CN107540624B

CN107540624B - Heat shock protein inhibitor and preparation method and application thereof

Info

Publication number: CN107540624B
Application number: CN201610509310.5A
Authority: CN
Inventors: 张健存; 林财; 李德耀; 邹晴安; 张袁超; 顾自强
Original assignee: Guangzhou Henovcom Bioscience Co ltd
Current assignee: Guangzhou Henovcom Bioscience Co ltd
Priority date: 2016-06-29
Filing date: 2016-06-29
Publication date: 2020-06-16
Anticipated expiration: 2036-06-29
Also published as: CN107540624A; WO2018001084A1

Abstract

The invention discloses a heat shock protein inhibitor and a preparation method and application thereof, belonging to the technical field of pharmaceutical chemistry. The heat shock protein inhibitor has the structural characteristics of a formula I. The compound can inhibit the activity of heat shock protein 90, and can be further used for preparing anti-tumor medicaments.

Description

Heat shock protein inhibitor and preparation method and application thereof

Technical Field

The invention relates to the technical field of pharmaceutical chemistry, in particular to a heat shock protein inhibitor and a preparation method and application thereof.

Background

HSP90 (HSP90) is an ATP-dependent molecular chaperone involved in activation and promotion of maturation of client proteins (client proteins) by HSP100(100-110kD), HSP90(83-90kD), HSP70(66-78kD), HSP60 and small HSP (15-30kD) according to molecular weight, wherein HSP90 is an ATP-dependent molecular chaperone involved in activation and promotion of maturation of client proteins (client proteins), maintains the conformation and function of various proteins of cells, and is closely associated with proliferation, apoptosis, canceration and tumor development of cells in normal cells, HSP90 expressed accounts for 1-2% of total intracellular protein, while 3% of these are found in the nucleus, which have a function of influencing nuclear regulation, under stress conditions, such as in cancer cells, the level of expressed HSP90 accounts for 4-6% of the entire protein 90 and 200 different client proteins interact with each other, these proteins are involved in the development of tumor cell signaling, and are involved in the cytoplasmic receptor subtypes of multiple HSP 58465, HSP 53, HSP 465, HSP 53, and the receptor subtypes of which are involved in the development of cancer cell, including the basal protein receptor subtypes of tumor, and the basal protein receptor, which are associated with the basal protein kinase receptor subtypes of tumor development of tumor, including the basal protein kinase receptor kinase, and the basal protein kinase receptor, which are classified into a tumor development of HSP90, and the basal protein receptor subtype of HSP90, and the basal protein receptor, which is classified into a tumor development of the basal protein, and the basal protein, so that the basal protein receptor, so that the basal protein, the basal protein of the basal protein receptor, the basal protein of HSP90, the basal protein of which is classified into the basal protein of the HSP90, the basal protein of the basal protein.

The HSP90 client protein needs to participate in the structural and functional stabilization of HSP, plays an important role in promoting cell growth, proliferation, survival and the like, is in an over-expression or sustained expression state in malignant tumor, and is closely related to the occurrence and development of tumor. Such as: the maintenance and regulation of the conformation and function of tumor-specific proteins such as mutant EGFR in non-small cell lung cancer cells (NSCLC), fusion protein BCR-ABL in chronic myelogenous leukemia cells, Her2 in breast cancer cells and the like require the participation of HSP 90.

The incidence of non-small cell lung cancer is 3/4-4/5 of lung cancer, whereas EGFR is highly expressed in about 3/5 non-small lung cancer cells. Many patients currently develop resistance to gefitinib and erlotinib, which are FDA approved drugs for the marketing of non-small cell lung cancer. Research shows that the HSP90 inhibitor 17-DMAG has an antiproliferative effect on 20 EGFR non-small cell lung cancer cell strains, and can obviously reduce the levels of p-EGFR, p-Akt, cyclinD1, Cdk4 and the like in EGFR mutant cell strains. Whereas 17-AAG down-regulates WT-EGFR levels, but requires higher concentrations and duration of action. The main reason for causing non-small cell lung cancer to be resistant to gefitinib and erlotinib is the K-Ras mutation. Although there are no specific inhibitors for their treatment, Jamie et al found that Ganetespib degraded the substrate C-Raf of K-Ras, inactivating some downstream signaling molecules. These results indicate that HSP90 inhibitors have significant inhibitory effects on non-small lung cancer cells, thereby antagonizing the resistance of non-small lung cancer cells to gefitinib and erlotinib.

20% -30% of breast cancers excessively express proto-oncogene Her2, and most of breast cancer patients who partially excessively express Her2/neu are clinically applied with herceptin monoclonal antibodies, but the majority of patients generate drug resistance. Since Her2 is most dependent on HSP90 and most sensitive to HSP90 inhibitors, Scaltriti et al studied the effect of HSP90 inhibitor IPI504 on treated BT47R (Her2+), BT477H104 1047R (Her2+) breast cancer cell lines in a primary or secondary drug resistance model caused by PI3K mutation activation or decreased PTEN expression. The results show that IPI504 can down-regulate the expression of Her2 in the above two cells, inhibit AKT and MAPK signal pathways, and inhibit cell proliferation in a dose-dependent manner. The main reason for resistance to Trastuzmab is the deletion of the extracellular domain of HER2 resulting in p95-HER2 and thus the loss of the domain to which it binds resulting in resistance. The research of Chandarlaycapacity finds that the HSP90 inhibitor SNX-2112 can degrade p95-Her2 in cells and simultaneously inhibit the activities of AKT and ERK; the cytotoxic test shows that the SNK-2112 can completely inhibit the proliferation of T47D cells, and is greatly higher than that of Trastuzmab; the use of the oral prodrug of SNX-2112, SNX-5422, in vivo, acts on a p95-Her2 expressing MEFS tumor model, which can completely inhibit tumor growth. Since triple negative breast cancer lacks therapeutic targets against Her2, no targeted inhibitor is currently available. However, experimental studies show that PU-H71 can inhibit the activity of AKT and Bcl-xl protein and down regulate the level of AKT and Bcl-xl protein, thereby inducing the apoptosis of triple-negative breast cancer cells. The cell death rates of the three-negative breast cancer cell strains HCC-1806, MAD-MB-231 and MAD-MB-468 are 80%, 65% and 80% respectively. From the above, it is known that HSP90 inhibitors have very important significance as clinical drug-resistant treatment for breast cancer.

Moreover, the occurrence of chronic myelogenous leukemia is closely related to the mutation of the ABL gene, and imatinib is widely used for the treatment of chronic myelogenous leukemia. Leukemia patients with resistance to imatinib mainly result from gene amplification or point mutation in the BCL-ABL kinase domain. This kinase is a HSP90 client protein and therefore HSP90 inhibitors should be able to act on it. Experimental research shows that tripterine, a natural HSP90 inhibitor, can cause apoptosis of imatinib-resistant KMB5-T315I cells caused by mutation of T315I, and immunohistochemical analysis shows that the tripterine inhibits expression of BCL-ABL. And 17-AAG, a synthetic small molecule HSP90 inhibitor, can degrade mutant and wild-type BCL-ABL, thereby inhibiting cell proliferation. Imatinib-resistant chronic myelogenous leukemia can therefore be antagonized using HSP90 inhibitors.

Compared with the traditional kinase inhibitor, the HSP90 inhibitor can degrade a plurality of similar kinases while inhibiting the activity of HSP90, and can generate better inhibition effect on some drug-resistant tumors. Experiments show that the HSP90 inhibitor has the antitumor effect by single or combined administration. HSP90 is therefore a research and development meaningful target for cancer therapy.

Disclosure of Invention

Based on the above, the invention provides a novel heat shock protein inhibitor, which can inhibit the activity of heat shock protein 90 and can be further used for preparing anti-tumor medicaments.

A heat shock protein inhibitor having the structural feature of formula I:

wherein:

R₁、R₇、R₈each independently selected from: h, C₁-C₆Alkyl radical, C₂-C₆Unsaturated alkyl, halogen, hydroxy, C₁-C₆Alkoxy, NHCOOR, SO₂NHR，NHSO₂R，CN，NHCOR，CONHR；

R is selected from: h, C₁-C₆Alkyl radical, C₃-C₆Unsaturated alkyl radical, C₃-C₈A cycloalkyl group;

R₂、R₃each independently selected from: h, D, C₁-C₆Alkyl radical, C₃-C₆Unsaturated alkyl radical, C₃-C₈Cycloalkyl, phenyl, substituted phenyl, heteroaryl, acyl;

R₄selected from: amino group, C₁-C₆Saturated alkylamines, C₂-C₆Saturated heterocyclic amines, C₁-C₆Alkylamido, arylamido, substituted arylamido, heteroarylamido, substituted heteroarylamido;

R₅、R₆is the same oxygen atom or is respectively and independently selected from: h, D, C₁-C₆Alkyl radical, C₃-C₈Cycloalkyl, carbonyl, cyano, C₁-C₆Alkoxy, COOR ', NHCOR ', CONHR ';

r' is selected from: h, C₁-C₆Alkyl radical, C₃-C₆An unsaturated alkyl group;

x, Y, Z, W are each independently selected from: c, NH, CH, N, O, S.

The present inventors have discovered in their research that the benzyl site of morpholine linkage in the structure of AUY922, a reported HSP90 inhibitor, may be readily metabolized and may produce toxic side effects. On the basis of the research, the inventor finds out through experimental groping and research that other groups are introduced into the benzyl site on the basis of the accumulation of long-term experience of the inventor, so that the activity is improved, and the metabolism of the site can be inhibited, and the potential toxic and side effects are eliminated.

In one embodiment, the five-membered heteroaromatic ring comprising X, Y, Z, W is selected from the following structures:

in one embodiment, the heat shock protein inhibitor is selected from the group consisting of compounds of formula II:

wherein:

R₇、R₈selected from: h, C₁-C₆Alkyl radical, C₂-C₆Unsaturated alkyl, halogen, hydroxy, C₁-C₆Alkoxy, CN;

R₁selected from: c₁-C₆Alkyl radical, C₂-C₆Unsaturated alkyl radical, C₁-C₆An alkoxy group.

In one embodiment, R is₂、R₃Each independently selected from: h, C₃-C₈Cycloalkyl radical, C₁-C₆Alkyl radical, C₃-C₆An unsaturated alkyl group.

In one embodiment, R₄Selected from: amino group, C₁-C₆Saturated alkylamines, C₂-C₆Saturated heterocyclic amines, C₁-C₆Alkylamido, arylamido, substituted arylamido, heteroarylamido, substituted heteroarylamido;

R₅selected from: h, or with R₆Are the same oxygen atom;

R₆selected from: CN, COOR ', CONHR', or with R₅Are the same oxygen atom;

wherein R' is selected from: h, C₁-C₆Alkyl radical, C₃-C₆Unsaturated alkyl radical, C₃-C₈A cycloalkyl group.

In one embodiment, R₄Selected from the group consisting of:

in one embodiment, the heat shock protein inhibitor is selected from the group consisting of compounds of formula III:

wherein:

R₁selected from: c₁-C₆An alkyl group;

R₇、R₈selected from: a hydroxyl group;

R₂selected from: h;

R₃selected from: c₁-C₆An alkyl group;

R₄selected from the group consisting of:

R₅selected from: h, or with R₆Are the same oxygen atom;

R₆selected from: CN, COOR ', CONHR', or the same oxygen atom as R5

The invention also discloses a preparation method of the heat shock protein inhibitor or the pharmaceutically acceptable salt thereof, which adopts the following route for synthesis:

wherein: r₁₀Is a hydroxyl protecting group.

R₁₀Is selected from: benzyl, trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, ditert-butylmethylsilyl, methyl, acetyl, p-methoxybenzyl, methoxymethyl.

The invention also discloses application of the heat shock protein inhibitor or the pharmaceutically acceptable salt thereof in preparing a medicament for preventing and treating diseases with the pathological characteristics of increased expression of the heat shock protein 90.

In one embodiment, the disease characterized by the pathology of increased expression of heat shock protein 90 is: cancer, metabolic disease, myelodysplastic syndrome, systemic mastocytosis, von hippel-lindau syndrome, multicentric Castleman disease, and psoriasis.

Compared with the prior art, the invention has the following beneficial effects:

the heat shock protein inhibitor or the pharmaceutically acceptable salt thereof is a novel heat shock protein inhibitor, compared with the conventional similar inhibitors, the heat shock protein inhibitor or the pharmaceutically acceptable salt thereof retains atoms and functional groups interacted with amino acid residues in an active pocket of an HSP90 receptor in an original molecule, and further modifies the position of a skeleton where acting force possibly exists, so that the inhibition effect of a control compound is optimized, and the heat shock protein inhibitor or the pharmaceutically acceptable salt thereof has the following advantages:

1. the introduction of effective modification can increase the binding force of target compound and amino acid residue of receptor protein, and inhibit two enzyme receptors of HSP90 α and HSP90 β and five tumor cell lines.

2. The inhibition capability of the positive control compound NVP/AUY-922 at an enzyme level and a cell level is further improved, and the possibility of promoting the drug development of the inhibitor is provided.

3. Compared with a single inhibitor, the simultaneous use of two drugs respectively acting on a single target point has the advantages that the drug patients acting on the two target points are convenient to use, and the interaction between the drugs can be avoided.

Detailed Description

The present invention is further illustrated by the following examples, which are not intended to limit the scope of the present invention in any way.

The compounds of the invention and their salts can also be prepared by methods known for the preparation of chemically related compounds, and the starting materials referred to in the examples can be obtained by analogous methods of the prior art.

The straight line pointing to the inside of the benzene ring outside the benzene ring in the general formula I of the invention represents the substitution with unfixed position.

The five-membered ring with dotted line in the general formula I is a five-membered aromatic heterocycle containing X, Y, Z and W.

"alkyl" refers to saturated hydrocarbon radicals including straight or branched chain alkyl radicals, such as C₁-C₆Alkyl means a saturated straight or branched chain alkyl group having 1 to 6 carbon atoms, wherein examples of the saturated straight chain alkyl group include, but are not limited to, ethyl, n-propyl, and the like, and examples of the saturated branched chain alkyl group include, but are not limited to, isopropyl, tert-butyl, and the like; "unsaturated alkyl" refers to a hydrocarbon group having an alkenyl or alkynyl group, including straight or branched chain unsaturated alkyl groups, wherein examples of unsaturated straight chain alkyl groups include, but are not limited to, ethenyl, propenyl, and the like, and examples of unsaturated branched chain alkyl groups include, but are not limited to, 2-methylpropenyl, and the like; "cycloalkyl" refers to an alkyl group having a cyclic structure, such as C₃-C₈The cyclic alkyl group means a saturated or unsaturated alkyl group having a cyclic structure having 3 to 8 carbon atoms, wherein examples of the saturated cyclic alkyl group include, but are not limited to, cyclopropyl, cyclopentyl, ethyl-substituted cyclohexyl, etc., and examples of the unsaturated cyclic alkyl group include, but are not limited to, cyclopentene, etc., with C being preferred in the present invention₃-C₆A cyclic alkane.

"substituted" refers to the replacement of a hydrogen radical in a particular structure with a radical of a specified substituent. When more than one position in any particular structure may be substituted with more than one substituent selected from a specified group, the substituents may be the same or different at each position. As used herein, the term "substituted" is intended to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. A heteroatom such as nitrogen may have a hydrogen substituent and/or any permissible substituents of organic compounds described herein that satisfy the valences of the heteroatom.

"heteroaryl" means a 4n +2 aromatic ring system containing a 5-6 membered monocyclic 6 ring and having from 1 to 4 ring heteroatoms and ring carbon atoms, wherein each ring heteroatom is independently selected from nitrogen, oxygen, and sulfur. Heteroaryl groups containing one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom if valency permits. The polycyclic ring system of heteroaryl groups can include one or more heteroatoms. "heteroaryl" also includes heteroaryl ring systems as defined above fused with one or more carbocyclic or heterocyclic groups in which the point of attachment is on the heteroaryl ring, and in which case the number of ring members includes only the number of members on the heteroaryl ring system. "heteroaryl" also includes heteroaryl ring systems as defined above fused with one or more aryl groups, where the point of attachment may be on an aryl or heteroaryl ring, and in which case the number of ring members includes only the number of ring members in the fused polycyclic (aryl/heteroaryl) ring system. Polycyclic heteroaryl groups, wherein one of the rings does not contain a heteroatom (e.g., indolyl, quinolinyl, carbazolyl, and the like), may have the point of attachment on either ring, that is, on the ring containing the heteroatom (e.g., 2-indolyl) or on the ring that does not contain the heteroatom (e.g., 5-indolyl). Exemplary 5-membered heteroaryl groups containing 2 heteroatoms include, but are not limited to, imidazole, pyrazole, oxazole, isoxazolyl, thiazolyl, and isothiazole. Exemplary 6-membered heteroaryl groups containing 1 heteroatom include, but are not limited to, pyridinyl. Exemplary 6-membered heteroaryl groups containing 2 heteroatoms include, but are not limited to, pyridazinyl, pyrimidinyl, and pyrazinyl.

"Heterocyclyl" refers to a cyclic alkyl group as defined herein, wherein the backbone further comprises 1 or more heteroatoms (e.g., oxygen, sulfur, nitrogen, boron, silicon, phosphorus).

The "amine group" refers to an organic compound in which a hydrogen atom of ammonia is replaced with an alkyl group, such as a heterocyclic amine, an aralkylamino group, and the like.

"amido" refers to derivatives of ammonia (or amine) wherein the hydrogen is replaced by an acyl group, such as cycloalkylamido, alkylamido, arylamido, benzoarylamido, heteroarylamido, benzoheteroarylamido, heterocyclylamido, sulfonamido, alkylureas, and the like.

Example 1

The following examples are prepared by reference to the following reaction schemes

Compound 1: preparation of 4- (4- (cyano (morpholino) methyl) phenyl) -5- (2, 4-dihydroxy-5-isopropylphenyl) -methylisoxazole-3-carboxamide).

The method is synthesized according to the above circuit, and comprises the following steps:

(1) preparation of 1- (2, 4-bis (benzyloxy) phenyl) ethanone (compound I-2).

To a 500mL two-necked flask equipped with a reflux condenser was added 2, 4-dihydroxy acetophenone I-1(9.12g, 60.00mmol), potassium carbonate (21.00g, 151.00mmol), and 200mL acetonitrile in an oil bath and heated at 80 deg.C under reflux for 1h, after which benzyl bromide (14.70mL, 144.00mmol) was injected by syringe and continued to reflux overnight. TLC (thin layer chromatography) detection reaction until the starting material completely reacts, cooling to room temperature, suction-filtering through a Buchner funnel, washing the residue with dichloromethane, collecting the filtrate, and removing the solvent by rotary evaporation to obtain a brown yellow oil. And adding diethyl ether into the oily substance, stirring to generate white flocculent precipitate, performing suction filtration by using a sand core funnel, washing by using the diethyl ether, and repeating the operations twice to obtain 15.80g of white solid, namely the compound I-2 with the yield of 79%. Mass Spectrometry ESI-MS M/z:333.0(M + H)⁺。

(2) Preparation of ((4-prop-1-en-2-yl) -1,3- (dioxy)) bis (methylene)) diphenyl (compound I-3).

A250 mL two-necked flask equipped with a dropping funnel was charged with potassium tert-butoxide (4.72g, 42.15mmol), triphenylphosphoriodomethane (17.88g, 42.15mmol), and anhydrous THF (tetrahydrofuran) was injected under nitrogen at 0 ℃ and stirred for 1 h. Then 1- (2, 4-bis (benzyloxy) phenyl) ethanone I-2(10.76g, 32,42mmol) is completely dissolved in anhydrous THF and injected into a dropping funnel, and then slowly added into the reaction system through the dropping funnel in a dropwise manner, after the dropwise addition is complete, the reaction system is stirred for 1h, and then the reaction system is stirred at room temperature overnight. After TLC monitoring starting materials completely react, rotary evaporation is carried out to remove reaction solvents, ethyl acetate is used for dissolving obtained residues, water is used for extracting for three times, saturated sodium chloride aqueous solution is used for extracting once, ethyl acetate layers are collected, drying is carried out through anhydrous sodium sulfate, rotary evaporation is carried out to remove organic solvents, and finally, silica gel column purification is carried out to obtain 8.45g of white solids, namely the compound I-3, wherein the yield is 79%.

The characterization data for this compound I-3 are: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-isopropylbenzene-1, 3-diol (Compound I-4).

A2L autoclave was charged with I-3(350.00g, 1.06mol), 35g of 10% palladium on carbon, 1mL of formic acid, 1L of ethanol, purged with hydrogen and heated to 78 ℃ under reflux for two days. And cooling to room temperature after the reaction is completed, performing suction filtration on the reaction solution by using a Buchner funnel filled with diatomite, washing with ethanol, collecting filtrate, performing rotary evaporation to remove the organic solvent, and finally purifying by using a silica gel column to obtain 130g of white solid, namely the compound I-4, wherein the yield is 80%.

The characterization data for this compound I-4 are: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-isopropylphenyl) -ethanone (Compound I-5).

A250 mL two-necked flask equipped with a reflux condenser was charged with 4-isopropylbenzene-1, 3-diol I-4(5.36g, 35.25mmol), followed by N₂Under the protection of (1), injecting 100mL of 47% boron trifluoride diethyl etherate solution, stirring for half an hour, injecting 4.03mL of acetic acid into an oil bath pot, heating and refluxing overnight, monitoring by TLC that the initial raw materials are completely reacted, cooling to room temperature, adding 70mL of 10% sodium acetate aqueous solution, stirring for 2h, adding water to dilute to 250mL, adding ethyl acetate to extract for three times, collecting an organic layer, drying by anhydrous sodium sulfate, and then performing rotary evaporation to remove the organic solventObtaining a brownish red solid, adding dichloromethane for pulping to generate a light yellow insoluble substance, performing suction filtration by using a sand core funnel, and washing by using dichloromethane to obtain 6.80g of light yellow solid, namely the compound I-5 with the yield of 99.4%.

The characterization data for this compound I-5 are: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- (2, 4-bis (benzyloxy) -5-isopropyl) -ethanone (compound I-6).

1- (2, 4-dihydroxy-5-isopropyl) acetophenone I-5(6.80g, 35.05mmol), potassium carbonate (12.09g, 87.63mmol), 100mL acetonitrile were added to a 250mL two-necked flask equipped with a reflux condenser, refluxed at 80 ℃ for 1h in an oil bath, and then injected with benzyl bromide (10mL, 84.15mmol) by syringe and refluxed overnight. And monitoring the reaction starting material by TLC, cooling the reaction system to room temperature after the reaction is completed, performing suction filtration by using a Buchner funnel, washing the residue by using dichloromethane, collecting filtrate, and performing rotary evaporation to remove the organic solvent to obtain a brownish red oily substance. Adding petroleum ether into the oily matter, stirring and pulping to generate white precipitate, performing suction filtration by using a sand core funnel, and washing by using petroleum ether to obtain 6.72g of white solid, namely the compound I-6, wherein the yield is 51.26%.

The characterization data for this compound I-6 are: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) preparation of methyl (Z) -ethyl-4- (2, 4-bis (benzyloxy) -5-isopropylphenyl) -2-hydroxy-4-oxobut-2-enoate (Compound I-7).

Adding metal sodium (0.90g, 39.00mmol) into a 250mL two-neck flask provided with a reflux condenser tube, stirring 100mL ethanol in an ice bath at 0 ℃ until the metal sodium completely reacts, adding 1- (2, 4-bis (benzyloxy) -5-isopropylphenyl) ethanone I-6(6.72g, 17.98mmol) at room temperature, stirring for 30min, and injecting diethyl oxalate (3.94mL, 29.11mmol) into an oil bath kettle to reflux for 4h at 80 ℃. And (3) monitoring by TLC (thin layer chromatography) until the substrate completely reacts, cooling to room temperature, adding 2M hydrochloric acid aqueous solution to adjust the pH value to be less than 7 to generate a light yellow precipitate, drying in vacuum to remove an organic solvent, adding dichloromethane to dissolve, extracting with water for three times, extracting with saturated sodium chloride solution for one time, drying with anhydrous sodium sulfate, and then performing rotary evaporation to remove dichloromethane to obtain 8.18g of a brown yellow solid, namely the compound I-7 with the yield of 96%.

The characterization data for this compound I-7 are: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) preparation of ethyl 5- (2, 4-bis (benzyloxy-5-isopropylphenyl) isoxazole-3-carboxylate (Compound I-8).

To a 250mL two-necked flask equipped with a reflux condenser were added (Z) -ethyl-4- (2, 4-bis (benzyloxy) -5-isopropylphenyl) -2-hydroxy-4-oxobut-2-enoic acid methyl ester I-7(8.18g, 17.26mmol), hydroxylamine hydrochloride (1.56g, 22.48mmol) and 100mL of ethanol in an oil bath and refluxed at 80 ℃ for 5 h. TLC after monitoring the reaction was complete, cooled to room temperature and dried in vacuo to give a white solid. Dissolving the solid with ethyl acetate, extracting with water for three times, extracting with saturated sodium chloride aqueous solution once, drying the organic layer with anhydrous sodium sulfate, and removing the organic solvent by rotary evaporation to obtain 6.74g of white solid, namely the compound I-8 with the yield of 83%.

The characterization data for this compound I-8 are: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- (2, 4-bis (benzyloxy) -5-isopropylphenyl) isoxazole-3-carboxamide (Compound I-9).

5- (2, 4-bis (benzyloxy-5-isopropylphenyl) isoxazole-3-carboxylic acid ethyl ester I-8(6.74g, 14.31mmol) and 100mL of ethanol were added into a 250mL two-necked flask equipped with a reflux condenser tube, heated and stirred to completely dissolve the solid, and then 70% aqueous solution of ethylamine (13.80mL, 215.73mmol) was injected into the flask and refluxed overnight at 80 ℃ in an oil bath, TLC was used to monitor the completion of the reaction and cooled to room temperature, and vacuum-dried to obtain a white solid, ethyl acetate-dissolved solid, 1M hydrochloric acid aqueous solution was extracted once, saturated sodium carbonate aqueous solution was extracted once, water was extracted once, saturated sodium chloride aqueous solution was extracted once, anhydrous sodium sulfate was used to dry the organic layer, and then the organic solvent was removed by rotary evaporation to obtain 6.46g of a white solid with a yield of 96.

The characterization data for this compound I-9 are: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) preparation of 5- (2, 4-bis (benzyloxy) -5-isopropylphenyl) -methylisoxazole-3-carboxamide (Compound I-10)

5- (2, 4-bis (benzyloxy) -5-isopropylphenyl) -N-ethylisoxazole-3-carboxamide I-9(6.46g, 13.47mmol) and 100mL of acetonitrile were added to a 250mL two-necked flask equipped with a reflux condenser and heated with stirring to dissolve the solid completely, and iodosuccinimide (3.71g, 16.49mmol) and ammonium cerium nitrate (0.74mg, 1.35mmol) were added to the flask and refluxed at 85 ℃ overnight in an oil bath. After the reaction was monitored by TLC, it was cooled to room temperature and dried in vacuo to give a reddish brown solid. Ethyl acetate dissolves the solid, water extraction is carried out for three times, saturated sodium chloride aqueous solution is extracted once, the organic layer is dried by anhydrous sodium sulfate, then rotary evaporation is carried out to remove the organic solvent, and finally, purification is carried out by a silica gel column to obtain 5.62g of brown white solid with the yield of 70%.

The characterization data for this compound I-10 are: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) preparation of 5- (2, 4-bis (benzyloxy) -5-isopropylphenyl) -N-methyl-4- (4-formylphenyl) isoxazole-3-carboxamide (Compound I-11).

In a 100mL two-necked flask equipped with a reflux condenser were placed 5- (2, 4-bis (benzyloxy) -5-isopropylphenyl) -N-ethyl-4-iodoisoxazole-3-carboxamide 1-10(1.35g, 2.27mmol), 4-formylphenylboronic acid (0.68g, 4.54mmol), ditriphenylphosphine dichloropalladium (0.08g, 0.12mmol), potassium carbonate (0.626mg, 4.54mmol), and 20mL of tetrahydrofuran and 2mL of distilled water, and they were refluxed in an oil bath at 80 ℃ overnight under nitrogen protection. And (3) monitoring the reaction by TLC, cooling to room temperature, removing the reaction solvent by rotary evaporation, adding ethyl acetate for dissolving, extracting for three times by water, extracting once by saturated sodium chloride aqueous solution, drying by anhydrous sodium sulfate, and purifying by a silica gel column after vacuum drying to obtain 1.02g of a reddish brown solid with the yield of 79%.

The characterization data for this compound I-11 are: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) preparation of 5- (2, 4-dihydroxy-5-isopropylphenyl) -N-methyl-4- (4-formylphenyl) isoxazole-3-carboxamide (Compound I-12)

A100 mL two-necked flask was taken, compound I-11(1g) was added, argon was used as a shield, anhydrous DCM (20mL) was added for dissolution, the mixture was placed at-5 ℃ and stirred for 15min, a solution of boron tribromide in dichloromethane (1M, 5.2mL, 3 equiv.) was added dropwise, stirred for 20min, and the mixture was placed at room temperature and stirred for 2 h. TLC tracking indicated the starting material was complete. At 0 deg.C, DCM (10mL) was added for dilution and saturated NaHCO was added₃The solution was quenched, extracted and the organic phase collected. EA was added to the aqueous phase, the resulting flocculent solid was dissolved, the organic phases were collected, mixed, dried over anhydrous sodium sulfate and concentrated over the column (DCM: acetone ═ 1: 1). 650mg of a brown solid are obtained in a yield of 94.8%. ESI-MSm/z:395.1(M + H)⁺。

(12) Preparation of 4- (4- (cyano (morpholino) methyl) phenyl) -5- (2, 4-dihydroxy-5-isopropylphenyl) -methylisoxazole-3-carboxamide (Compound 1).

A 25mL single vial was taken and compound I-12(50mg), methanol (2mL) dissolved, morpholine (17 μ L, 1.5 eq), guanidine hydrochloride (6mg,0.5 eq), stirred at 40 ℃ for 15min, compound TMSCN (47 μ L, 3 eq) was added, stirred at this temperature for 2h, TLC trace showed the starting material was reacted. EA dilution (10mL) was added and saturated NaHCO was added₃The solution was quenched, extracted with brine, dried over anhydrous sodium sulfate, and concentrated over the column (DCM: acetone ═ 1: 1). Pulping the obtained yellow viscous product, adding petroleum ether, standing at low temperature to separate out white solid, sucking yellow liquid, and pumping the obtained solid oil to obtain 25mg of white solid with a yield of 41.0%.

The characterization data for this compound 1 are: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)^-.

example 2

Compound 2: preparation of 4- (4- (cyano (2S,6R) -2, 6-dimethylmorpholine) methyl) -5- (2, 4-dihydroxy-5-isopropylphenyl) -N-ethylisoxazole-3-carboxamide.

A25 mL single vial was taken, compound I-12(30mg) and methanol (1mL) were added and dissolved, (2S,6R) -2, 6-dimethylmorpholine (15. mu.L, 1.5 equiv.), guanidine hydrochloride (4mg, 0.5 equiv.), stirred at 40 ℃ for 15min, compound TMSCN (29. mu.L, 3 equiv.) was added and TLC follow-up showed that the starting material was not completely reacted and was therefore directly purified. EA dilution (10mL) was added and saturated NaHCO was added₃The solution was quenched, extracted with brine, dried over anhydrous sodium sulfate, and concentrated over the column (DCM: acetone ═ 1: 1). The resulting yellow viscous product was purified by HPLC and the resulting solid oil pumped dry to give 9mg of a white solid with a yield of 23.1%.

The characterization data for this compound 2 are: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)^-.

example 3

Compound 3: preparation of 4- (4- (cyano (4-methylpiperazin-1-yl) methyl) phenyl) -5- (2, 4-dihydroxy-5-isopropylphenyl) -N-methylisoxazole-3-carboxamide the procedure of example 1 was referenced.

Characterization data for this compound 3 are: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)^-.

example 4

Compound 4: preparation of 4- (4- (cyano- (4-hydroxypiperidin-1-yl) methyl) phenyl) -5- (2, 4-dihydroxy-5-isopropylphenyl) -N-methylisoxazole-3-carboxamide the procedure of example 1 was referenced.

Characterization data for this compound 4 are: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)^-.

example 5

Compound 5: preparation of 4- (4- (cyano (2-oxa-6-azaspiro [3.3] hept-6-yl) methyl) phenyl) -5- (2, 4-dihydroxy-5-isopropylphenyl) -methylisoxazole-3-carboxamide reference is made to the procedure of example 1.

The characterization data for this compound 5 are: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-MSm/z:503.3(M+H)⁺,501.3(M-H)^-.

example 6

Compound 6: preparation of 4- (4- (cyano (4AR,7AS) -tetrahydro-2H- [1,4] dioxano [2,3-c ] pyrrol-6- (3H) -yl) methyl) phenyl) -5- (2, 4-hydroxy-5-isopropylphenyl) -N-methylisoxazole-3-carboxamide proceeds according to the method of example 1.

Characterization data for this compound 6 are: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).¹³CNMR(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)^-.

example 7

Compound 7: preparation of 4- (4- (cyano (3-methylpyrrolidin-1-yl) methyl) phenyl) -5- (2, 4-dihydroxy-5-isopropylphenyl) -N-methylisoxazole-3-carboxamide reference is made to the procedure of example 1.

Characterization data for this compound 7 are: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)^-.

example 8

Compound 8: preparation of 4- (4- (2-thia-5-azabicyclo [2.2.1] hept-5-yl (cyano) methyl) phenyl) -5- (2, 4-dihydroxy-5-isopropylphenyl) -methylisoxazole-3-carboxamide the procedure of example 1 was followed.

The characterization data for this 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)^-.

example 9

Compound 9: preparation of 4- (4- (4-methylpiperazin-1-yl) (cyano) methyl) phenyl) -5- (2, 4-dihydroxy-5-isopropylphenyl) -N-methylisoxazole-3-carboxamide the procedure of example 1 was referenced.

Characterization data for this compound 9 were: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)^-.

example 10

Compound 10: preparation of 4- (4- (cyano (2-methoxyethyl) amino) methyl) phenyl) -5- (2, 4-dihydroxy-5-isopropylphenyl) -methylisoxazole-3-carboxamide reference is made to the procedure of example 1.

Characterization data for this compound 10 are: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)^-.

example 11

Compound 11: preparation of 4- (4- (2-amino-1-morpholino-2-oxoethyl) phenyl) -5- (2, 4-dihydroxy-5-isopropylphenyl) -methylisoxazole-3-carboxamide.

A25 mL single vial was charged with Compound 1(30mg), dissolved in DMSO (1mL), and stirred at room temperature for 2h with 30% hydrogen peroxide (20mg, 3 eq.) and potassium carbonate (23mg, 3 eq.) and TLC follow-up indicating the starting material was reacted. EA was added to dilute (10mL) and extracted with water (10mL), the organic phase was extracted with saturated brine, dried over anhydrous sodium sulfate, and concentrated over the column (EA: PE ═ 2: 1). Pulping the obtained yellow viscous product, adding petroleum ether, standing at low temperature to separate out white solid, sucking yellow liquid, and pumping the obtained solid oil to obtain 3.4mg white solid with a yield of 11.0%.

Characterization data for this compound 11 are: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)-.

example 12

The following examples were prepared by reference to the following reaction schemes.

According to the circuit, the method comprises the following steps:

(1) preparation of methyl-2- (4- (5- (2, 4-bis (benzyloxy) -5-isopropylphenyl) -3- (methylcarbamoyl) isoxazol-4-yl) phenyl) -2-hydroxy (compound II-1).

A50 mL single vial was charged with Compound I-11(1g), a catalytic amount of iodine as a powder was added, dissolved by addition of anhydrous DCM (10mL), and TMSCN (0.26mL, 1.2 eq) was added dropwise, stirred at room temperature for 1h and monitored by TLC to show completion of the starting material reaction. Dilute with DCM (10mL), quench with water (5mL), extract the organic phase with saturated brine, dry over anhydrous sodium sulfate, concentrate and go to the next step.

The resulting compound was added to a solution of hydrogen chloride in methanol (20%, 15mL) and stirred at 40 ℃ overnight. TLC tracking indicated the starting material was complete. Diluting with EA (30mL), filtering the resulting solid, collecting the filtrate, concentrating, adding an EA (20mL) solution, adding saturated NaHCO₃The solution was neutralized, extracted with brine, dried over anhydrous sodium sulfate, and concentrated on a column (PE: EA ═ 4: 1). 1.05g of a white solid was obtained, the yield in two steps being 95.4%. ESI-MS M/z 635.1(M + H)⁺。

(2) Preparation of methyl-2- (4- (5- (2, 4-bis (benzyloxy) -5-isopropylphenyl) -3- (ethylcarbamoyl) isoxazol-4-yl) phenyl) -2- (toluenesulfonyloxy) acetate (Compound II-2).

A50 mL single vial was charged with Compound II-1 (0.5g), dissolved in anhydrous DCM (10mL), charged with dry triethylamine (0.33mL, 3 equivalents), stirred at 0 deg.C for 15min, charged with TsCl (0.46g, 3 equivalents), stirred for 15min, warmed to 40 deg.C, and stirred overnight. TLC tracing shows that the starting material reactsAnd (6) finishing. Adding saturated NaHCO₃The solution was neutralized, extracted with brine, dried over anhydrous sodium sulfate, and concentrated on a column (PE: EA ═ 6: 1). 0.34g of a white solid was obtained with a yield of 55.2%. ESI-MS M/z 789.2(M + H)⁺。

(3) Preparation of methyl-2- (4- (5- (2, 4-bis (benzyloxy) -5-isopropylphenyl) -3- (ethylcarbamoyl) isoxazol-4-yl) phenyl) -2-morpholinoacetate (Compound II-3).

A50 mL single vial was charged with Compound II-2 (0.33g), dissolved in toluene (5mL), and refluxed for 2h in an oil bath with dry triethylamine (0.12mL, 2 equiv.) and morpholine (55 μ L, 1.5 equiv.) following TLC to show the starting material was reacted. EA (15mL) was added for dilution, extracted with saturated brine, the organic phase was collected, dried over anhydrous sodium sulfate, and concentrated for the next step. 232mg of viscous product are obtained, the yield is 80%. ESI-MS M/z 7032(M + H)⁺。

(4) Preparation of methyl-2- (4- (5- (2, 4-dihydroxy-5-isopropylphenyl) -3- (ethylcarbamoyl) isoxazol-4-yl) phenyl) -2-morpholinoacetate (Compound 12).

Taking a 50mL two-mouth bottle, adding the compound II-3 (0.2g), protecting with argon, adding anhydrous DCM (10mL) for dissolution, placing at-5 ℃, stirring for 15min, dropwise adding a dichloromethane solution (1M, 0.9mL, 3 equivalents) of boron tribromide, stirring for 10min, placing at room temperature, and stirring for 1 h. TLC tracking indicated the starting material was complete. At 0 deg.C, DCM (10mL) was added for dilution and saturated NaHCO was added₃The solution was quenched, extracted and the organic phase collected. EA was added to the aqueous phase, the resulting flocculent solid was dissolved, the organic phases were collected, mixed, dried over anhydrous sodium sulfate and concentrated over the column (DCM: acetone ═ 1: 1). Pulping the obtained yellow viscous product, adding petroleum ether, standing at low temperature to separate out white solid, sucking yellow liquid, and pumping the obtained solid oil to obtain 35.7mg of white solid with a yield of 24.0%.

Characterization data for this compound 12 are: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)^-.

example 13

Compound 13: preparation of ethyl-2- (4- (5- (2, 4-dihydroxy-5-isopropylphenyl) -3- (ethylcarbamoyl) isoxazol-4-yl) phenyl) -2-morpholinoacetate according to the method of example 12.

Characterization data for this compound 13 are: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:

example 14

Compound 14: preparation of 5- (2, 4-dihydroxy-5-isopropylphenyl) -N-methyl-4- (4- (2- (dimethylamino) -1-morpholino-2-oxoethyl) phenyl) isoxazole-3-carboxamide.

(1) Preparation of 5- (2, 4-bis (benzyloxy) -5-isopropylphenyl) -N-methyl-4- (4- (2- (dimethylamino) -1-hydroxy-2-oxoethyl) phenyl) isoxazole-3-carboxamide (Compound II-5).

Get a 50mL single-necked bottleCompound II-4 (0.5g), aqueous ethylamine (68%, 0.2mL, 4 equiv.), and ethanol (5mL) were added and placed in an oil bath and the reaction was refluxed overnight, followed by TLC to show the completion of the reaction. Most of the solvent was removed by rotation, EA (20mL) and saturated brine were added for extraction, dried over anhydrous sodium sulfate, and concentrated to be used in the next step. The product was obtained in the form of a viscous mass (0.48g) with a yield of 96.2%. ESI-MS M/z 648.3(M + H)⁺。

(2) Compound 14: preparation of 5- (2, 4-dihydroxy-5-isopropylphenyl) -N-methyl-4- (4- (2- (dimethylamino) -1-morpholino-2-oxyethyl) phenyl) isoxazole-3-carboxamide reference was made to the procedure in example 12.

Characterization data for this compound 14 are: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)^-.

example 15

Compound 15: preparation of ethyl methyl-2- (4- (5- (2, 4-dihydroxy-5-isopropylphenyl) -3- (ethylcarbamoyl) isoxazol-4-yl) phenyl) -2- (4AR,7AS) -tetrahydro-2H- [1,4] dioxano [2,3-c ] pyrrol-6- (3H) -yl) acetate according to the procedure of example 12.

Characterization data for this compound 15 are: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:

example 16

Compound 16: preparation of ethyl-2- (4- (5- (2, 4-dihydroxy-5-isopropylphenyl) -3- (ethylcarbamoyl) isoxazol-4-yl) phenyl) -2- (4AR,7AS) -tetrahydro-2H- [1,4] dioxano [2,3-c ] pyrrol-6- (3H) -yl) acetate according to the procedure of example 12.

Characterization data for this compound 16 are: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-MSm/z:580.2(M+H)⁺,578.1(M-H)^-.

the structural formula of the compound 16 is:

example 17

According to the circuit, the method comprises the following steps:

(1) preparation of methyl 4- (5- (2, 4-bis (benzyloxy) -5-isopropylphenyl) -3- (methylcarbamoyl) isoxazol-4-yl) benzoate (Compound III-1).

A50 mL single-necked flask was charged with Compound I-10 (600mg, 1mmol), (4- (methoxycarbonyl) phenylboronic acid (235 m)g, 1.3mmol, 1.3 equiv.), Pd (PPh)₃)₂Cl₂(70mg, 0.1mmol, 10 mol%), sodium bicarbonate (252mg, 3mmol, 3 equiv.), exchange of argon, addition of DMF: h₂O10 mL: 2mL of the solution was stirred at 80 ℃ for 5 hours. And tracking and monitoring by TLC. Most of the solvent was removed by rotation and the mixture was purified by column chromatography (PE: EA: 4:1) to obtain 620mg of a tan product. ESI-MS M/z 605.2(M + H)⁺.

(2) Preparation of 4- (5- (2, 4-bis (benzyloxy) -5-isopropylphenyl) -3- (methylcarbamoyl) isoxazol-4-yl) benzoic acid (Compound III-2).

A25 mL single-neck flask was charged with Compound III-1 (600mg), dissolved in acetone (2mL), and then 35% sodium hydroxide solution (1mL) was added, followed by addition of methanol (4mL) to remove the layer and stirring at room temperature for 2 hours. And tracking and monitoring by TLC. Adding 1N HCl solution, adjusting to acidity, removing most of organic solution, adding EA and saturated saline solution for extraction, collecting organic phase, drying, and spin-drying. Used directly in the next step. ESI-MS M/z 591.1(M + H)⁺.

(3) Preparation of 5- (2, 4-bis (benzyloxy) -5-isopropylphenyl) -N-methyl-4- (4- (4AR,7AS) -hexahydro-2H- [1,4] dioxano [2,3-C ] pyrrole-6-carbonyl) phenyl) isoxazole-3-carboxamide (Compound III-3).

A25 mL single vial was charged with crude compound III-2 (400mg, 0.68mmol), HOBt (110mg, 0.816mmol, 1.2 equiv.), EDCI (195mg, 1.02mmol, 1.5 equiv.), DCM (5mL) dissolved, TEA (0.4mL, 2.72mmol, 4 equiv.) stirred for 15min, compound 7(87mg, 0.68mmol, 1 equiv.) stirred overnight. And tracking and monitoring by TLC. Extract with DCM and brine, dry, spin dry, and column pass (PE: EA ═ 2: 1). 160mg of a colorless sticky wall product is obtained. ESI-MS M/z:702.1(M + H)⁺.

(4) Preparation of 5- (2, 4-dihydroxy-5-isopropylphenyl) -N-methyl-4- (4- (4AR,7AS) -hexahydro-2H- [1,4] dioxano [2,3-C ] pyrrole-6-carbonyl) phenyl) isoxazole-3-carboxamide (Compound 17).

Adding crude compound III-3 (160mg,0.23mmol) into a 25mL two-neck flask, under the protection of argon, adding anhydrous DCM (10mL) to dissolve, standing at-5 deg.C, stirring for 15min, and adding dropwise a solution of boron tribromide in dichloromethane (1M, 0.6 mmol)9mL, 3 equivalents), stirred for 10min, left at room temperature, and stirred for 1 h. TLC tracking indicated the starting material was complete. At-5 deg.C, DCM (10mL) was added for dilution and saturated NaHCO was added₃The solution was quenched, extracted and the organic phase collected. EA was added to the aqueous phase, the resulting flocculent solid was dissolved, the organic phases were collected, mixed, dried over anhydrous sodium sulfate and concentrated over the column (DCM: acetone ═ 2: 1). Pulping the obtained yellow viscous product, adding petroleum ether, standing at low temperature to separate out white solid, sucking yellow liquid, and pumping the obtained solid oil to obtain 80mg white solid.

Characterization data for this compound 17 are: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)^-.

example 18

Compound 18: preparation of 5- (2, 4-dihydroxy-5-isopropylphenyl) -N-methyl-4- (4- (morpholine-4-carbonyl) phenyl) isoxazole-3-carboxamide according to the method of example 17.

Characterization data for this compound 18 were: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 this compound 18 is:

example 19

According to the circuit, the method comprises the following steps:

(1) preparation of (S) -ethyl-2-hydroxy-2-phenylacetate (Compound IV-1).

A100 mL single-neck bottle is taken, 3g of(s) - (+) -mandelic acid is added, ethanol (30mL) is used for dissolving, concentrated sulfuric acid (0.3mL) is added dropwise, the mixture is placed at 80 ℃ for refluxing and stirred for 3h, and TLC tracking shows that the raw materials are reacted completely. Adding saturated NaHCO₃The solution was spun off most of the ethanol solution until no bubbles were formed, EA (30mL) was added for extraction, washed with saturated brine, the organic phase was collected, dried over anhydrous sodium sulfate, and spun dry to give a colorless oily liquid (3.5g, 98.5%).

The characterization data of the compound IV-1 are as follows: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 ethyl (S) -ethyl-2-hydroxy-2- (4-iodophenyl) acetate (Compound IV-2).

A100 mL single vial was taken, compound IV-1 (3.2g,17.8mmol) and anhydrous DCM (60mL) were added and dissolved, the outer wall of the vial was wrapped with tinfoil and protected from light, iodine (4.5g, 17.7mmol) and silver triflate (4.56g, 17.7mmol) were added and stirred at room temperature for 4h, TLC trace showed the starting material was reacted. Adding sodium thiosulfate solution for quenching, filtering the precipitate, washing the precipitate by DCM, extracting, washing by saturated saline, collecting an organic phase, drying by anhydrous sodium sulfate, spin-drying, and passing through a column (PE: EA is 8:1) to obtain brown oily liquid.

The characterization data of the compound IV-2 are as follows: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) preparation of ethyl (S) -ethyl-2-hydroxy-2- (4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenyl) acetate (Compound IV-3).

A50 ml two-necked flask was charged with compound IV-2 (0.55g, 1.8mmol), pinacol diboron (0.55g, 2.16mmol), [1,1' -bis (diisopropylphosphine) ferrocene ] dichloropalladium (54mg, 0.09mmol), potassium acetate (529mg, 5.4mmol), argon exchanged, dissolved in DMF, placed at 90 ℃ and stirred for 1h, and TLC trace indicated that the starting material had reacted. Most of DMF was removed by spinning, EA was added, the precipitate was filtered, washed with EA, spun dry, and passed through a column (PE: EA: 8: 1). This gave an anhydrous oily liquid (390mg, 70.9%).

The characterization data of the compound IV-3 are as follows: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) preparation of methyl (S) -ethyl-2- (4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenyl) -2- (toluenesulfonyloxy) acetate (Compound IV-4).

A100 mL single vial was charged with Compound 3(1g, 3.27mmol) and dry DCM (10mL) and dried triethylamine (1.4mL, 9.8mmol, 3 equivalents) was added and TsCl (1.86g, 9.8mmol, 3 equivalents) was added slowly and allowed to stir at 50 deg.C overnight. TLC tracking indicated the starting material was complete. Water and DCM were added, extracted, washed with brine, dried over anhydrous sodium sulfate, spin dried and passed through a column (PE: EA ═ 10: 1). This gave an anhydrous oily liquid (1g, 66.7%).

(5) Preparation of ethyl (S) -ethyl 2-morpholinyl-2- (4- (4,4,5, 5-tetramethyl-1, 3, 2-dioxaborolan-2-yl) phenyl) acetate (Compound IV-5).

A50 mL single vial was charged with Compound IV-4 (0.5g, 1.09mmol), toluene (8mL) dissolved, and dry triethylamine (0.3mL, 2.17mmol, 2 equiv.), morpholine (0.14mL, 1.64mmol, 1.5 equiv.) placed in an oil bath under reflux. Stirring for 2h, TLC tracing to show the reaction of the raw materials is finished. Most of toluene was removed by rotation, EA and saturated brine were added, extracted, dried over anhydrous sodium sulfate, and dried by rotation (260mg, 64%) and used directly in the next step.

(6) Preparation of (S) - (4- (2-ethoxy-1-morpholino-2-oxoethyl) phenyl) boronic acid (compound IV-6).

Compound iv-5 (250mg, 0.67mmol) was added to a 50mL single neck flask, HCl solution 10mL (adjusted to pH 3) was added, n-hexane (10mL) was added, stirring was performed, phenylboronic acid (81mg, 0.67mmol, 1 eq) was added, stirring was vigorously continued until the phenylboronic acid was completely dissolved, stirring was continued for 0.5h, and TLC tracking indicated complete reaction of starting material. Extracting, reserving a water phase, adding normal hexane for washing once again, adding EA to wash out the phenylboronic acid which is not completely reacted, reserving the water phase, adding a saturated sodium bicarbonate solution, adjusting the water phase to be slightly alkaline, adding EA and 2 for extraction, drying with anhydrous sodium sulfate, spin-drying, and directly using in the next step. 160mg, 82% of colorless viscous product is obtained.

(7) Preparation of (S) -ethyl 2- (4- (5- (2, 4-bis (benzyloxy) -5-isopropylphenyl) -3- (ethylcarbamoyl) isoxazol-4-yl) phenyl) -2-morpholinoacetate (Compound IV-7).

A25 mL single-necked flask was charged with Compound I-10 (102mg, 0.17mmol), Compound IV-6 (60mg, 0.2mmol, 1.2 equiv.), Pd (PPh)₃)₂Cl₂(12mg, 0.02mmol, 10 mol%), sodium bicarbonate (43mg, 0.51mmol, 3 equiv.), exchange of argon, addition of DMF: h₂O10 mL: 2mL of the solution was stirred at 80 ℃ for 3 hours. And tracking and monitoring by TLC. Most of the solvent was removed by spinning and column chromatography (PE: EA: 4:1) gave 75mg of a tan product. ESI-MS M/z 718.1(M + H)⁺。

(8) Preparation of (S) -ethyl 2- (4- (5- (2, 4-dihydroxy-5-isopropylphenyl) -3- (ethylcarbamoyl) isoxazol-4-yl) phenyl) -2-morpholinoacetate (Compound 19).

Adding the crude compound IV-7 (68mg) into a 25mL two-mouth bottle, adding argon for protection, adding anhydrous DCM (10mL) for dissolution, placing at 0 ℃, stirring for 15min, dropwise adding a dichloromethane solution (1M, 0.28mL, 3 equivalents) of boron tribromide, stirring for 10min, placing at room temperature, and stirring for 1 h. TLC tracking indicated the starting material was complete. At 0 deg.C, DCM (10mL) was added for dilution and saturated NaHCO was added₃Solution quenchingAnd (5) killing, extracting and collecting an organic phase. EA was added to the aqueous phase, the resulting flocculent solid was dissolved, the organic phases were collected, mixed, dried over anhydrous sodium sulfate and concentrated over the column (DCM: acetone ═ 1: 1). Pulping the obtained yellow viscous product, adding petroleum ether, standing at low temperature to separate out white solid, sucking yellow liquid, and pumping the obtained solid oil to obtain 25mg white solid (S configuration).

Characterization data for this compound 19 are: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)^-.

example 20

Compound 20: preparation of ethyl (S) -ethyl-2- (4- (5- (2, 4-dihydroxy-5-isopropylphenyl) -3- (ethylcarbamoyl) isoxazol-4-yl) phenyl) -2- (4AR,7AS) -tetrahydro-2H- [1,4] dioxano [2,3-c ] pyrrol-6- (3H) -yl) acetate according to the procedure of example 19.

Characterization data for this compound 20 were: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:

example 21

Compound 21: preparation of ethyl (S) -isopropyl-2- (4- (5- (2, 4-dihydroxy-5-isopropylphenyl) -3- (ethylcarbamoyl) isoxazol-4-yl) phenyl) -2- (4AR,7AS) -tetrahydro-2H- [1,4] dioxano [2,3-c ] pyrrol-6- (3H) -yl) acetate according to the method of example 19.

Characterization data for this compound 21 are: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:

example 22

Compound 22: preparation of (S) -ethyl 2- (4- (5- (2, 4-dihydroxy-5-isopropylphenyl) -3- (ethylcarbamoyl) isoxazol-4-yl) phenyl) -2- ((2S,6R) -2, 6-dimethylmorpholine) acetate according to the method of example 19.

Characterization data for this compound 22 are: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:

example 23

Compound 23: preparation of (R) -ethyl-2- (4- (5- (2, 4-dihydroxy-5-isopropylphenyl) -3- (ethylcarbamoyl) isoxazol-4-yl) phenyl) -2-morpholinoacetate according to the method of example 19.

Characterization data for this compound 23 are: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).¹³CNMR(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:

example 24

Compound 24: preparation of ethyl (R) -ethyl-2- (4- (5- (2, 4-dihydroxy-5-isopropylphenyl) -3- (ethylcarbamoyl) isoxazol-4-yl) phenyl) -2- (4AR, AS) -tetrahydro-2H- [1,4] dioxano [2,3-c ] pyrrol-6- (3H) -yl) acetate according to the procedure of example 19.

Characterization data for this compound 24 are: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:

example 25

According to the circuit, the method comprises the following steps:

(1) preparation of (4-bromobenzyl) oxy) (tert-butyl) dimethylsilane (Compound VII-1).

A100 mL single neck flask was charged with 4-bromobenzyl alcohol (5g, 26.9mmol), imidazole (2.2g, 1.2 equiv., 32.3mmol) and 4-Dimethylaminopyridine (DMAP) (catalytic amount), DCM (40mL) was dissolved and tert-butyldimethylchlorosilane (TBSCl) (4.87g, 1.2 equiv., 32.3mmol) was added slowly and stirred overnight. TLC monitoring reaction, adding water, dissolving the generated solid, extracting, adding saturated salt water for washing, collecting organic phase, drying with anhydrous sodium sulfate, and rotary evaporating to remove organic solvent to obtain 7.7g colorless oily matter with yield of 95%. ESI-MS M/z 301.0(M + H)⁺。

(2) Preparation of 1- (4- (((tert-butyldimethylsilyl) oxy) methyl) phenyl) ethanone (Compound VII-2).

A50 mL two-necked flask was charged with Compound 2(4g, 13.3mmol) and palladium tetrakistriphenylphosphine (0.77g, 5 mmol%, 0.67mmol), argon exchanged, dissolved by DMF, charged with tributyl (1-ethoxyethylene) tin (5.4mL, 1.2 equiv., 16mmol), stirred at 100 ℃ for 2h, and TLC monitored for reaction completion. Decompressing by an oil pump, carrying out rotary evaporation in vacuum, removing most DMF, adding EA and 1N hydrochloric acid solution, stirring, monitoring the reaction by TLC, extracting, adding saturated salt water for washing, collecting an organic phase, drying by anhydrous sodium sulfate, carrying out rotary evaporation to remove an organic solvent, and carrying out sample mixing and column passing (PE: DCM ═ 8:1) to obtain 2.5g of colorless liquid, wherein the yield is 70%.

(3) Preparation of ethyl 3- (4- ((tert-butyldimethylsilyloxy) methyl) phenyl) -3-oxopropanoate (Compound VII-3).

A50 mL single vial was charged with Compound 3(1g, 3.8mmol), dissolved in toluene, diethyl carbonate (0.91mL, 2 equiv., 7.6mmol) was added, NaH (0.3g, 2 equiv., 7.6mmol) was added slowly, and the mixture was stirred at 100 deg.C under reflux overnight. TLC monitored the reaction was complete. Quenching the mixture at 0 ℃, adding water to quench the mixture, removing most of organic solvent by rotary evaporation, adding EA to extract the mixture twice, adding saturated saline solution, collecting an organic phase, drying the organic phase by anhydrous sodium sulfate, and concentrating and passing the mixture through a column (PE: DCM ═ 4: 1). 650mg of a colorless oil are obtained in 50% yield.

The characterization data of the compound VII-3 are as follows: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) preparation of ethyl-1- (2, 4-bis (benzyloxy) -5-isopropylphenyl) -5- (4- (((tert-butyldimethylsilyloxy) methyl) phenyl) -1H-1,2, 3-triazole-4-carboxylate (Compound VII-4).

A 50mL single vial was charged with compound vii-3 (600mg, 1.8mmol) and (((4-azido-6-isopropyl-1, 3-phenylenedi (oxy)) bis (methylene)) diphenyl (1g, 1.5 equivalents, 2.7mmol, mixture), dissolved in DMSO, DBU (0.4mL, 1.5 equivalents, 2.7mmol) was added, stirred at room temperature overnight, TLC monitored for completion of the reaction, water and EA were added, the organic phase was collected, water was added and extracted twice more, the organic phase was collected, dried over anhydrous sodium sulfate, and spun through column PE, EA ═ 8:1, giving 800mg of oil in 64% yield.

(5) Preparation of 1- (2, 4-bis (benzyloxy) -5-isopropylphenyl) -5- (4- (((tert-butyldimethylsilyloxy) methyl) phenyl) -N-ethyl-1H-1, 2, 3-triazole-4-carboxamide (Compound VII-5).

A50 mL single-neck flask was taken, the compound VII-4 (800mg, 1.16mmol) was added, ethanol was added for dissolution, an aqueous solution of ethylamine (10mL) was added, the mixture was left at 80 ℃ and stirred under reflux for 1 day, and the completion of the reaction was monitored by TLC. Most of the organic solvent was removed by rotary evaporation, EA was added, extracted twice, washed with saturated brine, dried over anhydrous sodium sulfate, and rotary dried to obtain 600mg of oily product with a yield of 75%.

(6) Preparation of 1- (2, 4-bis (benzyloxy) -5-isopropylphenyl) -N-methyl-5- (4- (hydroxymethyl) phenyl) -1H-1,2, 3-triazole-4-carboxamide (Compound VII-6).

A50 mL single-neck flask was charged with Compound VII-5 (600mg, 0.87mmol), dissolved in tetrabutylammonium fluoride (TBAF) in THF (10mL), and stirred at room temperature for 2 h. TLC monitored the reaction was complete. Most of the organic solvent is removed by rotary evaporation, EA is added, extraction is carried out twice, saturated salt solution is used for washing, anhydrous sodium sulfate is used for drying, and rotary drying is carried out, so that 500mg of oily product is obtained, and the yield is 99.8%.

(7) Preparation of 1- (2, 4-bis (benzyloxy) -5-isopropylphenyl) -N-methyl-5- (4-formylphenyl) -1H-1,2, 3-triazole-4-carboxamide (Compound VII-7).

A50 mL single vial was charged with Compound VII-6 (500mg, 0.87mmol), DCM and dissolved, 2,6, 6-tetramethylpiperidine oxide (TEMPO) and tetrabutylammonium bromide (TBAB) (catalytic amounts), sodium hypochlorite (NaOCl) solution (5mL) and NaHCO were added₃The solution (2mL) was stirred at room temperature for 1 h. The solution changed from reddish brown to pale yellow. TLC monitored the reaction was complete. DCM was added and extracted three times, washed with brine, dried over anhydrous sodium sulfate, spin dried and chromatographed (PE: EA ═ 5:1) to give 250mg of product as a viscous mass with 50% yield.

The characterization data of the compound VII-7 are as follows: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) preparation of ethyl-2- (4- (1- (2, 4-bis (benzyloxy) -5-isopropylphenyl) -4- (ethylcarbamoyl) -1H-1,2, 3-triazol-5-yl) phenyl) -2-hydroxyacetic acid (compound VII-8).

A50 mL single-necked flask was taken, compound VII-7 (250mg, 0.44mmol) was added, DCM was dissolved, and I was added₂(catalytic amount), trimethylsilyl cyanide (TMSCN) (0.11mL, 2 equiv., 0.87mmol) was added and stirred at room temperature for 30min, and the reaction was monitored by TLC for completion. Adding water for quenching, extracting, washing with saturated salt water, drying and spin-drying with anhydrous sodium sulfate. An ethanol solution of hydrogen chloride (10mL) was added thereto, and the mixture was stirred at room temperature overnight. TLC monitored the reaction was complete. Adding saturated sodium bicarbonate solution for quenching, performing rotary evaporation to remove most of organic solvent, adding EA, extracting twice, washing with saturated salt water, drying with anhydrous sodium sulfate, and performing rotary drying on column-passing PE: EA 4:1, 140mg, 49.1% yield. ESI-MS M/z 649.0(M + H)⁺。

(9) Preparation of ethyl-2- (4- (1- (2, 4-bis (benzyloxy) -5-isopropylphenyl) -4- (ethylcarbamoyl) -1H-1,2, 3-triazol-5-yl) phenyl) -2- (toluenesulfonyl) acetate (Compound VII-9).

A25 mL single vial was charged with Compound VII-8 (140mg, 0.22mmol), DCM and dissolved, TEA (92. mu.L, 3 equivalents, 0.66mmol) and TsCl (126mg, 3 equivalents, 0.66mmol) were added, placed at 40 ℃ and stirred for 4 h. TLC monitored the reaction was complete. Extracting with saturated saline, collecting organic phase, drying with anhydrous sodium sulfate, spin drying, and purifying with column PE (EA: 4:1) to obtain 50mg with 28% yield.

(10) Preparation of ethyl-2- (4- (1- (2, 4-bis (benzyloxy) -5-isopropylphenyl) -4- (ethylcarbamoyl) -1H-1,2, 3-triazol-5-yl) phenyl) -2-morpholinoacetate (Compound VII-10).

A25 mL single vial was charged with Compound 11(50mg, 0.06mmol), dissolved in toluene, and morpholine (20. mu.L, 4 equivalents, 0.24mmol) and TEA (33. mu.L, 4 equivalents, 0.24mmol) were added, and the mixture was stirred at 100 ℃ under reflux for 2 h. TLC monitored the reaction was complete. Removing most of organic solvent by rotary evaporation, adding saturated saline solution for extraction, collecting organic phase, drying with anhydrous sodium sulfate, and rotary drying to obtain 30mg with yield of 70%.

ESI-MS m/z:718.0(M+H)⁺。

(11) Preparation of ethyl-2- (4- (1- (2, 4-dihydroxy-5-isopropylphenyl) -4- (ethylcarbamoyl) -1H-1,2, 3-triazol-5-yl) phenyl) -2-morpholinoacetate (Compound 25).

Taking a 25mL single-neck bottle, adding the compound VII-10 (30mg, 0.04mmol), exchanging argon, adding anhydrous DCM for dissolution, placing at-5 ℃, stirring for 15min, dropwise adding a boron tribromide dichloromethane solution (0.12mL, 3 equivalents, 0.12mmol), stirring for 15min, placing at room temperature, and stirring for 2 h. Quench with saturated sodium bicarbonate solution, extract, wash with saturated brine, collect the organic phase, dry over anhydrous sodium sulfate, spin dry, and column pass (DCM: acetone ═ 2:1) to give 10mg of a solid in 48% yield.

Characterization data for this compound 25 are: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)^-.

examples of the experiments

(1) A method for screening an enzymatic activity of a target compound.

The principle is as follows: FITC-labeled geldanamycin, when bound to HSP90, produces a fluorescence polarization that is not detectable if the compound competes with geldanamycin for inhibition of the HSP90 enzyme.

Reagents and instruments-fluorescent labeled geldanamycin (Sigma), Hsp90 α or Hsp90 β enzyme solution (Stressgen Bioreagens Corp., cat. No. SPP-776), DTT (Promega), Bovine Serum Albumin (BSA) (Hyclone), DMSO (Sigma), Envision2104 fluorometer (Perkin Elmer, USA), sample applicator (Eppendorf), microplate (burning).

Each compound is provided with a plurality of different dosage groups, the high dosage group is prepared into mother liquor, the rest dosage groups are sequentially diluted to the lowest dosage group in a three-fold ratio, and all samples are dissolved in DMSO and stored at-20 ℃ for later use. Experimental buffer contained 20mmol/l HEPES (K), 50mmol/l KCl, 5mmol/l MgCl₂，20mmol/l Na₂MoO₄And 0.01% NP40, pH 7.3. mu.l of reaction buffer containing 40mM DTT and 2mg/ml BSA were added before each experiment, 2.5. mu.l of fluorescently labeled geldanamycin (reaction concentration 5nM) were added, then 10. mu.l of the 1000 Xgradient diluted compound was added to the reaction using the Compound transfer Instrument Liquid Handler Echo520, finally 2.5. mu.l of Hsp90 β or Hsp90 α enzyme solution (reaction concentration 35 ng/. mu.l) was added, the reaction was gently shaken at room temperature for 2 hours, and finally the reading was measured with a microplate reader, excitation light 485nM, emission 530nM, and data were processed with Graphpad Pri 5 software.

(2) A method for screening target compounds for anti-cancer cell activity.

The CCK kit has the working principle that: the Cell Counting Kit is called CCK Kit for short, and is a rapid high-sensitivity detection Kit widely applied to Cell proliferation and cytotoxicity based on WST-8 (chemical name: 2- (2-methoxy-4-nitrophenyl) -3- (4-nitrophenyl) -5- (2, 4-disulfophenyl) -2H-tetrazole monosodium salt). WST-8 belongs to an upgrading product of MTT, and the working principle is as follows: in the presence of an electron coupling reagent, this can be reduced by a mitochondrial dehydrogenase to produce a highly water-soluble orange-yellow formazan product (formazan) which precipitates in cells, whereas dead cells do not have this function. The shade of color is proportional to the proliferation of cells and inversely proportional to cytotoxicity. The absorbance was measured at 540/720nm using an enzyme linked detector to indirectly reflect the viable cell count.

Reagents and instrumentation: various cancer cell lines (ATCC company, USA), penicillin, streptomycin, DMSO (Sigma company), CCK8(CK04, Japan Dojindo chemical), RPMI1640(GIBCO company), BSA (Hyclone company), trypsin (Ginuo company), 384-well cell culture plates (Corning).

Each cancer cell is suspended in corresponding culture solution to be prepared into proper concentration,50 μ l/well volume was plated in 384 well plates and incubated at 37 ℃ with 5% CO₂The temperature in the incubator is kept constant for 24 hours. Compounds were dissolved in DMSO at 10mM stock and diluted in DMSO at 1:3 gradient to 10 concentration gradient stocks of 1000 × compound series concentration, and the stocks of 1000 × compound series concentration were transferred to corresponding wells of 384-well cells at 50nL per well using a compound transfer instrument, liquidhandle Echo520, and an equal volume of vehicle DMSO was added to blank wells. Gently mixing, and further culturing at 37 deg.C. After 72h, the medium was replaced and 3. mu.l of CCK8 cell proliferation-toxicity test reagent was added to each well. The incubation of the plates at 37 ℃ in the incubator was continued for 2h, and then the absorbance was measured at a wavelength of 540/720nm using an enzyme-linked detector.

The test adopts the method to respectively determine the inhibitory activity of the synthesized target compound on two subtypes HSP90 α and HSP90 β of HSP90 protein, and the inhibitory activity on human lung cancer cell line A549 with high HSP90 expression, human breast cancer cell line MCF-7, chronic myelogenous leukemia cell line K562, prostate cancer cell line DU145 and experimental proliferative epidermal cancer cell line Hela, and the results are shown in Table 1.

TABLE 1 inhibitory Activity of target Compounds IC against HSP90 protein and tumor cell lines₅₀(in nM)

According to the table 1, from the view of enzyme activity level, the compound has an inhibition effect on two HSP90 subtypes, and the binding capacity of most compounds and HSP90 α is stronger than that of HSP90 β. compared with a positive control compound NVP/AUY-922 (CAS: 747412-49-3), most compounds show stronger inhibition activity, and further prove that a target skeleton is more suitable for an active pocket of a target spot.

The technical features of the embodiments described above may be arbitrarily combined, and for the sake of brevity, all possible combinations of the technical features in the embodiments described above are not described, but should be considered as being within the scope of the present specification as long as there is no contradiction between the combinations of the technical features.

The above-mentioned embodiments only express several embodiments of the present invention, and the description thereof is more specific and detailed, but not construed as limiting the scope of the invention. It should be noted that, for a person skilled in the art, several variations and modifications can be made without departing from the inventive concept, which falls within the scope of the present invention. Therefore, the protection scope of the present patent shall be subject to the appended claims.

Claims

1. A heat shock protein inhibitor having the structural feature of formula I:

wherein:

R₄selected from the group consisting of:

R₅selected from: h, D, C₁-C₆Alkyl radical, C₃-C₈Cycloalkyl, carbonyl, cyano, C₁-C₆Alkoxy, COOR ', NHCOR ', CONHR ', or with R₆Are the same oxygen atom;

R₆selected from: cyano, COOR ', CONHR', or with R₅Are the same oxygen atom;

x, Y, Z, W are each independently selected from: c, NH, CH, N, O, S, and X, Y, Z are not simultaneously N.

2. The heat shock protein inhibitor or the pharmaceutically acceptable salt thereof according to claim 1, wherein the five-membered aromatic heterocycle comprising X, Y, Z, W is selected from the following structures:

3. the heat shock protein inhibitor or a pharmaceutically acceptable salt thereof of claim 1, wherein the heat shock protein inhibitor is selected from the group consisting of compounds of the following formula II:

wherein:

4. The heat shock protein inhibitor or the pharmaceutically acceptable salt thereof according to claim 1, wherein R is₂、R₃Each independently selected from: h, C₃-C₈Cycloalkyl radical, C₁-C₆Alkyl radical, C₃-C₆An unsaturated alkyl group.

5. The heat shock protein inhibitor or the pharmaceutically acceptable salt thereof according to any one of claims 1 to 4,

R₅selected from: h, or with R₆Are the same oxygen atom;

6. The heat shock protein inhibitor or the pharmaceutically acceptable salt thereof according to claim 5, wherein R is₄Selected from the group consisting of:

7. the heat shock protein inhibitor or a pharmaceutically acceptable salt thereof of claim 1, wherein the heat shock protein inhibitor is selected from the group consisting of compounds of the following formula III:

wherein:

R₁selected from: c₁-C₆An alkyl group;

R₇、R₈selected from: a hydroxyl group;

R₂selected from: h;

R₃selected from: c₁-C₆An alkyl group;

R₄is selected from the followingGroup (b):

R₅selected from: h, or with R₆Are the same oxygen atom;

8. The heat shock protein inhibitor or the pharmaceutically acceptable salt thereof according to claim 1, which is selected from the group consisting of:

9. the method for preparing a heat shock protein inhibitor or a pharmaceutically acceptable salt thereof according to any one of claims 1-8, wherein the synthesis is carried out by the following route:

wherein: r₁₀Is a hydroxyl protecting group.

10. Use of a heat shock protein inhibitor or a pharmaceutically acceptable salt thereof according to any one of claims 1-8 in the manufacture of a medicament for the prevention and treatment of a disease characterized by an increased expression of heat shock protein 90.

11. The use according to claim 10, wherein the disease characterized by the pathology of increased expression of heat shock protein 90 is: cancer, metabolic disease, myelodysplastic syndrome, systemic mastocytosis, von hippel-lindau syndrome, multicentric Castleman disease, and psoriasis.