-p-methoxy-phenyl) hexanamide

With 441mg (1mmol) 6-(5-(((5-fluoro-2-oxygen-1; 2-dihydro-indoles-3-subunit) methyl hydrazono-))-2; 4-dimethyl--1H-pyrroles-3-carboxamido-group) caproic acid is dissolved among the 8ml DMF, 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 (purchasing the company in Alfa Aesar) then.Mixture at room temperature stirred 24 hours.Dilute with 400ml salt solution then.Vacuum filtration is collected solid.Solid is used water washing.Vacuum-drying gets red solid 6-(5-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl--1H-pyrroles-3-carboxamido-group)-N-(2-amino-4-p-methoxy-phenyl) hexanamide (427mg, productive rate 76%).LC-MS(m/z)562(M+1)。

Embodiment 19

1H-pyrroles-3-carboxamido-group)-preparation of N-(2-amino-4-trifluoromethyl) hexanamide

With 441mg (1mmol) 6-(5-(((5-fluoro-2-oxygen-1; 2-dihydro-indoles-3-subunit) methyl hydrazono-))-2; 4-dimethyl--1H-pyrroles-3-carboxamido-group) caproic acid is dissolved among the 8ml DMF, 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 (purchasing the company in Alfa Aesar) then.Mixture at room temperature stirred 24 hours.Dilute with 400ml salt solution then.Vacuum filtration is collected solid.Solid is used water washing.Vacuum-drying gets red solid 6-(5-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl--1H-pyrroles-3-carboxamido-group)-N-(2-amino-4-trifluoromethyl) hexanamide (390mg, productive rate 65%).LC-MS(m/z)600(M+1)。

Embodiment 20

6-(5-(((2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2, the 4-dimethyl--

1H-pyrroles-3-carboxamido-group)-preparation of N-(2-aminophenyl) hexanamide

With 423mg (1mmol) 6-(5-(((2-oxygen-1; 2-dihydro-indoles-3-subunit) methyl hydrazono-))-2; 4-dimethyl--1H-pyrroles-3-carboxamido-group) caproic acid is dissolved among the 8ml DMF, adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 432mg (4mmol) O-Phenylene Diamine then.Mixture at room temperature stirred 24 hours.Dilute with 400ml salt solution then.Vacuum filtration is collected solid.Solid is used water washing.Vacuum-drying gets red solid 6-(5-(((2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl--1H-pyrroles-3-carboxamido-group)-N-(2-aminophenyl) hexanamide (395mg, productive rate 77%).LC-MS(m/z)514(M+1)。

Embodiment 21

6-(5-(((5-chloro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2, the 4-dimethyl--

1H-pyrroles-3-carboxamido-group)-preparation of N-(2-aminophenyl) hexanamide

With 458mg (1mmol) 6-(5-(((5-chloro-2-oxygen-1; 2-dihydro-indoles-3-subunit) methyl hydrazono-))-2; 4-dimethyl--1H-pyrroles-3-carboxamido-group) caproic acid is dissolved among the 8ml DMF, adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 432mg (4mmol) O-Phenylene Diamine then.Mixture at room temperature stirred 24 hours.Dilute with 400ml salt solution then.Vacuum filtration is collected solid.Solid is used water washing.Vacuum-drying gets red solid 6-(5-(((5-chloro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl--1H-pyrroles-3-carboxamido-group)-N-(2-aminophenyl) hexanamide (438mg, productive rate 80%).LC-MS(m/z)548(M+1)。

Embodiment 22

6-(5-(((4-methyl-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2, the 4-dimethyl--

1H-pyrroles-3-carboxamido-group)-preparation of N-(2-aminophenyl) hexanamide

With 437mg (1mmol) 6-(5-(((4-methyl-2-oxygen-1; 2-dihydro-indoles-3-subunit) methyl hydrazono-))-2; 4-dimethyl--1H-pyrroles-3-carboxamido-group) caproic acid is dissolved among the 8ml DMF, adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 432mg (4mmol) O-Phenylene Diamine then.Mixture at room temperature stirred 24 hours.Dilute with 400ml salt solution then.Vacuum filtration is collected solid.Solid is used water washing.Vacuum-drying gets red solid 6-(5-(((4-methyl-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl--1H-pyrroles-3-carboxamido-group)-N-(2-aminophenyl) hexanamide (427mg, productive rate 81%).LC-MS(m/z)528(M+1)。

Embodiment 23

6-(5-(((5-nitro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2, the 4-dimethyl--

1H-pyrroles-3-carboxamido-group)-preparation of N-(2-aminophenyl) hexanamide

With 468mg (1mmol) 6-(5-(((5-nitro-2-oxygen-1; 2-dihydro-indoles-3-subunit) methyl hydrazono-))-2; 4-dimethyl--1H-pyrroles-3-carboxamido-group) caproic acid is dissolved among the 8ml DMF, adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 432mg (4mmol) O-Phenylene Diamine then.Mixture at room temperature stirred 24 hours.Dilute with 400ml salt solution then.Vacuum filtration is collected solid.Solid is used water washing.Vacuum-drying gets red solid 6-(5-(((5-nitro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl--1H-pyrroles-3-carboxamido-group)-N-(2-aminophenyl) hexanamide (402mg, productive rate 72%).LC-MS(m/z)559(M+1)。

Embodiment 24

6-(5-(((6-methoxyl group-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2, the 4-dimethyl--

1H-pyrroles-3-carboxamido-group)-preparation of N-(2-aminophenyl) hexanamide

With 453mg (1mmol) 6-(5-(((6-methoxyl group-2-oxygen-1; 2-dihydro-indoles-3-subunit) methyl hydrazono-))-2; 4-dimethyl--1H-pyrroles-3-carboxamido-group) caproic acid is dissolved among the 8ml DMF, adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 432mg (4mmol) O-Phenylene Diamine then.Mixture at room temperature stirred 24 hours.Dilute with 400ml salt solution then.Vacuum filtration is collected solid.Solid is used water washing.Vacuum-drying gets red solid 6-(5-(((6-methoxyl group-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl--1H-pyrroles-3-carboxamido-group)-N-(2-aminophenyl) hexanamide (408mg, productive rate 75%).LC-MS(m/z)544(M+1)。

Embodiment 25

6-(5-(((6-trifluoromethyl-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-

Dimethyl--1H-pyrroles-3-carboxamido-group)-preparation of N-(2-aminophenyl) hexanamide

With 491mg (1mmol) 6-(5-(((6-trifluoromethyl-2-oxygen-1; 2-dihydro-indoles-3-subunit) methyl hydrazono-))-2; 4-dimethyl--1H-pyrroles-3-carboxamido-group) caproic acid is dissolved among the 8ml DMF, adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 432mg (4mmol) O-Phenylene Diamine then.Mixture at room temperature stirred 24 hours.Dilute with 400ml salt solution then.Vacuum filtration is collected solid.Solid is used water washing.Vacuum-drying gets red solid 6-(5-(((6-trifluoromethyl-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl--1H-pyrroles-3-carboxamido-group)-N-(2-aminophenyl) hexanamide (384mg, productive rate 66%).LC-MS(m/z)582(M+1)。

Embodiment 26

8-(5-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2, the 4-dimethyl--

1H-pyrroles-3-carboxamido-group) preparation of methyl caprylate

With 328mg (1mmol) 5-(((5-fluoro-2-oxygen-1; 2-dihydro-indoles-3-subunit) methyl hydrazono-))-2; 4-dimethyl--1H-pyrroles-3-carboxylic acid is dissolved among 8 milliliters of DMF, adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 251mg (1.2mmol) 8-aminocaprylic acid methyl ester hydrochloride then.Mixture at room temperature stirred 24 hours.Dilute with 400ml salt solution then.Vacuum filtration is collected solid.Solid is used water washing.Vacuum-drying gets red solid 8-(5-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl--1H-pyrroles-3-carboxamido-group) methyl caprylate (287mg, productive rate 59%).LC-MS(m/z)484(M+1)。

Embodiment 27

1H-pyrroles-3-carboxamido-group) sad preparation

483mg (1mmol) 8-(5-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl--1H-pyrroles-3-carboxamido-group) methyl caprylate is dissolved in the 300ml methyl alcohol, adds the lithium hydroxide aqueous solution of 25ml4M then.Mixture at room temperature stirred 24 hours.Using hydrochloric acid to be neutralized to the pH value in mixture is 7, and methyl alcohol is removed in decompression.Residue is with salt acid for adjusting pH value to 3.Vacuum filtration is collected solid, washing, and vacuum-drying gets red solid 8-(5-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl--1H-pyrroles-3-carboxamido-group) sad (159mg, productive rate 34%).LC-MS(m/z)470(M+1)。

Embodiment 28

1H-pyrroles-3-carboxamido-group)-preparation of N-hydroxy capryloyl amine

With 469mg (1mmol) 8-(5-(((5-fluoro-2-oxygen-1; 2-dihydro-indoles-3-subunit) methyl hydrazono-))-2; 4-dimethyl--1H-pyrroles-3-carboxamido-group) sad, 151mg (1.5mmol) triethylamine is dissolved among the 20ml DMF, adds 163mg (1.5mmol) Vinyl chloroformate in 0 ℃.Mixture adds the aqueous hydroxylamine of 1.32g (20mmol) 50% after stirring 2 hours under 0 ℃.Mixture at room temperature continued to stir 3 hours, diluted with 1000ml salt solution then.Vacuum filtration is collected solid, washing, and vacuum-drying must get red solid 8-(5-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl--1H-pyrroles-3-carboxamido-group)-N-hydroxy capryloyl amine (237mg, productive rate 49%).LC-MS(m/z)485(M+1)。

Embodiment 29

1H-pyrroles-3-carboxamido-group)-preparation of N-(2-amino-4-fluorophenyl) decoylamide

With 469mg (1mmol) 8-(5-(((5-fluoro-2-oxygen-1; 2-dihydro-indoles-3-subunit) methyl hydrazono-))-2; 4-dimethyl--1H-pyrroles-3-carboxamido-group) sad being dissolved among the 8ml DMF 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 then.Mixture at room temperature stirred 24 hours.Dilute with 400ml salt solution then.Vacuum filtration is collected solid.Solid is used water washing.Vacuum-drying gets red solid 8-(5-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl--1H-pyrroles-3-carboxamido-group)-N-(2-amino-4-fluorophenyl) decoylamide (386mg, productive rate 67%).LC-MS(m/z)578(M+1)。

Embodiment 30

1H-pyrroles-3-carboxamido-group)-preparation of N-(2-aminophenyl) decoylamide

With 469mg (1mmol) 8-(5-(((5-fluoro-2-oxygen-1; 2-dihydro-indoles-3-subunit) methyl hydrazono-))-2; 4-dimethyl--1H-pyrroles-3-carboxamido-group) sad being dissolved among the 8ml DMF adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 432mg (4mmol) O-Phenylene Diamine then.Mixture at room temperature stirred 24 hours.Dilute with 400ml salt solution then.Vacuum filtration is collected solid.Solid is used water washing.Vacuum-drying gets red solid 8-(5-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl)-2,4-dimethyl--1H-pyrroles-3-carboxamido-group)-N-(2-aminophenyl) decoylamide (402mg, productive rate 72%).LC-MS(m/z)560(M+1)。

Embodiment 31

6-(4-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl) benzoylamino)-

The preparation of methyl caproate

With 311mg (1mmol) 4-(((5-fluoro-2-oxygen-1; 2-dihydro-indoles-3-subunit) methyl hydrazono-)) phenylformic acid is dissolved among 8 milliliters of DMF, adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 219mg (1.2mmol) 6-aminocaprolc acid methyl ester hydrochloride then.Mixture at room temperature stirred 24 hours.Dilute with 400ml salt solution then.Vacuum filtration is collected solid.Solid is used water washing.Vacuum-drying gets brown solid 6-(4-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl) benzoylamino) methyl caproate (114mg, productive rate 28%).LC-MS(m/z)439(M+1)。

Embodiment 32

6-(4-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl) benzoylamino)

The preparation of caproic acid

438mg (1mmol) 6-(4-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl) benzoylamino) methyl caproate is dissolved in the 300ml methyl alcohol, adds the lithium hydroxide aqueous solution of 25ml4M then.Mixture at room temperature stirred 24 hours.Using hydrochloric acid to be neutralized to the pH value in mixture is 7, and methyl alcohol is removed in decompression.Residue is with salt acid for adjusting pH value to 3.Vacuum filtration is collected solid, washing, and vacuum-drying gets brown solid 6-(4-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl) benzoylamino) caproic acid (119mg, productive rate 28%).LC-MS(m/z)425(M+1)。

Embodiment 33

The preparation of N-hydroxyl hexanamide

424mg (1mmol) 6-(4-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl) benzoylamino) caproic acid, 151mg (1.5mmol) triethylamine are dissolved among the 20ml DMF, add 163mg (1.5mmol) Vinyl chloroformate in 0 ℃.Mixture adds the aqueous hydroxylamine of 1.32g (20mmol) 50% after stirring 2 hours under 0 ℃.Mixture at room temperature continued to stir 3 hours, diluted with 1000ml salt solution then.Vacuum filtration is collected solid, washing, and vacuum-drying must get brown solid 6-(4-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl) benzoylamino)-N-hydroxyl hexanamide (224mg, productive rate 51%).LC-MS(m/z)440(M+1)。

Embodiment 34

The preparation of N-(2-aminophenyl) hexanamide

With 424mg (1mmol) 6-(4-(((5-fluoro-2-oxygen-1; 2-dihydro-indoles-3-subunit) benzoylamino methyl hydrazono-))) caproic acid is dissolved among the 8ml DMF, adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 432mg (4mmol) O-Phenylene Diamine then.Mixture at room temperature stirred 24 hours.Dilute with 400ml salt solution then.Vacuum filtration is collected solid.Solid is used water washing.Vacuum-drying gets brown solid 6-(4-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl) benzoylamino)-N-(2-aminophenyl) hexanamide (375mg, productive rate 76%).LC-MS(m/z)515(M+1)。

Embodiment 35

8-(4-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl) benzoylamino)

The preparation of methyl caprylate

With 311mg (1mmol) 4-(((5-fluoro-2-oxygen-1; 2-dihydro-indoles-3-subunit) methyl hydrazono-)) phenylformic acid is dissolved among 8 milliliters of DMF, adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 251mg (1.2mmol) 8-aminocaprylic acid methyl ester hydrochloride then.Mixture at room temperature stirred 24 hours.Dilute with 400ml salt solution then.Vacuum filtration is collected solid.Solid is used water washing.Vacuum-drying gets brown solid 8-(4-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl) benzoylamino) methyl caprylate (290mg, productive rate 62%).LC-MS(m/z)467(M+1)。

Embodiment 36

Sad preparation

466mg (1mmol) 8-(4-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl) benzoylamino) methyl caprylate is dissolved in the 300ml methyl alcohol, adds the lithium hydroxide aqueous solution of 25ml 4M then.Mixture at room temperature stirred 24 hours.Using hydrochloric acid to be neutralized to the pH value in mixture is 7, and methyl alcohol is removed in decompression.Residue is with salt acid for adjusting pH value to 3.Vacuum filtration is collected solid, washing, and vacuum-drying gets brown solid 8-(4-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl) benzoylamino) sad (149mg, productive rate 33%).LC-MS(m/z)453(M+1)。

Embodiment 37

8-(4-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl) benzoylamino)-

The preparation of N-(2-aminophenyl) decoylamide

With 452mg (1mmol) 8-(4-(((5-fluoro-2-oxygen-1; 2-dihydro-indoles-3-subunit) benzoylamino methyl hydrazono-))) sad being dissolved among the 8ml DMF adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 432mg (4mmol) O-Phenylene Diamine then.Mixture at room temperature stirred 24 hours.Dilute with 400ml salt solution then.Vacuum filtration is collected solid.Solid is used water washing.Vacuum-drying gets brown solid 8-(4-(((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) hydrazono-) methyl) benzoylamino)-N-(2-aminophenyl) decoylamide (423mg, productive rate 72%).LC-MS(m/z)543(M+1)。

Embodiment 38

The preparation of N-(2-amino-4-fluorophenyl) decoylamide

With 452mg (1mmol) 8-(4-(((5-fluoro-2-oxygen-1; 2-dihydro-indoles-3-subunit) sad being dissolved among 8ml DMF benzoylamino methyl hydrazono-))) 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 then.Mixture at room temperature stirred 24 hours.Dilute with 400ml salt solution then.Vacuum filtration is collected solid.Solid is used water washing.Vacuum-drying gets brown solid 8-(4-(((5-fluoro-2-oxygen-1,2-dihydro-hydrogen indoles-3-subunit) hydrazono-) methyl) benzoylamino)-N-(2-amino-4-fluorophenyl) decoylamide (358mg, productive rate 64%).LC-MS(m/z)561(M+1)。

Embodiment 39

The preparation of 6-(4-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl) benzoylamino) methyl caproate

With 283mg (1mmol) 4-((5-fluoro-2-oxygen-1; 2-dihydro-indoles-3-subunit) methyl) phenylformic acid is dissolved among 8 milliliters of DMF, adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 219mg (1.2mmol) 6-aminocaprolc acid methyl ester hydrochloride then.Mixture at room temperature stirred 24 hours.Dilute with 400ml salt solution then.Vacuum filtration is collected solid.Solid is used water washing.Vacuum-drying gets brown solid 6-(4-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl) benzoylamino) methyl caproate (362mg, productive rate 88%).LC-MS(m/z)411(M+1)。

Embodiment 40

The preparation of 6-(4-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl) benzoylamino) caproic acid

410mg (1mmol) 6-(4-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl) benzoylamino) methyl caproate is dissolved in the 300ml methyl alcohol, adds the lithium hydroxide aqueous solution of 25ml 4M then.Mixture at room temperature stirred 24 hours.Using hydrochloric acid to be neutralized to the pH value in mixture is 7, and methyl alcohol is removed in decompression.Residue is with salt acid for adjusting pH value to 3.Vacuum filtration is collected solid, washing, and vacuum-drying gets brown solid 6-(4-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl) benzoylamino) caproic acid (305mg, productive rate 77%).LC-MS(m/z)397(M+1)。

Embodiment 41

6-(4-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl) benzoylamino)-N-

The preparation of (2-aminophenyl) hexanamide

With 396mg (1mmol) 6-(4-((5-fluoro-2-oxygen-1; 2-dihydro-indoles-3-subunit) benzoylamino methyl)) caproic acid is dissolved among the 8mlDMF, adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 432mg (4mmol) O-Phenylene Diamine then.Mixture at room temperature stirred 24 hours.Dilute with 400ml salt solution then.Vacuum filtration is collected solid.Solid is used water washing.Vacuum-drying gets brown solid 6-(4-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl) benzoylamino)-N-(2-aminophenyl) hexanamide (394mg, productive rate 81%). ¹H?NMR(DMSO-d ₆)δ?1.38(m，2H，CH ₂)，1.54(m，2H，CH ₂)，1.60(m，2H，CH ₂)，2.32(t，J＝8.0Hz，2H，CH ₂CO)，3.28(m，2H，NCH ₂)，4.80(s，2H，benzene-NH ₂)，6.52(t，J＝8.0Hz，1H，Ar-H)，6.69(d，J＝8.0Hz，1H，Ar-H)，6.86(m，2H，Ar-H)，7.14(m，2H，Ar-H)，7.71(s，1H，vinyl-H)，7.76(m，1H，Ar-H)，7.92(m，1H，Ar-H)，7.96(m，1H，Ar-H)，8.39(d，J＝8.0Hz，1H，Ar-H)，8.50(m，1H，Ar-H)，9.09(s，1H，benzene-NH)，10.68(s，1H，CONH)。LC-MS(m/z)487(M+1)。

Embodiment 42

The preparation of (2-amino-4-fluorophenyl) hexanamide

With 396mg (1mmol) 6-(4-((5-fluoro-2-oxygen-1; 2-dihydro-indoles-3-subunit) benzoylamino methyl)) caproic acid is dissolved among the 8mlDMF, 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 then.Mixture at room temperature stirred 24 hours.Dilute with 400ml salt solution then.Vacuum filtration is collected solid.Solid is used water washing.Vacuum-drying gets brown solid 6-(4-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl) benzoylamino)-N-(2-amino-4-fluorophenyl) hexanamide (348mg, productive rate 69%). ¹H?NMR(DMSO-d ₆)δ?1.36(m，2H，CH ₂)，1.60(m，2H，CH ₂)，1.60(m，2H，CH ₂)，2.30(t，J＝8.0Hz，2H，CH ₂CO)，3.29(m，2H，NCH ₂)，5.12(s，2H，benzene-NH ₂)，6.27(t，J＝8.0Hz，1H，Ar-H)，6.46(dd，J＝4.0Hz?and?8.0Hz，1H，Ar-H)，6.88(m，1H，Ar-H)，7.10(m，2H，Ar-H)，7.71(s，1H，vinyl-H)，7.77(m，1H，Ar-H)，7.92(m，1H，Ar-H)，7.96(m，1H，Ar-H)，8.39(d，J＝8.0Hz，1H，Ar-H)，8.50(m，1H，Ar-H)，9.01(s，1H，benzene-NH)，10.68(s，1H，CONH)。LC-MS(m/z)505(M+1)。

Embodiment 43

The preparation of hydroxyl hexanamide

396mg (1mmol) 6-(4-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl) benzoylamino) caproic acid, 151mg (1.5mmol) triethylamine are dissolved among the 20ml DMF, add 163mg (1.5mmol) Vinyl chloroformate in 0 ℃.Mixture adds the aqueous hydroxylamine of 1.32g (20mmol) 50% after stirring 2 hours under 0 ℃.Mixture at room temperature continued to stir 3 hours, diluted with 1000ml salt solution then.Vacuum filtration is collected solid, washing, and vacuum-drying must get brown solid 6-(4-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl) benzoylamino)-N-hydroxyl hexanamide (189mg, productive rate 46%).LC-MS(m/z)412(M+1)。

Embodiment 44

The preparation of 6-(3-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl) benzene methanamine base) methyl caproate

With 283mg (1mmol) 3-((5-fluoro-2-oxygen-1; 2-dihydro-indoles-3-subunit) methyl) phenylformic acid is dissolved among 8 milliliters of DMF, adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 219mg (1.2mmol) 6-aminocaprolc acid methyl ester hydrochloride then.Mixture at room temperature stirred 24 hours.Dilute with 400ml salt solution then.Vacuum filtration is collected solid.Solid is used water washing.Vacuum-drying gets brown solid 6-(3-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl) benzoylamino) methyl caproate (332mg, productive rate 81%).LC-MS(m/z)411(M+1)。

Embodiment 45

The preparation of 6-(3-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl) benzoylamino) caproic acid

410mg (1mmol) 6-(3-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl) benzoylamino) methyl caproate is dissolved in the 300ml methyl alcohol, adds the lithium hydroxide aqueous solution of 25ml4M then.Mixture at room temperature stirred 24 hours.Using hydrochloric acid to be neutralized to the pH value in mixture is 7, and methyl alcohol is removed in decompression.Residue is with salt acid for adjusting pH value to 3.Vacuum filtration is collected solid, washing, and vacuum-drying gets brown solid 6-(3-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl) benzoylamino) caproic acid (289mg, productive rate 73%).LC-MS(m/z)397(M+1)。

Embodiment 46

6-(3-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl) benzoylamino)-N-

The preparation of (2-aminophenyl) hexanamide

With 396mg (1mmol) 6-(3-((5-fluoro-2-oxygen-1; 2-dihydro-indoles-3-subunit) benzoylamino methyl)) caproic acid is dissolved among the 8mlDMF, adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 432mg (4mmol) O-Phenylene Diamine then.Mixture at room temperature stirred 24 hours.Dilute with 400m1 salt solution then.Vacuum filtration is collected solid.Solid is used water washing.Vacuum-drying gets brown solid 6-(3-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl) benzoylamino)-N-(2-aminophenyl) hexanamide (379mg, productive rate 78%). ¹H?NMR(DMSO-d ₆)δ?1.36(m，2H，CH ₂)，1.59(m，4H，CH ₂)，2.30(t，J＝8.0Hz，2H，CH ₂CO)，3.29(t，J＝8.0Hz，2H，NCH ₂)，4.80(s，2H，benzene-NH ₂)，6.50(t，J＝8.0Hz，1H，Ar-H)，6.69(d，J＝8.0Hz，1H，Ar-H)，6.87(m，2H，Ar-H)，7.12(m，2H，Ar-H)，7.54(d，J＝8.0Hz，1H，Ar-H)，7.64(m，1H，Ar-H)，7.73(s，0.6H，vinyl-H)，7.83(m，1H，Ar-H)，7.94(m，1H，Ar-H)，8.15(s，0.4H，vinyl-H)，8.57(m，1H，Ar-H)，9.08(s，1H，benzene-NH)，10.68(s，1H，CONH)。LC-MS(m/z)487(M+1)。

Embodiment 47

The preparation of (2-amino-4-fluorophenyl) hexanamide

With 396mg (1mmol) 6-(3-((5-fluoro-2-oxygen-1; 2-dihydro-indoles-3-subunit) benzoylamino methyl)) caproic acid is dissolved among the 8mlDMF, 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 then.Mixture at room temperature stirred 24 hours.Dilute with 400ml salt solution then.Vacuum filtration is collected solid.Solid is used water washing.Vacuum-drying gets brown solid 6-(3-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl) benzoylamino)-N-(2-amino-4-fluorophenyl) hexanamide (373mg, productive rate 74%). ¹H?NMR(DMSO-d ₆)δ?1.36(m，2H，CH ₂)，1.59(m，4H，CH ₂)，2.30(t，J＝8.0Hz，2H，CH ₂CO)，3.28(t，J＝8.0Hz，2H，NCH ₂)，5.12(s，2H，benzene-NH ₂)，6.26(t，J＝8.0Hz，1H，Ar-H)，6.46(d，J＝8.0Hz，1H，Ar-H)，6.87(m，1H，Ar-H)，7.12(m，3H，Ar-H)，7.64(m，1H，Ar-H)，7.73(s，0.6×1H，vinyl-H)，7.83(m，1H，Ar-H)，7.94(m，1H，Ar-H)，8.16(s，0.4×1H，vinyl-H)，8.57(m，1H，Ar-H)，9.02(s，1H，benzene-NH)，10.68(s，1H，CONH)。LC-MS(m/z)505(M+1)。

Embodiment 48

The preparation of hydroxyl hexanamide

396mg (1mmol) 6-(3-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl) benzoylamino) caproic acid, 151mg (1.5mmol) triethylamine are dissolved among the 20ml DMF, add 163mg (1.5mmol) Vinyl chloroformate in 0 ℃.Mixture adds the aqueous hydroxylamine of 1.32g (20mmol) 50% after stirring 2 hours under 0 ℃.Mixture at room temperature continued to stir 3 hours, diluted with 1000ml salt solution then.Vacuum filtration is collected solid, washing, and vacuum-drying must get brown solid 6-(4-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl) benzoylamino)-N-hydroxyl hexanamide (168mg, productive rate 41%).LC-MS(m/z)412(M+1)。

Embodiment 49

5-(3-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl) benzoylamino)

The preparation of methyl valerate

With 283mg (1mmol) 3-((5-fluoro-2-oxygen-1; 2-dihydro-indoles-3-subunit) methyl) phenylformic acid is dissolved among 8 milliliters of DMF, adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 202mg (1.2mmol) 5-aminovaleric acid methyl ester hydrochloride then.Mixture at room temperature stirred 24 hours.Dilute with 400ml salt solution then.Vacuum filtration is collected solid.Solid is used water washing.Vacuum-drying gets brown solid 5-(3-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl) benzoylamino) methyl valerate (337mg, productive rate 89%).LC-MS(m/z)397(M+1)。

Embodiment 50

The preparation of 5-(3-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl) benzoylamino) valeric acid

396mg (1mmol) 5-(3-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl) benzoylamino) methyl valerate is dissolved in the 300ml methyl alcohol, adds the lithium hydroxide aqueous solution of 25ml 4M then.Mixture at room temperature stirred 24 hours.Using hydrochloric acid to be neutralized to the pH value in mixture is 7, and methyl alcohol is removed in decompression.Residue is with salt acid for adjusting pH value to 3.Vacuum filtration is collected solid, washing, and vacuum-drying gets brown solid 5-(3-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl) benzoylamino) valeric acid (294mg, productive rate 77%).LC-MS(m/z)383(M+1)。

Embodiment 51

5-(3-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl) benzoylamino)-N-

The preparation of (2-aminophenyl) valeramide

With 382mg (1mmol) 5-(3-((5-fluoro-2-oxygen-1; 2-dihydro-indoles-3-subunit) benzoylamino methyl)) valeric acid is dissolved among the 8mlDMF, adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 432mg (4mmol) O-Phenylene Diamine then.Mixture at room temperature stirred 24 hours.Dilute with 400ml salt solution then.Vacuum filtration is collected solid.Solid is used water washing.Vacuum-drying gets brown solid 5-(3-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl) benzoylamino)-N-(2-aminophenyl) valeramide (382mg, productive rate 81%).LC-MS(m/z)473(M+1)。

Embodiment 52

The preparation of (2-amino-4-fluorophenyl) valeramide

With 382mg (1mmol) 5-(3-((5-fluoro-2-oxygen-1; 2-dihydro-indoles-3-subunit) benzoylamino methyl)) valeric acid is dissolved among the 8mlDMF, 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 then.Mixture at room temperature stirred 24 hours.Dilute with 400ml salt solution then.Vacuum filtration is collected solid.Solid is used water washing.Vacuum-drying gets brown solid 5-(3-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl) benzoylamino)-N-(the amino 4-fluorophenyl of 2-) valeramide (372mg, productive rate 76%).LC-MS(m/z)491(M+1)。

Embodiment 53

The preparation of hydroxyl valeramide

382mg (1mmol) 5-(3-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl) benzoylamino) valeric acid, 151mg (1.5mmol) triethylamine are dissolved among the 20ml DMF, add 163mg (1.5mmol) Vinyl chloroformate in 0 ℃.Mixture adds the aqueous hydroxylamine of 1.32g (20mmol) 50% after stirring 2 hours under 0 ℃.Mixture at room temperature continued to stir 3 hours, diluted with 1000ml salt solution then.Vacuum filtration is collected solid, washing, and vacuum-drying must get brown solid 5-(3-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl) benzoylamino)-N-hydroxyl valeramide (186mg, productive rate 47%).LC-MS(m/z)398(M+1)。

Embodiment 54

8-(3-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl) benzoylamino)-

The preparation of methyl caprylate

With 283mg (1mmol) 3-((5-fluoro-2-oxygen-1; 2-dihydro-indoles-3-subunit) methyl) phenylformic acid is dissolved among 8 milliliters of DMF, adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 251mg (1.2mmol) 8-aminocaprylic acid methyl ester hydrochloride then.Mixture at room temperature stirred 24 hours.Dilute with 400ml salt solution then.Vacuum filtration is collected solid.Solid is used water washing.Vacuum-drying gets brown solid 8-(3-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl) benzoylamino) methyl caprylate (333mg, productive rate 76%).LC-MS(m/z)439(M+1)。

Embodiment 55

The sad preparation of 8-(3-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl) benzoylamino)

438mg (1mmol) 8-(3-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl) benzoylamino) methyl caprylate is dissolved in the 300ml methyl alcohol, adds the lithium hydroxide aqueous solution of 25ml 4M then.Mixture at room temperature stirred 24 hours.Using hydrochloric acid to be neutralized to the pH value in mixture is 7, and methyl alcohol is removed in decompression.Residue is with salt acid for adjusting pH value to 3.Vacuum filtration is collected solid, washing, and vacuum-drying gets brown solid 8-(3-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl) benzoylamino) sad (305mg, productive rate 72%).LC-MS(m/z)425(M+1)。

Embodiment 56

8-(3-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl) benzoylamino)-N-

The preparation of (2-aminophenyl) decoylamide

With 424mg (1mmol) 8-(3-((5-fluoro-2-oxygen-1; 2-dihydro-indoles-3-subunit) sad being dissolved among 8mlDMF benzoylamino methyl)) adds 384mg (2mmol) 1-ethyl-3-(3-dimethylamine propyl) carbodiimide hydrochloride, 162mg (1.2mmol) I-hydroxybenzotriazole, 404mg (4mmol) triethylamine and 432mg (4mmol) O-Phenylene Diamine then.Mixture at room temperature stirred 24 hours.Dilute with 400ml salt solution then.Vacuum filtration is collected solid.Solid is used water washing.Vacuum-drying gets brown solid 8-(3-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl) benzoylamino)-N-(2-aminophenyl) decoylamide (426mg, productive rate 83%).LC-MS(m/z)515(M+1)。

Embodiment 57

The preparation of (2-amino-4-fluorophenyl) decoylamide

With 424mg (1mmol) 8-(3-((5-fluoro-2-oxygen-1; 2-dihydro-indoles-3-subunit) sad being dissolved among 8mlDMF benzoylamino methyl)) 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 then.Mixture at room temperature stirred 24 hours.Dilute with 400ml salt solution then.Vacuum filtration is collected solid.Solid is used water washing.Vacuum-drying gets brown solid 8-(3-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl) benzoylamino)-N-(2-amino-4-fluorophenyl) decoylamide (436mg, productive rate 82%).LC-MS(m/z)533(M+1)。

Embodiment 58

The preparation of hydroxy capryloyl amine

424mg (1mmol) 8-(3-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl) benzoylamino) is sad, 151mg (1.5mmol) triethylamine is dissolved among the 20ml DMF, adds 163mg (1.5mmol) Vinyl chloroformate in 0 ℃.Mixture adds the aqueous hydroxylamine of 1.32g (20mmol) 50% after stirring 2 hours under 0 ℃.Mixture at room temperature continued to stir 3 hours, diluted with 1000ml salt solution then.Vacuum filtration is collected solid, washing, and vacuum-drying must get brown solid 8-(3-((5-fluoro-2-oxygen-1,2-dihydro-indoles-3-subunit) methyl) benzoylamino)-N-hydroxy capryloyl amine (224mg, productive rate 51%).LC-MS(m/z)440(M+1)。

The preparation of embodiment 59 tablets

Prescription (1000):

Compound 13:5g

Microcrystalline Cellulose: 90g

Sodium starch glycolate: 5g

4% 30 POVIDONE K 30 BP/USP, 30 ethanol solutions: 50g

Talcum powder: 0.5g

Preparation technology: compound 13 is crossed 100 mesh sieves; Microcrystalline Cellulose, sodium starch glycolate and talcum powder are crossed 80 mesh sieves, take by weighing the Microcrystalline Cellulose and the sodium starch glycolate of recipe quantity, mix; Then with compound 13 with its by the equivalent method mixing that progressively increases; Add 4% 30 POVIDONE K 30 BP/USP, 30 ethanol solutions and granulate in right amount, dry back adds recipe quantity talcum powder mixing, and compressing tablet promptly gets.

Embodiment 60 capsular preparations

Prescription (1000):

Compound 13:5g

Microcrystalline Cellulose: 55g

Lactose: 35g

Sodium starch glycolate: 5g

Magnesium Stearate: 0.5g

Preparation technology: compound 13 is crossed 100 mesh sieves; Microcrystalline Cellulose, lactose, sodium starch glycolate and Magnesium Stearate are crossed 80 mesh sieves; Take by weighing Microcrystalline Cellulose, lactose and the sodium starch glycolate of recipe quantity, mix, then with compound 13 with its by the equivalent method mixing that progressively increases; Add recipe quantity Magnesium Stearate mixing, the can capsule promptly gets.

The preparation of embodiment 61 injection liquids

Prescription:

Compound 11:1.00mg

Medicinal DMSO:0.10ml

Medicinal alcohol: 1.00ml

Preparation technology: compound 11 is dissolved in medicinal DMSO, adds medicinal alcohol and promptly get.

Embodiment 62

Compound 7-56 vitro inhibition HDAC total enzyme activity, vitro inhibition HDAC

Hypotype activity and the acetylize of intracellular canaliculus albumen and the acetylizad test of total protein

One, the 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 suppressor factor is tested.Experiment principle is following: a kind of special substrate Fluor deLys (adopts the nuclear extract of HeLa cell at histon deacetylase (HDAC) in the experiment; Be rich in the HDAC of multiple hypotype) effect under can remove an ethanoyl; Expose free amino, after adding Developer, can produce a kind of derivable fluorescence.The excitation wavelength of this fluorescence is 360nm, and emission wavelength is 460nm.The substrate deacetylation is abundant more, and the inductive fluorescent value is just high more, and the fluorescent value under unrestraint agent situation is contrast; And when suppressor factor existed, derivative fluorescent value can reduce, and when not having enzyme the fluorescent value of (being equivalent to enzymic activity is suppressed fully) 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, it is high more be worth more little then explanation inhibition activity.

Two, reporter gene is to the mensuration of suppressor factor HDAC subtype-selective

The HDAC different subtype can combine with different transcription factors, participates in the heterogeneic expression regulation.Select the controlling element of suitable transcription factor to be built into reporter gene, can be used for assessing the selective inhibitory of suppressor factor the HDAC hypotype.Transfection previous day with HeLa cell kind go into 96 orifice plates when making transfection density reach the fusion of 50-80%.Inserted the p21-promotor at the luciferase gene upper reaches, the gdf11-promotor, the MEF-binding member, the reporter gene plasmid of regulating and controlling sequences such as Nur77-promotor uses FuGene6 (Roche) transfection reagent respectively, 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.After 24 hours, collect and lysing cell, use luciferase (Promega) detection kit that the amount of luciferase is assessed according to operation instructions.Further measure the content of GFP simultaneously, transfection efficiency is proofreaied and correct.

Three, the mensuration of acetylize level in the cell

(1) ELISA of tubulin acetylize level detects

Material and reagent:

1.96 hole tissue culture blank

2.A549 clone

3. anti-acetylize-tubulin antibody (Upstate)

4.HRP-the sheep anti mouse 1gG (Uptate) of mark

5. stationary liquid: 95% ethanol, 5% acetate

6.TBS：0.15M?NaCl，0.02M?Tris-Cl?pH7.4

7.ADS：TBS+2％BSA+0.1％Triton?X-100

8. strengthen chemical illuminating reagent (ECL) (Amersham)

(2) ELISA of Methionin acetylize level detects

Material and reagent:

1.96 hole tissue culture blank

2.A549 clone

3. anti-Methionin acetylize-antibody (Upstate)

4.HRP-the sheep anti mouse 1gG (Uptate) of mark

5. stationary liquid: 95% ethanol, 5% acetate

6.TBS：0.15M?NaCl，0.02M?Tris-Cl?pH7.4

7.ADS：TBS+2％BSA+0.1％Triton?X-100

8. strengthen chemical illuminating reagent (ECL) (Amersham).

Experimental result is seen table 2.

Table 2

Embodiment 63

The compound 26-56 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.In perfect medium, cultivated 72 hours then.Method according to specification sheets adds MTS reagent (Promega), at 37 ℃ of CO ₂Cultivated 2 hours in the incubator, on the ELISA ELIASA, read the absorption value of 490nm then.

Experimental result is seen table 3.

Table 3

Nd ^-: undetermined

Claims

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

Wherein,

R ²Be fluorine;

R ¹, R ³And R ⁴Be respectively hydrogen;

R ⁵For-NHOH or

R ⁷Be hydrogen or fluorine;

R ⁶, R ⁸And R ⁹Be respectively hydrogen;

Ar is phenyl ring or 2,4-dimethyl--1H-pyrroles;

N is 1 to 7 integer;

The form that comprises its free form and salt.

2. preparation method who has the compound of general formula (I) according to claim 1, this method comprises the steps:

(b) compound 3 hydrolysis are obtained compound 4;

(d) compound 4 and compound 6 are carried out condensation reaction and obtain compound 5b;

Wherein,

R ²Be fluorine;

R ¹, R ³And R ⁴Be respectively hydrogen;

R ⁵For-NHOH or

R ⁷Be hydrogen or fluorine;

R ⁶, R ⁸And R ⁹Be respectively hydrogen;

Ar is phenyl ring or 2,4-dimethyl--1H-pyrroles;

N is 1 to 7 integer.

3. medicinal prepns that is used to treat rubinstein-Taybi syndrome, fragile X chromosome syndromes and cancer, it comprises the compound as the said general formula of the claim 1 of active ingredient (I), and pharmaceutical carrier or auxiliary material.

4. compound is used for treating the application of the medicine of rubinstein-Taybi syndrome, fragile X chromosome syndromes and cancer in preparation according to claim 1.