CN101184751A - Quinazolines and their use as AURORA kinase inhibitors - Google Patents

Abstract

The invention provided a compound of formula (I) for use in the treatment of disease, in particular proliferative diseases such as cancer and for use in the preparation of medicaments for use in the treatment of proliferative diseases; the invention also processes for the preparation of such compounds, as well as pharmaceutical compositions containing them as active ingredient.

Description

Quinazoline ditosylate salt and as the purposes of AURORA kinase inhibitor

The present invention relates to be used for for example quinazoline derivant of cancer therapy of disease, particularly proliferative disease, be used for the treatment of purposes in the medicine of proliferative disease in preparation, and relate to their method of preparation, and contain their pharmaceutical compositions as activeconstituents.

Cancer (with other excessively proliferative disease) is a feature with uncontrolled hyperplasia.It is the result who controls the cell pathway gene damage of process by the cell cycle that the forfeiture of this hyperplasia normal regulating shows as.

In eukaryotic cell, the order cascade of protein phosphorylation is considered to controlling the cell cycle.Determined that several protein kinase family is bringing into play requisite effect in this cascade.Compare with normal tissue, activity many in these kinases increase in people's tumour.The result of gene amplification (for example as) can take place in this result when this protein expression level increases, or produces by the expression that changes common activation factor or repressible protein.

These Cycle Regulation agent of broad research first sign and are cell cycle protein dependent kinase (or CDKs).More recent, identified the protein kinase that structure is different with CDK family, it is bringing into play requisite effect in the adjusting cell cycle.These kinases seem it also is important on tumour forms, comprise the Drosophila aurora and the S.cerevisiae Ipll albumen of people's homology type.Three people's homology type aurora A of these genes, the serine-threonine protein kinase enzyme of aurora B and aurora C (yet being called aurora 2, aurora 1 and aurora 3 respectively) Codocyte periodic adjustment (at Adams etc., 2001, Trends in Cell Biology.11 (2): summarize among the 49-54), it shows the peak and the kinase activity of expressing by G2 and mitotic division.Several observation prompters' aurora albumen relates to cancer.

Aurora A gene is positioned on the karyomit(e) 20q13, and this zone comprises often amplification in mammary gland and the colon tumor in people's tumour.Aurora A may be the main target gene of this amplicon because in surpassing primary people's colorectal cancer disease of 50% aurora A DNA be amplified with mRNA by overexpression.Compare with contiguous healthy tissues, obviously raising appears in these tumours aurora A protein level.In addition, personnel selection aurora A transfection rodent inoblast causes changing, and gives the ability of growing on soft agar, and form tumour (Bischoff etc., 1998, The EMBO Journal.17 (11): 3052-3065) on nude mouse.Other research work (Zhou etc., 1998, Nature Genetics.20 (2): 189-93) show that the people causes the increase of centrosome quantity and the increase of dysploidy for overexpression aurora A, this is a known incident in the cancer development.

Research also shows when comparing with normal cell, aurora B (Adams etc., 2001 in tumour cell, Chromsoma.110 (2): 65-74) with aurora C (Kimura etc., 1999, Journal ofBiological Chemistry, 274 (11): expression 7334-40) increases.Also overexpression in cancer cell of Aurora B has shown the increase and colorectal cancer disease in late period relevant (Katayama etc., (1999) J Natl Cancer Inst.91:1160) of Aurora B level.In addition, report prompting Aurora B overexpression is induced dysploidy by the phosphorylation that is increased in Serine 10 histone H 3s, and the cell of overexpression Aurora B forms and has more aggressive tumour (Ota, the T. etc. that develop into transfer, 2002, Cancer res 62:5168-5177).Aurora B is a karyomit(e) passenger albumen, itself and at least three other passenger's Protein S urvivin, and INCENP and Borealin are present in (Carmena M etc., 2003, Nat.Rev.Mol.Cell Biol.4:842-854) in the stabilized complex.Survivin also raises in cancer, and contains BIR (apoptotic proteins (IAP) multiple baculovirus inhibitor) structure function territory, and therefore may avoid playing a role in apoptosis and/or the mitotic division destruction at the protection tumour cell.

About Aurora C, its expression is considered to be limited in the testis, although have been found that its overexpression (Katayama H etc., 2003, Cancer and MetastasisReviews 22:451-464) in various cancer systems.

Importantly, verified by antisense oligonucleoside treatment human tumor cell line (WO97/22702 and WO 99/37788), aurora A expresses and the abolishment of function causes the cell cycle to be obstructed, and brings into play anti-proliferative effect in these tumor cell lines.In addition, the micromolecular inhibitor of aurora A and aurora B has been proved to be has anti-proliferative effect (Keen etc. to human tumor cells, 2001, Poster#2455, American Association of Cancer research annualmeeting), this expresses the same (Ditchfield etc., 2003 with treat the alternative aurora of abolishment B separately by siRNA, Journal of Cell Biology, 161 (2): 267-280).This shows that the function that suppresses aurora A and/or aurora B will have anti-proliferative effect, can be used for people's the tumour and the treatment of other excessively proliferative disease.Suppress one or more aurora kinases and have significant benefit for the signal path upstream of targeting cell-cycle (for example by growth factor receptor tyrosine kinase for example those of EGF-R ELISA (EGFR) or other receptor activation) as these treatment of diseases methods.

Because the cell cycle finally is all these unlike signal active downstreams, for example the kinase whose inhibition of one or more aurora is predicted is effectively in all hyperplasia tumour cells in the treatment of cell cycle guiding, is effective to the tumour cell hypotype of expressing those acceptors only though point to that the method for concrete signaling molecule (for example EGFR) is considered to.Also think between these signal paths, to have significant " dialogue (cross talk) ", this means that a kind of composition of inhibition can be by other compensation.

Many quinazoline derivants have been proposed for and have suppressed one or more aurora kinases.WO 04/94410 discloses by the cyclosubstituted quinazoline derivant of pyrazoles.These compounds suppress one or more aurora kinases, can suppress the cell growth of human tumor cell line SW620.Compound example is like this:

Have this active other more active compound need, if such compound in addition for the cell of known opposing chemotherapeutics, the cell that overexpression is flowed out transhipment is active especially, it will be favourable.The example that flows out transhipment comprises p-glycoprotein, albumen 1,2,3,4 and 5, BCRP, BSEP and sPgp that multidrug resistance is relevant.

We it has surprisingly been found that the compound of a little group selection, and in general, the selected subgroup of those that describe in WO04/94410 has higher activity for the aurora kinases, and it particularly is more effective in drug-resistant cell in cell.

Described compound suppresses aurora A kinases and/or the kinase whose effect of aurora B especially, therefore is used for the treatment of for example cancer of excessively proliferative disease.Especially, compound can be used for handling treatment entity or hematology tumour, more specifically is colorectal cancer, mammary cancer, lung cancer, prostate cancer, bladder cancer, kidney or carcinoma of the pancreas or leukemia or lymphadenomatous any or its arbitrary combination.

Therefore, one aspect of the present invention provides formula (I) compound

Formula (I)

Or its salt;

R wherein ¹For hydrogen or methyl and X are key or oxygen.

On the other hand, provide formula (I) compound or pharmaceutically acceptable salt thereof.

Compound of the present invention is in the auxiliary name (ACD/ title version 8.0) down of computer software.

Be to be understood that within the present invention compound of the present invention can demonstrate tautomerism, the molecular formula figure within this specification sheets only represents a kind of possible tautomeric form.Be to be understood that to the present invention includes any tautomeric form, it has kinase inhibiting activity, has aurora A and/or aurora B kinase inhibiting activity especially, any one tautomeric form that is not limited only to adopt in molecular formula figure.

It should also be understood that some compound of the present invention and its salt can exist with solvate and non-solvent compound form, for example, hydrate forms.Be to be understood that the present invention includes to have the aurora kinase inhibiting activity, particularly all such solvate form thereof of aurora A and/or aurora B kinase inhibiting activity.

The present invention relates to formula (I) compound and the salt thereof of this paper definition.The salt that is used for pharmaceutical composition should be pharmacologically acceptable salt, but other salt can be used for the preparation of formula (I) compound and their pharmacologically acceptable salts.Pharmacologically acceptable salt of the present invention for example can comprise the acid salt of formula (I) compound that this paper defines, and described compound has enough alkalescence to form salt.Such acid salt includes but not limited to fumarate (furmarate), methane sulfonates, hydrochloride, hydrobromate, Citrate trianion and maleate and the salt that forms with phosphoric acid and sulfuric acid.In addition, if formula (I) compound has enough acidity, salt is alkali salt, example includes but not limited to an alkali metal salt for example sodium salt or sylvite, alkaline earth salt is calcium salt or magnesium salts for example, or organic amine salt triethylamine, thanomin, diethanolamine, trolamine, morpholine, N-methyl piperidine, N-ethylpiperidine, dibenzyl amine or the amino acid salt of Methionin for example for example.

Formula (I) compound can also be applied with the form of prodrug, its formula that obtains (I) compound that is decomposed in human or animal's health.Therefore, the present invention also provides the prodrug of formula (I) compound.Various forms of prodrugs are known in this area.For example such prodrug derivant, referring to:

A) Design of Prodrugs, H.Bundgaard edits, (Elsevier, 1985) and Methods inEnzymology, 42 volumes, the 309-396 page or leaf, K.Widder etc. edit (Academic Press, 1985);

B) A Textbook of Drug Design and Development, Krogsgaard-Larsen and H.Bundgaard edit, the 5th chapter " Design and Application of Prodrugs ", HBundgaard, 113-191 (1991).

c)H Bundgaard，Advanced Drug Delivery Reviews，8，1-38(1992)；

D) H.Bundgaard, etc., Journal of Pharmaceutical Sciences, 77,285 (1988); With

E) N.Kakeya, etc., Chem Pharm Bull, 32,692 (1984).

In one aspect of the invention, X is a key.When X is key, the invention provides formula (IA) compound

Formula (IA)

Or its salt;

R wherein ¹Be hydrogen or methyl.

When X is key and R ¹During for hydrogen, formula (I) compound is N-(2, the 3-difluorophenyl)-2-[4-({ 7-oxyethyl group-5-[(2R)-tetramethyleneimine-2-ylmethoxy] quinazoline-4-yl } amino)-1H-pyrazol-1-yl] ethanamide.When X is key and R ¹During for methyl, formula (I) compound is N-(2, the 3-difluorophenyl)-2-{4-[(7-oxyethyl group-5-{[(2R)-1-methylpyrrolidin-2-yl] methoxyl group } quinazoline-4-yl) amino]-the 1H-pyrazol-1-yl } ethanamide.

In another aspect of this invention, X is an oxygen.When X is oxygen, the invention provides formula (IB) compound

Formula (IB)

Or its salt;

R wherein ¹Be hydrogen or methyl.

When X is oxygen and R ¹During for hydrogen, formula (I) compound is N-(2, the 3-difluorophenyl)-2-[4-({ 7-oxyethyl group-5-[(3R)-morpholine-3-ylmethoxy] quinazoline-4-yl } amino)-1H-pyrazol-1-yl] ethanamide.When X is oxygen and R ¹During for methyl, formula (I) compound is N-(2, the 3-difluorophenyl)-2-{4-[(7-oxyethyl group-5-{[(3R)-4-methylmorpholine-3-yl] methoxyl group } quinazoline-4-yl) amino]-the 1H-pyrazol-1-yl } ethanamide.

The present invention also provides wherein R ¹Be the preparation method of formula (I) compound of methyl, this method comprises R wherein ¹For formula (I) compound of hydrogen and formaldehyde in formic acid at elevated temperatures, for example from 50 ℃ under 100 ℃, for example 30 minutes to 2 hours for some time of reaction, after this, if necessary:

I) remove any protecting group; And/or

Ii) form its salt.

Prepare wherein R ¹Method for formula (I) compound of hydrogen comprises N-(2, the 3-difluorophenyl)-2-{4-[(7-oxyethyl group-5-hydroxyl quinazoline-4-yl) amino]-the 1H-pyrazol-1-yl } ethanamide and formula (II)

The alcohol reaction:

Wherein PG is suitable protecting group, for example tert-butoxycarbonyl (BOC), benzyloxycarbonyl (Z) or 9-fluorenyl methyl oxygen base carbonyl (Fmoc) and after this if necessary:

I) remove any protecting group; And/or

Ii) form its salt.

This reaction can be finished under the condition and range of document description, for example with N-(2, the 3-difluorophenyl)-and 2-{4-[(7-oxyethyl group-5-hydroxyl quinazoline-4-yl) amino]-the 1H-pyrazol-1-yl } alcohol of ethanamide and formula (II) is at solvent for example in the tetrahydrofuran (THF), for example azo-2-carboxylic acid's di tert butyl carbonate and suitable phosphine are 20 to 60 ℃ of couplings 1 to 5 hour in temperature for example in the presence of the triphenylphosphine at suitable coupling reagent.

Perhaps, R wherein ¹For formula (I) compound of hydrogen can prepare by the following method, this method comprises formula (III) compound

Wherein PG is suitable protecting group; for example tert-butoxycarbonyl (BOC), benzyloxycarbonyl (Z) or 9-fluorenyl methyl oxygen base carbonyl (Fmoc); with L be for example chlorine of suitable leaving group; with 2-(4-amino-1H-pyrazol-1-yl)-N-(2; the 3-difluorophenyl) ethanamide reaction, after this if necessary:

I) remove any protecting group; And/or

Ii) form its salt.

This reaction can be finished under the condition and range of document description, for example with formula (III) compound and 2-(4-amino-1H-pyrazol-1-yl)-N-(2, the 3-difluorophenyl) ethanamide is at solvent for example in Virahol or the N,N-DIMETHYLACETAMIDE, being with or without an acidic catalyst for example under the hydrochloric acid, be 20 to 100 ℃ of reactions 30 minutes to 24 hours in temperature.

The present invention also provides preparation N-(2, the 3-difluorophenyl)-and 2-{4-[(7-oxyethyl group-5-hydroxyl quinazoline-4-yl) amino]-the 1H-pyrazol-1-yl } method of ethanamide, this method comprises N-(2, the 3-difluorophenyl)-and 2-{4-[(7-oxyethyl group-5-methoxyl group quinazoline-4-yl) amino]-the 1H-pyrazol-1-yl } ethanamide and suitable demethylation reagent for example pyridine hydrochloride or magnesium bromide, at suitable solvent for example in pyridine or the tetrahydrofuran (THF), be 60 to 120 ℃ of reactions 5 to 48 hours down in temperature.

Described method can further comprise N-(2, the 3-difluorophenyl)-2-{4-[(7-oxyethyl group-5-methoxyl group quinazoline-4-yl) amino]-the 1H-pyrazol-1-yl } preparation method of ethanamide, this method comprises formula (IV) compound

Wherein L is suitable leaving group, and for example chlorine reacts with 2-(4-amino-1H-pyrazol-1-yl)-N-(2, the 3-difluorophenyl) ethanamide.Such reaction can be finished under the condition and range of document description, for example with formula (IV) compound and 2-(4-amino-1H-pyrazol-1-yl)-N-(2, the 3-difluorophenyl) ethanamide, at solvent for example in Virahol or the N,N-DIMETHYLACETAMIDE, have or or do not have acid catalyst for example under the hydrochloric acid, be 20 to 100 ℃ of heating 30 minutes to 24 hours in temperature.

Formula (IV) compound can be by conventional method preparation.Formula (IV) compound can prepare by the following method especially: with 7-oxyethyl group-5-methoxyl group quinazoline-4 (3H)-ketone and suitable chlorination reagent for example phosphorus oxychloride at suitable solvent for example 1, in 2-ethylene dichloride or the acetonitrile, for example in the presence of two-sec.-propyl ethylamine, is 0 to 80 ℃ reaction 2 to 24 hour in temperature at suitable alkali.

Compound for example 7-oxyethyl group-5-methoxyl group quinazoline-4 (3H)-ketone can be by conventional method preparation.7-oxyethyl group-5-methoxyl group quinazoline-4 (3H)-ketone can prepare by the following method especially: with 5,7-diethoxy quinazoline-4 (3H)-ketone and sodium methylate, at suitable solvent for example in N,N-DIMETHYLACETAMIDE, dimethyl formamide or the 1-Methyl-2-Pyrrolidone, in the reaction 6 to 24 hours down of 90 to 110 ℃ of temperature.

Compound for example 5,7-diethoxy quinazoline-4 (3H)-ketone can be by conventional method preparation.Especially 5,7-diethoxy quinazoline-4 (3H)-ketone can prepare by the following method: with 5,7-difluoro quinazoline-4 (3H)-ketone and sodium ethylate are at solvent for example in N,N-DIMETHYLACETAMIDE, dimethyl formamide or the 1-Methyl-2-Pyrrolidone, in temperature is under 90 to 110 ℃, reacts 2 to 12 hours.

5,7-difluoro quinazoline-4 (3H)-ketone is known in this area.

The present invention also provides preparation 2-(4-amino-1H-pyrazol-1-yl)-N-(2, the 3-difluorophenyl) method of ethanamide, this method comprises N-(2, the 3-difluorophenyl)-and 2-{4-[(phenylbenzene methylene radical) amino]-the 1H-pyrazol-1-yl } ethanamide is at suitable solvent for example in the ethyl acetate, in for example hydrolysis in the presence of the hydrochloric acid of the concentrated acid aqueous solution.

N-(2, the 3-difluorophenyl)-2-{4-[(phenylbenzene methylene radical) amino]-the 1H-pyrazol-1-yl } ethanamide can prepare by the following method, and this method comprises the formula V compound

Wherein X is a for example bromine or iodine of halogen, reacts with benzophenone imine.This reaction can be finished under the condition and range of document description, for example with formula V compound and benzophenone imine at solvent for example 1, in the 4-two  alkane, at suitable alkaline catalysts sodium tert-butoxide for example, cesium carbonate, salt of wormwood or potassiumphosphate exist down, at suitable part for example (9,9-dimethyl-9H-xanthene-4,5-two bases) two (diphenylphosphines), 1,1 '-dinaphthalene-2,2 '-two bases two (diphenylphosphine) or 1,1 '-two (diphenylphosphino) ferrocene exist down and appropriate catalyst for example three (dibenzalacetones), two palladiums (0) or palladium down, temperature be for example 90 ℃ descend to heat 30 minutes to 6 hours.

This method can further comprise the formula V compounds process for production thereof, and this method comprises formula (VI) compound:

Wherein X is a for example bromine or iodine of halogen, reacts with formula (VII) compound:

Wherein L is a for example chlorine or bromine of leaving group.This reaction can be finished under the condition and range of document description, for example with formula (VI) compound and formula (VII) compound, alkali for example salt of wormwood in the presence of, at solvent for example in the N,N-DIMETHYLACETAMIDE, in for example 20 ℃ of reactions 14 to 48 hours down of temperature.

Formula (VI) and (VII) compound or be known in this area perhaps can obtain from other compound deriving known in the art by the ordinary method that document occurs.

Perhaps, 2-(4-amino-1H-pyrazol-1-yl)-N-(2, the 3-difluorophenyl) ethanamide can be by reduction N-(2, the 3-difluorophenyl)-2-(4-nitro-1H-pyrazol-1-yl) ethanamide preparation.This reaction can be finished under the condition and range of document description, for example under 1 to 5 crust hydrogen pressure, appropriate catalyst for example platinum oxide or palladium on carbon in the presence of, at suitable solvent for example in ethanol and/or the ethyl acetate, at suitable temperature 20 ℃ of following reduction N-(2, the 3-difluorophenyl)-2-(4-nitro-1H-pyrazol-1-yl) ethanamide 0.5 to 5 hour for example.

N-(2, the 3-difluorophenyl)-2-(4-nitro-1H-pyrazol-1-yl) ethanamide can pass through (4-nitro-1H-pyrazol-1-yl) acetate and 2,3-difluoroaniline prepared in reaction.This reaction can be finished under the condition and range of document description, and for example with (4-nitro-1H-pyrazol-1-yl) acetate and 2, the 3-difluoroaniline with phosphorus oxychloride and pyridine, for example in the methylene dichloride, is 0 to 20 ℃ following coupling 2-3 hour in temperature at solvent.

(4-nitro-1H-pyrazol-1-yl) acetate and 2, the 3-difluoroaniline is known in this area.

The present invention further provides the method for preparation formula (III) compound, method comprises formula (VIII) compound

Wherein PG is for example tert-butoxycarbonyl (BOC), benzyloxycarbonyl (Z) or a 9-fluorenyl methyl oxygen base carbonyl (Fmoc) of suitable protecting group; with suitable chlorination reagent phosphorus oxychloride for example; at suitable solvent for example 1; in 2-ethylene dichloride or the acetonitrile; at suitable alkali for example in the presence of two-sec.-propyl ethylamine, be 0 to 80 ℃ of reaction 2 to 24 hours down in temperature.

This method can further comprise the method for preparation formula (VIII) compound; wherein PG is for example tert-butoxycarbonyl (BOC), benzyloxycarbonyl (Z) or a 9-fluorenyl methyl oxygen base carbonyl (Fmoc) of suitable protecting group; this method comprises 7-oxyethyl group-5-fluquinconazole quinoline-4 (3H)-ketone and the reaction of formula (II) compound, and protecting group changes subsequently.Wherein 7-oxyethyl group-5-fluquinconazole quinoline-4 (3H)-ketone and formula (II) compound reaction, PG or be hydrogen or be for example benzyl of suitable protecting group.This reaction can be finished under the condition and range of document description; for example with 7-oxyethyl group-5-fluquinconazole quinoline-4 (3H)-ketone; with PG wherein or be hydrogen or be the suitable protecting group formula of benzyl (II) compound for example; at solvent for example in tetrahydrofuran (THF), dimethyl formamide, N,N-DIMETHYLACETAMIDE or the 1-Methyl-2-Pyrrolidone; with alkali for example sodium hydride or potassium tert.-butoxide, be 20 to 100 ℃ of reactions 2 to 24 hours down in temperature.

Wherein PG or be hydrogen or for the suitable protecting group formula of benzyl (II) compound for example perhaps is known in the art, and perhaps can obtain from other compound deriving known in the art by the ordinary method in the document.

7-oxyethyl group-5-fluquinconazole quinoline-4 (3H)-ketone can be by with 2-amino-4-oxyethyl group-6-fluorine benzonitrile and formic acid, with the mineral acid of catalytic amount sulfuric acid for example, in for example 100 ℃ of reaction preparations in 2 to 24 hours down of temperature.

Compound is 2-amino-4-oxyethyl group-6-fluorine benzonitrile or be known in the art for example, perhaps can obtain from other compound deriving known in the art by the ordinary method in the document.

Should be understood that in the various ring substituents of The compounds of this invention some can be before or after method mentioned above at once, introduce by the substitution reaction of standard fragrance, or by conventional modified with functional group generation, such method is also included within method of the present invention aspect.Such reaction and modification for example comprise introduces substituting group, substituent reduction, substituent alkylation and substituent oxidation by the method for fragrant substitution reaction.The reagent of this quadrat method and reaction conditions are well-known in chemical field.The specific example of fragrance substitution reaction comprises use concentrated nitric acid introducing nitro, uses for example carboxylic acid halides and Lewis acid (for example aluminum chloride) introducing acyl group under Friedel Crafts condition; Under Friedel Crafts condition, use halogenated alkyl thing and Lewis acid (for example aluminum chloride) to introduce alkyl; With the introducing halogen group.The particular instance of modifying comprises by for example using the nickel catalyzator catalytic hydrogenation, or exists under the heating at hydrochloric acid and to handle nitroreduction to amino with iron; Alkylthio is oxidized to alkyl sulphinyl or alkyl sulphonyl.

It should also be understood that the group of some reactions that this paper mentions any sensitivity in may the essential/compound that needs protection.Wherein protection is essential or the example of needs and suitable guard method are known for those skilled in the art.Conventional protecting group can be used (for example referring to T.W.Green, Protective Groups in Organic Synthesis, John Wileyand Sons, 1991) according to standard operating instructions.Therefore, if reactant comprises group for example amino, carboxyl or hydroxyl, it may need protection in some reactions that this paper mentions.

The appropriate protection base of amino or alkylamino is for example acyl group, for example alkyloyl, for example ethanoyl; alkoxy carbonyl, for example methoxycarbonyl, ethoxy carbonyl or tert-butoxycarbonyl, the aryl methoxy carbonyl is benzyloxycarbonyl for example; or aroyl, for example benzoyl.The deprotection condition of above-mentioned protecting group must change along with the selection of protecting group.Therefore, acyl group for example, for example alkyloyl or alkoxy carbonyl or aroyl can be for example by at suitable alkali for example alkali metal hydroxide, for example lithium hydroxide or sodium hydroxide hydrolysis.Perhaps for example tert-butoxycarbonyl can be for example by removing with suitable sour example hydrochloric acid, sulfuric acid or phosphoric acid or trifluoroacetic acid processing for acyl group; with aryl methoxy carbonyl benzyloxycarbonyl for example; can by for example catalyzer for example on the palladium on carbon hydrogenation remove, or by with Lewis acid for example three (trifluoroacetic acid) boron handle.The suitable selectable protecting group of primary amino for example is a phthaloyl, and it can be by with alkylamine dimethylaminopropyl amine for example, or handles with hydrazine and to remove.

The appropriate protection base of hydroxyl for example is an acyl group, alkyloyl for example, and ethanoyl for example, aroyl is benzoyl for example, or arylmethyl, for example benzyl.The deprotection condition of above-mentioned protecting group must change along with the selection of protecting group.Therefore, acyl group for example, for example alkyloyl or aroyl can be for example by with suitable alkali alkali metal hydroxide for example, for example lithium hydroxide or sodium hydroxide hydrolysis are removed.Perhaps arylmethyl, for example benzyl can by with catalyzer for example palladium on carbon hydrogenation remove.

The appropriate protection base that is used for carboxyl for example is esterified group; for example methyl or ethyl ester; its can be for example by with alkali for example sodium hydroxide hydrolysis remove; or for example be tertiary butyl ester; its can by with acid for example organic acid for example trifluoroacetic acid handle and to remove; or for example be benzyl ester, its can be for example by catalyzer for example on the palladium on carbon hydrogenation remove.

Protecting group can use in chemical field well-known routine techniques in office what easily synthesis phase remove.

Further aspect provides pharmaceutical composition according to the present invention, and it comprises formula (I) compound or pharmaceutically acceptable salt thereof of this paper definition and blended pharmaceutically acceptable diluent or carrier with it.

Composition of the present invention can be the form that is fit to orally use (tablet for example, lozenge (lozenge), hard or soft capsule, water-based or oiliness suspensoid, emulsion, dispersible pulvis or granule, syrup or elixir), be used for the local (creme for example that uses, paste, gel, or water-based or oily solution or suspensoid), (for example pulverizing pulvis or the liquid aerosol) used by suction, (for example be used for intravenously by what be blown into (for example pulverizing pulvis) used or be used for parenteral administration, subcutaneous, the sterilization water-based of intramuscular or intramuscular administration or oily solution or conduct are used for the suppository of rectal administration).

The present composition can use the drug excipient of routine well-known in the art to obtain by the method for routine.Therefore, the purpose composition that is used to orally use can contain for example one or more tinting materials, sweeting agent, sweetener and/or sanitas.

The suitable pharmaceutically acceptable vehicle that is used for Tabules comprises for example inert thinner, and for example lactose, yellow soda ash, calcium phosphate or lime carbonate are granulated and for example W-Gum or algenic acid of disintegrating agent; Tackiness agent is starch for example; Lubricant is Magnesium Stearate, stearic acid or talcum powder for example; Sanitas is ethyl p-hydroxybenzoate or propyl ester for example, and antioxidant, for example xitix.Tabules can for dressing not or dressing, with the disintegration and the absorption of activeconstituents within gi tract subsequently of modifying them, or improve their stability and/or outward appearance, in all cases, use in conventional Drug coating well-known in the art and method.

The composition that is used to orally use can be with the form of hard gelatin capsule, wherein for example lime carbonate, calcium phosphate or kaolin mix with the inert solid diluent with activeconstituents, or be soft gelatin capsule, wherein for example peanut oil, whiteruss, soybean oil, Oleum Cocois or preferred sweet oil or any other acceptable medium are mixed with activeconstituents and water or oil.

Aqueous suspension contains activeconstituents and one or more following reagent of pulverizing form in general: suspension agent, for example sodium carboxy methyl cellulose, methylcellulose gum, HYDROXY PROPYL METHYLCELLULOSE, sodium alginate, polyvinylpyrrolidone, tragakanta and Sudan Gum-arabic; Dispersion agent or moistening agent, the condensation product of Yelkin TTS or alkylene oxide and lipid acid (for example polyoxyethylene stearate) for example, or the condensation product of ethylene oxide and long-chain fat family alcohol, 17 carbon ethyleneoxy group cetyl alcohols for example, or ethylene oxide and derived from the condensation product of the partial ester of lipid acid and hexitol, polyoxyethylene sorbitol monooleate for example, or the condensation product of ethylene oxide and long-chain fat family alcohol 17 carbon ethyleneoxy group cetyl alcohols for example, or ethylene oxide and derived from the condensation product of the partial ester of lipid acid and hexitol, polyoxyethylene sorbitol monooleate for example, or ethylene oxide and derived from the condensation product of the partial ester of lipid acid and hexitol acid anhydrides, for example poly-ethylidene dehydrated sorbitol mono-fatty acid ester.Aqueous suspension can also contain one or more sanitass (for example ethyl p-hydroxybenzoate or propyl ester, antioxidant (for example xitix), tinting material, sweetener and/or sweeting agent (for example sucrose, asccharin or aspartame).

The oiliness suspensoid can be by being suspended in activeconstituents vegetables oil (for example peanut oil, sweet oil, sesame oil or Oleum Cocois) or preparation in mineral oil (for example whiteruss).The oiliness suspensoid can also contain thickening material for example beeswax, paraffinum durum or hexadecyl alcohol.Sweeting agent for example listed above those, can add sweetener so that agreeable to the taste oral preparations to be provided.These compositions can be by adding for example xitix preservation of antioxidant.

Dispersible or freeze dried pulvis and granule are suitable for preparing aqueous suspension or solution, realize by adding entry, contain dispersion agent or moistening agent, suspension agent and one or more sanitass with activeconstituents in general.Those example explanations of suitable dispersion agent or moistening agent and suspension agent by above having mentioned.Can also there be other vehicle for example sweeting agent, sweetener and tinting material.

Pharmaceutical composition of the present invention can also be with the form of oil-in-water emulsion.Oil phase can be vegetables oil for example sweet oil or peanut oil, or the mineral oil mixture of whiteruss or any of these for example.Suitable emulsifying agent can be for example naturally occurring colloid, for example Sudan Gum-arabic or tragakanta, naturally occurring phosphatide for example soybean oil, Yelkin TTS, from the condensation product of the ester of lipid acid and hexitol acid anhydrides or partial ester (for example dehydrated sorbitol mono-fatty acid ester) and described partial ester and ethylene oxide polyoxyethylene dehydrated sorbitol mono-fatty acid ester for example.This emulsion can also contain sweeting agent, sweetener and sanitas.

Syrup and elixir can also contain negative catalyst, sanitas, sweetener and/or tinting material with for example glycerine, propylene glycol, Sorbitol Powder, aspartame or sucrose preparation of sweeting agent.

Pharmaceutical composition can also be injectable water-based or oiliness suspensoid, the solution of sterilizing, emulsion or specific system form, and it can use one or more suitable dispersion agents mentioned above or moistening agent and suspension agent preparation according to known method.Sterilizing injectable preparation can also be at nontoxic parenteral acceptable diluent or solvent injectable solution of sterilization or the suspensoid in the polyglycol solution for example.

Suppository formulation can be by being mixed with activeconstituents and suitable non-irritating excipient, and described vehicle is a solid under typical temperature, but is liquid under rectal temperature, therefore discharges medicine in the rectum fusing.Suitable vehicle comprises for example theobroma oil and polyethylene glycols.

Topical formulations is creme, paste, gel and water-based or oily solution or suspensoid for example, in general can be by using ordinary method preparation well-known in the art to obtain activeconstituents and conventional, local acceptable medium or thinner.

Can be pulverizing powder form by being blown into the composition of using, the particulate mean diameter that contains for example is 30 μ m or littler, preferred 5 μ m or littler, more preferably between 5 μ m and 1 μ m, pulvis itself comprises activeconstituents separately, perhaps with for example lactose dilution of one or more physiology acceptable carriers.The pulvis that is used for then being blown into is retained in capsule easily, and for example 1 to 50mg activeconstituents is used for the use of turboinhalator equipment, for example is used to be blown into known reagent Sodium Cromoglicate.

The composition of using by suction can be the pressure aerosol form of routine, is used for activeconstituents or is assigned as the aerosol that contains pulverizing solid or liquid droplets.Conventional aerosol propellant for example can use volatile hydrofluoric ether or hydrocarbon, and aerosol device is advantageously used in the activeconstituents of distribution and computation quantity.

For the more information of formulation, ask the reader with reference to Comprehensive MedicinalChemistry (Corwin Hansch; The chairman of editorial committee), 25.2 chapters of the 5th of Pergamon Press 1990 the volume.

Therefore the present invention further aspect, the formula that is provided for treating (I) compound or pharmaceutically acceptable salt thereof.Formula (I) as medicine compound or pharmaceutically acceptable salt thereof further is provided.Others of the present invention provide formula (I) compound or pharmaceutically acceptable salt thereof as treatment excessively proliferative disease cancer and be used for the treatment of colorectal cancer, mammary cancer, lung cancer, prostate cancer, bladder cancer, kidney or carcinoma of the pancreas especially or the medicine of leukemia or lymphadenomatous any one or any combination for example.

In addition, provide formula (I) compound or pharmaceutically acceptable salt thereof to be used for being used for the treatment of for example people's method of warm-blooded animal by treatment.Be provided for treating for example cancer and treat colorectal cancer, mammary cancer, lung cancer, prostate cancer, bladder cancer, kidney or carcinoma of the pancreas especially or formula (I) compound or pharmaceutically acceptable salt thereof of the method for leukemia or lymphadenomatous any one or any combination of excessively proliferative disease in others of the present invention.

In another aspect of this invention, provide formula (I) compound or pharmaceutically acceptable salt thereof to be used to prepare the purposes for the treatment of disease medicament, it is useful wherein suppressing one or more aurora kinases.Imagine aurora A kinases especially and/or the kinase whose inhibition of aurora B may be useful.The preferred kinase whose inhibition of aurora B is useful.In another aspect of this invention, provide formula (I) compound or pharmaceutically acceptable salt thereof to be used to prepare the purposes of medicine, described medicine is used for the treatment of for example cancer and treat colorectal cancer, mammary cancer, lung cancer, prostate cancer, bladder cancer, kidney or carcinoma of the pancreas especially or leukemia or lymphadenomatous any one or any combination of excessively proliferative disease.

According on the other hand, be provided for treating formula (I) compound or pharmaceutically acceptable salt thereof of the people's who suffers from disease method, it is useful suppressing one or more aurora kinases in described disease, comprises formula (I) compound or pharmaceutically acceptable salt thereof to people's administering therapeutic significant quantity of needs treatment.Imagination inhibition aurora A kinases and/or aurora B kinases may be useful especially.Preferred inhibition aurora B kinases is useful.Further be provided for treatment and suffer from for example cancer of excessively proliferative disease, formula (I) compound or pharmaceutically acceptable salt thereof of suffering from the people of colorectal cancer, mammary cancer, lung cancer, prostate cancer, bladder cancer, kidney or carcinoma of the pancreas or leukemia or lymphadenomatous any one or any combination especially comprises the step to formula (I) compound or pharmaceutically acceptable salt thereof of people's administering therapeutic significant quantity of needs treatment.Formula (I) compound or pharmaceutically acceptable salt thereof the treatment above-described people any method in purposes also constitute of the present invention aspect.

The present invention further aspect, people's the method that provides treatment to suffer from disease, it is useful suppressing one or more aurora kinases in described disease, comprises the step to formula (I) compound or pharmaceutically acceptable salt thereof of people's administering therapeutic significant quantity of needs treatment.

Imagination inhibition aurora A kinases and/or aurora B kinases may be useful especially.Preferred inhibition aurora B kinases is useful.Further provide treatment to suffer from for example cancer and suffer from colorectal cancer, mammary cancer, lung cancer, prostate cancer, bladder cancer, kidney or carcinoma of the pancreas especially or the people's of leukemia or lymphadenomatous any one or any combination method of excessively proliferative disease, comprise the step of formula (I) compound or pharmaceutically acceptable salt thereof of people's administering therapeutic significant quantity for the treatment of to needs.

For the above-mentioned therepic use of mentioning, the dosage of using will be along with the compound that adopts, method of application, required treatment, adaptation illness and variations such as animal or patient's age and sex.Therefore should calculate the size of dosage according to well-known medicine principle.

Be used for the treatment of or during the purpose of preventing at formula (I) compound, the per daily dose scope of using in general is for for example accepting 0.05mg/kg to 50mg/kg body weight (with 0.05mg/kg to 15mg/kg body weight especially), the dosage that then separates if desired.Usually when adopting parenteral route, use lower dosage.Therefore, for example use for intravenously, dosage range for example generally uses 0.05mg/kg to 25mg/kg body weight (with 0.05mg/kg to 15mg/kg body weight especially).Similarly, the dosage range of using by suction for example will use 0.05mg/kg to 25mg/kg body weight (with 0.05mg/kg to 15mg/kg body weight especially).

The treatment of this paper definition can maybe can comprise conventional surgical operation or radiotherapy or chemotherapy for being applied as independent treatment except that compound of the present invention.Such chemotherapy can comprise the anti-tumor agents of one or more following kinds:

(i) be used for other anti-hyperplasia/antitumor drug of medical science oncology and its combination, for example alkylating reagent (for example cis-platinum, oxaliplatin, carboplatin, endoxan, mustargen, alkeran, Chlorambucil, busulfan, Temozolomide and nitrosourea); Metabolic antagonist (for example gemcitabine and antifol for example fluorine pyrimidine such as 5 FU 5 fluorouracil and Tegafur, Raltitrexed, Rheumatrex, cytosine arabinoside and hydroxyurea); Antitumor antibiotics (for example anthracycline antibiotics, as Zorubicin, bleomycin, Dx, daunorubicin, epirubicin, idarubicin, Mitomycin-C, dactinomycin and Plicamycin); Antimitotic agent (for example vinca alkaloids, as vincristine(VCR), vinealeucoblastine(VLB), vindesine and vinorelbine and taxanes such as taxol and taxotere and polokinase inhibitor); And topoisomerase enzyme inhibitor (for example etoposide toxin, as Etoposide and teniposide, amsacrine, Hycamtin and camptothecine);

(ii) cytostatic agent antiestrogen (tamoxifen for example for example, fulvestrant, toremifene, raloxifene, droloxifene and iodoxyfene), the downward conditioning agent of estrogen receptor (for example fulvestratrant), antiandrogen (bicalutamide for example, flutamide, Nilutamide and cyproterone acetate), lhrh antagonist or LHRH agonist (goserelin for example, Leuprolide and buserelin), progestogen (for example Magace), aromatase inhibitor (Anastrozole for example, letrozole, vorazole and Exemestane) and the 5 inhibitor for example Fei Nasi carry;

(iii) anti-invade medicine (c-Src kinases man group inhibitor for example is as 4-(6-chloro-2,3-methylenedioxyphenyl amino)-7-[2-(4-methylpiperazine-1-yl) oxyethyl group]-5-tetrahydropyran-4-base oxygen base quinazoline (AZD0530; International Patent Application WO 01/94341) and N-(2-chloro-6-aminomethyl phenyl)-2-{6-[4-(2-hydroxyethyl) piperazine-1-yl]-2-methylpyrimidine-4-base is amino thiazole-5-carboxylic acid amides (dasatinib, BMS-354825; J.Med.Chem., 2004, 47, 6658-6661), and inhibitors of metalloproteinase, as marimastat, urokinase plasminogen activator function of receptors inhibitor or heparanase antibody);

(iv) somatomedin depressant of functions, for example such inhibitor comprise growth factor antibodies, growth factor receptor antibody (anti--erbB2 antibody trastuzumab [Herceptin for example ^TM], anti-EGFR-antibodies panitumumab, anti--erbB1 antibody Cetuximab [Erbitux, C225] and any by Stern etc. at oncology/haematology2005,54 volumes, crucial disclosed somatomedin of summary of 11-29 page or leaf or growth factor receptor antibody; Such inhibitor also comprises tyrosine kinase inhibitor, epidermal growth factor family inhibitor (EGFR family tyrosine kinase inhibitor for example for example, N-(3-chloro-4-fluorophenyl)-7-methoxyl group-6-(3-morpholino propoxy-) quinazoline-4-amine (gefitinib for example, ZD1839), N-(3-ethynyl phenyl)-6, two (2-methoxy ethoxy) quinazolines of 7--4-amine (erlotinib, OSI-774) and 6-propenyl acid amides-N-(3-chloro-4-fluorophenyl)-7-(3-morpholino propoxy-)-quinazoline-4-amine (CI 1033), the erbB2 tyrosine kinase inhibitor is lapatinib for example, pHGF man group inhibitor, platelet-derived growth factor family inhibitor is imatinib for example, (for example Ras/Raf signal suppressing agent of serine/threonine kinase inhibitor, farnesyl tranfering enzyme inhibitor for example, sorafenib (BAY 43-9006) for example), by MEK and/or the kinase whose cell signal inhibitor of AKT, pHGF man group inhibitor, the c-kit inhibitor, the abl kinase inhibitor, IGF acceptor (insulin-like growth factor) kinase inhibitor; Aurora kinase inhibitor (for example PH739358, VX-680, MLN8054, R763, MP235, MP529, VX-528 and AX39459) and cell cycle protein dependent kinase inhibitor be CDK2 and/or CDK4 inhibitor for example;

(v) anti-angiogenic agent for example suppresses those (anti-vascular endothelial cell growth factor antibody rhuMAb-VEGF [Avastin for example of vascular endothelial growth factor effect ^TM] and vegf receptor tyrosine kinase inhibitor, for example 4-(4-bromo-2-fluoroanilino)-6-methoxyl group-7-(1-methyl piperidine-4-ylmethoxy) quinazoline (ZD6474; Embodiment 2 within the WO 01/32651), 4-(4-fluoro-2 methyl indole-5-base oxygen base)-6-methoxyl group-7-(3-tetramethyleneimine-1-base propoxy-) quinazoline (AZD2171; Embodiment 240 within the WO00/47212), vatalanib (PTK787; WO 98/35985) and SU11248 (sunitinib; WO 01/60814), for example those are in International Patent Application WO 97/22596, and WO 97/30035, disclosed compound among WO 97/32856 and the WO 98/13354, with compound by other machine-processed onset (linomide for example, beta 2 integrin alpha v β 3 depressant of functions and blood vessel he spit of fland);

(vi) the blood vessel injury agent for example combretastatin A4 and in International Patent Application WO 99/02166, WO00/40529, WO00/41669, WO01/92224, WO02/04434 and WO02/08213 disclosed compound;

(for example ISIS 2503 for vii) antisense therapy, guiding those of listed target above for example, for anti--ras antisense;

(viii) gene therapy method, comprise and for example replace for example unusual p53 of aberrant gene or unusual BRCA1 or BRCA2, the method of GDEPT (the enzyme prodrug treatment of gene targeting), those that in cytosine(Cyt) deaminase, Thymine deoxyriboside kinases or bacterium nitroreductase enzyme, use for example, with the method for increase patient, for example many-gene therapy of drug resistance disease to chemotherapy or radiotherapy tolerance;

(ix) immunotherapy, comprise and for example exsomatizing (ex-vivo) and the interior method of body, to increase the patient tumors cell immunogenicity, for example with for example interleukin-22, interleukin 4 or rHuGM-CSF transfection of cytokine, reduce the method that T cell strain power lacks, use the method for transfection immunocyte, for example the method for the dendritic cell of cytokine transfection is used the method for cytokine transfection tumor cell line and the method for use anti-id AB.

In addition, compound or pharmaceutically acceptable salt thereof of the present invention can be united use with one or more cell cycle inhibitors.Unite use with the cell cycle inhibitor that suppresses bub1, bubR1 or CDK especially.

Such combination therapy can be by simultaneously, in succession or the mode of separating the single composition for the treatment of realize.Such combination product adopts the The compounds of this invention within dosage range described herein, and the other medicines active agent is within its approved dosage range.

Except that they the purposes of medicine, formula (I) compound and its pharmacologically acceptable salt also can be used as pharmacological tool, be used for laboratory animal for example cat, dog, rabbit, monkey, rat and mouse estimate the exploitation and the stdn of experimental system in the external and body of cell cycle activity inhibitor effect, described experiment is used to seek new therapeutical agent.

In above-mentioned other medicines composition, process, method, purposes and medication preparation feature, also can use the selectable and embodiment preferred of compound of the present invention described herein.

Compound of the present invention suppresses the kinase whose serine-threonine kinase activity of aurora, suppresses aurora A kinases and/or the kinase whose activity of aurora B especially, therefore suppresses cell cycle and hyperplasia.It is interested especially suppressing the kinase whose compound of aurora B.Compound also is active in drug-resistant cell, has favourable physical properties.One or more evaluations that these character can for example use method hereinafter to list.

(a) external aurora A kinase inhibition experiment

This is analyzed the confirmed test compound and suppresses the active ability of serine-threonine kinase.The DNA of coding aurora A is can be by gene complete synthesis or obtain by the clone.This DNA can express to obtain to have the active polypeptide of serine-threonine kinase in appropriate expression system then.Under the situation of aurora A, separate encoding sequence by polymerase chain reaction (PCR) from cDNA, be cloned into BamH1 and the Not1 restriction endonuclease site of rhabdovirus expression vector pFastBac HTc (GibcoBRL/Life technologies).5 ' PCR primer contains the recognition sequence of restriction endonuclease BamH1 5 ' to aurora A encoding sequence.This makes aurora A gene be inserted into to have in 6 histidine residues, transcribed spacer and the framework by the rTEV protease cracking site of pFastBac HTc vector encoded.3 ' PCR primer is replaced aurora A with other encoding sequence and is stopped codon, is to stop codon and restriction endonuclease not1 recognition sequence after the described other encoding sequence.This other encoding sequence (5 ' TAC CCA TAC GAT GTT CCAGAT TAC GCT TCT TAA 3 ') coded polypeptide sequence YPYDVPDYAS.This sequence through being commonly used for unlabelled antigen determination section bit sequence, can be used specific monoclonal antibody identification from the influenza hemagglutinin.Reorganization pFastBac carrier so the terminal Aurora-A albumen 6 his marks, C-terminal influenza hemagglutinin epitope mark of the N-that encodes.The method of recombinant DNA molecules assembling can be found in received text, Sambrook etc. 1989 for example, Molecular Clonmg-A Laboratory Manual, the 2nd edition, Cold Spring HarborLaboratory press and Ausubel etc. 1999, Current Protocols in MolecularBiology, John Wiley and Sons Inc..

The preparation of recombinant virus can be according to finishing from GibcoBRL manufacturer's scheme.In brief, the pFastBac-1 carrier that will have aurora A gene is transferred in the E.coli DH10Bac cell that contains baculovirus genome (bacmid DNA), by cell displacement activity, will contain the pFastBac carrier of gentamicin drug resistant gene and comprise that the aurora A gene of baculovirus polyhedrin body protein promotor directly is shifted among the bacmid DNA.By selecting gentamicin, kantlex, tsiklomitsin and X-gal, the containing of aurora A that obtain encoding the recombinate blank of bacmidDNA clone.From the blank clone's of several BH10Bac short run substratum, extract BacmidDNA, according to manufacturer's instruction transfection to using cell FECTIN reagent (GibcoBRL) to contain in the Spodoptera frugiperda Sf21 cell of growing in the TC100 medium (GibcoBRL) of 10% serum.By collecting transfection cell culture medium medium results virom crosome after 72 hours.The 0.5ml medium is used to infect 100ml contains 1 * 10 ⁷The Sf21s substratum suspension of cell/ml.Harvested cell substratum medium after infecting 48 hours uses the patch analytical procedure of standard to determine virus titer.Using standby virus infection Sf9 and " High 5 " cell is 3 to infecting multiple degree (MOI), to determine the proteic expression of reorganization aurora A.

For the aurora A kinase activity of expressing in enormous quantities, with the Sf21 insect cell in the TC100 medium that is supplemented with 10% foetal calf serum (Viralex) and 0.2%F68 polyethers (Sigma) on the Wheaton shaking table at 3r.p.m. 28 ℃ of cultivations.When cell density reaches 1.2 * 10 ⁶Cells ^-1The time, be 1 time to infect 48 hour after results with the aurora A recombinant virus of patch-pure (plaque-pure) infecting multiple degree with them.All subsequent purificn steps are finished at 4 ℃.To contain sum 2.0 * 10 ⁸The freezing insect cell bead fusing of individual cell, with dissolving damping fluid (25mMHEPES (N-[2-hydroxyethyl] piperazine-N '-[2 ethane sulfonic aicd]), pH7.4 is at 4 ℃, 100mM KCl, 25mM NaF, 1mM Na ₃VO ₄, 1mM PMSF (the phenyl methyl alkylsulfonyl is fluoridized thing), 2mM2-mercaptoethanol, 2mM imidazoles, 1 μ g/ml Trypsin inhibitor,Trasylol, 1 μ g/ml pepstatin, 1 μ g/ml leupeptin) and dilution, use 1.0ml per 3 * 10 ⁷Cell.Use the dounce homogenizer to implement dissolving, subsequently with lysate 41, centrifugal 35 minutes of 000g.On the 5mm diameter chromatographic column that contains 500 μ l Ni NTA (nitrilo-tri-acetate) agaroses (Qiagen, production number 30250), this post is with dissolving damping fluid balance with the supernatant liquid pump of sucking-off.With 12ml dissolving damping fluid washing column, use 7ml lavation buffer solution (25mM HEPES pH7.4 at 4 ℃, 100mM KCl, 20mM imidazoles, 2mM 2 mercapto ethanol) washing subsequently after, the UV that reaches elutriant absorbs the horizontal base line level.Use elution buffer (25mM HEPES pH7.4 at 4 ℃, 100mM KCl, 400mM imidazoles, 2mM 2 mercapto ethanol) with bonded aurora A albumen wash-out from the post.Collection is corresponding to the wash-out part (2.5ml) of UV absorption peak.To contain the fully dialysis in dialysis buffer liquid (25mM HEPES pH7.4 at 4 ℃, 45% glycerine (v/v), 100mM KCl, 0.25%Nonidet P40 (v/v), 1mM dithiothreitol dithio) once more of the kinase whose wash-out of active auroraA part.

Every batch of new aurora A enzyme is diluted titrimetry by using enzyme thinner (25mM Tris-HCl pH7.5,12.5mM KCl, 0.6mM DTT).For typical batch (Upstate provides), alternation enzyme dilutes the every ml of 1 μ m with the enzyme thinner, and each is analyzed the hole and uses 20 μ l dilution enzyme.Dilute with water experimental compound (10mM is in methyl-sulphoxide (DMSO)) is transferred to the compound of 10 μ l dilution in the hole of analysis plates." fully " and " blank " control wells contains 2.5%DMSO rather than compound.The enzyme of the fresh dilution of 20 microlitres is joined in all holes, except " blank " hole.20 microlitre enzyme thinners are joined in " blank " hole.Then 20 microlitres are contained 0.2 μ Ci[γ ³³P] ATP (Amersham Pharmacia, reaction mixture (25mM Tris-HCl, 78.4mM KCl, 2.5mM NaF, 0.6mM dithiothreitol dithio, the 6.25mM MnCl of given activity 〉=2500Ci/mmol) ₂, 25mM ATP, 7.5 μ M peptide substrates [vitamin H-LRRWSLGLRRWSLGLRRWSLGLRRWSLG]) and join initiation reaction in all experimental ports.With plate incubated at room temperature 60 minutes.In all holes, add 100 μ l 20%v/v ortho-phosphoric acid stopped reaction.Use 96-orifice plate collector (TomTek) that peptide substrates is captured on the positive charge Nitrocellulose P30 filter pad (Whatman), analyze the combination of 33P then with β plate counter." blank " (no enzyme) and " fully " (no compound) control value are used for the dilution range of confirmed test mixture, provide the value (IC50 value) of inhibitory enzyme activity 50%.Compound of the present invention generally provides the IC50 value and is 0.1nM to 5 μ M.

(b) external aurora B kinase inhibition experiment

This is analyzed and determines that experimental compound suppresses the active ability of serine-threonine kinase.Synthetic or by full gene by clone can obtain the to encode DNA of aurora.This DNA can be expressed in appropriate expression system then to obtain to have the active polypeptide of serine-threonine kinase.Under the situation of aurora B, separate encoding sequence by polymerase chain reaction (PCR) from cDNA, with above the similar mode of the description of aurora A is cloned in the pFastBac system (promptly directly the aurora B albumen of expression 6-histidine mark).

For great expression aurora B kinase activity, the Sf21 insect cell is cultivated at 3r.p.m on the Wheaton shaking table at 28 ℃ in the TC100 medium additional with 10% foetal calf serum (Viralex) and 0.2%F68 Pluronic (Sigma).When cell density reaches 1.2 * 10 ⁶Cells ^-1The time, be 1 time infection with patch-pure aurora B recombinant virus infecting multiple degree with them, results after 48 hours.All purification steps are subsequently finished at 4 ℃.To contain sum 2.0 * 10 ⁸The freezing insect cell bead fusing of cell, with dissolving damping fluid (50mM HEPES (N-[2-hydroxyethyl] piperazine-N '-[2 ethane sulfonic aicd]), pH7.5 is at 4 ℃, 1mM Na ₃VO ₄, 1mM PMSF (the phenyl methyl alkylsulfonyl is fluoridized thing), 1mM dithiothreitol dithio, 1 μ g/ml Trypsin inhibitor,Trasylol, 1 μ g/ml pepstatin, 1 μ g/ml leupeptin) and dilution, use 1.0ml per 2 * 10 ⁷Cell.Use ultrasonic homogenizer to realize dissolving, subsequently with lysate 41, centrifugal 35 minutes of 000g.On the 5mm diameter chromatographic column that contains 1.0ml CM high flow rate agarose (Sepharose FastFlow) (Amersham Pharmacia Biotech), this post is balance in the dissolving damping fluid with the supernatant liquid pump of sucking-off.With 12ml dissolving damping fluid washing column, use 7ml lavation buffer solution (50mM HEPES pH7.4 at 4 ℃, 1mM dithiothreitol dithio) washing subsequently after, the UV of elutriant absorbs and reaches baseline values.Use gradient elution damping fluid (50mM HEPES pH7.4 is at 4 ℃, 0.6M NaCl, the 1mM dithiothreitol dithio, from 0% elution buffer to 100% elution buffer, experience is 15 minutes under the 0.5ml/min flow velocity) aurora B albumen of elution of bound from post.Collection is corresponding to the wash-out part (1.0ml) of UV absorption peak.With wash-out part to dialysis buffer liquid (25mM HEPES pH7.4 at 4 ℃, 45% glycerine (v/v), 100mM KCl, 0.05% (v/v) IGEPAL CA630 (Sigma Aldrich), 1mM dithiothreitol dithio) fully dialysis once more.Analyze the aurora B kinase activity of dialysis part.

By at 50mM Tris-HCl pH7.5,0.1mM EGTA, the 0.1%2-mercaptoethanol, 0.1mM vandate sodium, the 10mM magnesium acetate contains 0.1mg/ml GST-INCENP[826-919] 0.1mM ATP at 30 ℃ of activation aurora B (5 μ M) 30 minutes preparation Aurora B-INCENP enzyme (Upstate provides).

Every batch of new aurora B-INCENP enzyme is diluted titrimetry by using enzyme thinner (25mM Tris-HClpH7.5,12.5mM KCl, 0.6mM DTT).For typical batch, alternation enzyme uses 20 μ l dilution enzyme with enzyme thinner dilution 1: 40, each analysis hole.Dilute with water experimental compound (10mM is in methyl-sulphoxide (DMSO)) is transferred to the compound of 10 μ l dilution in the hole of analysis plates." fully " and " blank " control wells contains 2.5%DMSO rather than compound.The enzyme of the fresh dilution of 20 microlitres is joined in all holes, except " blank " hole.20 microlitre enzyme thinners are joined in " blank " hole.To contain 0.2 μ Ci[γ then ³³P] ATP (Amersham Pharmacia, 20 microlitre reaction mixtures (25mM Tris-HCl, 12.7mM KCl, 2.5mM NaF, 0.6mM dithiothreitol dithio, the 6.25mM MnCl of specific activity 〉=2500Ci/mmol) ₂, 15mM ATP, 6.25 μ M peptide substrates [vitamin H-LRRWSLGLRRWSLGLRRWSLGLRRWSLG])) join initiation reaction in all experimental ports.Plate was at room temperature cultivated 60 minutes.In all holes, add 100 μ l 20%v/v ortho-phosphoric acid stopped reaction.Use 96-orifice plate collector (TomTek) that peptide substrates is captured on the positive charge Nitrocellulose P30 filter pad, analyze the combination of 33P then with β plate counter." blank " (no enzyme) and " fully " (no compound) control value are used for the dilution range of confirmed test mixture, provide the value (IC50 value) of inhibitory enzyme activity 50%.It is 0.05 to 10nM that compound of the present invention generally provides the IC50 value.Especially, compound 1 provides IC50 0.5nM, and compound 2 is that 0.5nM and compound 3 are 0.1nM.

(c) cell in vitro phenotype and substrate phosphorylation analysis

This analysis is used for determining the external effect to SW620 human colon tumor cell of compound.Compound typically causes the increase of inhibition of phosphorus histone H 3 level and nuclear area.

With every hole 10 ⁴The SW620 cell is implanted in the 100 μ l DMEM media (containing 10%FCS and 1% glutamine) (DMEM is Dulbecco ' s Modified Eagle ' sMedium (Sigma D6546)) in costar 96 orifice plates, at 37 ℃ and 5%CO ₂The middle placement spent the night to adhesion.Then cell is used in the compound administration of (add among the 50 μ l in each hole and obtain 0.00015 μ-1 μ M compound concentration) of dilution in the medium, is using compound treatment after 24 hours, fixed cell.

At first use the light microscopy cell, any morphological change of record cell.Add 100 μ l, 3.7% formaldehyde then to each hole, plate was placed 30 minutes in room temperature at least.Decantation and rap plate and remove stationary liquid on paper handkerchief uses automatic plate washer that plate is washed once in PBS (Dulbecco ' s phosphate buffer saline (Sigma D8537)) then.Add 100 μ l PBS and 0.5%triton X-100, plate is placed on mixing tank last 5 minute.Plate is washed in 100 μ lPBS, and solution raps to be removed.Be added in the original antibody of 50 μ l in PBS 1%BSA (bovine serum albumin) and 0.5% tween, rabbit resisted-the phosphorus histone H 3 in 1: 500.Buy anti-phosphorus histone H 3 rabbit polyclone 06-750 from UpstateBiotechnology.Plate was at room temperature placed 1 hour in mixing tank.

Second day, antibody is clean, with plate PBS washed twice.In unit surface, be added in PBS 1%BSA, 50 μ l second antibody in 0.5% tween, 1: 10,000 Hoechst and 1: 200 sub-IgGA of Alexa Fluor 488 goat antirabbits (article No. 11008 molecular probes).Plate is wrapped in the tinfoil paper thin slice, at room temperature shook 1 hour.Antibody is clean, use twice of PBS wash plate.Each hole adds 200 μ l PBS, and plate was shaken 10 minutes, removes PBS, adds 100 μ l PBS in each hole, and plate sealing etc. is to be analyzed.Cell levels and the nuclear area change of using array scanning target activation algorithm (Arrayscan Target Activation algorithm) to measure the phosphorus histone H 3 are finished analysis.The result is reported to and requires to obtain effective concentration that phosphorus histone H 3 level 50% suppresses and the effective concentration (EC50 value) that increases of nuclear area 50% similarly.The EC50 value that the phosphorus histone H 3 level that generally provides compound of the present invention suppresses is 0.5nM to 0.1 μ M.The IC50 of compound 1 is 5.5nM especially, compound 2 be 2nM and compound 3 for 2nM.

(d) external mdr cell phenotype and substrate phosphorylation analysis.

This analysis is used for the cytosis of external definite compound to resistance MCF7-ADR HBT cell.

With MCF7 cell multiple doses adriomycin pre-treatment (Dr.Hickinson, MolecularOncology lab, ICRF, University of Oxford Institute of Molecular Medicine, Headington, Oxford), this method causes cell transition to express drug-resistant protein.Compound typically causes the inhibition of phosphorus histone H 3 level and the increase of treatment nuclear area.But if compound is the proteic substrate of outflow of overexpression, they littler activity occurs in will analyzing than former SW620.

With every hole 0.8 * 10 ⁴The MCF7-ADR cell is implanted in costar 96 orifice plates in the 100 μ l DMEM media (containing 10%FCS (foetal calf serum) and 1% glutamine), at 37 ℃ and 5%CO ₂Spend the night to adhesion.

Those of all other method and above-mentioned use SW620 cell analysis are identical.

It is 0.5nM to 0.1 μ M that compound of the present invention generally has the phosphorus of inhibition histone H 3 EC50 value level.Compound 1 provides IC50 value 17nM especially, and compound 2 is that 20nM and compound 3 are 4nM.

The present invention illustrates with the following example now, unless otherwise indicated, and the known standard technique of chemical technology personnel and can adopt in suitable place wherein with those similar techniques of describing in these embodiments:

(i) evaporate into evaporation by rotation under vacuum, for example finish post-processing step behind the siccative by removing by filter residual solid;

(ii), typically under 18-25 ℃ of scope, operate in the air and carry out, except as otherwise noted, or remove non-technical personnel and will for example carry out under the argon gas at inert atmosphere especially in envrionment temperature;

(iii) column chromatography (passing through fast method) and medium pressure liquid chromatography method (MPLC) are finished on Merck Kiesel gel silica gel (Art.9385):

The productive rate that (iv) provides only illustrates, need not obtainable maximum;

(v) generally by nuclear (generally being proton) mr (NMR) and mass-spectrometric technique conclusive evidence, the proton resonance chemical displacement value uses a kind of at deuterated dimethyl sulfoxide (DMSO d of following 4 kinds of instruments to the structure of formula (I) final product ₆) (except as otherwise noted) measure (from the ppm of tetramethyl-silicomethane with end (downfield)) with δ and measure:

-Varian Gemini 2000 spectrographs are operated under intensity of field 300MHz

-Bruker DPX300 spectrograph is operated under intensity of field 300MHz

-JEOL EX 400 spectrographs are operated under intensity of field 400 MHz

-Bruker Avance 500 spectrographs, operation peak multiplicity shows below under intensity of field 500MHz: s, unimodal; D, bimodal; Dd, double doublet; T, triplet; Q, quartet; Qu, quintet; M, multiplet; Br s, wide unimodal;

(vi) automation synthetic (robotic synthesis) uses Zymate XP robot to finish, and adds solution by Zymate Master cut-and-try work station, by Stem RS5000 reaction-workstation 25 ℃ of stirrings;

(vii) finish as follows from the aftertreatment and the purifying of the synthetic reaction mixture that obtains of automation: evaporation uses Genevac HT 4 to finish under vacuum; AnachemSympur MPLC system on column chromatography use or the silica gel or use diameter 27mm Merck silica gel (60 μ m, 25g) pillar of Tian Chonging is finished; The structure of final product is used following condition conclusive evidence by LCMS (liquid chromatography mass) at the little mass spectrometer system of Waters2890/ZMD, and retention time (RT) is minute to provide:

Post: waters symmetry C183.5 μ m 4.6 * 50mm

Solvent orange 2 A: H ₂O

Solvent B:CH ₃CN

Solvent C: MeOH+5%HCOOH

Flow velocity: 2.5ml/min

Working time: 5 minutes, 4.5 minutes gradients from 0-100%C

Wavelength: 254nm, bandwidth 10nm

The little mass spectrum of mass detector: ZMD

Volume injected 0.005ml

(viii) the LCMS for the analysis of the synthetic preparation of automation of no use compound finishes in WatersAlliance HT system, uses following condition, and retention time (RT) is minute to provide:

Post: 2.0mm * 5Gm Phenomenex Max-RP 80A

Solvent orange 2 A: water

Solvent B: acetonitrile

Solvent C: methyl alcohol/1% formic acid or water/1% formic acid

Flow velocity: 1.1ml/min

Working time: 5 minutes, 4.5 minutes gradients from 0-95%B+ constant 5% solvent C

Wavelength: 254nm, bandwidth 10nm

Volume injected 0.005ml

Mass detector: Micromass ZMD

(ix) high performance liquid chromatography of preparation type (HPLC) or finish as follows:

-Waters prepares the LCMS instrument of type, minute to measure a retention time (RT):

Post: β-alkaline Hypercil (21 * 100mm) 5 μ m

Solvent orange 2 A: water/0.1% volatile salt

Solvent B: acetonitrile

Flow velocity: 25ml/min

Working time: 10 minutes, 7.5 minutes from the 0-100%B gradient

Wavelength: 254nm, bandwidth 10nm

Volume injected 1-1.5ml

Mass detector: Micromass ZMD perhaps is:

-Gilson prepares the HPLC instrument of type, uses a minute mensuration retention time (RT):

Post: 21mm * 15cm Phenomenex Luna2 C18

Solvent orange 2 A: water+0.1% trifluoroacetic acid,

Solvent B: acetonitrile+0.1% trifluoroacetic acid

Flow velocity: 21ml/min

Working time: 20 minutes, the gradient that changed from 5-100%B in 10 minutes

Wavelength: 254nm, bandwidth 10nm

Volume injected 0.1-4.0ml

(x) intermediate does not generally all characterize, and purity is by tlc (TLC), HPLC, infrared (IR), MS or NMR assay.

Table 1

Compound	R 1
		1	Me
2	H

Embodiment 1: compound 1-N in the table 1 (2, the 3-difluorophenyl)-2-{4-[(7-oxyethyl group-5-{[(2R)-1-methylpyrrolidin-2-yl] methoxyl group } quinazoline-4-yl) amino]-the 1H-pyrazol-1-yl } preparation of ethanamide

With N-(2, the 3-difluorophenyl)-2-[4-({ 7-oxyethyl group-5-[(2R)-tetramethyleneimine-2-ylmethoxy] quinazoline-4-yl } amino)-1H-pyrazol-1-yl] ethanamide (0.800g, 1.53mmol) and the mixture of formaldehyde (the 15ml37% aqueous solution) in formic acid (30ml) 90 ℃ of heating 2.5 hours.Make the solution that obtains be cooled to room temperature, then vaporising under vacuum.Resistates is handled with the solution (5%wt/v) of yellow soda ash in water (50ml), then with the dichloromethane mixture extracting twice that contains 10% methyl alcohol.

With the extraction liquid dried over mgso that merges, evaporation obtains the light brown solid, by the silica gel chromatography purifying, uses the dichloromethane mixture wash-out that contains 2-5% methyl alcohol to obtain the compound 1 of table 1 it, is colorless solid (0.624g, 76% productive rate):

¹H-NMR(DMSO d ₆)：10.28(s，1H)，10.11(s，1H)，8.45(s，1H)，8.28(s，1H)，7.72(m，1H)，7.57(s，1H)，7.20(m，2H)，6.75(d，1H)，6.52(d，1H)，5.16(s，2H)，4.43(dd，1H)，4.24(dd，1H)，4.1 8(q，2H)，3.16(m，1H)，2.56(m，1H)，2.37(s，3H)，2.27(q，1H)，2.02-1.93(m，1H)，1.85-1.62(m，3H)，1.39(t，3H).

MS(+ve ESI)：53 8(M+H) ⁺

Embodiment 2: compound 2-N-in the table 1 (2, the 3-difluorophenyl)-2-[4-({ 7-oxyethyl group-5-[(2R)-tetramethyleneimine-2-ylmethoxy] quinazoline-4-yl } amino)-1H-pyrazol-1-yl] preparation of ethanamide

At room temperature trifluoroacetic acid (8ml) is once added (the 2R)-2-[({4-[(1-{2-[(2 in stirring, the 3-difluorophenyl) amino]-the 2-oxoethyl }-1H-pyrazoles-4-yl) amino]-7-oxyethyl group quinazoline-5-yl } the oxygen base) methyl] (2.0g is 3.21mmol) in the solution in methylene dichloride (30ml) for tetramethyleneimine-1-t-butyl formate.The solution that obtains was at room temperature stirred 1 hour, then evaporation.Resistates is handled with the solution (5%wt/v) of yellow soda ash in water (30ml), then with the dichloromethane mixture extracting twice that contains 10% methyl alcohol.The organic moiety dried over mgso that merges, evaporation obtains pale solid then, and it by the silica gel chromatography purifying, is obtained the compound 2 of table 1 with the dichloromethane mixture wash-out that contains 5% methyl alcohol that contains 0-2% methanol ammonia (7M), be colorless solid (1.35g, 80% productive rate):

¹H-NMR(DMSO d ₆)：10.43(s，1H)，10.27(s，1H)，8.45(s，1H)，8.38(s，1H)，7.77(s，1H)，7.72(m，1H)，7.19(m，2H)，6.74(d，1H)，6.70(d，1H)，5.15(s，2H)，4.31(dd，1H)，4.17(q，2H)，3.93(t，1H)，3.63-3.57(m，1H)，2.96-2.81(m，3H)，1.91-1.83(m，1H)，1.80-1.63(m，2H)，1.50-1.42(m，1H)，1.38(t，3H).MS(+ve ESI)：524(M+H) ⁺

As (the 2R)-2-[({4-[(1-{2-[(2 of raw material, 3-difluorophenyl) amino]-the 2-oxoethyl-1H-pyrazoles-4-yl) amino]-7-oxyethyl group quinazoline-5-yl the oxygen base) methyl] tetramethyleneimine-1-carboxylic acid tert-butyl ester be prepared as follows described:

A) with 5,7-difluoro quinazoline-4 (3H)-ketone (2.0g, and 11.0mmol) (referring to: Hennequin, Laurent Francois Andre; Ple, Patrick.Preparation of 4-anilinoquinazolinederivatives for the treatment of tumors.PCT Int.Appl.WO01/094341) and sodium ethylate (3.7g, 54.4mmol) solution in dimethyl formamide 90 ℃ the heating 6 hours.Make this mixture be cooled to room temperature, be poured into then in ammonium chloride (100ml) solution.The precipitation that filtration obtains washes with water, and drying obtains 5 under high vacuum then, 7-diethoxy quinazoline-4 (3H)-ketone (1.36g, 53% productive rate):

MS(+ve ESI)：235(M+H) ⁺

B) with 5,7-diethoxy quinazoline-4 (3H)-ketone (0.700g, 2.99mmol) and sodium methylate (2.66ml 28%w/v methanol solution, 13.8mmol) solution in dimethyl formamide (7ml) 110 ℃ the heating 18 hours.Make mixture be cooled to room temperature, be adjusted to acidity by adding concentrated hydrochloric acid then,, be used in gradient acetonitrile (0.2% trifluoroacetic acid) wash-out in the water (0.2% trifluoroacetic acid) then by hplc (C18 silicagel column) purifying of preparation type.Merge the part that contains product, transfer to alkalescence, under vacuum, concentrate then by adding solid sodium bicarbonate.The precipitation that filtration obtains washes with water, and subsequently with the acetonitrile washing, with the ether washing, final dry air obtains 7-oxyethyl group-5-methoxyl group quinazoline-4 (3H)-ketone (0.1 98g, 30% productive rate) then:

¹NMR(DMSO d ₆)：7.94(s，1H)，6.64(s，1H)，6.53(s，1H)，4.15(q，2H)，3.83(s，3H)，1.37(t，3H).

MS(+ve ESI)：221(M+H) ⁺

C) (4.76g, (2.0g, 9.1mmol) (4.26g is 33.0mmol) 1, in the mixture in the 2-ethylene dichloride (50ml) with two-sec.-propyl ethylamine 31.0mmol) to join 7-oxyethyl group-5-methoxyl group quinazoline-4 (3H)-ketone with phosphorus oxychloride.Mixture was heated 6 hours at 80 ℃.Make mixture be cooled to room temperature, then vaporising under vacuum.Resistates is distributed between methylene dichloride and saturated sodium bicarbonate solution.

Separate organic layer, use dried over mgso, then vaporising under vacuum.Crude product obtains 4-chloro-7-oxyethyl group-5-methoxyl group quinazoline (2.0g, 92% productive rate) by fast silica gel chromatogram method purifying with eluent ethyl acetate:

MS(+ve ESI)：23 9/24 1(M+H) ⁺

D) with 4-chloro-7-oxyethyl group-5-methoxyl group quinazoline (1.90g, 7.97mmol) and 2-(4-amino-1H-pyrazol-1-yl)-N-(2, the 3-difluorophenyl) ethanamide (2.02g, 8.01mmol) mixture in 2-propyl alcohol (70ml) 90 ℃ the heating 45 minutes.Make reaction be cooled to room temperature, dilute with methyl tertiary butyl ether then.Filtering mixt obtains N-(2, the 3-difluorophenyl)-2-{4-[(7-oxyethyl group-5-methoxyl group quinazoline-4-yl) amino]-the 1H-pyrazol-1-yl } acetamide hydrochloride, be faint yellow solid (3.91g, 100% productive rate):

¹H-NMR(DMSO d ₆)：10.63(s，1H)，10.44(s，1H)，8.81(s，1H)，8.35(s，1H)，7.95(s，1H)，7.69(m，1H)，7.22(m，2H)，6.95(d，1H)，6.90(d，1H)，5.21(s，2H)，4.24(q，2H)，4.14(s，3H)，1.43(t，3H).

MS(+ve ESI)：455(M+H) ⁺

E) with N-(2, the 3-difluorophenyl)-and 2-{4-[(7-oxyethyl group-5-methoxyl group quinazoline-4-yl) amino]-the 1H-pyrazol-1-yl } ethanamide (5.0g, 10.2mmol) and pyridine hydrochloride (7.2g, 62.3mmol) mixture in pyridine (60ml) 120 ℃ the heating 18 hours.(4.0g 34.6mmol), continues heating 24 hours again to add another part pyridine hydrochloride.The solution that obtains is cooled to room temperature, then cancellation in water (150ml).The precipitation that filtration obtains washes with water, and drying obtains N-(2, the 3-difluorophenyl)-2-{4-[(7-oxyethyl group-5-hydroxyl quinazoline-4-yl under high vacuum then) amino]-the 1H-pyrazol-1-yl } ethanamide, be faint yellow solid (4.16g, 93% productive rate):

MS(+ve ESI)：441(M+H) ⁺

F) at room temperature in batches with azo-2-carboxylic acid's di tert butyl carbonate (1.25g, 5.44mmol) join the N-(2 in the stirring, the 3-difluorophenyl)-and 2-{4-[(7-oxyethyl group-5-hydroxyl quinazoline-4-yl) amino]-the 1H-pyrazol-1-yl } ethanamide (2.0g, 4.54mmol), (2R)-2-(hydroxymethyl) tetramethyleneimine-1-carboxylic acid tert-butyl ester (1.28g, 6.37mmol) and triphenylphosphine (1.43g is 5.46mmol) in the suspension in tetrahydrofuran (THF) (30ml).Mixture was at room temperature stirred 2 hours, add then another part triphenylphosphine (0.700g, 2.67mmol) and azo-2-carboxylic acid's di tert butyl carbonate (0.600g, 2.61mmol).At room temperature stirred the mixture 3 hours, then evaporating mixture.Resistates is by the silica gel chromatography purifying, obtain (2R)-2-[({4-[(1-{2-[(2 with the dichloromethane mixture wash-out that contains 2-5% methyl alcohol, the 3-difluorophenyl) amino]-the 2-oxoethyl }-1H-pyrazoles-4-yl) amino]-7-oxyethyl group quinazoline-5-yl } the oxygen base) methyl] tetramethyleneimine-1-carboxylic acid tert-butyl ester, be colorless solid (2.25g, 79% productive rate):

¹H-NMR(DMSO d ₆)：10.26(s，1H)，9.82(s)&9.52(s)(1H)，8.45(s，1H)，8.38(s，1H)，7.86(s，1H)，7.73(m，1H)，7.20(m，2H)，6.76(s，2H)，5.14(s，2H)，4.50-4.31(m，2H)，4.30-4.08(m，1H)，4.17(q，2H)，3.42-3.26(m，2H)，2.16-2.03(m，1H)，2.03-1.77(m，3H)，1.38(s，9H)，1.38(t，3H).

MS(+ve ESI)：624(M+H) ⁺

As raw material 2-(4-amino-1H-pyrazol-1-yl)-N-(2, the 3-difluorophenyl) ethanamide is prepared as follows described:

A) with 2,3-difluoroaniline (12.9g, 100mmol) the 1M aqueous sodium hydroxide solution (98ml of the solution in ether (100ml), 98mmol) handle, and vigorous stirring, dripped chloroacetyl chloride (13.3g, 117mmol) solution in ether (100ml) through 20 minutes 5 ℃ of whiles.Make mixture be warming up to 20 ℃, add ethyl acetate (100ml) then through 1 hour.Separate organic phase, with the washing of 20% potassium bicarbonate aqueous solution, drying, evaporation obtains white solid then.Solid is dissolved in the ebullient tetrahydrofuran (THF) (20ml), uses the dilution of hexanaphthene (300ml) and isohexane (100ml) then.Enriched mixture is to about 250ml, and cooling and filtration obtain 2-chloro-N-(2, the 3-difluorophenyl) ethanamide, are white crystal (18.48g, 90% productive rate):

¹H-NMR(DMSO d ₆)：10.26(br s，1H)，7.67(m，1H)，7.19(m，2H)，4.36(s，2H).MS(+ve ESI)：206，204(M+H) ⁺

B) (10.28g, 50mmol) (7 35g, 50mmol) (8.29g 60mmol) handles the solution in N,N-DIMETHYLACETAMIDE (20ml), stirs 18 hours under nitrogen atmosphere at 20 ℃ with salt of wormwood with 4-bromine pyrazoles with 2-chloro-N-(2, the 3-difluorophenyl) ethanamide.In mixture impouring water (300ml), filter and water (500ml) washing solid, and dry air.Solid is dissolved in the ebullient tetrahydrofuran (THF) (80ml), filters,, be evaporated to about 100ml then with hexanaphthene (100ml) dilution.The slurries that obtain obtain 2-(4-bromo-1H-pyrazol-1-yl)-N-(2, the 3-difluorophenyl) ethanamide with isohexane (100ml) dilution, cooling and filtration, are white solid (13.09g, 83% productive rate):

¹H-NMR(DMSO d ₆)：10.32(br s，1H)，8.00(s，1H)，7.70(m，1H)，7.59(s，1H)，7.19(m，2H)，5.14(s，2H).

MS(+ve ESI)：316，318(M+H) ⁺

C) with 2-(4-bromo-1H-pyrazol-1-yl)-N-(2, the 3-difluorophenyl) ethanamide (9.48g, 30mmol) with (9,9-dimethyl-9H-xanthene-4,5-two bases) two (diphenylphosphines) (1.74g, 3mmol) anhydrous 1, the solution in the 4-two  alkane (50ml) is with three (dibenzalacetones), two palladiums (0) (1.37g, 1.5mmol) handle, mixture was stirred 5 minutes under nitrogen.Once add benzophenone imine (5.7g, 31.5mmol), add subsequently sodium tert-butoxide (8.64g, 90mmol).Make the mixture degassing with nitrogen, under nitrogen, be heated to then 90 ℃ 4 hours.With the mixture cooling,, be poured into then in the saturated aqueous ammonium chloride solution (100ml) with ether (100ml) dilution.By the diatomite filtration mixture, separate each layer then.Use the dried over mgso organic phase, simmer down to oily matter is with ebullient hexanaphthene (200ml, 100ml) extracting twice.Evaporating cyclohexane solution obtains colloid, with it from isohexane: crystallization the ether 1: 1 obtains N-(2, the 3-difluorophenyl)-2-{4-[(phenylbenzene methylene radical) amino]-the 1H-pyrazol-1-yl ethanamide, be faint yellow solid (5.50g, 44% productive rate):

¹H-NMR(DMSO d ₆)：10.21(br s，1H)，7.66(m，3H)，7.56(m，3H)，7.44(m，3H)，7.35(s，1H)，7.24(m，2H)，7.18(m，2H)，6.48(s，1H)，4.98(s，2H).

MS(+ve ESI)：417(M+H) ⁺

D) N-(2 at room temperature will fully stirring, the 3-difluorophenyl)-and 2-{4-[(phenylbenzene methylene radical) amino]-the 1H-pyrazol-1-yl } ethanamide (2.08g, 5mmol) solution in ethyl acetate (25ml) dropwise added 37% aqueous hydrochloric acid (0.496ml, 6mmol) processing through 1 minute.Mixture was stirred 1 hour, filter then.Resistates washs with ethyl acetate and ether, and dry air obtains 2-(4-amino-1H-pyrazol-1-yl)-N-(2, the 3-difluorophenyl) acetamide hydrochloride then, is white powder (1.35g, 93% productive rate):

¹H NMR(DMSO d ₆)：10.48(s，1H)；10.22(br s，3H)；8.03(s，1H)；7.68(m，1H)；7.60(s，1H)；7.19(m，2H)；5.20(s，2H).

MS(+ve ESI)：253(M+H) ⁺

E) (1.685g 5.84mmol) is suspended in the mixture of ethyl acetate (70ml) and saturated sodium bicarbonate aqueous solution (35ml), stirs then 1 hour with 2-(4-amino-1H-pyrazol-1-yl)-N-(2, the 3-difluorophenyl) acetamide hydrochloride.Each of separating clarifying layer contains water with ethyl acetate (4 * 30ml) washings.The organic solution dried over mgso that merges is evaporated to solid then, obtains 2-(4-amino-1H-pyrazol-1-yl)-N-(2, the 3-difluorophenyl) ethanamide with the ether washing, is pink solid (1.377g, 94%).

¹H NMR(DMSO d ₆)：10.06(br s，1H)；7.70(m，1H)；7.17(m，2H)；7.08(s，1H)；6.98(s，1H)：4.90(s，2H)；3.84(br s，2H).

MS(+ve ESI)：253(M+H) ⁺

Compound 2 in the table 1 also is prepared as follows:

Embodiment 2ii: the compound 2-N-in the table 1 (2, the 3-difluorophenyl)-2-[4-({ 7-oxyethyl group-5-[(2R)-tetramethyleneimine-2-ylmethoxy] quinazoline-4-yl } amino)-1H-pyrazol-1-yl] preparation of ethanamide

Trifluoroacetic acid (5ml) is once added (2R)-2-[({4-[(1-{2-[(2 in stirring, the 3-difluorophenyl) amino]-the 2-oxoethyl }-1H-pyrazoles-4-yl) amino]-7-oxyethyl group quinazoline-5-yl } the oxygen base) methyl] (0.800g is 1.21mmol) in the suspension in methylene dichloride (20ml) for tetramethyleneimine-1-carboxylic acid tert-butyl ester hydrochloride.Mixture was at room temperature stirred 30 minutes, then evaporation.Resistates with the solution-treated of yellow soda ash (5%wt/v) in water (40ml), is extracted with the dichloromethane mixture that contains 10% methyl alcohol then.The organic moiety dried over mgso that merges, evaporation obtains faint yellow solid then, by the silica gel chromatography purifying, obtains the compound 2 of table 1 with the dichloromethane mixture wash-out that contains 5% methyl alcohol that contains 0-2% methanol ammonia (7M), be colorless solid (0.591g, 93%):

¹H-NMR(DMSO d ₆)：10.43(s，1H)，10.27(s，1H)，8.45(s，1H)，8.38(s，1H)，7.77(s，1H)，7.72(m，1H)，7.19(m，2H)，6.74(d，1H)，6.70(d，1H)，5.15(s，2H)，4.31(dd，1H)，4.17(q，2H)，3.93(t，1H)，3.63-3.57(m，1H)，2.96-2.81(m，3H)，1.91-1.83(m，1H)，1.80-1.63(m，2H)，1.50-1.42(m，1H)，1.38(t，3H).

MS(+ve ESI)：524(M+H) ⁺

As (the 2R)-2-[({4-[(1-{2-[(2 of raw material, 3-difluorophenyl) amino]-the 2-oxoethyl }-1H-pyrazoles-4-yl) amino]-7-oxyethyl group quinazoline-5-yl } the oxygen base) methyl] tetramethyleneimine-1-carboxylic acid tert-butyl ester hydrochloride preparation as described below:

A) Xiang Zaibing/water-bath refrigerative 3, the 5-difluorophenol (13.0g, 0.10mol) and salt of wormwood (20.9g, 0.15mol) add in the solution in dimethyl formamide (200ml) ethyl sulfate (13.1ml, 0.10mol).With mixture heating up to 80 ℃ 1.5 hours.Add another part ethyl sulfate (3.3ml, 25mmol) and salt of wormwood (5.2g, 37.5mmol), mixture further heated 2 hours.Make the solution that obtains be cooled to room temperature, be poured into then in the water, use extracted with diethyl ether then twice.The ether extraction liquid that merges washes with water, uses dried over mgso, and evaporation obtains 1-oxyethyl group-3 then, and 5-two fluorobenzene are yellow oil (13.59g, 86% productive rate):

¹H-NMR(DMSO d ₆)：6.77-6.65(m，3H)，4.06(q，2H)，1.32(t，3H).MS(+ve EI)：158(M ^·+)

B) (13.5ml 1.6M hexane solution 21.6mmol) is added drop-wise to 1-oxyethyl group-3 in the stirring, and (3.42g is 21.6mmol) in the solution in tetrahydrofuran (THF) (30ml) for 5-two fluorobenzene with n-Butyl Lithium at-78 ℃ under nitrogen atmosphere.Mixture was stirred 2 hours at-78 ℃, add excessive solidified carbon dioxide bead then.Make reaction mixture be warming up to room temperature, the solution that obtains is poured in the water.By adding aqueous sodium hydroxide solution, use the extracted with diethyl ether mixture then with mixture furnishing alkalescence.Separating mixture is by adding dilute hydrochloric acid with water layer furnishing acidity.With twice in extracted with diethyl ether mixture.Wash the ethereal extract of merging with water, use dried over mgso, evaporation obtains 4-oxyethyl group-2 then, and the 6-difluoro-benzoic acid is colorless solid (3.87g, 89% productive rate):

¹H-NMR(DMSO d ₆)：13.36(br.s，1H)，6.82-6.76(m，2H)，4.11(q，2H)，1.33(t，3H).

MS(+ve CI)：203(M+H) ⁺

C) (3.17ml 36.4mmol) is added drop-wise to 4-oxyethyl group-2 in the stirring, in 6-difluoro-benzoic acid (3.5g, 1 7.3mmol) and the suspension of dimethyl formamide (5) in methylene dichloride (50ml) with oxalyl chloride.The solution that obtains was at room temperature stirred 4 hours, then evaporation.Resistates is dissolved in the tetrahydrofuran (THF), is added drop-wise to then in 35% ammonia soln (60ml) of vigorous stirring.Filtering mixt is used the icy water debris, and drying obtains 4-oxyethyl group-2 under vacuum then, and the 6-difluorobenzamide is colorless solid (3.23g, 93% productive rate):

¹H-NMR(DMSO d ₆)：7.91(br.s，1H)，7.65(br.s，1H)，6.78-6.71(m，2H)，4.08(q，2H)，1.32(t，3H).

MS(+ve EI)：201(M ^·+)

D) at the 0-5 ℃ of 4-oxyethyl group-2 in stirring, the 6-difluorobenzamide (3.18g, 15.8mmol) and triethylamine (4.44ml, 31.6mmol) add in the suspension in methylene dichloride (25ml) trichoroacetic chloride (1.94ml, 17.4mmol).At 0-5 ℃ mixture was stirred 5 minutes.Water, dilute hydrochloric acid, dilute sodium hydroxide, dilute hydrochloric acid and final water wash the solution that obtains successively.Use dried over mgso organic solution, evaporation obtains 4-oxyethyl group-2 then, and 6-difluoro benzonitrile is faint yellow solid (2.56g 89% productive rate):

¹H-NMR(DMSO d ₆)：7.06-7.03(m，2H)，4.17(q，2H)，1.34(t，3H).MS(+ve CI)：184(M+H) ⁺

E) with 4-oxyethyl group-2,6-difluoro benzonitrile (8.0g, 44mmol) mixture in the saturated solution of ammonia in ethanol (270ml) in autoclave, be heated to 150 ℃ 16 hours.The solution that evaporation obtains is dissolved in resistates in the methylene dichloride, washes with water then.Use dried over mgso organic solution, under vacuum, concentrate, by the silica gel chromatography purifying, obtain 2-amino-4-oxyethyl group-6-fluorine benzonitrile with the methylene dichloride wash-out, colorless solid (6.07g, 77% productive rate) then:

¹H-NMR(DMSO d ₆)：6.32(s，2H)，6.14-6.10(m，2H)，3.99(q，2H)，1.30(t，3H).

MS(+ve EI)：180(M ^·+)

F) (2.5g 13.9mmol) joined in the mixture of the formic acid (20ml) of 100 ℃ of heating and the vitriol oil (5) in batches with 2-amino-4-oxyethyl group-6-fluorine benzonitrile through 20 minutes.Mixture makes it be cooled to room temperature 100 ℃ of heating 5 hours then.Mixture is poured in ice/water (80ml), collects the precipitation that obtains by filtering, with the ether washing, drying obtains 7-oxyethyl group-5-fluquinconazole quinoline-4 (3H)-ketone to water under vacuum then, is colorless solid (2.02g, 70% productive rate) subsequently:

¹H-NMR(DMSO d ₆)：12.06(br.S.1H)，8.01(s，1H)，6.91-6.85(m，2H)，4.17(q，2H)，1.36(t，3H).

MS(+ve ESI)：209(M+H) ⁺

G) will be in dimethyl formamide (7ml) under nitrogen atmosphere [(2R)-and 1-benzyl-pyrrole alkane-2-yl] methyl alcohol (1.1g, 57.6mmol) (0.461g 60% oil dispersant is 11.52mmol) in the suspension in dimethyl formamide (8ml) to be added drop-wise to sodium hydride in the stirring.The solution that obtains at room temperature stirred 30 minutes, and (1.0g 4.8mmol), further at room temperature stirred mixture 5 hours then to add 7-oxyethyl group-5-fluquinconazole quinoline-4 (3H)-ketone then.Add then another part [(2R)-1-benzyl-pyrrole alkane-2-yl] methyl alcohol (0.220g, 1.15mmol) and sodium hydride (0.092g, 2.3mmol), with reaction mixture be heated to 60 ℃ 1 hour.Make mixture be cooled to room temperature, be poured into then in the saturated ammonium chloride solution.The precipitation that filtration obtains, with the ether washing, drying obtains 5-{[(2R under vacuum then)-1-benzyl-pyrrole alkane-2-yl] methoxyl group }-7-oxyethyl group quinazoline-4 (3H)-ketone, be colorless solid (1.24g, 68% productive rate):

¹H-NMR(DMSO d ₆)：11.61(br.s，1H)，7.89(s，1H)，7.31-7.17(m，5H)，6.63(d，1H)，6.52(d，1H)，4.49(d，1H)，4.14(q，2H)，4.05-3.94(m，2H)，3.44(d，1H)，3.12-3.00(m，1H)，2.87-2.79(m，1H)，2.31-2.20(m，1H)，2.03-1.96(m，1H)，1.69(m，3H)，1.36(t，3H).

MS(+ve ESI)：380(M+H) ⁺

Described as being prepared as follows of [(2R)-1-benzyl-pyrrole alkane-2-yl] methyl alcohol of raw material:

With (brooethyl) benzene (2.36ml, 19.8mmol) and salt of wormwood (8.20g 59.3mmol) joins (2R)-tetramethyleneimine-(2.00g is 19.8mmol) in the solution in ethanol (40ml) and water (6ml) for 2-base methyl alcohol.With the solution that obtains be heated to 80 ℃ 4 hours, then the evaporation.Resistates water (150ml) is handled, and uses twice of extracted with diethyl ether.The organic extract that merges washes with water, uses dried over mgso, then evaporation.Resistates is by the silica gel chromatography purifying, obtains [(2R)-1-benzyl-pyrrole alkane-2-yl] methyl alcohol with the dichloromethane mixture wash-out that contains 5% methyl alcohol that contains 0-1% methanol ammonia solution (7M), is yellow oil (2.38g, 63% productive rate):

¹H NMR(DMSO d ₆)：7.33-7.27(m，4H)，7.25-7.20(m，1H)，4.37(t，1H)，4.04(d，1H)，3.48-3.43(m，1H)，3.34-3.26(m，2H)，2.78-2.74(m，1H)，2.59-2.53(m，1H)，2.17-2.11(m，1H)，1.88-1.79(m，1H)，1.65-1.53(m，3H).]

H) to 5-{[(2R)-1-benzyl-pyrrole alkane-2-yl] methoxyl group }-7-oxyethyl group quinazoline-4 (3H)-ketone (1.2g, 3.16mmol) and tert-Butyl dicarbonate (0.758g, 3.48mmol) add 10% palladium on carbon in the suspension in dimethyl formamide (15ml), at room temperature under nitrogen atmosphere, stirred the mixture 5 hours then.By the diatomite filtration mixture, evaporation then.Resistates is by the silica gel chromatography purifying, obtain (2R)-2-{[(7-oxyethyl group-4-oxo-3 with the methylene dichloride elution mixture that contains 5% methyl alcohol, 4-dihydroquinazoline-5-yl) oxygen base] methyl } tetramethyleneimine-1-carboxylic acid tert-butyl ester, be colorless solid (1.00g, 81% productive rate):

¹H-NMR(DMSO d ₆)：7.83(s，1H)，6.64(d，1H)，6.55(d，1H)，4.19-4.03(m，5H)，3.40-3.30(m，2H)，2.16-2.05(m，2H)，2.03-1.94(m，1H)，1.79-1.69(m，1H)，1.40(s，9H)，1.63(t，3H).

MS(+ve ESI)：390(M+H) ⁺

I) with (2R)-2-{[(7-oxyethyl group-4-oxo-3,4-dihydroquinazoline-5-yl) oxygen base] methyl } tetramethyleneimine-1-carboxylic acid tert-butyl ester (0.950g, 2.44mmol), N, N-diisopropyl ethyl amine (1.44ml, 8.30mmol) and phosphorus oxychloride (0.72ml, 7.81mmol) 1, the mixture in the 2-ethylene dichloride (20ml) was 80 ℃ of heating 2 hours.Make the solution that obtains be cooled to room temperature, then evaporation.Resistates is dissolved in the methylene dichloride, with the sodium bicarbonate aqueous solution washing, uses dried over mgso, then evaporation.Resistates obtains (2R)-2-{[(4-chloro-7-oxyethyl group quinazoline-5-yl by the silica gel chromatography purifying with eluent ethyl acetate) the oxygen base] methyl } tetramethyleneimine-1-carboxylic acid uncle ester, be yellow solid (0.621g, 63% productive rate):

MS(+ve ESI)：408(M+H) ⁺

J) with (2R)-2-{[(4-chloro-7-oxyethyl group quinazoline-5-yl) the oxygen base] methyl } tetramethyleneimine-1-carboxylic acid tert-butyl ester (0.615g, 1.51mmol) and 2-(4-amino-1H-pyrazol-1-yl)-N-(2, the 3-difluorophenyl) (0.380g, 1.51mmol) mixture in 2-propyl alcohol (20ml) obtained stiff precipitation in 30 minutes 70 ℃ of heating to ethanamide.Make mixture be cooled to room temperature,, filter then with the ether dilution.Use the ether debris, drying obtains (2R)-2-[({4-[(1-{2-[(2 under vacuum then, the 3-difluorophenyl) amino]-the 2-oxoethyl }-1H-pyrazoles-4-yl) amino]-7-oxyethyl group quinazoline-5-yl } the oxygen base) methyl] tetramethyleneimine-1-carboxylic acid tert-butyl ester hydrochloride, be emulsifiable paste shape solid (0.832g, 83% productive rate):

¹H-NMR(DMSOd ₆)：8.69(s，1H)，8.33(s，1H)，7.94(s，1H)，7.70-7.66(m，1H)，7.17-7.10(m，2H)，6.96(d，1H)，6.88(d，1H)，5.13(s，2H)，4.48(dd，1H)，4.42-4.37(m，1H)，4.30(dd，1H)，4.27(q，2H)，3.47-3.42(m，1H)，3.34-3.29(m，1H)，2.16-2.08(m，1H)，2.00-1.83(m，3H)，1.42(t，3H)，1.35(s，9H).

MS(+ve ESI)：624(M+H) ⁺

Embodiment 3: compound 3-N-(2, the 3-difluorophenyl)-2-[4-({ 7-oxyethyl group-5-[(3R)-morpholine-3-ylmethoxy] quinazoline-4-yl } amino)-1H-pyrazol-1-yl] preparation of ethanamide

With similarly reaction is described at embodiment 2ii, but be to use (3R)-3-[({4-[(1-{2-[(2, the 3-difluorophenyl) amino]-the 2-oxoethyl }-1H-pyrazoles-4-yl) amino]-7-oxyethyl group quinazoline-5-yl } the oxygen base) methyl] morpholine-4-carboxylic acid tert-butyl ester hydrochloride (540mg, 0.84mmol) obtain compound 3 (380 mg, 84% productive rate).

¹H NMR(DMSO d ₆)：10.1(br s，1H)，9.85(br s，1H)，8.43(s，1H)，8.27(s，1H)，7.74(s，1H)，7.71-7.66(m，1H)，7.19-7.11(m，2H)，6.77(d，1H)，6.65(d，1H)，5.08(s，2H)，4.35-4.22(m，3H)，4.17(q，3H)，3.88-3.84(m，1H)，3.73-3.68(m，1H)，3.52-3.44(m，2H)，3.38-3.33(m，1H)，3.03-2.88(m，2H)，1.37(t，3H).

MS(+ve ESI)：540(M+H) ⁺

As (the 3R)-3-[({4-[(1-{2-[(2 of raw material, 3-difluorophenyl) amino]-the 2-oxoethyl }-1H-pyrazoles-4-yl) amino]-7-oxyethyl group quinazoline-5-yl } the oxygen base) methyl] morpholine-4-carboxylic acid tert-butyl ester is prepared as follows described:

A) with in embodiment 2ii description similarly react, but be to use [(3S)-and 4-benzyl morpholine-3-yl] methylate hydrochlorate (843mg, 3.46mmol) and sodium hydride (392mg 60% oil dispersant, 9.79mmol) obtain 5-{[(3R)-4-benzyl morpholine-3-yl] methoxyl group }-7-oxyethyl group quinazoline-4 (3H)-ketone, it is not further purified and uses at next step.

As [(3S)-4-benzyl morpholine-3-yl] methylate hydrochlorate of raw material according to G.R.Brown et al.J.Chem.Soc.Perkins Trans.I, 1985,2577-2580. describes preparation.

B) with in embodiment 2ii description similarly react, but all 5-{[(3R)-and 4-benzyl morpholine-3-yl] methoxyl group }-7-oxyethyl group quinazoline-4 (3H)-ketone (approximately 2.9mmol) begins to obtain (3R)-3-{[(7-oxyethyl group-4-oxo-3,4-dihydroquinazoline-5-yl) oxygen base] methyl } morpholine-4-carboxylic acid tert-butyl ester (570mg is 49% through two step productive rates).

¹H NMR(DMSO d ₆)：7.84(s，1H)，6.65(dd，2H)，4.32(t，1H)，4.23-4.15(m，4H)，4.10-4.07(m 1H)，3.82-3.79(m，1H)，3.69-3.65(m，1H)，3.51-3.47(m，1H)，3 42-3.37(m 1H)，3.21-3.15(m，1H)，1.41(s，9H)，1.37(t，3H).

MS(+ve ESI)：406(M+H) ⁺

C) with in embodiment 2ii description similarly react, but with (3R)-3-{[(7-oxyethyl group-4-oxo-3,4-dihydroquinazoline-5-yl) oxygen base] methyl } morpholine-4-carboxylic acid tert-butyl ester (560mg, 1.4mmol) begin to obtain (3R)-3-{[(4-chloro-7-oxyethyl group quinazoline-5-yl) the oxygen base] methyl morpholine-4-carboxylic acid tert-butyl ester (420mg, 71% productive rate).

MS(+ve ESI)：424(M+H) ⁺

D) with in embodiment 2iij description similarly react, but with (3R)-3-{[(4-chloro-7-oxyethyl group quinazoline-5-yl) the oxygen base] methyl } morpholine-4-carboxylic acid tert-butyl ester (420mg, 0.99mmol) beginning, obtain (3R)-3-[({4-[(1-{2-[(2, the 3-difluorophenyl) amino]-the 2-oxoethyl }-1H-pyrazoles-4-yl) amino]-7-oxyethyl group quinazoline-5-yl } the oxygen base) methyl] morpholine-4-carboxylic acid tert-butyl ester hydrochloride (561mg, 84% productive rate).

¹H NMR(DMSO d ₆)：10.38(s，1H)，10.01(s，1H)，8.69(s，1H)，8.31(s，1H)，7.92(s，1H)，7.69-7.64(m，1H)，7.20-7.11(m，2H)，7.01(m，2H)，5.15(s，2H)，4.83-4.79(m，1H)，4.61-4.58(m，1H)，4.54-4.50(m，1H)，4.27(q，2H)，4.03(d，1H)，3.86-3.81(m，1H)，3.75-3.71(m，1H)，3.68-3.63(m，1H)，3.47-3.32(m，2H)，1.42(t，3H)，1.26(s，9H).

MS(+ve ESI)：640(M+H) ⁺

Claims (18)

1. the compound of formula (I)

Or its salt;

R wherein ¹For hydrogen or methyl and X are key or oxygen.

2. the compound or its salt of claim 1, wherein X is a key.

3. the compound or its salt of claim 2, it is N-(2, the 3-difluorophenyl)-2-[4-({ 7-oxyethyl group-5-[(2R)-tetramethyleneimine-2-ylmethoxy] quinazoline-4-yl } amino)-1H-pyrazol-1-yl] ethanamide.

4. the compound or its salt of claim 2, it is N-(2, the 3-difluorophenyl)-2-{4-[(7-oxyethyl group-5-{[(2R)-1-methylpyrrolidin-2-yl] methoxyl group } quinazoline-4-yl) amino]-the 1H-pyrazol-1-yl } ethanamide.

5. the compound or its salt of claim 1, wherein X is an oxygen.

6. the compound or its salt of claim 5, it is N-(2, the 3-difluorophenyl)-2-[4-({ 7-oxyethyl group-5-[(3R)-morpholine-3-ylmethoxy] quinazoline-4-yl } amino)-1H-pyrazol-1-yl] ethanamide.

7. the compound or its salt of claim 5, it is N-(2, the 3-difluorophenyl)-2-{4-[(7-oxyethyl group-5-{[(3R)-4-methylmorpholine-3-yl] methoxyl group } quinazoline-4-yl) amino]-the 1H-pyrazol-1-yl } ethanamide.

8. comprise pharmaceutical composition, mix mutually with pharmaceutically acceptable diluent or carrier as formula (I) compound or pharmaceutically acceptable salt thereof of claim 1 definition.

9. as formula (I) compound or pharmaceutically acceptable salt thereof of claim 1 definition, be used as medicine.

10. be used for the treatment of purposes in the excessively proliferative disease medicine as formula (I) compound or pharmaceutically acceptable salt thereof of claim 1 definition in preparation.

11. the purposes of claim 10, wherein excessively proliferative disease is a cancer.

12. the purposes of claim 11, wherein excessively proliferative disease is colorectal cancer, mammary cancer, lung cancer, prostate cancer, bladder cancer, kidney or pancreatic cancer or leukemia or lymphadenomatous any or its arbitrary combination.

13. treatment suffers from the people's of excessively proliferative disease method, comprises the step to formula (I) compound or pharmaceutically acceptable salt thereof of people's administering therapeutic significant quantity that this demand is arranged.

14. the method for claim 13, wherein excessively proliferative disease is a cancer.

15. the method for claim 14, wherein excessively proliferative disease is colorectal cancer, mammary cancer, lung cancer, prostate cancer, bladder cancer, kidney or carcinoma of the pancreas or leukemia or lymphadenomatous any or its arbitrary combination.

16. prepare wherein R ¹Be the method for formula (I) compound of methyl, comprise R wherein ¹For formula (I) compound of hydrogen and formaldehyde in formic acid, in the temperature that raises for example under 50 ℃ to 100 ℃, for example 30 minutes to 2 hours for some time of reaction, after this if necessary:

I) remove any protecting group; And/or

Ii) form its salt.

17. prepare wherein R ¹Method for formula (I) compound of hydrogen comprises N-(2, the 3-difluorophenyl)-2-{4-[(7-oxyethyl group-5-hydroxyl quinazoline-4-yl) amino]-the 1H-pyrazol-1-yl } the alcohol reaction of ethanamide and formula (II):

Wherein PG is suitable protecting group, for example tert-butoxycarbonyl (BOC), benzyloxycarbonyl (Z) or 9-fluorenyl methyl oxygen base carbonyl (Fmoc), after this if necessary:

I) remove any protecting group; And/or

Ii) form its salt.

18. prepare wherein R ¹Method for formula (I) compound of hydrogen comprises formula (III) compound

Wherein PG is suitable protecting group; for example tert-butoxycarbonyl (BOC), benzyloxycarbonyl (Z) or 9-fluorenyl methyl oxygen base carbonyl (Fmoc); with L be for example chlorine of suitable leaving group; with 2-(4-amino-1H-pyrazol-1-yl)-N-(2; the 3-difluorophenyl) ethanamide reaction, after this if necessary:

I) remove any protecting group; And/or

Ii) form its salt.