CN107540624A - Heat shock protein inhibitors and its preparation method and application - Google Patents

Abstract

The invention discloses a kind of heat shock protein inhibitors and its preparation method and application, belong to field of pharmaceutical chemistry technology.The heat shock protein inhibitors have Formulas I architectural feature.Such compound can suppress the activity of heat shock protein 90, and then can be used for preparing in anti-tumor drug.

Description

Heat shock protein inhibitors and its preparation method and application

Technical field

The present invention relates to field of pharmaceutical chemistry technology, more particularly to a kind of heat shock protein inhibitors and preparation method thereof And application.

Background technology

Heat shock protein (heat shock protein, HSP) is the highly conserved albumen of generally existing in organism Matter.According to molecular size range, heat shock protein is divided into：HSP100 (100-110kD), HSP90 (83-90kD), HSP70 (66- 78kD), HSP60 and small molecule HSP (15-30kD).Wherein, heat shock protein 90 (HSP90) is the molecule companion of ATP dependences Companion, participate in client protein (client protein) and activate and promote it ripe, maintain the conformation and function of cell multiple protein, Propagation, apoptosis, canceration and tumor development with cell is closely related.In ordinary cells, the HSP90 of expression accounts for intracellular total egg White 1-2%, and 3% among these is found in nucleus, it has the function of influenceing nucleus regulation.Stress shape Under state, such as in cancer cell, the HSP90 of expression level increases to the 4-6% for accounting for whole proteomics.More than HSP90 and 200 The different client protein interaction of kind, these client proteins are related to the conduction of signal, the transport of protein, recipient adult and fitted Answering property is immunized.And substantial amounts of critical tumorogenic albumen just belongs to client protein, such as：Her2、AKT、CDK4、VEGF、MET、ALK、 Mutation p53 etc..HSP90 mainly has four hypotypes：HSP90 α and HSP90 β (in cytoplasm), Grp94 hypotypes (are located at endoplasm In net), TRAP1 hypotypes (are located in mitochondrial matrix).HSP90 main molecules companion include Aha1, Hip, Hop, HSP70, CDC37/P50 etc..In cancer cell, HSP90 is in activated state, itself and the shape such as client protein and total molecule companion HSP70, p23 Into compound, client protein is protected not degraded by proteasome.The generation of cancer, development by HSP90 receptor proteins more ways Footpath, too many levels influence, so as to become the novel targets of cancer therapy drug effect.HSP90 inhibitor is different according to its binding site N- terminal inhibitors, intermediate ends inhibitor and C- terminal inhibitors can be divided into.

The stabilization of HSP90 client protein 26S Proteasome Structure and Functions needs HSP participation, and client protein then plays and promotes cell life The critical function such as long, propagation and survival, while it is in overexpression or continuous expression state in malignant tumour, with tumour Occurrence and development have close ties.Such as：Mutation EGFR, chronic myelogenous leukemia in non-small cell lung cancer cell (NSCLC) The maintenance of the conformation and function of the Tumor-specific proteins such as the Her2 in fusion protein BCR-ABL, breast cancer cell in cell HSP90 participation is needed with regulation and control.

The incidence of non-small cell lung cancer accounts for 3/4-the 4/5 of lung cancer, and EGFR has in about 3/5 non-small lung cancers cell Height expression.Current many patients are general to the treatment non-small cell lung cancer drug-Gefitinib and Erlotinib of FDA approval listings All over generation resistance.Research shows that HSP90 inhibitor 17-DMAG has antiproliferative to 20 kinds of EGFR non-small cell lung cancer cell strains Effect, it can substantially lower the level of p-EGFR, p-Akt, CyclinD1, Cdk4 etc. in EGFR mutant clones.And 17-AAG energy WT-EGFR level is lowered, simply needs higher concentration and action time.Cause non-small cell lung cancer to Gefitinib and angstrom Sieve is that main cause is K-Ras mutation for Buddhist nun's resistance.Although currently there are no the specific inhibitor for its treatment, Jamie etc. has found that Ganetespib can degrade K-Ras substrate C-Raf, inactivate some downstream signaling molecules.These all show HSP90 inhibitor has obvious inhibiting effect to non-small lung cancers cell, so as to antagonism non-small lung cancers cell to Gefitinib and angstrom Sieve replaces the resistance of Buddhist nun.

Proto-oncogene Her2, the clinical breast that Her2/neu is overexpressed to part are over-expressed in 20%-30% breast cancer Adenocarcinoma patients apply Trastuzumab monoclonal antibody more, but most of patients can produce resistance.Because Her2 is most relied on HSP90 and right HSP90 inhibitor is most sensitive, therefore Scaltriti etc. is with primary or secondary caused by PI3K mutation activation or PTEN expression declines Resistant models, research HSP90 inhibitor IPI504 are thin to the BT47R (Her2+) of processing, BT477H1047R (Her2+) breast cancer The effect of born of the same parents' strain.As a result show that IPI504 leads to that can lower in both the above cell Her2 expression and suppress AKT, MAPK signal Road, it is in dose dependent to suppress cell propagation.The main reason for producing resistance to Trastuzmab is HER2 extracellular regions missing P95-Her2 is produced so as to lose region in connection and then produce resistance.Chandarlapaty research finds HSP90 suppressions P95-Her2 in preparation SNX-2112 energy degradation of cell, while suppress AKT and ERK activity；Cytotoxicity test shows SNK- 2112 can completely inhibit T47D cells propagation, be much higher than Trastuzmab；SNX-2112 oral prodrugs are applied in vivo SNX-5422 effect expression p95-Her2 MEFS tumor models, it can completely inhibit the growth of tumour.Due to three negative breasts Gland cancer lacks anti-Her2 therapy target, and there is presently no obtain targetedly inhibitor.But experimental study shows that PU-H71 can Suppress AKT and Bcl-xl protein actives and lower its level, so as to induce triple negative breast cancer Apoptosis.It is to three negative breasts Adenocarcinoma cell strain HCC-1806, MAD-MB-231 and MAD-MB-468 cell mortality are respectively 80%, 65% and 80%.With Upper to understand, HSP90 inhibitor is of great significance as clinical breast cancer resistance treatment tool.

Also, the generation of chronic granulocytic leukemia and the mutation of abl gene are closely related, and Imatinib extensively should Treatment for chronic granulocytic leukemia.The leukaemic of resistance is produced to Imatinib mainly because of BCL-ABL kinases Area's producer amplification or point mutation cause.And this kinases is a kind of HSP90 client proteins, therefore HSP90 inhibitor should be able to It is acted on.Experimental study shows that natural HSP90 inhibitor Celastrol can make the Imatinib caused by T315I is mutated resistance to The KMB5-T315I apoptosis of medicine, immunohistochemical analysis show the expression which inhibits BCL-ABL.And synthesized micromolecule HSP90 inhibitor 17-AAG can degrade saltant type and wild type BCL-ABL, so as to suppress cell propagation.Therefore to Imatinib The chronic granulocytic leukemia of resistance can use HSP90 inhibitor to carry out antagonism.

Compared with traditional kinase inhibitor, HSP90 inhibitor suppresses a variety of similar kinases of being degraded while HSP90 activity, Preferable inhibition can be produced to some drug-resistant type tumours.Experiment show that HSP90 inhibitor is single or drug combination all With antineoplastic action.Therefore, HSP90 is a kind of target with research and development meaning for the treatment of of cancer.

The content of the invention

Based on this, the present invention provides a kind of new heat shock protein inhibitors, and such compound can suppress heat shock protein White 90 activity, and then can be used for preparing in anti-tumor drug.

A kind of heat shock protein inhibitors or its pharmaceutically acceptable salt with Formulas I architectural feature：

Wherein：

R₁、R₇、R₈It is respectively and independently selected from：H, C₁-C₆Alkyl, C₂-C₆Unsaturated alkyl, halogen, hydroxyl, C₁-C₆Alkoxy, NHCOOR, SO₂NHR, NHSO₂R, CN, NHCOR, CONHR；

R is selected from：H, C₁-C₆Alkyl, C₃-C₆Unsaturated alkyl, C₃-C₈Cycloalkyl；

R₂、R₃It is respectively and independently selected from：H, D, C₁-C₆Alkyl, C₃-C₆Unsaturated alkyl, C₃-C₈Cycloalkyl, phenyl are substituted Phenyl, heteroaryl, acyl group；

R₄It is selected from：Amino, C₁-C₆Saturated alkyl amine, C₂-C₆Saturated heterocyclic amine, C₁-C₆Alkylamidoalkyl, aryl amido group, Substituted aryl amido group, heteroaryl amide base, substituted heteroaryl amide base；

R₅、R₆For same oxygen atom or it is respectively and independently selected from：H, D, C₁-C₆Alkyl, C₃-C₈Cycloalkyl, carbonyl, cyano group, C₁-C₆Alkoxy, COOR ', NHCOR ', CONHR '；

R ' is selected from：H, C₁-C₆Alkyl, C₃-C₆Unsaturated alkyl；

X, Y, Z, W are respectively and independently selected from：C, NH, CH, N, O, S.

The present inventor has found that morpholine links in AUY922 (for the HSP90 inhibitor of a report) structure under study for action Benzyl site may be easily metabolized so as to produce toxic side effect.On the basis of the studies above, the present inventor is from height On the basis of phase experience accumulation, groped by experiment and found after study, in the other groups of benzyl site introducing, not only improved Activity is also possible to suppress the metabolism in the site so as to eliminate potential toxic side effect.

In one of the embodiments, five yuan of heteroaromatics comprising X, Y, Z, W are selected from following structure：

In one of the embodiments, the heat shock protein inhibitors are selected from below formula II compound：

Wherein：

R₇、R₈It is selected from：H, C₁-C₆Alkyl, C₂-C₆Unsaturated alkyl, halogen, hydroxyl, C₁-C₆Alkoxy, CN；

R₁It is selected from：C₁-C₆Alkyl, C₂-C₆Unsaturated alkyl, C₁-C₆Alkoxy.

In one of the embodiments, the R₂、R₃It is respectively and independently selected from：H, C₃-C₈Cycloalkyl, C₁-C₆Alkyl, C₃-C₆No Saturated alkyl.

In one of the embodiments, R₄It is selected from：Amino, C₁-C₆Saturated alkyl amine, C₂-C₆Saturated heterocyclic amine, C₁-C₆Alkane Base amide groups, aryl amido group, substituted aryl amido group, heteroaryl amide base, substituted heteroaryl amide base；

R₅It is selected from：H, or and R₆For same oxygen atom；

R₆It is selected from：CN, COOR ', CONHR ', or and R₅For same oxygen atom；

Wherein, R ' is selected from：H, C₁-C₆Alkyl, C₃-C₆Unsaturated alkyl, C₃-C₈Cycloalkyl.

In one of the embodiments, R₄Selected from following group：

In one of the embodiments, the heat shock protein inhibitors are selected from below formula III compound：

Wherein：

R₁It is selected from：C₁-C₆Alkyl；

R₇、R₈It is selected from：Hydroxyl；

R₂It is selected from：H；

R₃It is selected from：C₁-C₆Alkyl；

R₄Selected from following group：

R₅It is selected from：H, or and R₆For same oxygen atom；

R₆It is selected from：CN, COOR ', CONHR ', or with R5 be same oxygen atom

Wherein, R ' is selected from：H, C₁-C₆Alkyl, C₃-C₆Unsaturated alkyl, C₃-C₈Cycloalkyl.

The invention also discloses the preparation method of above-mentioned heat shock protein inhibitors or its pharmaceutically acceptable salt, adopt With following circuit combination：

Wherein：R₁₀For hydroxyl protecting group.

R₁₀It is selected from：Benzyl, trimethylsilyl, three second silicon substrates, t-Butyldimethylsilyl, tert-butyl diphenyl silicon substrate, two is special Butyl methyl silicon substrate, methyl, acetyl group, to methoxy-benzyl, methoxy.

The invention also discloses a kind of above-mentioned heat shock protein inhibitors or its pharmaceutically acceptable salt to prepare in advance Application in anti-and disease of the treatment with the increased pathological characteristicses of expression of heat shock protein 90 medicine.

In one of the embodiments, the disease with the increased pathological characteristicses of expression of heat shock protein 90 is： Cancer, metabolic disease, myelodysplastic syndrome, systemic mastocytosis, Xi Peier-lindau's syndrome, more in Cardioid Castleman diseases, and at least one of psoriasis.

Compared with prior art, the invention has the advantages that：

The heat shock protein inhibitors or its pharmaceutically acceptable salt of the present invention, it is a kind of new heat shock protein suppression Preparation, compared with conventional similar inhibitor, remain in original molecule with amino acid residue phase interaction in HSP90 receptor active pockets Atom and functional group, the position that active force is further there may be to skeleton are modified, so as to optimize control chemical combination The suppression effect of thing, makes it possess advantages below：

1st, effective modification is introduced, increases the adhesion of the amino acid residue of target compound and receptor protein, at the same it is right Two kinds of enzyme acceptors of HSP90 α and HSP90 β, and five class tumor cell lines are inhibited.

2nd, positive reference compound NVP/AUY-922 is further improved in enzyme level and the rejection ability of cellular level, is Promote the patent medicine process offer of such inhibitor may.

3rd, compared with single inhibitor, while the medicine for being respectively acting on using two single target spot is compared, and acts on two The medicine patient of individual target spot uses more convenient, can also avoid the interaction of medicine and medicine.

Embodiment

The present invention is described further with reference to embodiments, but does not cause any restrictions to the present invention.

The compound and its salt of the present invention can also be prepared by becoming known for preparing the method for chemical related compound, The raw material being related in embodiment can be obtained by the similar approach of prior art.

The unfixed substitution in position is represented by the straight line pointed to outside phenyl ring in phenyl ring in formula I of the present invention.

The five-membered ring with dotted line is to include X, Y, Z, W five yuan of heteroaromatics in formula I of the present invention.

" alkyl " refers to saturated hydrocarbyl, includes the alkyl of straight or branched, such as C₁-C₆Alkyl refers to there is 1 to 6 carbon original The saturated straight chain of son or the alkyl of side chain, the example of wherein straight chain saturated alkyl include but is not limited to ethyl, n-propyl etc., saturation The example of branched alkyl includes but is not limited to isopropyl, tert-butyl group etc.；" unsaturated alkyl " refers to the hydrocarbon with alkenyl or alkynyl Base, include the unsaturated alkyl of straight or branched, wherein the example of unsaturated straight chained alkyl includes but is not limited to vinyl, propylene Base etc., the example of unsaturated side chain alkyl include but is not limited to 2- methylpropenyls etc.；" cycloalkyl " refers to cyclic structure Alkyl, such as C₃-C₈Cyclic alkyl refers to saturation or the undersaturated alkyl with cyclic structure with 3 to 8 carbon atoms, its The example of middle saturated cyclic alkyls includes but is not limited to cyclopropyl, cyclopenta, ethyl substituted cyclohexyl etc., unsaturated cyclic alkyl Example include but is not limited to cyclopentene etc., preferred C in the present invention₃-C₆Ring-type alkane.

" substituted " refers to the hydrogen-based in the group displacement specific structure for specifying substituent.When in any specific structure When the substituent that more than one position can be selected from designated group through more than one substitutes, the substituent can be identical in each position It is or different.As used herein, term " substitution " expection, which includes all of organic compound, allows substituent.It is extensive one Aspect, the acyclic and cyclic for allowing substituent to include organic compound, branch and non-branch, carbocyclic ring and heterocycle, virtue Fragrant race and non-aromatic substituent.The hetero atoms such as nitrogen can have hydrogen substituent and/or organic compound as described herein Any to allow substituent, the substituent meets heteroatomic valence mumber.

" heteroaryl " refers to the 4n+2 aromatic rings systems containing 5-6 unit monocycles 6, and aromatic ring system have ring carbon atom and 1-4 ring hetero atom, wherein each ring hetero atom is independently selected from nitrogen, oxygen and sulphur.Include the heteroaryl of one or more nitrogen-atoms Base, tie point can be carbon or nitrogen-atoms, if crossing chemical valence allows.The multi-loop system of heteroaryl can include one or more Hetero atom." heteroaryl " also includes described heteroaryl ring-member as defined above, and fusion has one or more carbocyclic rings or heterocyclic radical Group, wherein tie point be on heteroaryl ring, and in this case, the quantity of ring memberses only include on heteroaryl ring system into The number of member." heteroaryl " also includes described heteroaryl ring-member as defined above, and fusion has one or more aromatic yl groups, its Middle tie point can be on aryl or heteroaryl ring, and in this case, the quantity of ring memberses only is included in fused polycycle Ring memberses number in (aryl/hetaryl) member ring systems.Polyheteroaromatic, one of them does not contain heteroatomic ring (for example, Yin Diindyl base, quinolyl, carbazyl, and the like), its tie point can be on any one ring, that is to say, that can wrap Containing (for example, 2- indyls) on heteroatomic ring or on heteroatomic ring is not contained (for example, 5- indyls).Containing 2 miscellaneous originals Exemplary 5 unit's heteroaryl of son includes but is not limited to, imidazoles, pyrazoles, oxazole, isoxazolyl, thiazolyl, and isothiazole.Contain 1 Individual heteroatomic exemplary 6 unit's heteroaryl includes but is not limited to, pyridine radicals.Include containing 2 heteroatomic exemplary 6 unit's heteroaryls But it is not limited to, pyridazinyl, pyrimidine radicals, and pyrazinyl.

" heterocyclic radical " refers to cyclic alkyl as herein defined, and wherein main chain also includes one or more hetero atoms (such as Oxygen, sulphur, nitrogen, boron, silicon, phosphorus).

" amido " refer to the hydrogen atom of ammonia replaced by alkyl after organic compound, such as heterocyclic amine, aralkyl amido.

" amide groups " refers to the derivative that the hydrogen of ammonia (or amine) is substituted by acyl group, as cyclic alkyl amides base, alkylamidoalkyl, Aryl amido group, benzo aryl amido group, heteroaryl amide base, benzo heteroaryl amide base, heterocyclyl amides base, sulfonamide Base, ureine etc..

Embodiment 1

Following examples are prepared with reference to following reaction schemes

Compound 1：4- (4- (cyano group (morpholino) methyl) phenyl) -5- (2,4- dihydroxy -5- isopropyl phenyls)-methyl Isoxazole -3- formamides) preparation.

Synthesize, comprise the following steps according to Above Transmission Lines：

(1) preparation of 1- (double (benzyloxy) phenyl of 2,4-) ethyl ketone (compound I-2).

Added in the 500mL two-mouth bottles equipped with reflux condensing tube 2,4-dihydroxyacetophenone I-1 (9.12g, 60.00mmol), potassium carbonate (21.00g, 151.00mmol), acetonitrile 200mL are heated to reflux 1h in oil bath pan at 80 DEG C, so Cylite (14.70mL, 144.00mmol) is injected with syringe continue backflow overnight afterwards.TLC (thin-layer chromatography) detections reaction is extremely After initiation material reaction completely, room temperature is cooled to, Buchner funnel filters, and dichloromethane debris collects filtrate, and revolving is removed Solvent is gone to obtain pale tan oil.Ether is added into grease, stirring produces white flock precipitate, and sand core funnel filters right Ether washs afterwards, repeats operation above and obtains white solid 15.80g, as compound I-2, yield 79% twice.Mass spectrum is supervised Survey ESI-MS m/z:333.0(M+H)⁺。

(2) preparation of (((4- propyl- 1- alkene -2- bases) -1,3- (dioxygen base)) double (methylene)) hexichol (compound I-3).

Potassium tert-butoxide (4.72g, 42.15mmol), triphenyl phosphorus iodine are added in the 250mL two-mouth bottles equipped with dropping funel Methane (17.88g, 42.15mmol), anhydrous THF (tetrahydrofuran) stirrings 1h is injected under 0 DEG C and nitrogen protective effect.Then 1- (2,4- double (benzyloxy) phenyl) ethyl ketone I-2 (10.76g, 32,42mmol) is dissolved completely in anhydrous THF and injects dropping liquid It is slowly added dropwise again through dropping funel in reaction system in funnel, 1h is stirred after being added dropwise completely, then reaction is stirred at room temperature Mix overnight.After TLC monitoring initiation material reactions completely, revolving removes reaction dissolvent, ethyl acetate dissolving gained residue, uses water Three times, saturated sodium-chloride water solution extracts once for extraction, collects ethyl acetate layer, then removed with anhydrous sodium sulfate drying, revolving Organic solvent, most purify through silicagel column to obtain white solid 8.45g, as compound I-3, yield 79% afterwards.

Compound I-3 characterize data is：¹H NMR(400MHz,Chloroform-d,δ_ppm):7.38-7.28(m, 10H), 7.13 (d, J=8.4Hz, 1H), 6.59 (s, 1H), 6.52 (d, J=8.4Hz, 1H), 5.08 (s, 2H), 5.03 (s, 2H),5.00(s,2H),2.12(s,3H)。

(3) preparation of 4- cumenes -1,3- glycol (compound I-4).

The addition I-3 (350.00g, 1.06mol) in 2L autoclave, 35g 10% palladium carbon, 1mL formic acid, 1L ethanol, It is passed through hydrogen and is heated to 78 DEG C and flows back two days.Room temperature is cooled to after reaction completely, reaction solution is used diatomaceous Bu Shi is housed Funnel filters, and ethanol washing, collects filtrate, and revolving removes organic solvent, finally purified to obtain white solid 130g with silicagel column, As compound I-4, yield 80%.

Compound I-4 characterize data is：¹H NMR(400MHz,Chloroform-d,δ_ppm):7.01 (d, J= 8.4Hz, 1H), 6.39 (d, J=8.4Hz, 1H), 6.32 (s, 1H), 5.50 (brs, 1H), 5.33 (brs, 1H), 3.12 (m, 1H), 1.21 (d, J=6.4Hz, 6H)

(4) preparation of 1- (2,4- dihydroxy -5- isopropyl phenyls)-ethyl ketone (compound I-5).

Added in the 250mL two-mouth bottles for be provided with reflux condensing tube 4- cumene -1,3- glycol I-4 (5.36g, 35.25mmol), then in N₂Protective effect under inject 100mL 47% boron trifluoride ether solutions stirring half an hour after, note Enter 4.03mL acetic acid heated overnight at reflux in oil bath pan, be cooled to room temperature after TLC monitoring initiation material reactions completely, add 70mL10% sodium acetate aqueous solutions stir 2h, are diluted with water to 250mL, add ethyl acetate extraction three times, collected organic layer, Revolving removes organic solvent and obtains brown-red solid after anhydrous sodium sulfate drying, and it is faint yellow insoluble to add dichloromethane mashing generation Thing, sand core funnel filter and are washed to obtain faint yellow solid 6.80g, as compound I-5, yield 99.4%. with dichloromethane

Compound I-5 characterize data is：¹H NMR(400MHz,Chloroform-d,δ_ppm):12.56(s,1H), 7.50 (s, 1H), 6.31 (s, 1H), 5.89 (s, 1H), 3.14 (m, 1H), 2,57 (s, 3H), 1.25 (d, J=6.0Hz, 6H)

(5) preparation of 1- (double (the benzyloxy) -5- isopropyls of 2,4-)-ethyl ketone (compound I-6).

1- (the double hydroxyl -5- isopropyls of 2,4-) acetophenone I-5 is added in the 250mL two-mouth bottles equipped with reflux condensing tube (6.80g, 35.05mmol), potassium carbonate (12.09g, 87.63mmol), 100mL acetonitriles, 80 DEG C of backflow 1h in oil bath pan, so Afterwards cylite (10mL, 84.15mmol) backflow is injected with syringe overnight.After TLC monitoring reaction initiation material reactions completely, instead System is answered to be cooled to room temperature, Buchner funnel is filtered, and residue is washed with dichloromethane, collects filtrate, and revolving removes organic solvent Obtain brownish red grease.Petroleum ether is added into stirring to pulp in grease, produces white precipitate, sand core funnel filters, oil Ether washs, and obtains white solid 6.72g, as compound I-6, yield 51.26%.

Compound I-6 characterize data is：¹H NMR(400MHz,Chloroform-d,δ_ppm):7.76(s,1H), 7.32-7.42(m,10H),6.51(s,1H),5.10(s,2H),5.09(s,2H),3.29(m,1H),2,56(s,3H),1.22 (d, J=6.8Hz, 6H)

(6) (Z)-ethyl -4- (double (the benzyloxy) -5- isopropyl phenyls of 2,4-) -2- hydroxyl -4- oxos but-2-ene acid The preparation of methyl esters (compound I-7).

Metallic sodium (0.90g, 39.00mmol), 100mL second are added in the 250mL two-mouth bottles for be provided with reflux condensing tube Alcohol is stirred in 0 DEG C of ice bath until after metallic sodium reaction completely, adds 1- (2,4- double (benzyloxy) -5- isopropyls at room temperature Base phenyl) ethyl ketone I-6 (6.72g, 17.98mmol) stirring 30min, injection diethy-aceto oxalate (3.94mL, 29.11mmol) is in oil 80 DEG C of backflow 4h in bath.TLC monitor to substrate reactions it is complete after be cooled to room temperature, add 2M aqueous hydrochloric acid solution regulation pH value Light yellow precipitate is produced to being less than 7, vacuum drying removes organic solvent, then adds dichloromethane dissolving, and water extracts three times, Saturated nacl aqueous solution extracts once, and revolving removes dichloromethane after anhydrous sodium sulfate drying, obtains yellow-brown solid 8.18g, As compound I-7 yields 96%.

Compound I-7 characterize data is：¹H NMR(400MHz,Chloroform-d,δ_ppm):7.87(s,1H), 7.45-7.35 (m, 11H), 6.53 (s, 1H), 5.13 (s, 2H), 5.11 (s, 2H), 4.28 (q, J=14.0Hz, 2H), 3.30 (m, 1H), 1.28 (t, J=6.8Hz, 3H), 1.23 (d, J=6.8Hz, 6H)

(7) 5- (systems of double (benzyloxy -5- isopropyl phenyls) isoxazole -3- carboxylic acid, ethyl esters (compound I-8) of 2,4- It is standby.

(Z)-ethyl -4- (double (benzyloxy) -5- of 2,4- are added in the 250mL two-mouth bottles for be provided with reflux condensing tube Isopropyl phenyl) -2- hydroxyl -4- oxo but-2-ene acid methyl esters I-7 (8.18g, 17.26mmol), hydroxylamine hydrochloride (1.56g, 22.48mmol) and 100mL ethanol in oil bath pan 80 DEG C backflow 5h.Room temperature is cooled to after TLC monitoring reactions completely, vacuum is done It is dry to obtain white solid.Ethyl acetate dissolved solid, water extract three times, and saturated sodium-chloride water solution extracts once, anhydrous slufuric acid Sodium dries organic layer, and revolving removes organic solvent and obtains white solid 6.74g, as compound I-8, yield 83%.

Compound I-8 characterize data is：¹H NMR(400MHz,Chloroform-d,δ_ppm):7.83(s,1H), 7.41-7.33 (m, 10H), 7.00 (s, 1H), 6.58 (s, 1H), 5.16 (s, 2H), 5.07 (s, 2H), 4.42 (q, J=7.2Hz, 2H), 3.34 (m, 1H), 1.41 (t, J=7.2Hz, 3H), 1.23 (d, J=6.8Hz, 6H)

(8) preparation of 5- (double (the benzyloxy) -5- isopropyl phenyls of 2,4-) isoxazole -3- formamides (compound I-9).

5- is added in the 250mL two-mouth bottles for be provided with reflux condensing tube, and (2,4- is double (benzyloxy -5- isopropyl phenyls) Isoxazole -3- carboxylic acid, ethyl esters I-8 (6.74g, 14.31mmol), 100mL ethanol heating stirring, make solid be injected after being completely dissolved 70% ethylamine solution (13.80mL, 215.73mmol), 80 DEG C of backflows in oil bath pan are stayed overnight.It is cold after TLC monitoring reactions completely But white solid is obtained to room temperature, vacuum drying.Ethyl acetate dissolved solid, 1M hydrochloric acid water extract once, saturated sodium carbonate Once, water extracts once aqueous solution extraction, and saturated sodium-chloride water solution extracts once, anhydrous sodium sulfate drying organic layer, then Revolving removes organic solvent and obtains white solid 6.46g, yield 96%.

Compound I-9 characterize data is：¹H NMR(400MHz,Chloroform-d,δ_ppm):7.79(s,1H), 7.33-7.39(m,10H),7.07(s,1H),6.81(brs,1H),6.55(s,1H),5.17(s,2H),5.14(s,2H), 3.48 (m, 2H), 3.33 (m, 1H), 1.27 (t, J=6.8Hz, 3H), 1.24 (d, J=6.8Hz, 6H)

(9) 5- (double (the benzyloxy) -5- isopropyl phenyls of 2,4-)-methylisoxazole -3- formamides (compound I-10) Preparation

5- (double (benzyloxy) -5- cumenes of 2,4- are added in the 250mL two-mouth bottles for be provided with reflux condensing tube Base)-N- ethyl isoxazole -3- formamides I-9 (6.46g, 13.47mmol), 100mL acetonitrile heating stirrings, make solid completely molten N-iodosuccinimide (3.71g, 16.49mmol) and ammonium ceric nitrate (0.74mg, 1.35mmol) are added after solution in oil bath pan 85 DEG C of backflows are overnight.Room temperature is cooled to after TLC monitoring reactions completely, vacuum drying obtains brown-red solid.Ethyl acetate dissolves Solid, water extract three times, and saturated sodium-chloride water solution extracts once, anhydrous sodium sulfate drying organic layer, and then rotating removing has Solvent, most purify through silicagel column to obtain brown white solid 5.62g, yield 70% afterwards.

Compound I-10 characterize data is：¹H NMR(400MHz,Chloroform-d,δ_ppm):7.40-7.29(m, 11H),6.81(brs,1H),6.59(s,1H),5.08(s,2H),5.05(s,2H),3.48(m,2H),3.34(m,1H),1.27 (t, J=6.8Hz, 3H), 1.23 (d, J=6.8Hz, 6H)

(10) 5- (2,4- double (benzyloxy) -5- isopropyl phenyls)-N- methyl -4- (4- Fonnylphenyls) isoxazole - The preparation of 3- formamides (compound I-11).

5- (double (benzyloxy) -5- cumenes of 2,4- are added in the 100mL two-mouth bottles for be provided with reflux condensing tube Base)-N- ethyl -4- iodo isoxazole -3- formamides 1-10 (1.35g, 2.27mmol), 4- formylphenylboronic acids (0.68g, 4.54mmol), two triphenyl phosphorus palladium chloride (0.08g, 0.12mmol), potassium carbonate (0.626mg, 4.54mmol), and 20mL tetrahydrofurans and 2mL distilled water, flowed back overnight in 80 DEG C of oil bath pans under nitrogen protective effect.TLC monitorings have been reacted Room temperature is cooled to after complete, revolving removes reaction dissolvent, adds ethyl acetate dissolving, and water extracts three times, saturated sodium-chloride water solution Extraction once, anhydrous sodium sulfate drying, purifies through silicagel column to obtain red brown solid 1.02g, yield 79% after vacuum drying.

Compound I-11 characterize data is：¹H NMR(400MHz,Chloroform-d,δ_ppm):9.96(s,1H), 7.70 (d, J=6.4Hz, 2H), 7.41 (d, J=6.4Hz, 2H), 7.39-7.33 (m, 5H), 7.27 (s, 2H) 7.19 (s, 1H), 7.00 (s, 2H), 6.82 (t, J=2.2Hz, 1H), 6.43 (s, 1H), 4.98 (s, 2H), 4.69 (s, 2H), 3.45 (m, 2H), 3.26 (m, 1H), 1.24 (t, J=5.6Hz, 3H), 1.11 (d, J=6.0Hz, 6H)

(11) 5- (2,4- dihydroxy -5- isopropyl phenyls)-N- methyl -4- (4- Fonnylphenyls) isoxazole -3- formyls The preparation of amine (compound I-12)

A 100mL two-mouth bottles are taken, compound I-11 (1g) is added, argon gas protection, adds anhydrous DCM (20mL) dissolvings, put At -5 DEG C, 15min is stirred, the dichloromethane solution (1M, 5.2mL, 3 equivalent) of Boron tribromide is added dropwise, 20min is stirred, is placed in At room temperature, 2h is stirred.The reaction of TLC tracking displays raw material finishes.At 0 DEG C, DCM (10mL) dilutions are added, add saturation NaHCO₃ Solution is quenched, and extraction, collects organic phase.EA is added into aqueous phase, the fluffy solid of generation is dissolved, collects organic phase, is mixed, Anhydrous sodium sulfate drying, concentrated pillar (DCM:Acetone=1:1).650mg brown solids, yield 94.8% can be obtained. ESI-MS m/z:395.1(M+H)⁺。

(12) 4- (4- (cyano group (morpholino) methyl) phenyl) -5- (2,4- dihydroxy -5- isopropyl phenyls) different evil of-methyl The preparation of azoles -3- formamides (compound 1).

A 25mL single port bottles are taken, add compound I-12 (50mg), methanol (2mL) dissolving, adding morpholine, (17 μ L, 1.5 work as Amount), guanidine hydrochloride (6mg, 0.5 equivalent), 15min is stirred at 40 DEG C, add compound TMSCN (47 μ L, 3 equivalents), the temperature Lower stirring 2h, TLC tracking display raw material reaction finishes.EA dilutions (10mL) are added, add saturation NaHCO₃Solution is quenched, saturation Saline solution extracts, anhydrous sodium sulfate drying, concentrated pillar (DCM:Acetone=1:1).Gained yellow, viscous product is beaten Slurry, petroleum ether is added, is stood under low temperature, white solid is separated out, sucks yellow liquid, gained solid oil pumping is done, and can obtain 25mg White solid, yield 41.0%.

The characterize data of the compound 1 is：¹H NMR(400MHz,Chloroform-d,δ_ppm):7.58 (d, J= 8.0Hz, 2H), 7.44 (d, J=8.0Hz, 2H), 6.80 (s, 1H), 6.32 (s, 1H), 4.80 (s, 1H), 3.72 (d, J= 3.6Hz, 4H), 3.57-3.40 (m, 2H), 2.99-2.92 (m, 1H), 2.61 (d, J=3.6Hz, 4H), 1.25 (t, J= 7.2Hz, 3H), 0.88 (d, J=6.8Hz, 6H)¹³C NMR(125MHz,Chloroform-d,δ_ppm):167.7,159.3, 157.1,156.6,154.0,132.8,131.4,130.7,128.5,128.1,127.6,115.3,105.8,104.5,67.0, 62.6,51.2,50.4,34.9,30.0,29.7,26.3,22.6,14.9.ESI-MS m/z:491.3(M+H)⁺,489.3(M- H)^-.

Embodiment 2

Compound 2：4- (4- (cyano group (2S, 6R) -2,6- thebaines) methyl) -5- (2,4- dihydroxy -5- isopropyls Phenyl)-N- ethyl isoxazole -3- formamides preparation.

A 25mL single port bottles are taken, compound I-12 (30mg) is added, methanol (1mL) dissolving, adds (2S, 6R) -2,6- bis- Methyl morpholine (15 μ L, 1.5 equivalents), guanidine hydrochloride (4mg, 0.5 equivalent), 15min is stirred at 40 DEG C, add compound TMSCN (29 μ L, 3 equivalents), TLC tracking display raw materials fail reaction completely and finished, therefore direct purification.EA dilutions (10mL) are added, are added Saturation NaHCO₃Solution is quenched, saturated aqueous common salt extraction, anhydrous sodium sulfate drying, concentrated pillar (DCM:Acetone=1: 1).Gained yellow, viscous product is purified by HPLC, gained solid oil pumping is done, and can obtain 9mg white solids, yield is 23.1%.

The characterize data of the compound 2 is：¹H NMR(400MHz,CD₃OD,δ_ppm):7.47 (d, J=8.0Hz, 2H), 7.37 (d, J=8.0Hz, 2H), 6.80 (s, 1H), 6.35 (s, 1H), 5.11 (s, 1H), 3.78 (t, J=7.6Hz, 1H), 3.59 (t, J=6.4Hz, 1H), 3.39,3.31 (dd, J=7.2,7.6Hz, 2H), 3.11-3.04 (m, 1H), 2.91 (d, J= 10.8Hz, 1H), 2.45 (d, J=10.4Hz, 1H), 2.21 (t, J=10.4Hz, 1H), 1.86 (t, J=10.4Hz, 1H), 1.21 (t, J=7.2Hz, 6H), 1.08 (d, J=6.0Hz, 3H), 0.99 (d, J=7.2Hz, 6H)¹³C NMR(125MHz, CD₃OD,δ_ppm):170.0,163.4,160.1,159.4,156.9,134.4,133.3,132.0,130.0,129.8,129.1, 117.2,117.0,107.3,104.8,73.8,73.7,63.3,60.1,60.1,55.0,54.9,36.3,28.1,24.0, 23.9,20.1,20.0,15.5.ESI-MS m/z:519.3(M+H)⁺,517.3(M-H)^-.

Embodiment 3

Compound 3：4- (4- (cyano group (4- methylpiperazine-1-yls) methyl) phenyl) -5- (2,4- dihydroxy -5- isopropyls Phenyl)-N- methylisoxazole -3- formamides preparation, with reference to embodiment 1 method.

The characterize data of the compound 3 is：¹H NMR(400MHz,CD₃OD,δ_ppm):7.477 (d, J=8.0Hz, 2H), 7.35 (d, J=8.4Hz, 2H), 6.81 (s, 1H), 6.36 (s, 1H), 5.06 (s, 1H), 3.39,3.36 (dd, J=7.2, 7.6Hz, 2H), 3.10-3.04 (m, 1H), 2.99 (d, J=11.2Hz, 1H), 2.62 (d, J=11.2Hz, 1H), 2.47 (t, J =8.8Hz, 1H), 2.10 (t, J=10.8Hz, 1H), 1.74 (d, J=8Hz, 1H), 1.61 (d, J=13.2Hz, 1H), 1.22 (t, J=12Hz, 3H), 0.98,0.95 (dd, J=7.2,6.4Hz, 9H)¹³C NMR(125MHz,CD₃OD,δ_ppm):169.9, 163.4,160.1,159.4,156.8,135.3,133.0,132.1,131.2,130.0,129.9,129.7,129.1, 117.6,117.0,107.3,104.8,63.9,54.9,36.3,36.3,35.9,32.6,28.1,23.9,23.8,23.8, 22.9,15.5.ESI-MS m/z:503.2(M+H)⁺,501.3(M-H)^-.

Embodiment 4

Compound 4：4- (4- (cyano group-(4- hydroxy piperidine -1- bases) methyl) phenyl) -5- (2,4- dihydroxy -5- isopropyls Phenyl)-N- methylisoxazole -3- formamides preparation, with reference to embodiment 1 method.

The characterize data of the compound 4 is：¹H NMR(400MHz,CD₃OD,δ_ppm):7.47 (d, J=8.0Hz, 2H), 7.36 (d, J=8.4Hz, 2H), 6.81 (s, 1H), 6.36 (s, 1H), 5.10 (s, 1H), 3.66-3.62 (m, 1H), 3.39, 3.55 (dd, J=7.6,7.2Hz, 2H), 3.10-3.04 (m, 1H), 2.92 (t, J=5.6Hz, 1H), 2.69 (t, J=5.6Hz, 1H), 2.49-2.43 (m, 1H), 2.27 (t, J=9.2Hz, 1H), 1.90 (t, J=12.8Hz, 2H), 1.67-1.58 (m, 1H), 1.53-1.29 (m, 1H), 1.21 (t, J=7.2Hz, 3H), 0.90 (d, J=6.8Hz, 6H)¹³C NMR(125MHz,CD₃OD, δ_ppm):169.9,163.4,160.1,159.4,156.9,135.3,133.0,132.0,130.0,129.7,129.1,117.5, 117.0,107.3,104.8,63.5,48.1,36.3,36.1,35.8,28.1,23.9,23.8,15.5.ESI-MS m/z: 505.3(M+H)⁺,503.3(M-H)^-.

Embodiment 5

Compound 5：4- (4- (cyano group (2- oxa- -6- azaspiros [3.3] hept- 6- yls) methyl) phenyl) -5- (2,4- dihydroxies Base -5- isopropyl phenyls)-methylisoxazole -3- formamides preparation, with reference to embodiment 1 method.

The characterize data of the compound 5 is：¹H NMR(400MHz,CD₃OD,δ_ppm):7.41 (d, J=8.0Hz, 2H), 7.37 (d, J=8.4Hz, 2H), 6.85 (s, 1H), 6.36 (s, 1H), 4.90 (s, 1H), 4.78 (t, J=7.6Hz, 4H), 3.55 (d, J=7.6Hz, 2H), 3.50 (d, J=7.6Hz, 2H), 3.41,3.37 (dd, J=7.2,7.2Hz, 2H), 3.14-3.07 (m, 1H), 1.23 (t, J=7.2Hz, 3H), 0.99 (d, J=7.2Hz, 6H)¹³C NMR(125MHz,CD₃OD,δ_ppm): 169.9,163.4,160.1,159.4,156.8,134.5,133.5,132.1,131.2,130.0,129.4,129.1, 118.1,117.0,107.3,104.7,82.8,62.4,62.1,41.0,36.3,28.1,23.9,23.8,15.5.ESI-MS m/z:503.3(M+H)⁺,501.3(M-H)^-.

Embodiment 6

Compound 6：4- (4- (cyano group (4AR, 7AS)-tetrahydrochysene -2H- [1,4] dioxanes simultaneously [2,3-c] pyrroles -6- (3H) - Base) methyl) phenyl) and -5- (2,4- hydroxyl -5- isopropyl phenyls)-N- methylisoxazole -3- formamides preparation, with reference to implement The method of example 1.

The characterize data of the compound 6 is：¹H NMR(400MHz,CD₃OD,δ_ppm):7.50 (d, J=8.4Hz, 2H), 7.38 (d, J=6.4Hz, 2H), 6.86 (s, 1H), 6.38 (s, 1H), 5.25 (s, 1H), 4.18-4.09 (m, 2H), 3.85- 3.79 (m, 2H), 3.61-3.57 (m, 2H), 3.42,3.39 (dd, J=7.2,7.2Hz, 2H), 3.14-3.07 (m, 1H), 3.00-2.99 (m, 2H), 2.86-2.83 (m, 1H), 1.24 (t, J=7.6Hz, 3H), 1.03 (d, J=6.8Hz, 6H)¹³C NMR(125MHz,CD₃OD,δ_ppm):169.9,163.5,160.1,159.5,156.8,135.4,133.2,132.0,130.0, 129.5,129.1,127.1,118.5,117.1,107.3,104.8,75.2,75.1,64.7,64.3,61.0,53.7,53.4, 36.3,31.6,28.1,24.6,23.9,15.5,15.3.ESI-MS m/z:533.1(M+H)⁺,531.2(M-H)^-.

Embodiment 7

Compound 7：4- (4- (cyano group (3- methylpyrrolidin- 1- yls) methyl) phenyl) -5- (2,4- dihydroxy -5- isopropyls Base phenyl)-N- methylisoxazole -3- formamides preparation, with reference to embodiment 1 method.

The characterize data of the compound 7 is：¹H NMR(400MHz,CD₃OD,δ_ppm):7.48 (d, J=8.0Hz, 2H), 7.37 (d, J=8.4Hz, 2H), 6.85 (d, J=2.4Hz, 1H), 6.37 (d, J=0.8Hz, 1H), 5.23 (s, 1H), 4.02- 3.96 (m, 1H), 3.41,3.37 (dd, J=7.2,7.2Hz, 2H), 3.29 (d, J=8.4Hz, 3H), 3.15-3.06 (m, 1H), 2.90-2.80 (m, 2H), 2.67-2.57 (m, 4H), 2.16-2.09 (m, 1H), 1.85-1.79 (m, 1H), 1.22 (t, J= 7.6Hz, 3H), 1.01 (d, J=1.2Hz, 6H) .ESI-MS m/z:505.1(M+H)⁺,503.2(M-H)^-.

Embodiment 8

Compound 8：4- (4- (2- thia -5- azabicyclos [2.2.1] hept- 5- bases (cyano group) methyl) phenyl) -5- (2,4- Dihydroxy -5- isopropyl phenyls)-methylisoxazole -3- formamides preparation, with reference to embodiment 1 method.

The characterize data of the compound 8 is：¹H NMR(400MHz,CD₃OD,δ_ppm):7.55 (d, J=8.4Hz, 1H), 7.50 (d, J=8.0Hz, 1H), 7.40 (t, J=6.4Hz, 2H), 6.86 (d, J=2.8Hz, 1H), 6.38 (s, 1H), 5.24 (s,0.5H),5.08(s,0.5H),3.99(s,0.5H),3.79(s,0.5H),3.56-3.51(m,1H),3.42,3.39(dd, J=7.2,7.6Hz, 2H), 3.25 (d, J=5.2Hz, 1H), 3.12-3.06 (m, 2H), 3.02 (t, J=10Hz, 1H), 2.90, 2.86 (dd, J=9.2,10Hz, 1H), 2.42,2.38 (dd, J=11.2,9.6Hz, 1H), 1.83 (t, J=13.6Hz, 1H), 1.22 (t, J=4Hz, 3H), 1.03 (d, J=6.8Hz, 6H)¹³C NMR(125MHz,CD₃OD,δ_ppm):170.0,163.4, 160.1,159.4,156.8,136.6,136.1,133.2,132.1,130.0,129.7,129.5,129.1,120.4, 117.3,107.3,104.8,64.6,64.3,63.9,62.3,60.1,59.7,47.0,46.9,41.7,41.3,39.6, 39.0,36.3,31.6,28.1,23.9,15.5.ESI-MS m/z:519.0(M+H)⁺,517.3(M-H)^-.

Embodiment 9

Compound 9：4- (4- (4- methylpiperazine-1-yls) (cyano group) methyl) phenyl) -5- (2,4- dihydroxy -5- isopropyls Phenyl)-N- methylisoxazole -3- formamides preparation, with reference to embodiment 1 method.

The characterize data of the compound 9 is：¹H NMR(400MHz,CD₃OD,δ_ppm):7.49 (d, J=7.2Hz, 2H), 7.38 (d, J=7.2Hz, 2H), 6.83 (s, 1H), 6.35 (s, 1H), 5.19 (s, 1H), 3.58 (s, 4H), 3.38 (d, J= 7.6Hz, 2H), 3.10-3.07 (m, 1H), 2.60-2.53 (m, 4H), 2.10 (s, 3H), 1.21 (t, J=7.2Hz, 3H), 1.00 (dd, J=6.4Hz, 6H)¹³C NMR(125MHz,CD₃OD,δ_ppm):172.1,171.8,169.8,162.9,162.1, 158.8,156.3,132.0,129.8,129.4,128.9,116.8,112.0,107.0,104.6,95.6,63.1,51.4, 50.8,47.9,43.1,36.1,36.3,31.3,27.7,23.8,22.0,15.5.ESI-MS m/z:532.3(M+H)⁺, 530.3(M-H)^-.

Embodiment 10

Compound 10：4- (4- (cyano group (2- methoxy ethyls) amino) methyl) phenyl) -5- (2,4- dihydroxy -5- isopropyls Base phenyl)-methylisoxazole -3- formamides preparation, with reference to embodiment 1 method.

The characterize data of the compound 10 is：¹H NMR(400MHz,CD₃OD,δ_ppm):7.47 (d, J=8.0Hz, 2H), 7.36 (d, J=8.0Hz, 2H), 6.89 (s, 1H), 6.34 (s, 1H), 5.00 (s, 1H), 3.53 (t, J=4.8Hz, 1H), 3.38,3.35 (dd, J=2.0,3.6Hz, 2H), 3.34 (s, 3H), 3.10-3.07 (m, 1H), 2.87 (t, J=5.2Hz, 2H), 1.21 (t, J=7.2Hz, 3H), 1.02 (d, J=6.8Hz, 6H) .ESI-MS m/z:479.1(M+H)⁺,477.2(M-H)^-.

Embodiment 11

Compound 11：4- (4- (2- amino -1- morpholino -2- oxygen ethyl) phenyl) -5- (2,4- dihydroxy -5- isopropyls Phenyl)-methylisoxazole -3- formamides preparation.

A 25mL single port bottles are taken, add compound 1 (30mg), add DMSO (1mL) dissolvings, add 30% hydrogen peroxide (20mg, 3 equivalents) and potassium carbonate (23mg, 3 equivalents), is stirred at room temperature 2h, and the reaction of TLC tracking displays raw material finishes.It is dilute to add EA Release (10mL) and extracted with water (10mL), organic phase is extracted with saturated aqueous common salt, anhydrous sodium sulfate drying, concentrated pillar (EA: PE=2:1).Gained yellow, viscous product is beaten, petroleum ether is added, is stood under low temperature, separate out white solid, suck yellow Liquid, gained solid oil pumping are done, and can obtain 3.4mg white solids, yield 11.0%.

The characterize data of the compound 11 is：¹H NMR(400MHz,CD₃OD,δ_ppm):7.45 (d, J=8.4Hz, 2H), 7.31 (d, J=8.4Hz, 2H), 6.81 (s, 1H), 6.35 (s, 1H), 3.74 (s, 1H), 3.73 (t, J=4.4Hz, 4H), 3.40,3.37 (dd, J=7.2,7.2Hz, 2H), 3.09-3.06 (m, 1H), 2.46-2.37 (m, 4H), 1.21 (t, J= 7.2Hz, 3H), 0.99 (d, J=6.8Hz, 6H) .ESI-MS m/z:509.1(M+H)⁺,507.2(M-H)-.

Embodiment 12

Following examples are prepared with reference to following reaction schemes.

According to Above Transmission Lines, comprise the following steps：

(1) ((5- (double (the benzyloxy) -5- isopropyl phenyls of 2,4-) -3- (methylcarbamoyl) is different by 4- by methyl -2- Oxazole -4- bases) phenyl) -2- hydroxyls (compound ii -1) preparation.

A 50mL single port bottles are taken, compound I-11 (1g) is added, adds the powdered iodine of catalytic amount, add anhydrous DCM (10mL) dissolves, and TMSCN (0.26mL, 1.2 equivalents) is added dropwise, and 1h is stirred at room temperature, and the reaction of TLC tracking displays raw material finishes.Add DCM dilutes (10mL), adds water (5mL) and is quenched, and organic phase is extracted with saturated aqueous common salt, anhydrous sodium sulfate drying, and concentration is direct Input is in next step.

Gained compound is added to the methanol solution (20%, 15mL) of hydrogen chloride, is placed at 40 DEG C and is stirred overnight.TLC with Track shows that raw material reaction finishes.EA (30mL) dilutions are added, the solid of generation is filtered, filtrate concentration is collected, adds EA (20mL) solution, add saturation NaHCO₃Solution neutralizes, saturated aqueous common salt extraction, anhydrous sodium sulfate drying, concentrated pillar (PE:EA=4:1).White solid 1.05g is obtained, two step yields are 95.4%.ESI-MS m/z:635.1(M+H)⁺。

(2) ((5- (double (the benzyloxy) -5- isopropyl phenyls of 2,4-) -3- (ethylaminocarbonyl) is different by 4- by methyl -2- Oxazole -4- bases) phenyl) -2- (tosyloxy) methyl acetate (compound ii -2) preparation.

A 50mL single port bottles are taken, add compound ii -1 (0.5g), anhydrous DCM (10mL) dissolvings is added, adds dry Triethylamine (0.33mL, 3 equivalents), 0 DEG C is placed in, stirs 15min, add TsCl (0.46g, 3 equivalents), stirred 15min, be warming up to 40 DEG C, it is stirred overnight.The reaction of TLC tracking displays raw material finishes.Add saturation NaHCO₃Solution neutralizes, saturated aqueous common salt extraction, Anhydrous sodium sulfate drying, concentrated pillar (PE:EA=6:1).Obtain white solid 0.34g, yield 55.2%.ESI-MS m/ z:789.2(M+H)⁺。

(3) ((5- (double (the benzyloxy) -5- isopropyl phenyls of 2,4-) -3- (ethylaminocarbonyl) is different by 4- by methyl -2- Oxazole -4- bases) phenyl) -2- morpholinoes acetic acid esters (compound ii -3) preparation.

A 50mL single port bottles are taken, add compound ii -2 (0.33g), toluene (5mL) dissolving is added, adds three dry second Amine (0.12mL, 2 equivalents), morpholine (55 μ L, 1.5 equivalents) is added, be placed in oil bath pan backflow 2h, the reaction of TLC tracking displays raw material Finish.EA (15mL) dilutions are added, saturated aqueous common salt extraction is added, collects organic phase, anhydrous sodium sulfate drying, concentrate under being used for One step.Obtain thick product 232mg, yield 80%.ESI-MS m/z:7032(M+H)⁺。

(4) methyl -2- (4- (5- (2,4- dihydroxy -5- isopropyl phenyls) -3- (ethylaminocarbonyl) isoxazoles -4- Base) phenyl) -2- morpholinoes acetic acid esters (compound 12) preparation.

A 50mL two-mouth bottles are taken, compound ii -3 (0.2g) is added, argon gas protection, adds anhydrous DCM (10mL) dissolvings, put At -5 DEG C, 15min is stirred, the dichloromethane solution (1M, 0.9mL, 3 equivalent) of Boron tribromide is added dropwise, 10min is stirred, is placed in At room temperature, 1h is stirred.The reaction of TLC tracking displays raw material finishes.At 0 DEG C, DCM (10mL) dilutions are added, add saturation NaHCO₃ Solution is quenched, and extraction, collects organic phase.EA is added into aqueous phase, the fluffy solid of generation is dissolved, collects organic phase, is mixed, Anhydrous sodium sulfate drying, concentrated pillar (DCM:Acetone=1:1).Gained yellow, viscous product is beaten, adds oil Ether, standing under low temperature, separate out white solid, suck yellow liquid, gained solid oil pumping is done, and can obtain 35.7mg white solids, Yield is 24.0%.

The characterize data of the compound 12 is：¹H NMR(400MHz,CD₃OD,δ_ppm):7.40 (d, J=8.4Hz, 2H), 7.30 (d, J=8.4Hz, 2H), 6.76 (s, 1H), 6.36 (s, 1H), 4.02 (s, 1H), 3.68 (d, J=4.4Hz, 4H), 3.66 (s, 3H), 3.39,3.35 (dd, J=7.2,7.6Hz, 2H), 3.09-3.02 (m, 1H), 2.45 (d, J=4.4Hz, 4H), 1.20 (t, J=7.6Hz, 3H), 0.95 (d, J=6.8Hz, 6H)¹³C NMR(125MHz,CD₃OD,δ_ppm):174.1,169.8, 163.4,160.0,159.4,156.9,136.7,132.8,132.0,130.8,130.1,129.0,117.1,107.3, 104.8,76.0,68.5,53.6,53.4,36.3,28.0,26.1,23.9,15.5.ESI-MS m/z:524.3(M+H)⁺, 523.3(M-H)^-.

Embodiment 13

Compound 13：((5- (2,4- dihydroxy -5- isopropyl phenyls) -3- (ethylaminocarbonyl) is different by 4- by ethyl -2- Oxazole -4- bases) phenyl) -2- morpholino acetic acid esters preparation, with reference to embodiment 12 method.

The characterize data of the compound 13 is：¹H NMR(400MHz,CD₃OD,δ_ppm):7.41 (d, J=8.0Hz, 2H), 7.31 (d, J=8.0Hz, 2H), 6.77 (s, 1H), 6.36 (s, 1H), 4.20-4.05 (m, 2H), 4.00 (s, 1H), 3.69 (t, J =4.8Hz, 3H), 3.39,3.35 (dd, J=7.2,7.2Hz, 2H), 3.14-3.02 (m, 1H), 2.46 (d, J=4.4Hz, 4H), 1.21,1.18 (dd, J=7.2,6.8Hz, 6H), 0.95 (d, J=7.2Hz, 6H)¹³C NMR(125MHz,CD₃OD, δ_ppm):173.6,169.8,163.5,160.0,159.5,156.9,131.9,130.8,130.0,129.0,117.1,107.3, 104.8,76.1,68.5,53.1,53.4,36.3,28.0,23.9,15.5,15.2.ESI-MS m/z:538.3(M+H)⁺, 536.3(M-H)^-.

The structural formula of the compound 13 is：

Embodiment 14

Compound 14：5- (2,4- dihydroxy -5- isopropyl phenyls)-N- methyl -4- (4- (2- (dimethylamino) -1- Quinoline generation -2- oxygen ethyl) phenyl) isoxazole -3- formamides preparation.

(1) 5- (double (the benzyloxy) -5- isopropyl phenyls of 2,4-)-N- methyl -4- (4- (2- (dimethylamino) -1- hydroxyls Base -2- oxoethyls) phenyl) isoxazole -3- formamides (compound ii -5) preparation.

A 50mL single port bottles are taken, add compound ii -4 (0.5g), ethylamine solution (68%, 0.2mL, 4 equivalent), ethanol (5mL), is placed in oil bath pan, and back flow reaction is overnight, and the reaction of TLC tracking displays raw material finishes.Most of solvent is removed in rotation, adds EA (20mL) and saturated aqueous common salt are extracted, anhydrous sodium sulfate drying, and concentration is directly used in next step.The thick product of gained (0.48g), yield 96.2%.ESI-MS m/z:648.3(M+H)⁺。

(2) compound 14：5- (2,4- dihydroxy -5- isopropyl phenyls)-N- methyl -4- (4- (2- (dimethylamino) - 1- morpholino -2- oxygen ethyl) phenyl) isoxazole -3- formamides preparation, with reference to embodiment 12 method.

The characterize data of the compound 14 is：¹H NMR(400MHz,CD₃OD,δ_ppm):7.40 (d, J=8.4Hz, 2H), 7.29 (d, J=8.0Hz, 2H), 6.79 (s, 1H), 6.35 (s, 1H), 3.70 (t, J=4.4Hz, 5H), 3.38,3.42 (dd, J =7.2,7.6Hz, 2H), 3.24-3.14 (m, 2H), 3.09-3.02 (m, 1H), 2.40-2.34 (m, 4H), 1.19 (t, J= 7.2Hz, 3H), 1.10 (t, J=7.2Hz, 3H), 0.96 (d, J=6.8Hz, 6H)¹³C NMR(125MHz,CD₃OD,δ_ppm): 174.1,169.7,163.5,160.0,159.5,156.8,137.9,132.3,131.7,130.6,130.0,129.0, 117.2,107.4,104.8,78.1,68.6,54.1,36.3,36.0,28.1,23.1,23.9,15.6,15.5.ESI-MS m/ z:537.3(M+H)⁺,535.3(M-H)^-.

Embodiment 15

Compound 15：((5- (2,4- dihydroxy -5- isopropyl phenyls) -3- (ethylaminocarbonyl) is different by 4- by methyl -2- Azoles -4- bases) phenyl) -2- (4AR, 7AS)-tetrahydrochysene -2H- [1,4] dioxanes simultaneously [2,3-c] pyrroles -6- (3H)-yl) ethyl acetate Preparation, with reference to embodiment 12 method.

The characterize data of the compound 15 is：¹H NMR(400MHz,CD₃OD,δ_ppm):7.41 (d, J=8.4Hz, 2H), 7.30 (d, J=8.1Hz, 2H), 6.77 (s, 1H), 6.36 (s, 1H), 4.25 (s, 1H), 4.09-4.04 (m, 2H), 3.80- 3.75 (m, 2H), 3.65 (s, 3H), 3.57-3.49 (m, 2H), 3.39,3.33 (dd, J=7.2,7.2Hz, 2H), 3.09-2.99 (m, 2H), 2.84-2.71 (m, 2H), 2.76-2.68 (m, 1H), 1.20 (t, J=7.2Hz, 3H), 0.96 (d, J=2.8Hz), 0.95 (d, J=2.8Hz)¹³C NMR(125MHz,CD₃OD,δ_ppm):174.2,169.8,163.5,160.0,159.5, 156.9,138.1,132.7,131.9,130.4,130.1,129.1,117.1,107.3,104.8,75.5,75.3,75.0, 64.7,64.2,55.5,54.1,53.5,36.3,31.6,31.2,28.0,23.9,15.5.ESI-MS m/z:566.2(M+H )⁺,564.1(M-H)^-.

The structural formula of the compound 15 is：

Embodiment 16

Compound 16：((5- (2,4- dihydroxy -5- isopropyl phenyls) -3- (ethylaminocarbonyl) is different by 4- by ethyl -2- Azoles -4- bases) phenyl) -2- (4AR, 7AS)-tetrahydrochysene -2H- [1,4] dioxanes simultaneously [2,3-c] pyrroles -6- (3H)-yl) ethyl acetate Preparation, with reference to embodiment 12 method.

The characterize data of the compound 16 is：¹H NMR(400MHz,CD₃OD,δ_ppm):7.44 (d, J=8.4Hz, 2H), 7.33 (d, J=8.4Hz, 2H), 6.81 (s, 1H), 6.39 (s, 1H), 4.23 (s, 1H), 4.21-4.13 (m, 1H), 4.10- 4.08 (m, 3H), 3.65 (s, 3H), 3.82,3.80 (dd, J=4,3.2Hz, 2H), 3.59-3.52 (m, 2H), 3.41,3.38 (dd, J=5.6,6Hz, 2H), 3.11-3.04 (m, 2H), 2.87-2.84 (m, 2H), 2.75-2.72 (m, 1H), 1.20 (t, J= 7.2Hz, 3H), 1.23,1.21 (dd, J=5.6,5.6Hz, 6H), 1.00,0.98 (dd, J=2.8,2.8Hz, 6H) .ESI-MS m/z:580.2(M+H)⁺,578.1(M-H)^-.

The structural formula of the compound 16 is：

Embodiment 17

Following examples are prepared with reference to following reaction schemes

According to Above Transmission Lines, comprise the following steps：

(1) methyl 4- (5- (2,4- double (benzyloxy) -5- isopropyl phenyls) -3- (methylcarbamoyl) isoxazole - 4- yls) methyl benzoate (compound III -1) preparation.

A 50mL single port bottles are taken, add chemical compounds I -10 (600mg, 1mmol), (4- (methoxycarbonyl) phenylboric acid (235mg, 1.3mmol, 1.3 equivalent), Pd (PPh₃)₂Cl₂(70mg, 0.1mmol, 10mol%), sodium acid carbonate (252mg, 3mmol, 3 equivalents), argon gas exchanges, and adds DMF：H₂O=10mL：2mL solution, 80 DEG C are placed in, stir 5h.TLC tracking and monitorings. Most of solvent is removed in rotation, crosses post (PE：EA=4:1) brown color product 620mg, is obtained.ESI-MS m/z:605.2(M+H)⁺.

(2) 4- (5- (double (the benzyloxy) -5- isopropyl phenyls of 2,4-) -3- (methylcarbamoyl) isoxazoles -4- Base) benzoic acid (compound III -2) preparation.

A 25mL single port bottles are taken, add compound III -1 (600mg), acetone (2mL) dissolving is added, adds 35% hydroxide Sodium solution (1mL), adding methanol (4mL) makes layering disappear, and 2h is stirred at room temperature.TLC tracking and monitorings.1N HCl solutions are added, are adjusted To acidity, most of organic solution is gone in rotation, is added EA and saturated aqueous common salt extraction, is collected organic phase, dries, be spin-dried for.Directly use In in next step.ESI-MS m/z:591.1(M+H)⁺.

(3) 5- (2,4- double (benzyloxy) -5- isopropyl phenyls)-N- methyl -4- (4- (4AR, 7AS)-hexahydro -2H- [1,4] dioxanes simultaneously [2,3-C] pyrroles -6- carbonyls) phenyl) isoxazole -3- formamides (compound III -3) preparation.

Take a 25mL single port bottles, add crude compound III -2 (400mg, 0.68mmol), HOBt (110mg, 0.816mmol, 1.2 equivalents), EDCI (195mg, 1.02mmol, 1.5 equivalent), DCM (5mL) dissolving, adding TEA, (0.4mL, 2.72mmol, 4 work as Amount), 15min is stirred, compound 7 (87mg, 0.68mmol, 1 equivalent) is added, is stirred overnight.TLC tracking and monitorings.Add DCM and Saturated aqueous common salt extracts, and dries, is spin-dried for, and crosses post (PE：EA=2:1).Obtain colourless viscous wall-like product 160mg.ESI-MS m/z: 702.1(M+H)⁺.

(4) 5- (2,4- dihydroxy -5- isopropyl phenyls)-N- methyl -4- (4- (4AR, 7AS)-hexahydro -2H- [1,4] two Oxane simultaneously [2,3-C] pyrroles -6- carbonyls) phenyl) isoxazole -3- formamides (compound 17) preparation.

A 25mL two-mouth bottles are taken, crude compound III -3 (160mg, 0.23mmol) is added, argon gas protection, adds anhydrous DCM (10mL) dissolves, and is placed at -5 DEG C, stirs 15min, and the dichloromethane solution (1M, 0.69mL, 3 equivalent) of Boron tribromide is added dropwise, 10min is stirred, is placed at room temperature, stirs 1h.The reaction of TLC tracking displays raw material finishes.At -5 DEG C, DCM (10mL) dilutions are added, Add saturation NaHCO₃Solution is quenched, and extraction, collects organic phase.EA is added into aqueous phase, the fluffy solid of generation is dissolved, is received Collect organic phase, mixing, anhydrous sodium sulfate drying, concentrated pillar (DCM:Acetone=2:1).Gained yellow, viscous is produced Thing is beaten, and is added petroleum ether, is stood under low temperature, separates out white solid, suck yellow liquid, gained solid oil pumping is done, can obtained 80mg white solids.

The characterize data of the compound 17 is：¹H NMR(400MHz,CD₃OD,δ_ppm):7.51 (d, J=6.4Hz, 2H), 7.42 (d, J=6.4Hz, 2H), 6.94 (s, 1H), 6.36 (s, 1H), 4.32 (s, 1H), 4.20 (s, 1H), 3.88-3.59 (m, 8H), 3.42,3.39 (dd, J=5.6,5.6Hz, 2H), 3.34-3.12 (m, 1H), 1.24 (t, J=6.0Hz, 3H), 1.08 (d, J=5.6Hz, 6H)¹³C NMR(125MHz,CD₃OD,δ_ppm):173.3,169.9,163.3,160.2,159.4,156.7, 136.8,134.9,131.6,130.0,129.1,128.9,117.0,107.3,104.7,75.0,74.2,64.4,64.2, 62.4,51.4,36.4,28.2,23.9,23.9,15.5.ESI-MS m/z:522.1(M+H)⁺,520.1(M-H)^-.

Embodiment 18

Compound 18：5- (2,4- dihydroxy -5- isopropyl phenyls)-N- methyl -4- (4- (morpholine -4- carbonyls) phenyl) is different The preparation of oxazole -3- formamides, with reference to the method for embodiment 17.

The characterize data of the compound 18 is：¹H NMR(400MHz,CD₃OD,δ_ppm):7.40-7.35(m,4H),6.91 (s, 1H), 6.32 (s, 1H), 3.72-3.70 (m, 8H), 3.39,3.36 (dd, J=7.2,7.2Hz, 2H), 3.14-3.07 (m, 1H), 1.21 (t, J=7.2Hz, 3H), 1.05 (d, J=6.8Hz, 6H)¹³C NMR(125MHz,CD₃OD,δ_ppm):173.2, 170.0,163.3,160.2,159.4,156.7,136.2,134.6,131.7,130.0,129.1,128.8,117.1, 107.3,104.7,68.6,74.2,36.3,28.2,23.9,15.5.ESI-MS m/z:480.1(M+H)⁺,478.3(M-H)^-.

The structure of the compound 18 is：

Embodiment 19

Following examples are prepared with reference to following reaction schemes

According to Above Transmission Lines, comprise the following steps：

(1) preparation of (S)-ethyl -2- hydroxyls -2- phenylacetic acids ester (compounds Ⅳ -1).

A 100mL single port bottles are taken, add 3g (s)-(+)-mandelic acid, are dissolved with ethanol (30mL), the concentrated sulfuric acid is added dropwise (0.3mL), it is placed at 80 DEG C and flows back, stir 3h, the reaction of TLC tracking displays raw material finishes.Add saturation NaHCO₃Solution until Bubble-free generates, and most of ethanol solution is removed in rotation, adds EA (30mL) extractions, saturated common salt washing, collects organic phase, anhydrous sulphur Sour sodium is dried, and is spin-dried for, is obtained colourless oil liquid (3.5g, 98.5%).

The characterize data of the compounds Ⅳ -1 is：¹H NMR(400MHz,Chloroform-d,δ_ppm):7.42–7.27(m, 5H), 5.15 (d, J=4.0Hz, 1H), 4.27-4.10 (m, 2H), 3.68 (d, J=4.0Hz, 1H), 1.20 (t, J=8.0Hz, 3H)

(2) preparation of (S)-ethyl -2- hydroxyls -2- (4- iodophenyls) ethyl acetate (compounds Ⅳ -2).

A 100mL single port bottles are taken, add compounds Ⅳ -1 (3.2g, 17.8mmol), anhydrous DCM (60mL) dissolving, by bottle Sub- outer wall wraps up lucifuge with tinfoil, adds iodine (4.5g, 17.7mmol) and silver trifluoromethanesulfonate (4.56g, 17.7mmol), room temperature 4h is stirred, the reaction of TLC tracking displays raw material finishes.Add hypo solution to be quenched, filtering precipitation, DCM rinses precipitation, extraction Take, saturated common salt washing, collect organic phase, anhydrous sodium sulfate drying, be spin-dried for, cross post (PE：EA=8:1) brown oil liquid, is obtained Body.

The characterize data of the compounds Ⅳ -2 is：¹H NMR(400MHz,Chloroform-d,δ_ppm):7.67 (d, J= 8.0Hz, 2H), 7.16 (d, J=8.4Hz, 2H), 5.10 (d, J=4.8Hz), 4.29-4.12 (m, 2H), 3.59 (d, J= 5.2Hz), 1.24 (t, J=7.8Hz, 3H)

(3) (S)-ethyl -2- hydroxyls -2- (4- (4,4,5,5- tetramethyl -1,3,2- dioxy boron pentane -2- bases) phenyl) second The preparation of acetoacetic ester (compounds Ⅳ -3).

Take the two-mouth bottle of a 50ml, add compounds Ⅳ -2 (0.55g, 1.8mmol), connection boric acid pinacol ester (0.55g, 2.16mmol), [1,1'- double (diisopropyl phosphine) ferrocene] dichloro palladium (54mg, 0.09mmol), potassium acetate (529mg, 5.4mmol), argon gas exchanges, and adds DMF dissolvings, is placed at 90 DEG C, stirs 1h, and the reaction of TLC tracking displays raw material finishes.Rotation is gone Most of DMF, EA, filtering precipitation are added, EA is rinsed, is spin-dried for, and crosses post (PE：EA=8:1).Anhydrous oily liquids (390mg, 70.9%).

The characterize data of the compounds Ⅳ -3 is：¹H NMR(400MHz,Chloroform-d,δ_ppm):7.81 (d, J= 7.6Hz, 2H), 7.44 (d, J=7.2Hz, 2H), 5.16 (s, 1H), 4.23-4.09 (m, 2H), 3.56 (s, 1H), 1.33 (s, 12H), 1.22 (t, J=7.2Hz, 3H)

(4) (S)-ethyl 2- (4- (4,4,5,5- tetramethyl -1,3,2- dioxaborolane -2- bases) phenyl) -2- (first Phenylsulfonyloxy) methyl acetate (compounds Ⅳ -4) preparation.

A 100mL single port bottles are taken, compound 3 (1g, 3.27mmol) is added, anhydrous DCM (10mL) dissolving, adds and dry three Ethamine (1.4mL, 9.8mmol, 3 equivalent), TsCl (1.86g, 9.8mmol, 3 equivalent) is slowly added to, is placed at 50 DEG C, it is stirred Night.The reaction of TLC tracking displays raw material finishes.Water and DCM are added, extraction, saturated common salt washing, anhydrous sodium sulfate drying, is spin-dried for, Cross post (PE：EA=10:1).Obtain anhydrous oily liquids (1g, 66.7%).

(5) (S)-ethyl 2- morpholinyls -2- (4- (4,4,5,5- tetramethyl -1,3,2- dioxy boron pentane -2- bases) phenyl) The preparation of ethyl acetate (compounds Ⅳ -5).

A 50mL single port bottles are taken, compounds Ⅳ -4 (0.5g, 1.09mmol) is added, toluene (8mL) dissolving, adds and dry three Ethamine (0.3mL, 2.17mmol, 2 equivalent), morpholine (0.14mL, 1.64mmol, 1.5 equivalent), is placed in oil bath pan and flows back.Stir 2h is mixed, the reaction of TLC tracking displays raw material finishes.Most of toluene is removed in rotation, adds EA and saturated aqueous common salt, extraction, anhydrous slufuric acid Sodium is dried, and is spin-dried for (260mg, 64%), is directly used in next step.

(6) preparation of (S)-(4- (2- ethyoxyl -1- morpholino -2- oxygen ethyl) phenyl) boric acid (compounds Ⅳ -6).

A 50mL single port bottles are taken, add compounds Ⅳ -5 (250mg, 0.67mmol), HCl solution 10mL is added and (is adjusted to pH =3) n-hexane (10mL), is added, stirring, phenyl boric acid (81mg, 0.67mmol, 1 equivalent) is added, is stirred vigorously, until benzene boron Sour all dissolvings, are stirred for 0.5h, and the reaction of TLC tracking displays raw material finishes.Extraction, retain aqueous phase, add n-hexane and wash one again It is secondary, add EA and wash away the phenyl boric acid for reacting endless, retain aqueous phase, add saturated sodium bicarbonate solution, aqueous phase is adjusted to meta-alkalescence, EA*2 extractions are added, anhydrous sodium sulfate drying, are spin-dried for, are directly used in next step.Colorless viscous shape product 160mg, 82%.

(7) (S)-ethyl 2- (4- (5- (double (the benzyloxy) -5- isopropyl phenyls of 2,4-) -3- (ethylaminocarbonyl) Isoxazole -4- bases) phenyl) -2- morpholinoes acetic acid esters (compounds Ⅳ -7) preparation.

Take a 25mL single port bottles, add chemical compounds I -10 (102mg, 0.17mmol), compounds Ⅳ -6 (60mg, 0.2mmol, 1.2 equivalents), Pd (PPh₃)₂Cl₂(12mg, 0.02mmol, 10mol%), sodium acid carbonate (43mg, 0.51mmol, 3 Equivalent), argon gas exchanges, and adds DMF：H₂O=10mL：2mL solution, 80 DEG C are placed in, stir 3h.TLC tracking and monitorings.Big portion is gone in rotation Divide solvent, cross post (PE：EA=4:1) brown color product 75mg, is obtained.ESI-MS m/z:718.1(M+H)⁺。

(8) (S)-ethyl 2- (4- (5- (2,4- dihydroxy -5- isopropyl phenyls) -3- (ethylaminocarbonyl) different evils Azoles -4- bases) phenyl) -2- morpholinoes acetic acid esters (compound 19) preparation.

A 25mL two-mouth bottles are taken, crude compound IV -7 (68mg) is added, argon gas protection, adds anhydrous DCM (10mL) dissolvings, It is placed at 0 DEG C, stirs 15min, the dichloromethane solution (1M, 0.28mL, 3 equivalent) of Boron tribromide is added dropwise, stirs 10min, put At room temperature, 1h is stirred.The reaction of TLC tracking displays raw material finishes.At 0 DEG C, DCM (10mL) dilutions are added, add saturation NaHCO₃Solution is quenched, and extraction, collects organic phase.EA is added into aqueous phase, the fluffy solid of generation is dissolved, is collected organic Phase, mixing, anhydrous sodium sulfate drying, concentrated pillar (DCM:Acetone=1:1).Gained yellow, viscous product is beaten, Petroleum ether is added, is stood under low temperature, white solid is separated out, sucks yellow liquid, gained solid oil pumping is done, and can obtain 25mg whites Solid (S configurations).

The characterize data of the compound 19 is：¹H NMR(400MHz,CD₃OD,δ_ppm):7.44 (d, J=6Hz, 2H), 7.33 (d, J=5.6Hz, 2H), 6.79 (s, 1H), 6.39 (s, 1H), 4.22 (d, J=8Hz, 1H), 4.19 (d, J=5.6Hz, 1H), 4.12 (d, J=5.6Hz, 1H), 4.02 (s, 4H), 3.40 (d, J=5.6Hz, 2H), 3.10 (t, J=5.2Hz, 1H), 2.47 (s, 4H), 1.22 (d, J=5.6Hz, 6H), 0.98 (d, J=5.2Hz, 6H)¹³C NMR(125MHz,CD₃OD,δ_ppm): 173.6,169.8, 163.4,160.0,159.4,156.9,132.7,132.1,131.9,130.8,130.0,129.0, 117.3,117.1,107.3,104.7,76.1,68.5,63.1,53.6,53.6,36.3,28.0,23.9,23.9,15.5, 15.2.ESI-MS m/z:538.1(M+H)⁺,536.1(M-H)^-.

Embodiment 20

Compound 20：(S)-ethyl -2- (4- (5- (2,4- dihydroxy -5- isopropyl phenyls) -3- (ethylcarbamoyls Base) isoxazole -4- bases) phenyl) -2- (4AR, 7AS)-tetrahydrochysene -2H- [1,4] dioxanes simultaneously [2,3-c] pyrroles -6- (3H)-yl) The preparation of ethyl acetate, with reference to the method for embodiment 19.

The characterize data of the compound 20 is：¹H NMR(400MHz,CD₃OD,δ_ppm):7.44 (d, J=6.4Hz, 2H), 7.33 (d, J=6.4Hz, 2H), 6.81 (s, 1H), 6.81 (s, 1H), 4.26 (s, 1H), 4.21-4.16 (m, 1H), 4.12- 4.09 (m, 3H), 3.82-3.79 (m, 2H), 3.59-3.54 (m, 2H), 3.41,3.38 (dd, J=5.6,6.0Hz, 2H), 3.11-3.05 (m, 2H), 2.87-2.84 (m, 1H), 2.81-2.72 (m, 1H), 1.23,1.20 (dd, J=5.6,5.6Hz, 6H), 0.99 (t, J=2.4Hz, 6H)¹³C NMR(125MHz,CD₃OD,δ_ppm):173.7,169.7,163.5,160.0, 159.5,156.9,138.1,134.0,132.6,131.9,130.4,130.0,129.0,117.1,107.3,104.7,75.4, 75.3,75.2,64.7,64.1,63.1,55.5,54.1,36.3,28.1,23.9,15.5,15.2.ESI-MS m/z:580.2 (M+H)⁺,578.1(M-H)^-.

The structural formula of the compound 20 is：

Embodiment 21

Compound 21：(S)-isopropyl -2- (4- (5- (2,4- dihydroxy -5- isopropyl phenyls) -3- (ethylcarbamoyls Base) isoxazole -4- bases) phenyl) -2- (4AR, 7AS)-tetrahydrochysene -2H- [1,4] dioxanes simultaneously [2,3-c] pyrroles -6- (3H)-yl) The preparation of ethyl acetate, with reference to the method for embodiment 19.

The characterize data of the compound 21 is：¹H NMR(400MHz,CD₃OD,δ_ppm):7.44 (d, J=8.4Hz, 2H), 7.33 (d, J=8.4Hz, 2H), 6.81 (s, 1H), 6.39 (s, 1H), 5.02-4.97 (m, 1H), 4.21 (s, 1H), 4.09 (s, 2H), 3.82-3.80 (m, 2H), 3.59-3.53 (m, 2H), 3.41,3.38 (dd, J=5.6,5.6Hz, 2H), 3.11-3.04 (m, 2H), 2.86-2.78 (m, 2H), 2.75-2.72 (m, 1H), 1.27 (d, J=4.8Hz, 3H), 1.22 (t, J=5.6Hz, 3H), 1.14 (d, J=4.8Hz, 3H), 1.00 (t, J=3.2Hz, 6H)¹³C NMR(125MHz,CD₃OD,δ_ppm):173.3, 169.7,163.5,160.0,159.6,156.9,138.2,132.5,131.8,130.4,130.0,129.0,117.1, 107.3,104.7,75.5,75.3,70.9,64.7,64.1,55.5,54.1,36.4,28.1,24.0,22.8,22.6, 15.5.ESI-MS m/z:594.2(M+H)⁺,592.1(M-H)^-.

The structural formula of the compound 21 is：

Embodiment 22

Compound 22：(S)-ethyl 2- (4- (5- (2,4- dihydroxy -5- isopropyl phenyls) -3- (ethylaminocarbonyl) Isoxazole -4- bases) phenyl) -2- ((2S, 6R) -2,6- thebaines) acetic acid esters preparation, with reference to embodiment 19 method.

The characterize data of the compound 22 is：¹H NMR(400MHz,CD₃OD,δ_ppm):7.39 (d, J=8.4Hz, 2H), 7.31 (d, J=8.0Hz, 2H), 6.77 (s, 1H), 6.37 (s, 1H), 4.19-4.03 (m, 2H), 3.97 (s, 1H), 3.77- 3.63 (m, 2H), 3.39,3.35 (dd, J=7.2,7.2Hz, 2H), 3.09-3.02 (m, 1H), 2.86 (d, J=6.8Hz, 1H), 2.57 (d, J=11.2Hz, 1H), 1.94 (t, J=6.8Hz, 1H), 1.66 (t, J=10.8Hz, 1H), 1.20-1.16 (s, 6H), 1.22 (d, J=6.4Hz, 3H), 1.03 (d, J=6.4Hz, 3H), 0.96 (d, J=6.4Hz, 6H)¹³C NMR (125MHz,CD₃OD,δ_ppm):173.6,169.7,163.4,159.9,159.4,156.9,136.6,132.5,131.8, 130.7,130.0,128.9,117.0,107.2,104.7,75.7,73.6,73.4,63.0,59.5,58.6,36.3,28.8, 28.0,23.9,23.8,20.0,15.4,15.2.ESI-MS m/z:566.2(M+H)⁺,564.1(M-H)^-.

The structural formula of the compound 22 is：

Embodiment 23

Compound 23：(R)-ethyl -2- (4- (5- (2,4- dihydroxy -5- isopropyl phenyls) -3- (ethylcarbamoyls Base) isoxazole -4- bases) phenyl) and -2- morpholino acetic acid esters preparation, with reference to embodiment 19 method.

The characterize data of the compound 23 is：¹H NMR(400MHz,CD₃OD,δ_ppm):7.40 (d, J=6.4Hz, 2H), 7.30 (d, J=6.4Hz, 2H), 6.77 (s, 1H), 6.36 (s, 1H), 4.26 (s, 1H), 4.29-4.10 (m, 1H), 4.09- 4.04 (m, 1H), 3.99 (s, 1H), 3.67 (s, 4H), 3.39,3.35 (dd, J=5.6,5.6Hz, 2H), 3.08-3.03 (m, 1H), 2.45 (d, J=3.6Hz, 4H), 1.20,1.17 (dd, J=5.6,6.0Hz, 6H), 0.95 (d, J=5.6Hz, 6H)¹³C NMR(125MHz,CD₃OD,δ_ppm):173.6,169.8,163.4,160.0,159.4,156.9,126.7,132.7,132.0, 131.9,130.8,130.2,129.0,117.1,117.1,107.3,104.7,104.6,76.1,68.6,68.5,63.1, 53.6,36.3,28.0,23.9,23.9,15.5,15.2.ESI-MS m/z:538.1(M+H)⁺,536.1(M-H)^-.

The structural formula of the compound 23 is：

Embodiment 24

Compound 24：(R)-ethyl -2- (4- (5- (2,4- dihydroxy -5- isopropyl phenyls) -3- (ethylcarbamoyls Base) isoxazole -4- bases) phenyl) -2- (4AR, AS)-tetrahydrochysene -2H- [1,4] dioxanes simultaneously [2,3-c] pyrroles -6- (3H)-yl) second The preparation of acetoacetic ester, with reference to the method for embodiment 19.

The characterize data of the compound 24 is：¹H NMR(400MHz,CD₃OD,δ_ppm):7.41 (d, J=6.4Hz, 2H), 7.30 (d, J=6.4Hz, 2H), 6.78 (s, 1H), 6.37 (s, 1H), 4.24 (s, 1H), 4.18-4.15 (m, 1H), 4.09- 4.06 (m, 1H), 3.79-3.76 (m, 2H), 3.56-3.49 (m, 2H), 3.38,3.35 (dd, J=5.6,5.6Hz, 2H), 3.08-3.01 (m, 2H), 2.85-2.76 (m, 2H), 2.73-2.69 (m, 1H), 1.20,1.17 (dd, J=5.2,5.6Hz, 6H), 0.97 (dd, J=2.8,2.8Hz, 6H)¹³C NMR(125MHz,CD₃OD,δ_ppm):173.6,169.8,163.5, 160.0,159.5,156.9,138.0,132.6,131.9,130.4,130.1,130.1,129.7,129.0,117.1, 107.3,104.8,75.4,75.3,75.2,64.7,64.1,63.1,55.5,54.1,36.3,28.1,24.0,23.8,15.5, 15.2.ESI-MS m/z:580.2(M+H)⁺,578.1(M-H)^-.

The structural formula of the compound 24 is：

Embodiment 25

Following examples are prepared with reference to following reaction schemes

According to Above Transmission Lines, comprise the following steps：

(1) (4- bromobenzyls) epoxide) (tert-butyl group) dimethylsilane (compound VII -1) preparation.

A 100mL single port bottles are taken, add 4- bromobenzyls alcohol (5g, 26.9mmol), imidazoles (2.2g, 1.2 equivalents, 32.3mmol) With DMAP (DMAP) (catalytic amount), DCM (40mL) dissolvings, tert-butyl chloro-silicane is slowly added to (TBSCl) (4.87g, 1.2 equivalents, 32.3mmol), is stirred overnight.TLC monitoring reactions finish, and add water, dissolve consolidating for generation Body, extraction, add saturated common salt washing, collect organic phase, revolving removes organic solvent after anhydrous sodium sulfate drying, obtain 7.7g without Color grease, yield 95%.ESI-MS m/z:301.0(M+H)⁺。

(2) system of 1- (4- (((t-butyldimethylsilyl) epoxide) methyl) phenyl) ethyl ketone (compound VII -2) It is standby.

Take a 50mL two-mouth bottles, add compound 2 (4g, 13.3mmol) and tetra-triphenylphosphine palladium (0.77g, 5mmol%, 0.67mmol), argon gas exchanges, and adds DMF and dissolves, addition tributyl (1- ethoxy ethylenes) tin (5.4mL, 1.2 equivalents, 16mmol), 100 DEG C are placed in, stirs 2h, TLC monitoring reactions finish.Depressurized with oil pump, vacuum revolving, remove most of DMF, add Enter EA and 1N hydrochloric acid solutions, stir, TLC monitoring reactions finish, and extract, and add saturated common salt washing, collect organic phase, anhydrous sulphur Revolving removes organic solvent after sour sodium is dried, and mixes sample and crosses post (PE：DCM=8:1) 2.5g colourless liquids, yield 70%, are obtained.

(3) 3- (4- ((tertiary butyl dimethyl Si base) methyl) phenyl) -3- oxopropanoates (compound VII -3) Prepare.

A 50mL single port bottles are taken, compound 3 (1g, 3.8mmol) is added, toluene dissolving, adds diethyl carbonate (0.91mL, 2 equivalents, 7.6mmol), NaH (0.3g, 2 equivalents, 7.6mmol) is slowly added to, is placed in 100 DEG C, return stirring mistake Night.TLC monitoring reactions finish.It is placed in 0 DEG C, adds water quenching and go out, revolving removes most of organic solvent, adds EA extractions two It is secondary, saturated aqueous common salt is added, collects organic phase, anhydrous sodium sulfate drying, concentrated post (PE：DCM=4:1).It is colourless to obtain 650mg Grease, yield 50%.

The characterize data of the compound VII -3 is：¹H NMR(400MHz,CD₃Cl,δ_ppm):7.93 (d, J=8.1Hz, 2H), 7.44 (d, J=8.1Hz, 2H), 4.80 (s, 2H), 4.24 (dd, J=7.2,7.2Hz, 2H), 3.98 (s, 2H), 0.95 (s, 9H),0.11(s,6H).

(4) ethyl -1- (double (the benzyloxy) -5- isopropyl phenyls of 2,4-) -5- (4- (((tertiary butyl dimethyl Si base) Methyl) phenyl) -1H-1,2,3- triazole -4- carboxylic acid, ethyl esters (compound VII -4) preparation.

Take a 50mL single port bottles, add compound VII -3 (600mg, 1.8mmol) and (((4- azido -6- isopropyl -1, 3- phenylenes two (epoxide)) double (methylene)) hexichol (1g, 1.5 equivalents, 2.7mmol, mixture), DMSO dissolvings, add DBU (0.4mL, 1.5 equivalents, 2.7mmol), is stirred overnight at room temperature.TLC monitoring reactions finish.Water and EA are added, organic phase is collected, adds Enter water to be extracted twice again, collect organic phase, anhydrous sodium sulfate drying, be spin-dried for post PE：EA=8:1,800mg grease is obtained, is produced Rate is 64%.

(5) 1- (double (the benzyloxy) -5- isopropyl phenyls of 2,4-) -5- (4- (((tertiary butyl dimethyl Si base) methyl) Phenyl)-N- ethyl -1H-1,2,3- triazole -4- formamides (compound VII -5) preparation.

A 50mL single port bottles are taken, add compound VII -4 (800mg, 1.16mmol), ethanol dissolving is added, adds ethamine water Solution (10mL), 80 DEG C are placed in, return stirring 1d, TLC monitoring reaction finishes.Revolving removes most of organic solvent, adds EA, It is extracted twice, saturated common salt washing, anhydrous sodium sulfate drying, is spin-dried for, obtains 600mg oil products, yield 75%.

(6) 1- (double (the benzyloxy) -5- isopropyl phenyls of 2,4-)-N- methyl -5- (4- (hydroxymethyl) phenyl) -1H- The preparation of 1,2,3- triazole -4- formamides (compound VII -6).

A 50mL single port bottles are taken, add compound VII -5 (600mg, 0.87mmol), add tetrabutyl amine fluoride (TBAF) THF solution (10mL) dissolving, 2h is stirred at room temperature.TLC monitoring reactions finish.Revolving removes most of organic solvent, adds EA, It is extracted twice, saturated common salt washing, anhydrous sodium sulfate drying, is spin-dried for, obtains 500mg oil products, yield 99.8%.

(7) 1- (double (the benzyloxy) -5- isopropyl phenyls of 2,4-)-N- methyl -5- (4- Fonnylphenyls) -1H-1,2, The preparation of 3- triazole -4- formamides (compound VII -7).

A 50mL single port bottles are taken, compound VII -6 (500mg, 0.87mmol) is added, DCM dissolvings, adds 2,2,6,6- tetra- Methyl piperidine oxide (TEMPO) and TBAB (TBAB) (catalytic amount), add sodium hypochlorite (NaOCl) solution (5mL) and NaHCO₃Solution (2mL), is stirred at room temperature 1h.Solution becomes light yellow from rufous.TLC monitoring reactions finish.Add DCM, extraction three times, saturated common salt washing, anhydrous sodium sulfate drying, are spin-dried for, cross post (PE：EA=5:1) the thick productions of 250mg are obtained Thing, yield 50%.

The characterize data of the compound VII -7 is：¹H NMR(400MHz,CD₃Cl,δ_ppm):9.99(s,1H),7.75(d,J =8Hz, 2H), 7.46 (d, J=8.4Hz, 2H), 7.39-7.21 (m, 8H), 6.94 (t, J=5.6Hz, 2H), 6.39 (s, 1H), 4.96 (s, 2H), 4.72 (s, 2H), 3.82 (t, J=6.4Hz, 1H), 3.51-3.44 (m, 2H), 3.31-3.26 (m, 1H), 1.26 (d, J=2.8Hz, 3H), 1.18 (d, J=6.8Hz, 6H) .ESI-MS m/z:575.0(M+H)⁺。

(8) ethyl -2- (4- (1- (2,4- double (benzyloxy) -5- isopropyl phenyls) -4- (ethylaminocarbonyl) - 1H-1,2,3- triazole -5- bases) phenyl) -2- hydroxyacetic acids (compound VII -8) preparation.

A 50mL single port bottles are taken, compound VII -7 (250mg, 0.44mmol) is added, DCM dissolvings, adds I₂(catalytic amount), Trimethylsilyl cyanide (TMSCN) (0.11mL, 2 equivalents, 0.87mmol) is added, 30min is stirred at room temperature, TLC monitoring reactions finish. Add water quenching to go out, extract, saturated common salt washing, dry, anhydrous sodium sulfate is spin-dried for.Add ethanol solution of hydrogen chloride (10mL), room Temperature is stirred overnight.TLC monitoring reactions finish.Add saturated sodium bicarbonate solution to be quenched, revolving removes most of organic solvent, adds Enter EA, be extracted twice, saturated common salt washing, anhydrous sodium sulfate drying, be spin-dried for post PE：EA=4:1,140mg is obtained, yield is 49.1%.ESI-MS m/z:649.0(M+H)⁺。

(9) ethyl -2- (4- (1- (2,4- double (benzyloxy) -5- isopropyl phenyls) -4- (ethylaminocarbonyl) - 1H-1,2,3- triazole -5- bases) phenyl) -2- (tosyl) acetic acid esters (compound VII -9) preparation.

Take a 25mL single port bottles, add compound VII -8 (140mg, 0.22mmol), DCM dissolvings, add TEA (92 μ L, 3 Equivalent, 0.66mmol) and TsCl (126mg, 3 equivalents, 0.66mmol), it is placed in 40 DEG C, stirs 4h.TLC monitoring reactions finish. Saturated aqueous common salt extraction is added, organic phase is collected, anhydrous sodium sulfate drying, is spin-dried for, crosses post PE:EA=4:1, obtain 50mg, yield 28%.

(10) ethyl -2- (4- (1- (2,4- double (benzyloxy) -5- isopropyl phenyls) -4- (ethylaminocarbonyl) - 1H-1,2,3- triazole -5- bases) phenyl) -2- morpholinoes acetic acid esters (compound VII -10) preparation.

A 25mL single port bottles are taken, add compound 11 (50mg, 0.06mmol), toluene dissolving, adding morpholine, (20 μ L, 4 work as Amount, 0.24mmol) and TEA (33 μ L, 4 equivalents, 0.24mmol), 100 DEG C are placed in, return stirring 2h.TLC monitoring reactions finish. Revolving removes most of organic solvent, adds saturated aqueous common salt extraction, collects organic phase, anhydrous sodium sulfate drying, be spin-dried for, obtain 30mg, yield 70%.

ESI-MS m/z:718.0(M+H)⁺。

(11) ethyl -2- (4- (1- (2,4- dihydroxy -5- isopropyl phenyls) -4- (ethylaminocarbonyl) -1H-1,2, 3- triazole -5- bases) phenyl) -2- morpholinoes acetic acid esters (compound 25) preparation.

A 25mL single port bottles are taken, add compound VII -10 (30mg, 0.04mmol), argon gas exchanges, and it is molten to add anhydrous DCM Solution, is placed at -5 DEG C, stirs 15min, and Boron tribromide dichloromethane solution (0.12mL, 3 equivalents, 0.12mmol), stirring is added dropwise 15min, it is placed in and 2h is stirred at room temperature.Add saturated sodium bicarbonate solution to be quenched, extract, saturated common salt washing, collect organic phase, nothing Aqueous sodium persulfate is dried, and is spin-dried for, and crosses post (DCM：Acetone=2:1) 10mg solids, yield 48%, are obtained.

The characterize data of the compound 25 is：¹H NMR(400MHz,CD₃OH,δ_ppm):7.43 (d, J=8.4Hz, 2H), 7.39 (d, J=8.4Hz, 2H), 6.85 (s, 1H), 6.37 (s, 1H), 4.19-4.08 (m, 2H), 4.06 (s, 1H), 3.72- 3.65 (m, 4H), 3.45,3.42 (dd, J=6,6.4Hz, 2H), 3.16-3.09 (m, 1H), 2.48-2.41 (m, 4H), 1.27 (t, J=7.2Hz, 3H), 1.18 (t, J=7.2Hz, 3H), 1.09 (d, J=6.8Hz, 6H)¹³C NMR(125MHz,CD₃OD, δ_ppm):194.4,176.9,164.2,163.7,159.3,157.4,153.5,142.4,140.2,137.5,132.15, 130.3,129.1,127.7,117.0,104.6,83.4,75.7,69.2,68.3,63.3,53.5,48.0,35.9,28.2, 23.8,15.9,15.2.ESI-MS m/z:538.2(M+H)⁺,536.2(M-H)^-.

Experimental example

(1) the activity screen method of target compound.

Principle：After the geldanamycin of FITC marks is combined with HSP90, generation fluorescence polarization can be made, if compound energy With geldanamycin Reverse transcriptase HSP90 enzymes, then fluorescence polarization is can't detect.

Reagent and instrument：The geldanamycin (Sigma companies) of fluorescence labeling, Hsp90 α or Hsp90 β enzyme solutions (Stressgen Bioreagents Corp companies, cat.no.SPP-776), DTT (Promega companies), bovine serum albumin(BSA) (BSA) (Hyclone companies), DMSO (Sigma companies).(U.S. Perkin Elmer are public for Envision2104 fluor testers Department), sample injector (Eppendorf companies), microwell plate (corning companies).

Each compound is respectively provided with multiple various dose groups, and high dose, which assembles, is set to mother liquor, three times successively of doses remaining group Than being diluted to lowest dose level group, whole samples are dissolved in DMSO and saved backup at -20 DEG C.Experiment buffer includes 20mmol/l HEPES (K), 50mmol/l KCl, 5mmol/l MgCl₂, 20mmol/l Na₂MoO₄And 0.01%NP40, PH are 7.3.5 μ l DTT containing 40mM and 2mg/ml BSA reaction buffer is added before experiment every time, adds 2.5 μ l fluorescence labelings Geldanamycin (reaction density 5nM).Then compound transferring apparatus Liquid Handler Echo520 are utilized by 10nl The compound that 1000 × gradient concentration has diluted is added in reaction solution, and it is molten to be eventually adding 2.5 μ l Hsp90 β or Hsp90 α enzymes Liquid (reaction density 35ng/ μ l).Room temperature slight oscillatory reacts 2 hours, finally determines reading with microwell plate plate reader, and exciting light is 485nm, transmitting light are 530nm, data Graphpad Prism5 software processings.

(2) target compound inhibiting tumor cell method for screening active ingredients.

CCK kit operation principles：Cell Counting Kit abbreviation CCK kits are a kind of based on WST-8 (chemistry Name：2- (2- methoxyl group -4- nitre phenyl) -3- (4- nitre phenyl) -5- (2,4- disulfobenzenes) -2H- tetrazolium monosodium salts) extensively should For cell propagation and the fast high-sensitive degree detection kit of cytotoxicity.WST-8 belongs to MTT upgrading products, and work is former Manage and be：In the presence of electronics coupled reagent, the orange of generation high water soluble can be reduced by Intramitochondrial dehydrogenase The first a ceremonial jade-ladle, used in libation product (formazan) of yellow, and be deposited in cell, and dead cell is without this function.The depth of color and the increasing of cell Grow directly proportional, be inversely proportional with cytotoxicity.Light absorption value is determined at 540/720nm wavelength using enzyme detector, indirectly reflection Living cells quantity.

Reagent and instrument：Various cancerous cell lines (ATCC companies of the U.S.), penicillin, streptomysin, DMSO (Sigma companies), CCK8 (CK04, Japanese colleague's chemical industry), RPMI1640 (GIBCO companies), BSA (Hyclone companies), trypsase (Ji Nuogong Department), 384 porocyte culture plates (Corning).

Each cancer cell, which is suspended in corresponding nutrient solution, is configured to suitable concentration, and 50 μ l/ pore volumes are planted in 384 orifice plates, training Support in 37 DEG C, 5%CO₂24h in constant incubator.Compound is dissolved into 10mM mother liquors with DMSO, and with DMSO 1:3 gradients 1000 × series of compounds concentration storing liquid of 10 concentration gradients is diluted to, utilizes compound transferring apparatus Liquid 1000 × series concentration compound storing liquid is transferred in the respective aperture of the orifice plate of cell 384 by Handler Echo520, per hole 50nL, isometric solvent DMSO is added in blank control wells.Soft to mix, 37 DEG C are continued to cultivate.Re-replaced after 72h corresponding Culture medium, and breed to 3 μ l of every hole addition CCK8 cells --- toxicity detection reagent.Continue culture plate in 37 DEG C of incubators 2h is incubated, light absorption value is then determined at 540/720nm wavelength using enzyme detector.

This experiment determines two kind hypotypes of the synthesized target compound to HSP90 albumen respectively using the above method HSP90 α and HSP90 β inhibitory activity；The human lung cancer cell line A549 of expression high to HSP90, human breast carcinoma cell lines MCF-7, Chronic myeloid leukemia cells system K562, prostate cancer cell line DU145 and experiment are with proliferating epidermal cancerous cell line Hela's Inhibitory activity.As a result it is as shown in table 1.

The target compound of table 1 is to HSP90 albumen and the inhibitory activity IC of tumor cell line₅₀(unit nM)

According to table 1, from the point of view of enzyme activity level, above-claimed cpd is respectively provided with inhibitory action to two kinds of HSP90 hypotypes simultaneously, and Majority of compounds and HSP90 α binding ability are relatively better than HSP90 β.Compared to positive reference compound NVP/AUY-922 (CAS： 747412-49-3), most compounds show stronger inhibitory activity, further prove that target skeleton is more suitable for target spot Active pocket.From the point of view of cellular level, above-claimed cpd has stronger inhibitory activity, most of chemical combination to five quasi-cancer cell systems Thing can reach several or tens nanomoles level, especially have preferable inhibition to epidermal carcinoma cell lines Hela.

Each technical characteristic of embodiment described above can be combined arbitrarily, to make description succinct, not to above-mentioned reality Apply all possible combination of each technical characteristic in example to be all described, as long as however, the combination of these technical characteristics is not deposited In contradiction, the scope that this specification is recorded all is considered to be.

Embodiment described above only expresses the several embodiments of the present invention, and its description is more specific and detailed, but simultaneously Can not therefore it be construed as limiting the scope of the patent.It should be pointed out that come for one of ordinary skill in the art Say, without departing from the inventive concept of the premise, various modifications and improvements can be made, these belong to the protection of the present invention Scope.Therefore, the protection domain of patent of the present invention should be determined by the appended claims.

Claims (11)

1. a kind of heat shock protein inhibitors or its pharmaceutically acceptable salt with Formulas I architectural feature：

Wherein：

R₁、R₇、R₈It is respectively and independently selected from：H, C₁-C₆Alkyl, C₂-C₆Unsaturated alkyl, halogen, hydroxyl, C₁-C₆Alkoxy, NHCOOR, SO₂NHR, NHSO₂R, CN, NHCOR, CONHR；

R is selected from：H, C₁-C₆Alkyl, C₃-C₆Unsaturated alkyl, C₃-C₈Cycloalkyl；

R₂、R₃It is respectively and independently selected from：H, D, C₁-C₆Alkyl, C₃-C₆Unsaturated alkyl, C₃-C₈Cycloalkyl, phenyl, substituted phenyl, Heteroaryl, acyl group；

R₄It is selected from：Amino, C₁-C₆Saturated alkyl amine, C₂-C₆Saturated heterocyclic amine, C₁-C₆Alkylamidoalkyl, aryl amido group, substitution Aryl amido group, heteroaryl amide base, substituted heteroaryl amide base；

R₅、R₆For same oxygen atom or it is respectively and independently selected from：H, D, C₁-C₆Alkyl, C₃-C₈Cycloalkyl, carbonyl, cyano group, C₁-C₆ Alkoxy, COOR ', NHCOR ', CONHR '；

R ' is selected from：H, C₁-C₆Alkyl, C₃-C₆Unsaturated alkyl；

X, Y, Z, W are respectively and independently selected from：C, NH, CH, N, O, S.

2. heat shock protein inhibitors according to claim 1 or its pharmaceutically acceptable salt, it is characterised in that described Comprising X, Y, Z, W five yuan of heteroaromatics are selected from following structure：

3. heat shock protein inhibitors according to claim 1 or its pharmaceutically acceptable salt, it is characterised in that described Heat shock protein inhibitors are selected from below formula II compound：

Wherein：

R₇、R₈It is selected from：H, C₁-C₆Alkyl, C₂-C₆Unsaturated alkyl, halogen, hydroxyl, C₁-C₆Alkoxy, CN；

R₁It is selected from：C₁-C₆Alkyl, C₂-C₆Unsaturated alkyl, C₁-C₆Alkoxy.

4. heat shock protein inhibitors according to claim 1 or its pharmaceutically acceptable salt, it is characterised in that described R₂、R₃It is respectively and independently selected from：H, C₃-C₈Cycloalkyl, C₁-C₆Alkyl, C₃-C₆Unsaturated alkyl.

5. heat shock protein inhibitors or its pharmaceutically acceptable salt according to claim any one of 1-4, its feature It is, R₄It is selected from：Amino, C₁-C₆Saturated alkyl amine, C₂-C₆Saturated heterocyclic amine, C₁-C₆Alkylamidoalkyl, aryl amido group, take The aryl amido group in generation, heteroaryl amide base, substituted heteroaryl amide base；

R₅It is selected from：H, or and R₆For same oxygen atom；

R₆It is selected from：CN, COOR ', CONHR ', or and R₅For same oxygen atom；

Wherein, R ' is selected from：H, C₁-C₆Alkyl, C₃-C₆Unsaturated alkyl, C₃-C₈Cycloalkyl.

6. heat shock protein inhibitors according to claim 5 or its pharmaceutically acceptable salt, it is characterised in that R₄Choosing From following group：

7. heat shock protein inhibitors according to claim 1 or its pharmaceutically acceptable salt, it is characterised in that described Heat shock protein inhibitors are selected from below formula III compound：

Wherein：

R₁It is selected from：C₁-C₆Alkyl；

R₇、R₈It is selected from：Hydroxyl；

R₂It is selected from：H；

R₃It is selected from：C₁-C₆Alkyl；

R₄Selected from following group：

R₅It is selected from：H, or and R₆For same oxygen atom；

R₆It is selected from：CN, COOR ', CONHR ', or and R₅For same oxygen atom；

Wherein, R ' is selected from：H, C₁-C₆Alkyl, C₃-C₆Unsaturated alkyl, C₃-C₈Cycloalkyl.

8. heat shock protein inhibitors according to claim 1 or its pharmaceutically acceptable salt, it is characterised in that be selected from Following compound：

9. the preparation method of the heat shock protein inhibitors or its pharmaceutically acceptable salt described in claim any one of 1-8, Characterized in that, using following circuit combination：

Wherein：R₁₀For hydroxyl protecting group.

10. the heat shock protein inhibitors or its pharmaceutically acceptable salt described in claim any one of 1-8 are preparing prevention With the application in the medicine of disease of the treatment with the increased pathological characteristicses of expression of heat shock protein 90.

11. application according to claim 10, it is characterised in that described that there is the increased pathology of expression of heat shock protein 90 Learn feature disease be：Cancer, metabolic disease, myelodysplastic syndrome, systemic mastocytosis, Xi Peier- Lindau's syndrome, multicenter type Castleman diseases, and at least one of psoriasis.