WO2018001084A1

WO2018001084A1 - Heat shock protein inhibitor, and manufacturing method and application thereof

Info

Publication number: WO2018001084A1
Application number: PCT/CN2017/088019
Authority: WO
Inventors: 张健存; 林财; 李德耀; 邹晴安; 张袁超; 顾自强
Original assignee: 广州市恒诺康医药科技有限公司
Priority date: 2016-06-29
Filing date: 2017-06-13
Publication date: 2018-01-04
Also published as: CN107540624A; CN107540624B

Abstract

The present invention relates to the field of pharmaceutical chemistry. Disclosed are a heat shock protein inhibitor, and a manufacturing method and application thereof. The heat shock protein inhibitor has a characteristic structure represented by formula I. The compound can inhibit an activity of heat shock protein 90 and can be used for preparing an antitumor pharmaceutical product.

Description

Heat shock protein inhibitor and preparation method and application thereof

Technical field

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

Background technique

Heat shock protein (HSP) is a highly conserved protein that is ubiquitous in organisms. According to the molecular weight, heat shock proteins are divided into: HSP100 (100-110kD), HSP90 (83-90kD), HSP70 (66-78kD), HSP60 and small molecule HSP (15-30kD). Among them, heat shock protein 90 (HSP90) is an ATP-dependent molecular chaperone, involved in the activation of client protein and promotes its maturation, maintains the conformation and function of various proteins, and promotes cell proliferation, apoptosis, and cancer. Tumor development is closely related. In normal cells, the expressed HSP90 accounts for 1-2% of the total protein in the cell, and 3% of this is found in the nucleus, which has a function of affecting nuclear regulation. Under stress, as in cancer cells, the level of expressed HSP90 is increased to 4-6% of the entire proteomics. HSP90 interacts with more than 200 different client proteins involved in signal transduction, protein trafficking, receptor maturation, and adaptive immunity. A large number of key oncogenic proteins belong to the client protein, such as: Her2, AKT, CDK4, VEGF, MET, ALK, mutant p53 and so on. HSP90 has four subtypes: HSP90α and HSP90β (located in the cytoplasm), Grp94 subtype (located in the endoplasmic reticulum), and TRAP1 subtype (located in the mitochondrial matrix). The main molecular chaperones of HSP90 include Aha1, Hip, Hop, HSP70, CDC37/P50 and the like. In cancer cells, HSP90 is in an activated state, which forms a complex with the client protein and the co-molecular chaperones HSP70, p23, etc., to protect the client protein from degradation by the proteasome. The occurrence and development of cancer is affected by multiple pathways and multiple links of HSP90 receptor protein, making it a new target for anticancer drugs. HSP90 inhibitors can be classified into N-terminal inhibitors, intermediate-end inhibitors, and C-terminal inhibitors depending on their binding sites.

The stability of HSP90 client protein structure and function requires the participation of HSP, while the client protein plays an important role in promoting cell growth, proliferation and survival, and it is overexpressed or continuously expressed in malignant tumors, and has the development of tumors. close relationship. For example, mutations in non-small cell lung cancer cells (NSCLC), fusion protein BCR-ABL in chronic myelogenous leukemia cells, and maintenance and regulation of tumor-specific proteins such as Her2 in breast cancer cells require HSP90. participate.

The incidence of non-small cell lung cancer accounts for 3/4-4/5 of lung cancer, while EGFR is highly expressed in about 3/5 non-small lung cancer cells. Many patients are currently generally resistant to FDA-approved non-small cell lung cancer drugs, gefitinib and erlotinib. Studies have shown that HSP90 inhibitor 17-DMAG has anti-proliferative effect on 20 EGFR non-small cell lung cancer cell lines, and can significantly down-regulate the levels of p-EGFR, p-Akt, CyclinD1, Cdk4 in EGFR mutant cell lines. 17-AAG can down-regulate the level of WT-EGFR, but requires a higher concentration and duration of action. The main cause of resistance to gefitinib and erlotinib in non-small cell lung cancer is the K-Ras mutation. Although there are no specific inhibitors for its treatment, Jamie et al. found that Ganetespib can degrade the substrate C-Raf of K-Ras and inactivate some downstream signaling molecules. These indicate that HSP90 inhibitors have a significant inhibitory effect on non-small lung cancer cells, thereby antagonizing the resistance of non-small lung cancer cells to gefitinib and erlotinib.

Over 20%-30% of breast cancers overexpress the proto-oncogene Her2, and clinically, Herceptin monoclonal antibodies are used in some breast cancer patients overexpressing Her2/neu, but most patients develop resistance. Since Her2 is most dependent on HSP90 and most sensitive to HSP90 inhibitors, Scaltriti et al. used primary or secondary resistance models induced by PI3K mutation activation or decreased PTEN expression. The HSP90 inhibitor IPI504 was treated with BT47R (Her2+) and BT477H1047R. (Her2+) role of breast cancer cell lines. The results showed that IPI504 could down-regulate the expression of Her2 in the above two cells and inhibit the AKT and MAPK signaling pathways, and inhibit cell proliferation in a dose-dependent manner. The main reason for resistance to Trastuzmab is that the extracellular region of HER2 is deleted to produce p95-Her2, thereby losing the region to which it binds and producing resistance. Chandarlapaty's study found that HSP90 inhibitor SNX-2112 can degrade p95-Her2 in cells and inhibit the activity of AKT and ERK. Cytotoxicity test showed that SNK-2112 can completely inhibit the proliferation of T47D cells, which is much higher than Trastuzmab; The oral prodrug SNX-5422 of 2112 acts to express the MEFS tumor model of p95-Her2, which can completely inhibit tumor growth. Due to the lack of anti-Her2 therapeutic targets in triple-negative breast cancer, no targeted inhibitors have been obtained. However, experimental studies have shown that PU-H71 can inhibit the activity of AKT and Bcl-xl proteins and down-regulate their levels, thereby inducing apoptosis in triple-negative breast cancer cells. It is a triple negative breast cancer cell line HCC-1806, The cell death rates of MAD-MB-231 and MAD-MB-468 were 80%, 65%, and 80%, respectively. As can be seen above, HSP90 inhibitors are of great significance as drug-resistant treatments for clinical breast cancer.

Moreover, the occurrence of chronic myeloid leukemia is closely related to the mutation of ABL gene, and imatinib is widely used in the treatment of chronic myeloid leukemia. Leukemia patients who are resistant to imatinib are mainly caused by gene amplification or point mutations in the BCL-ABL kinase domain. This kinase is a HSP90 client protein, so HSP90 inhibitors should be able to act on it. Experimental studies have shown that the natural HSP90 inhibitor tripterine can cause apoptosis of imatinib-resistant KMB5-T315I cells induced by T315I mutation, and immunohistochemical analysis showed that it inhibited the expression of BCL-ABL. The synthetic small molecule HSP90 inhibitor 17-AAG can degrade mutant and wild-type BCL-ABL, thereby inhibiting cell proliferation. Therefore, imatinib-resistant chronic myeloid leukemia can be antagonized with HSP90 inhibitors.

Compared with traditional kinase inhibitors, HSP90 inhibitors can inhibit the activity of HSP90 and degrade a variety of similar kinases, which can produce better inhibitory effects on some drug-resistant tumors. Experiments have shown that HSP90 inhibitors have anti-tumor effects either singly or in combination. Therefore, HSP90 is a research and development target for cancer therapy.

Summary of the invention

Based on this, the present invention provides a novel heat shock protein inhibitor which is capable of inhibiting the activity of heat shock protein 90 and which can be used for the preparation of an antitumor drug.

A heat shock protein inhibitor having the structural features of Formula I, or a pharmaceutically acceptable salt thereof:

among them:

R ₁ , R ₇ and R ₈ are each independently selected from: H, C ₁ -C ₆ alkyl, C ₂ -C ₆ unsaturated alkyl, halogen, hydroxy, C ₁ -C ₆ alkoxy, NHCOOR, SO ₂ NHR, NHSO ₂ R, CN, NHCOR, CONHR;

R is selected from the group consisting of: H, C ₁ -C ₆ alkyl, C ₃ -C ₆ unsaturated alkyl, C ₃ -C ₈ cycloalkyl;

R ₂ and R ₃ are each independently selected from the group consisting of: H, D, C ₁ -C ₆ alkyl, C ₃ -C ₆ unsaturated alkyl, C ₃ -C ₈ cycloalkyl, phenyl, substituted phenyl, hetero Aryl group, acyl group;

R _{4 is} selected from the group consisting of: an amino group, a C ₁ -C ₆ saturated alkylamine, a C ₂ -C ₆ saturated heterocyclic amine, a C ₁ -C ₆ alkylamido group, an arylamide group, a substituted arylamide group, a hetero Arylamide group, substituted heteroarylamide group;

R ₅ and R ₆ are the same oxygen atom or are each independently selected from: H, D, C ₁ -C ₆ alkyl, C ₃ -C ₈ cycloalkyl, carbonyl, cyano, C ₁ -C ₆ alkoxy ,COOR',NHCOR',CONHR';

R' is selected from the group consisting of: H, C ₁ -C ₆ alkyl, C ₃ -C ₆ unsaturated alkyl;

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

The present inventors have found in the study that the morphine-linked benzyl site in the structure of AUY922 (which is a reported HSP90 inhibitor) may be easily metabolized and may cause toxic side effects. Based on the above research, the inventors discovered through experiments and research, based on their long-term experience, that the introduction of other groups at the benzyl site not only enhances the activity but also inhibits the metabolism of the site. Thereby eliminating potential toxic side effects.

In one embodiment, the five-membered aromatic heterocycle 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 the compounds of Formula II:

among them:

R ₇ and R _{8 are} selected from the group consisting of: H, C ₁ -C ₆ alkyl, C ₂ -C ₆ unsaturated alkyl, halogen, hydroxy, C ₁ -C ₆ alkoxy, CN;

R _{1 is} selected from the group consisting of C ₁ -C ₆ alkyl, C ₂ -C ₆ unsaturated alkyl, C ₁ -C ₆ alkoxy.

In one embodiment, the R ₂ and R ₃ are each independently selected from the group consisting of: H, C ₃ -C ₈ cycloalkyl, C ₁ -C ₆ alkyl, C ₃ -C ₆ unsaturated alkyl.

In one embodiment, R _{4 is} selected from the group consisting of: amino, C ₁ -C ₆ saturated alkylamine, C ₂ -C ₆ saturated heterocyclic amine, C ₁ -C ₆ alkylamido, arylamide, substituted Arylamide group, heteroarylamide group, substituted heteroarylamide group;

R _{5 is} selected from the group consisting of: H or the same oxygen atom as R ₆ ;

R _{6 is} selected from the group consisting of CN, COOR', CONHR', or the same oxygen atom as R ₅ ;

Wherein R' is selected from the group consisting of H, C ₁ -C ₆ alkyl, C ₃ -C ₆ unsaturated alkyl, C ₃ -C ₈ cycloalkyl.

In one embodiment, R _{4 is} selected from the group consisting of:

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

among them:

R _{1 is} selected from the group consisting of: C ₁ -C ₆ alkyl;

R ₇ and R _{8 are} selected from the group consisting of: a hydroxyl group;

R _{2 is} selected from: H;

R _{3 is} selected from the group consisting of: C ₁ -C ₆ alkyl;

R _{4 is} selected from the group consisting of:

R _{6 is} selected from: CN, COOR', CONHR', or the same oxygen atom as R5

The invention also discloses a preparation method of the above heat shock protein inhibitor or a pharmaceutically acceptable salt thereof, which is synthesized by the following route:

Wherein: R ₁₀ is a hydroxy protecting group.

R _{10 is} selected from the group consisting of: benzyl, trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl, tert-butyldiphenylsilyl, di-tert-butylmethylsilyl, methyl, acetyl, para Oxybenzyl, methoxymethyl.

The present invention also discloses the use of the above heat shock protein inhibitor or a pharmaceutically acceptable salt thereof for the preparation of a medicament for preventing and treating a disease having an pathological characteristic of increased expression of heat shock protein 90.

In one embodiment, the disease having increased pathological characteristics of heat shock protein 90 expression is: cancer, metabolic disease, myelodysplastic syndrome, systemic mastocytosis, Hippel-Lindau syndrome, Multi-centered Castleman's disease, and at least one of psoriasis.

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

The heat shock protein inhibitor of the present invention or a pharmaceutically acceptable salt thereof is a novel heat shock protein inhibitor, which retains the amino acid residue in the original molecule and the HSP90 receptor active pocket compared with the conventional inhibitor of the same type. The atoms and functional groups that interact with each other further modify the position at which the backbone may have a force, thereby optimizing the inhibitory potency of the control compound, giving it the following advantages:

1. Introduce effective modification to increase the binding ability of the target compound to the amino acid residue of the receptor protein, and inhibit the two enzyme receptors of HSP90α and HSP90β, and the five types of tumor cell lines.

2. Further improving the inhibitory ability of the positive control compound NVP/AUY-922 at the enzyme level and the cellular level, and providing a possibility for advancing the drug-forming process of the inhibitor.

3. Compared with a single inhibitor, compared with two drugs that act on a single target at the same time, the drug patients acting on the two targets are more convenient to use and can avoid the interaction between the drug and the drug.

detailed description

The invention is further illustrated by the following examples, without any limitation of the invention.

The compounds of the present invention and salts thereof can also be prepared by methods known for the preparation of chemically related compounds, and the starting materials involved in the examples can be obtained by similar methods in the prior art.

The straight line from the benzene ring to the benzene ring in the general formula I of the present invention indicates a substitution in which the position is not fixed.

The five-membered ring with a dotted line in the general formula I of the present invention is a five-membered aromatic heterocyclic ring containing X, Y, Z, W.

"Alkyl" means a saturated hydrocarbon group, including a straight or branched alkyl group, for example, a C ₁ -C ₆ alkyl group means a saturated straight or branched alkyl group having 1 to 6 carbon atoms, wherein the saturated Examples of the alkyl group include, but are not limited to, an ethyl group, a n-propyl group and the like, and examples of the saturated branched alkyl group include, but are not limited to, an isopropyl group, a t-butyl group and the like; and an "unsaturated alkyl group" means an alkenyl group or an alkyne. The hydrocarbon group of the group includes a linear or branched unsaturated alkyl group, and examples of the unsaturated linear alkyl group include, but are not limited to, a vinyl group, a propenyl group and the like, and examples of the unsaturated branched alkyl group include, but are not limited to, 2- a methacryl group or the like; "cycloalkyl" means an alkyl group having a cyclic structure, such as a C ₃ -C ₈ cyclic alkyl group means a saturated or unsaturated ring structure having 3 to 8 carbon atoms Examples of the alkyl group in which the saturated cyclic alkyl group includes, but are not limited to, a cyclopropyl group, a cyclopentyl group, an ethyl-substituted cyclohexyl group, and the like, and examples of the unsaturated cyclic alkyl group include, but are not limited to, cyclopentene, etc., in the present invention A C ₃ -C ₆ cyclic alkane is preferred.

"Substituted" refers to the replacement of a hydrogen group in a particular structure with a group of a given substituent. When more than one position in any particular structure may be substituted with more than one substituent selected from the 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 an organic compound. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and non-aromatic substituents of the organic compound. A heteroatom such as nitrogen can have a hydrogen substituent and/or any permissible substituent of an organic compound described herein that satisfies the valency of the hetero atom.

"Heteroaryl" means a 4n+2 aromatic ring system containing 5-6 membered monocyclic 6 and the aromatic ring system has a ring carbon atom and 1-4 ring heteroatoms, wherein each ring heteroatom is independently selected from Nitrogen, oxygen and sulfur. A heteroaryl group containing one or more nitrogen atoms, the point of attachment may be a carbon or nitrogen atom, if the chemical price permits. Heteroaryl polycyclic systems can include one or more heteroatoms. "Heteroaryl" also includes heteroaryl ring systems as defined above, fused with one or more carbocyclic or heterocyclic groups wherein the point of attachment is on the heteroaryl ring, and in this 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, wherein the point of attachment may be on an aryl or heteroaryl ring, and in this case The number of ring members includes only the number of ring members in the fused polycyclic (aryl/heteroaryl) ring system. a polycyclic heteroaryl group, one of which does not contain a hetero atom (for example, an anthracenyl group, a quinolyl group, a carbazolyl group, and the like), and the point of attachment thereof may be on any ring, that is, It is on a ring containing a hetero atom (for example, 2-fluorenyl) or on a ring containing no hetero atom (for example, 5-fluorenyl). Example with 2 heteroatoms The 5-membered heteroaryl group includes, but is not limited to, imidazole, pyrazole, oxazole, isoxazolyl, thiazolyl, and isothiazole. Exemplary 6-membered heteroaryl groups containing 1 heteroatom include, but are not limited to, pyridyl. 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 includes one or more heteroatoms (eg, oxygen, sulfur, nitrogen, boron, silicon, phosphorus).

The "amino group" means an organic compound in which a hydrogen atom of ammonia is replaced by an alkyl group, such as a heterocyclic amine, an aralkylamino group or the like.

"Amido" means a derivative in which the hydrogen of ammonia (or an amine) is substituted with an acyl group, such as a cycloalkylamide group, an alkylamide group, an arylamide group, a benzoarylamide group, a heteroarylamide group, A benzoheteroarylamide group, a heterocyclic amide group, a sulfonamide group, an alkyl urea or the like.

Example 1

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

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

According to the above line synthesis, the following steps are included:

(1) Preparation of 1-(2,4-bis(benzyloxy)phenyl)ethanone (Compound I-2).

2,4-dihydroxyacetophenone I-1 (9.12 g, 60.00 mmol), potassium carbonate (21.00 g, 151.00 mmol), and 200 mL of acetonitrile in an oil bath were placed in a 500 mL two-necked flask equipped with a reflux condenser. Heat at reflux at 80 ° C for 1 h, then benzyl bromide (14.70 mL, 144.00 mmol) was injected with a syringe and refluxed overnight. After the reaction was detected by TLC (TLC), the reaction mixture was cooled to room temperature, filtered, filtered, filtered, washed with dichloromethane, and the filtrate was evaporated. Ethyl ether was added to the oil, and the mixture was stirred to give a white smudge, which was filtered, and then washed with diethyl ether and then diethyl ether. The above procedure was repeated twice to give a white solid, 15.80 g, as compound I-2, yield 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).

Potassium tert-butoxide (4.72 g, 42.15 mmol), triphenylphosphinoiodomethane (17.88 g, 42.15 mmol) was added to a 250 mL two-necked flask equipped with a dropping funnel, and anhydrous THF was poured at 0 ° C under nitrogen atmosphere. (Tetrahydrofuran) was stirred for 1 h. Then 1-(2,4-bis(benzyloxy)phenyl)ethanone I-2 (10.76 g, 32,42 mmol) was completely dissolved in anhydrous THF and poured into a dropping funnel and then slowly dripped through a dropping funnel. After the addition to the reaction system, the mixture was stirred for 1 h, and the reaction was stirred at room temperature overnight. After the TLC was used to monitor the reaction of the starting material, the reaction solvent was evaporated, and the residue was purified by ethyl acetate. The organic solvent was evaporated to give a white solid (yield: mp.

The characterization data of the compound I-3 is: ¹ H NMR (400 MHz, Chloroform-d, δ _ppm ): 7.38-7.28 (m, 10H), 7.13 (d, J = 8.4 Hz, 1H), 6.59 (s, 1H) ), 6.52 (d, J = 8.4 Hz, 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).

I-2 (350.00 g, 1.06 mol), 35 g of 10% palladium on carbon, 1 mL of formic acid, 1 L of ethanol were added to a 2 L autoclave, and hydrogen gas was introduced and heated to reflux at 78 ° C for two days. After the reaction was completed, the mixture was cooled to room temperature, and the reaction mixture was filtered with celite, celite, and washed with ethanol, and the filtrate was collected, and the organic solvent was evaporated to remove the residue. 4, the yield is 80%.

The characterization data for this compound I-4 is: ¹ H NMR (400 MHz, Chloroform-d, δ _ppm ): 7.01 (d, J = 8.4 Hz, 1H), 6.39 (d, J = 8.4 Hz, 1H), 6.32 ( s, 1H), 5.50 (brs, 1H), 5.33 (brs, 1H), 3.12 (m, 1H), 1.21 (d, J = 6.4 Hz, 6H).

(4) Preparation of 1-(2,4-dihydroxy-5-isopropylphenyl)-ethanone (Compound I-5).

4-isopropylbenzene-1,3-diol I-4 (5.36 g, 35.25 mmol) was added to a 250 mL two-necked flask equipped with a reflux condenser, and then 100 mL of 47% trifluoride was injected under the protection of N ₂ . After stirring for half an hour, the boronic acid solution was stirred and heated to reflux in an oil bath with a solution of 4.03 mL of acetic acid. The reaction of the starting material was completely cooled by TLC, then cooled to room temperature, and then added with 70 mL of 10% aqueous sodium acetate solution and stirred for 2 h, diluted with water to 250 mL. The mixture was extracted with EtOAc (3 mL). EtOAc was evaporated, evaporated, evaporated, evaporated. 6.00g of yellow solid, which is compound I-5, the yield is 99.4%.

The characterization data for this compound I-5 is: ¹ H NMR (400 MHz, 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 = 6.0 Hz, 6H).

(5) Preparation of 1-(2,4-bis(benzyloxy)-5-isopropyl)-ethanone (Compound I-6).

Add 1-(2,4-bishydroxy-5-isopropyl)acetophenone I-5 (6.80 g, 35.05 mmol), potassium carbonate (12.09 g, 87.63 mmol) to a 250 mL two-necked flask equipped with a reflux condenser. 100 mL of acetonitrile was refluxed in an oil bath at 80 ° C for 1 h, then benzyl bromide (10 mL, 84.15 mmol) was injected with a syringe and refluxed overnight. After the reaction of the reaction starting material was completed by TLC, the reaction mixture was cooled to room temperature, filtered with Buchner funnel, and the residue was washed with methylene chloride. The filtrate was collected, and the organic solvent was evaporated to give a brown-brown oil. The petroleum ether was added to the oil and stirred and beaten to produce a white precipitate. The mixture was filtered with celite funnel and washed with petroleum ether to give 6.72 g of white solid, which was compound I-6, yield 51.26%.

The characterization data of the compound I-6 is: ¹ H NMR (400 MHz, 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 = 6.8 Hz, 6H).

(6) (Z)-Ethyl-4-(2,4-bis(benzyloxy)-5-isopropylphenyl)-2-hydroxy-4-oxobut-2-enoate Preparation of (Compound I-7).

Add sodium metal (0.90 g, 39.00 mmol) to a 250 mL two-necked flask equipped with a reflux condenser, and stir 100 mL of ethanol in an ice bath at 0 ° C until the sodium metal reaction is complete. Add 1-(2,4-double) at room temperature. (Benzyloxy)-5-isopropylphenyl)ethanone I-6 (6.72 g, 17.98 mmol) was stirred for 30 min, and diethyl oxalate (3.94 mL, 29.11 mmol) was poured in an oil bath at 80 ° C reflux. 4h. After TLC was monitored until the substrate reaction was completed, it was cooled to room temperature, and a 2 M aqueous solution of hydrochloric acid was added to adjust the pH to less than 7 to give a pale yellow precipitate. The organic solvent was removed by vacuum drying, then dissolved in dichloromethane and extracted three times with saturated sodium chloride. The solution was extracted once, dried over anhydrous sodium sulfate and then evaporated to remove methylene chloride to afford 8.18 g of a brown solid.

The characterization data for this compound I-7 is: ¹ H NMR (400 MHz, 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 = 14.0 Hz, 2H), 3.30 (m, 1H), 1.28 (t, J = 6.8 Hz, 3H), 1.23 (d, J = 6.8 Hz) , 6H).

(7) Preparation of 5-(2,4-bis(benzyloxy-5-isopropylphenyl)isoxazol-3-carboxylate (Compound I-8).

Add (Z)-ethyl-4-(2,4-bis(benzyloxy)-5-isopropylphenyl)-2-hydroxy-4-oxo to a 250 mL two-necked flask equipped with a reflux condenser Methyl methylbutan-2-enoate I-7 (8.18 g, 17.26 mmol), hydroxylamine hydrochloride (1.56 g, 22.48 mmol) and 100 mL of ethanol were refluxed in an oil bath at 80 ° C for 5 h. The reaction was completely monitored by TLC, cooled to room temperature and dried in vacuo to give a white solid. The solid was dissolved in ethyl acetate, extracted with water three times, and extracted with a saturated aqueous solution of sodium chloride. The organic layer was dried over anhydrous sodium sulfate, and then evaporated to remove the organic solvent to afford 6.74 g of white solid as compound I-8.

The characterization data for this compound I-8 is: ¹ H NMR (400 MHz, 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 = 7.2 Hz, 2H), 3.34 (m, 1H), 1.41 (t, J = 7.2 Hz, 3H), 1.23 (d, J = 6.8 Hz, 6H).

(8) Preparation of 5-(2,4-bis(benzyloxy)-5-isopropylphenyl)isoxazole-3-carboxamide (Compound I-9).

Add 5-(2,4-bis(benzyloxy-5-isopropylphenyl)isoxazol-3-carboxylic acid ethyl ester I-8 (6.74 g) to a 250 mL two-necked flask equipped with a reflux condenser. , 14.31 mmol), 100 mL of ethanol was stirred with heating, and the solid was completely dissolved, and then a 70% aqueous solution of ethylamine (13.80 mL, 215.73 mmol) was refluxed overnight at 80 ° C in an oil bath. The reaction was completely monitored by TLC, cooled to room temperature and dried in vacuo to give a white solid. Ethyl acetate dissolved solid, extracted with 1M hydrochloric acid water once, extracted once with saturated aqueous sodium carbonate solution, extracted once with water, once with saturated aqueous sodium chloride solution, dried over anhydrous sodium sulfate, and then evaporated to remove organic solvent to give white solid 6.46 g, yield 96%.

The characterization data for this compound I-9 is: ¹ H NMR (400 MHz, 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 = 6.8 Hz, 3H) , 1.24 (d, J = 6.8 Hz, 6H).

(9) Preparation of 5-(2,4-bis(benzyloxy)-5-isopropylphenyl)-methylisoxazole-3-carboxamide (Compound I-10)

Add 5-(2,4-bis(benzyloxy)-5-isopropylphenyl)-N-ethylisoxazol-3-carboxamide I- to a 250 mL two-necked flask equipped with a reflux condenser. 9 (6.46 g, 13.47 mmol), 100 mL of acetonitrile was stirred with heating, and the solid was completely dissolved, then iodine succinimide (3.71 g, 16.49 mmol) and ammonium cerium nitrate (0.74 mg, 1.35 mmol) were added to the oil bath. Reflux at 85 ° C overnight. The reaction was monitored by TLC, then cooled to room temperature and dried in vacuo to give a brown solid. The solid was dissolved in ethyl acetate, extracted with water three times, and extracted with a saturated aqueous solution of sodium chloride. The organic layer was dried over anhydrous sodium sulfate, and then evaporated to remove organic solvent, and then purified by silica gel column to afford 5.62 g of brown white solid.

The characterization data for this compound I-10 is: ¹ H NMR (400 MHz, 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 = 6.8 Hz, 3H), 1.23 (d, J = 6.8 Hz, 6H).

(10) 5-(2,4-Bis(benzyloxy)-5-isopropylphenyl)-N-methyl-4-(4-formylphenyl)isoxazole-3-carboxamide Preparation of (Compound I-11).

Add 5-(2,4-bis(benzyloxy)-5-isopropylphenyl)-N-ethyl-4-iodoisoxazole-3 to a 100 mL two-necked flask equipped with a reflux condenser. -formamide 1-10 (1.35 g, 2.27 mmol), 4-formylbenzeneboronic acid (0.68 g, 4.54 mmol), ditriphenylphosphine palladium dichloride (0.08 g, 0.12 mmol), potassium carbonate (0.626 mg) , 4.54 mmol), and 20 mL of tetrahydrofuran and 2 mL of distilled water were refluxed overnight in an 80 ° C oil bath under nitrogen. After TLC was monitored, the reaction was completed and then cooled to room temperature. The solvent was evaporated to dryness. The solvent was evaporated to ethyl ether. The mixture was dissolved in ethyl acetate. Water was extracted three times, extracted with saturated aqueous sodium chloride, dried over anhydrous sodium sulfate, dried in vacuo. 1.02 g, yield 79%.

The characterization data for this compound I-11 is: ¹ H NMR (400 MHz, Chloroform-d, δ _ppm ): 9.96 (s, 1H), 7.70 (d, J = 6.4 Hz, 2H), 7.41 (d, J = 6.4 Hz, 2H), 7.39-7.33 (m, 5H), 7.27 (s, 2H) 7.19 (s, 1H), 7.00 (s, 2H), 6.82 (t, J = 2.2 Hz, 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 = 5.6 Hz, 3H), 1.11 (d, J = 6.0) Hz, 6H).

(11) 5-(2,4-Dihydroxy-5-isopropylphenyl)-N-methyl-4-(4-formylphenyl)isoxazole-3-carboxamide (Compound I-12 Preparation

Take a 100 mL two-necked bottle, add compound I-11 (1 g), argon-protected, add anhydrous DCM (20 mL) to dissolve, place at -5 ° C, stir for 15 min, add dropwise a solution of boron tribromide in dichloromethane ( 1 M, 5.2 mL, 3 eq.), stirred for 20 min, placed at room temperature and stirred for 2 h. TLC tracking showed that the reaction of the starting material was completed. Dilute with DCM (10 mL) at 0 ° C, quench with saturated NaHCO ₃ solution, extract and collect organic phase. EA was added to the aqueous phase, and the resulting flocculent solid was dissolved, and the organic phase was collected, mixed, dried over anhydrous sodium sulfate, and concentrated to a column (DCM: acetone = 1:1). 650 mg of a brown solid was obtained in a yield of 94.8%. ESI-MS m/z: 395.1 (M+H) ⁺ .

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

A 25 mL single-mouth bottle was added, and compound I-12 (50 mg) was added, and methanol (2 mL) was dissolved. Morpholine (17 μL, 1.5 eq.), hydrazine hydrochloride (6 mg, 0.5 eq.) was stirred at 40 ° C for 15 min, and the compound TMSCN was added. (47 μL, 3 equivalents), stirred at this temperature for 2 h, TLC tracking showed that the starting material was completed. EA dilution was added (10 mL), saturated NaHCO ₃ solution was added quenched with saturated brine and extracted, dried over anhydrous sodium sulfate, and concentrated through the column (DCM: acetone = 1: 1 ). The obtained yellow viscous product was beaten, petroleum ether was added thereto, and the mixture was allowed to stand at a low temperature to precipitate a white solid. The yellow liquid was taken up, and the obtained solid oil pump was drained to obtain 25 mg of a white solid in a yield of 41.0%.

The characterization data for this compound 1 is: ¹ H NMR (400 MHz, Chloroform-d, δ _ppm ): 7.58 (d, J = 8.0 Hz, 2H), 7.44 (d, J = 8.0 Hz, 2H), 6.80 (s, 1H), 6.32 (s, 1H), 4.80 (s, 1H), 3.72 (d, J = 3.6 Hz, 4H), 3.57-3.40 (m, 2H), 2.99-2.92 (m, 1H), 2.61 (d) , J = 3.6 Hz, 4H), 1.25 (t, J = 7.2 Hz, 3H), 0.88 (d, J = 6.8 Hz, 6H). ¹³ C NMR (125 MHz, 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 (MH) ^- .

Example 2

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

Take a 25mL single-mouth bottle, add compound I-12 (30mg), dissolve in methanol (1mL), add (2S,6R)-2,6-dimethylmorpholine (15μL, 1.5 equivalents), guanidine hydrochloride (4mg, 0.5 Equivalent), stirring at 40 ° C for 15 min, adding the compound TMSCN (29 μL, 3 equivalents), TLC tracking showed that the starting material was not completely reacted, so it was directly purified. EA dilution was added (10 mL), saturated NaHCO ₃ solution was added quenched with saturated brine and extracted, dried over anhydrous sodium sulfate, and concentrated through the column (DCM: acetone = 1: 1 ). The obtained yellow viscous product was purified by HPLC, and the obtained solid oil pump was dried to give 9 mg of white solid.

The characterization data for this compound 2 is: ¹ H NMR (400 MHz, CD ₃ OD, δ _ppm ): 7.47 (d, J = 8.0 Hz, 2H), 7.37 (d, J = 8.0 Hz, 2H), 6.80 (s, 1H), 6.35 (s, 1H), 5.11 (s, 1H), 3.78 (t, J = 7.6 Hz, 1H), 3.59 (t, J = 6.4 Hz, 1H), 3.39, 3.31 (dd, J = 7.2 , 7.6 Hz, 2H), 3.11-3.04 (m, 1H), 2.91 (d, J = 10.8 Hz, 1H), 2.45 (d, J = 10.4 Hz, 1H), 2.21 (t, J = 10.4 Hz, 1H) ), 1.86 (t, J = 10.4 Hz, 1H), 1.21 (t, J = 7.2 Hz, 6H), 1.08 (d, J = 6.0 Hz, 3H), 0.99 (d, J = 7.2 Hz, 6H). ¹³ C NMR (125 MHz, CD ₃ OD, 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 (MH) ^- .

Example 3

Compound 3: 4-(4-(cyano(4-methylpiperazin-1-yl)methyl)phenyl)-5-(2,4-dihydroxy-5-isopropylphenyl)-N For the preparation of methylisoxazole-3-carboxamide, refer to the method of Example 1.

The characterization data for this compound 3 is: ¹ H NMR (400 MHz, CD ₃ OD, δ _ppm ): 7.47 (d, J = 8.0 Hz, 2H), 7.35 (d, J = 8.4 Hz, 2H), 6.81 (s, 1H), 6.36 (s, 1H), 5.06 (s, 1H), 3.39, 3.36 (dd, J = 7.2, 7.6 Hz, 2H), 3.10-3.04 (m, 1H), 2.99 (d, J = 11.2 Hz) , 1H), 2.62 (d, J = 11.2 Hz, 1H), 2.47 (t, J = 8.8 Hz, 1H), 2.10 (t, J = 10.8 Hz, 1H), 1.74 (d, J = 8 Hz, 1H) , 1.61 (d, J = 13.2 Hz, 1H), 1.22 (t, J = 12 Hz, 3H), 0.98, 0.95 (dd, J = 7.2, 6.4 Hz, 9H). ¹³ C NMR (125 MHz, CD ₃ OD, 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 (MH) ^- .

Example 4

Compound 4: 4-(4-(Cyano-(4-hydroxypiperidin-1-yl)methyl)phenyl)-5-(2,4-dihydroxy-5-isopropylphenyl)-N For the preparation of methylisoxazole-3-carboxamide, refer to the method of Example 1.

The characterization data for this compound 4 are: ¹ H NMR (400 MHz, CD ₃ OD, δ _ppm ): 7.47 (d, J = 8.0 Hz, 2H), 7.36 (d, J = 8.4 Hz, 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, 7.2 Hz, 2H), 3.10-3.04 (m, 1H) , 2.92 (t, J = 5.6 Hz, 1H), 2.69 (t, J = 5.6 Hz, 1H), 2.49 - 2.43 (m, 1H), 2.27 (t, J = 9.2 Hz, 1H), 1.90 (t, J = 12.8 Hz, 2H), 1.67-1.58 (m, 1H), 1.53-1.29 (m, 1H), 1.21 (t, J = 7.2 Hz, 3H), 0.90 (d, J = 6.8 Hz, 6H). ¹³ C NMR (125 MHz, CD ₃ OD, 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 (MH) ^- .

Example 5

Compound 5: 4-(4-(cyano(2-oxa-6-azaspiro[3.3]hept-6-yl)methyl)phenyl)-5-(2,4-dihydroxy-5- For the preparation of isopropylphenyl)-methylisoxazole-3-carboxamide, refer to the method of Example 1.

The characterization data for this compound 5 is: ¹ H NMR (400 MHz, CD ₃ OD, δ _ppm ): 7.41 (d, J = 8.0 Hz, 2H), 7.37 (d, J = 8.4 Hz, 2H), 6.85 (s, 1H), 6.36 (s, 1H), 4.90 (s, 1H), 4.78 (t, J = 7.6 Hz, 4H), 3.55 (d, J = 7.6 Hz, 2H), 3.50 (d, J = 7.6 Hz, 2H), 3.41, 3.37 (dd, J=7.2, 7.2 Hz, 2H), 3.14-3.07 (m, 1H), 1.23 (t, J = 7.2 Hz, 3H), 0.99 (d, J = 7.2 Hz, 6H) ¹³ C NMR (125 MHz, CD ₃ OD, 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 (MH) ^- .

Example 6

Compound 6: 4-(4-(cyano(4AR,7AS)-tetrahydro-2H-[1,4]dioxane[2,3-c]pyrrole-6-(3H)-yl)methyl The preparation of phenyl)-5-(2,4-hydroxy-5-isopropylphenyl)-N-methylisoxazole-3-carboxamide was carried out according to the method of Example 1.

The characterization data for this compound 6 is: ¹ H NMR (400 MHz, CD ₃ OD, δ _ppm ): 7.50 (d, J = 8.4 Hz, 2H), 7.38 (d, J = 6.4 Hz, 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, 7.2 Hz, 2H), 3.14-3.07 (m, 1H), 3.00-2.99 (m, 2H), 2.86-2.83 (m, 1H), 1.24 (t, J = 7.6 Hz, 3H), 1.03 (d, J = 6.8 Hz, 6H). ¹³ C NMR (125 MHz, CD ₃ OD, 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 (MH) ^- .

Example 7

Compound 7: 4-(4-(cyano(3-methylpyrrolidin-1-yl)methyl)phenyl)-5-(2,4-dihydroxy-5-isopropylphenyl)-N For the preparation of methylisoxazole-3-carboxamide, refer to the method of Example 1.

The characterization data for this compound 7 is: ¹ H NMR (400 MHz, CD ₃ OD, δ _ppm ): 7.48 (d, J = 8.0 Hz, 2H), 7.37 (d, J = 8.4 Hz, 2H), 6.85 (d, J = 2.4 Hz, 1H), 6.37 (d, J = 0.8 Hz, 1H), 5.23 (s, 1H), 4.02-3.96 (m, 1H), 3.41, 3.37 (dd, J = 7.2, 7.2 Hz, 2H), 3.29 (d, J = 8.4 Hz, 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 = 7.6 Hz, 3H), 1.01 (d, J = 1.2 Hz, 6H). ESI-MS m/z: 505.1 (M+H) ⁺ , 503.2 (MH) ^- .

Example 8

Compound 8: 4-(4-(2-thia-5-azabicyclo[2.2.1]hept-5-yl(cyano)methyl)phenyl)-5-(2,4-dihydroxy- The preparation of 5-isopropylphenyl)-methylisoxazole-3-carboxamide was carried out in the same manner as in Example 1.

The characterization data for this compound 8 is: ¹ H NMR (400 MHz, CD ₃ OD, δ _ppm ): 7.55 (d, J = 8.4 Hz, 1H), 7.50 (d, J = 8.0 Hz, 1H), 7.40 (t, J = 6.4 Hz, 2H), 6.86 (d, J = 2.8 Hz, 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, 7.6 Hz, 2H), 3.25 (d, J = 5.2 Hz, 1H), 3.12-3.06 ( m, 2H), 3.02 (t, J = 10 Hz, 1H), 2.90, 2.86 (dd, J = 9.2, 10 Hz, 1H), 2.42, 2.38 (dd, J = 11.2, 9.6 Hz, 1H), 1.83 (t , J = 13.6 Hz, 1H), 1.22 (t, J = 4 Hz, 3H), 1.03 (d, J = 6.8 Hz, 6H). ¹³ C NMR (125 MHz, CD ₃ OD, 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 (MH) ^- .

Example 9

Compound 9: 4-(4-(4-methylpiperazin-1-yl)(cyano)methyl)phenyl)-5-(2,4-dihydroxy-5-isopropylphenyl)- For the preparation of N-methylisoxazole-3-carboxamide, refer to the method of Example 1.

The characterization data for this compound 9 are: ¹ H NMR (400 MHz, CD ₃ OD, δ _ppm ): 7.49 (d, J = 7.2 Hz, 2H), 7.38 (d, J = 7.2 Hz, 2H), 6.83 (s, 1H), 6.35 (s, 1H), 5.19 (s, 1H), 3.58 (s, 4H), 3.38 (d, J = 7.6 Hz, 2H), 3.10-3.07 (m, 1H), 2.60-2.53 (m) , 4H), 2.10 (s, 3H), 1.21 (t, J = 7.2 Hz, 3H), 1.00 (dd, J = 6.4 Hz, 6H). ¹³ C NMR (125 MHz, CD ₃ OD, 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 (MH) ^- .

Example 10

Compound 10: 4-(4-(cyano(2-methoxyethyl)amino)methyl)phenyl)-5-(2,4-dihydroxy-5-isopropylphenyl)-methyl For the preparation of isoxazole-3-carboxamide, refer to the method of Example 1.

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

Example 11

Compound 11: 4-(4-(2-Amino-1-morpholino-2-oxoethyl)phenyl)-5-(2,4-dihydroxy-5-isopropylphenyl)-methyl Preparation of isoxazole-3-carboxamide.

Take a 25mL single-mouth bottle, add compound 1 (30mg), add DMSO (1mL) to dissolve, add 30% hydrogen peroxide (20mg, 3 equivalents) and potassium carbonate (23mg, 3 equivalents), stir at room temperature for 2h, TLC tracking shows the reaction of raw materials Finished. It was diluted with EA (10 mL) and extracted with water (10 mL). The obtained yellow viscous product was beaten, petroleum ether was added thereto, and the mixture was allowed to stand at a low temperature to precipitate a white solid. The yellow liquid was taken up, and the obtained solid oil pump was drained to obtain 3.4 mg of a white solid in a yield of 11.0%.

The characterization data for this compound 11 is: ¹ H NMR (400 MHz, CD ₃ OD, δ _ppm ): 7.45 (d, J = 8.4 Hz, 2H), 7.31 (d, J = 8.4 Hz, 2H), 6.81 (s, 1H), 6.35 (s, 1H), 3.74 (s, 1H), 3.73 (t, J = 4.4 Hz, 4H), 3.40, 3.37 (dd, J = 7.2, 7.2 Hz, 2H), 3.09-3.06 (m , 1H), 2.46-2.37 (m, 4H), 1.21 (t, J = 7.2 Hz, 3H), 0.99 (d, J = 6.8 Hz, 6H). ESI-MS m/z: 509.1 (M+H) ⁺ , 507.2 (MH) ^- .

Example 12

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

Follow the above steps, including the following steps:

(1) Methyl-2-(4-(5-(2,4-bis(benzyloxy)-5-isopropylphenyl)-3-(methylcarbamoyl)isoxazole-4 Preparation of -yl)phenyl)-2-hydroxy (Compound II-1).

Take a 50mL single-mouth bottle, add compound I-11 (1g), add a catalytic amount of powdered iodine, add anhydrous DCM (10mL) to dissolve, add TMSCN (0.26mL, 1.2 equivalent), stir at room temperature for 1h, TLC tracking display The reaction of the raw materials is completed. The mixture was diluted with EtOAc (EtOAc) (EtOAc)

The obtained compound was added to a solution of hydrogen chloride in methanol (20%, 15 mL) and stirred at 40 ° C overnight. TLC tracking showed that the reaction of the starting material was completed. After adding EA (30 mL), the resulting solid was filtered, and the filtrate was concentrated. The filtrate was concentrated, then EA (20 mL) was added, and the mixture was added to a saturated NaHCO ₃ solution, extracted with saturated brine, dried over anhydrous sodium sulfate and concentrated. EA=4:1). A white solid of 1.05 g was obtained in a two-step yield of 95.4%. ESI-MS m/z: 635.1 (M+H) ⁺ .

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

Take a 50 mL single-mouth bottle, add compound II-1 (0.5 g), add anhydrous DCM (10 mL) to dissolve, add dry triethylamine (0.33 mL, 3 equivalents), set to 0 ° C, stir for 15 min, add TsCl ( 0.46 g, 3 eq.), stirred for 15 min, warmed to 40 ° C and stirred overnight. TLC tracking showed that the reaction of the starting material was completed. The mixture was neutralized with a saturated NaHCO ₃ solution, extracted with brine, dried over anhydrous sodium sulfate and evaporated. A white solid was obtained in an amount of 0.34 g, yield 55.2%. ESI-MS m/z: 789.2 (M+H) ⁺ .

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

Take a 50 mL single-mouth bottle, add compound II-2 (0.33 g), add toluene (5 mL) to dissolve, add dry triethylamine (0.12 mL, 2 equivalents), add morpholine (55 μL, 1.5 equivalents), and place in oil The bath was refluxed for 2 h, and TLC tracking showed that the reaction of the starting material was completed. It was diluted with EA (15 mL), extracted with brine, and then evaporated. The viscous product was 232 mg in a yield of 80%. ESI-MS m/z: 7032 (M+H) ⁺ .

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

Take a 50mL two-necked bottle, add compound II-3 (0.2g), argon protection, add anhydrous DCM (10mL) to dissolve, set at -5 ° C, stir for 15min, add dropwise boron tribromide solution in dichloromethane (1 M, 0.9 mL, 3 eq.), stirred for 10 min. TLC tracking showed that the reaction of the starting material was completed. Dilute with DCM (10 mL) at 0 ° C, quench with saturated NaHCO ₃ solution, extract and collect organic phase. EA was added to the aqueous phase, and the resulting flocculent solid was dissolved, and the organic phase was collected, mixed, dried over anhydrous sodium sulfate, and concentrated to a column (DCM: acetone = 1:1). The obtained yellow viscous product was beaten, petroleum ether was added thereto, and the mixture was allowed to stand at a low temperature to precipitate a white solid. The yellow liquid was taken up, and the obtained solid oil pump was drained to obtain 35.7 mg of a white solid in a yield of 24.0%.

The characterization data for this compound 12 is: ¹ H NMR (400 MHz, CD ₃ OD, δ _ppm ): 7.40 (d, J = 8.4 Hz, 2H), 7.30 (d, J = 8.4 Hz, 2H), 6.76 (s, 1H), 6.36 (s, 1H), 4.02 (s, 1H), 3.68 (d, J = 4.4 Hz, 4H), 3.66 (s, 3H), 3.39, 3.35 (dd, J = 7.2, 7.6 Hz, 2H) ), 3.09-3.02 (m, 1H), 2.45 (d, J = 4.4 Hz, 4H), 1.20 (t, J = 7.6 Hz, 3H), 0.95 (d, J = 6.8 Hz, 6H). ¹³ C NMR (125 MHz, CD ₃ OD, 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 (MH) ^- .

Example 13

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

The characterization data for this compound 13 are: ¹ H NMR (400 MHz, CD ₃ OD, δ _ppm ): 7.41 (d, J = 8.0 Hz, 2H), 7.31 (d, J = 8.0 Hz, 2H), 6.77 (s, 1H), 6.36 (s, 1H), 4.20-4.05 (m, 2H), 4.00 (s, 1H), 3.69 (t, J = 4.8 Hz, 3H), 3.39, 3.35 (dd, J = 7.2, 7.2 Hz) , 2H), 3.14 - 3.02 (m, 1H), 2.46 (d, J = 4.4 Hz, 4H), 1.21, 1.18 (dd, J = 7.2, 6.8 Hz, 6H), 0.95 (d, J = 7.2 Hz, 6H). ¹³ C NMR (125 MHz, CD ₃ OD, 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 (MH) ^- .

The structural formula of the compound 13 is:

Example 14

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

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

Take a 50mL single-mouth bottle, add compound II-4 (0.5g), ethylamine solution (68%, 0.2mL, 4 equivalents), ethanol (5mL), placed in an oil bath, reflux reaction overnight, TLC tracking shows raw materials The reaction is completed. Most of the solvent was stirred, extracted with EA (20 mL) and brine, dried over anhydrous sodium sulfate The obtained viscous product (0.48 g) was obtained in a yield of 96.2%. ESI-MS m/z: 648.3 (M+H) ⁺ .

(2) Compound 14: 5-(2,4-dihydroxy-5-isopropylphenyl)-N-methyl-4-(4-(2-(dimethylamino)-1-morpholino) The preparation of 2-oxoethyl)phenyl)isoxazole-3-carboxamide was carried out in the same manner as in Example 12.

The characterization data for this compound 14 is: ¹ H NMR (400 MHz, CD ₃ OD, δ _ppm ): 7.40 (d, J = 8.4 Hz, 2H), 7.29 (d, J = 8.0 Hz, 2H), 6.79 (s, 1H), 6.35 (s, 1H), 3.70 (t, J = 4.4 Hz, 5H), 3.38, 3.42 (dd, J = 7.2, 7.6 Hz, 2H), 3.24 - 3.14 (m, 2H), 3.09 - 3.02 (m, 1H), 2.40-2.34 (m, 4H), 1.19 (t, J = 7.2 Hz, 3H), 1.10 (t, J = 7.2 Hz, 3H), 0.96 (d, J = 6.8 Hz, 6H) ¹³ C NMR (125 MHz, CD ₃ OD, 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 (MH) ^- .

Example 15

Compound 15: methyl-2-(4-(5-(2,4-dihydroxy-5-isopropylphenyl)-3-(ethylcarbamoyl)isoxazol-4-yl)phenyl) Preparation of ethyl-2-(4AR,7AS)-tetrahydro-2H-[1,4]dioxane[2,3-c]pyrrole-6-(3H)-yl)acetate, refer to Example 12 Methods.

The characterization data for this compound 15 is: ¹ H NMR (400 MHz, CD ₃ OD, δ _ppm ): 7.41 (d, J = 8.4 Hz, 2H), 7.30 (d, J = 8.1 Hz, 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, 7.2 Hz, 2H), 3.09-2.99 (m, 2H), 2.84-2.71 (m, 2H), 2.76-2.68 (m, 1H), 1.20 (t, J = 7.2 Hz, 3H), 0.96 (d, J = 2.8 Hz), 0.95 (d, J = 2.8 Hz). ¹³ C NMR (125 MHz, CD ₃ OD, 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. MS m/z: 566.2 (M + H) ⁺ , 564.1 (MH) ^- .

The structural formula of the compound 15 is:

Example 16

Compound 16: ethyl-2-(4-(5-(2,4-dihydroxy-5-isopropylphenyl)-3-(ethylcarbamoyl)isoxazol-4-yl)phenyl) Preparation of ethyl-2-(4AR,7AS)-tetrahydro-2H-[1,4]dioxane[2,3-c]pyrrole-6-(3H)-yl)acetate, refer to Example 12 Methods.

The characterization data for this compound 16 is: ¹ H NMR (400 MHz, CD ₃ OD, δ _ppm ): 7.44 (d, J = 8.4 Hz, 2H), 7.33 (d, J = 8.4 Hz, 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, 3.2 Hz, 2H), 3.59-3.52 (m, 2H), 3.41, 3.38 (dd, J = 5.6, 6 Hz, 2H), 3.11-3.04 (m, 2H), 2.87-2.84 (m, 2H), 2.75-2.72 (m, 1H), 1.20 (t, J = 7.2 Hz, 3H), 1.23, 1.21 (dd, J = 5.6, 5.6 Hz, 6H), 1.00, 0.98 (dd, J = 2.8, 2.8 Hz, 6H). ESI-MS m/z: 580.2 (M+H) ⁺ , 578.1 (MH) ^- .

The structural formula of the compound 16 is:

Example 17

Follow the above steps, including the following steps:

(1) Methyl 4-(5-(2,4-bis(benzyloxy)-5-isopropylphenyl)-3-(methylcarbamoyl)isoxazol-4-yl)benzene Preparation of methyl formate (Compound III-1).

A 50 mL vial was added and compound I-10 (600 mg, 1 mmol), (4-(methoxycarbonyl)phenylboronic acid (235 mg, 1.3 mmol, 1.3 eq.), Pd(PPh ₃ ) ₂ Cl ₂ (70 mg, 0.1 mmol, 10 mol%), sodium hydrogencarbonate (252 mg, 3 mmol, 3 equivalents), argon exchange, addition of DMF: H ₂ O = 10 mL: 2 mL solution, placed at 80 ° C, stirred for 5 h. TLC tracking monitoring. portion of the solvent, through a column (PE: EA = 4: 1 ), to give a tan product 620mg.ESI-MS m / z: 605.2 (m + H) +.

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

A 25 mL single-mouth bottle was taken, compound III-1 (600 mg) was added, dissolved in acetone (2 mL), 35% sodium hydroxide solution (1 mL) was added, and methanol (4 mL) was added to dissolve the layer, and the mixture was stirred at room temperature for 2 h. TLC tracking monitoring. Add 1N HCl solution, adjust to acidity, spin off most of the organic solution, add EA and saturated brine to extract, collect the organic phase, dry and spin dry. Used directly in the next step. ESI-MS m/z: 591.1 (M+H) ⁺ .

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

A 25 mL one-necked flask was taken, and crude compound III-2 (400 mg, 0.68 mmol), HOBt (110 mg, 0.816 mmol, 1.2 eq.), EDCI (195 mg, 1.02 mmol, 1.5 eq.), dissolved in DCM (5 mL), 0.4 mL, 2.72 mmol, 4 eq.), stirred for 15 min, compound 7 (87 mg, <RTIgt; TLC tracking monitoring. It was extracted with DCM and saturated brine, dried, dried, and then passed to a column (PE: EA = 2:1). A colorless, sticky product of 160 mg was obtained. ESI-MS m/z: 702.1 (M+H) ⁺ .

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

Take a 25mL two-necked flask, add the crude compound III-3 (160mg, 0.23mmol), argon-protected, add anhydrous DCM (10mL) to dissolve, set at -5 ° C, stir for 15min, add boron tribromide two Methyl chloride solution (1 M, 0.69 mL, 3 eq.), stirred for 10 min, rt. TLC tracking showed that the reaction of the starting material was completed. Dilute with DCM (10 mL) at -5 ° C, quench with saturated NaHCO ₃ solution, extract and collect organic phase. EA was added to the aqueous phase, and the resulting flocculent solid was dissolved, and the organic phase was collected, mixed, dried over anhydrous sodium sulfate, and concentrated to a column (DCM:acetone = 2:1). The obtained yellow viscous product was beaten, petroleum ether was added, and the mixture was allowed to stand at a low temperature to precipitate a white solid, and the yellow liquid was taken up, and the obtained solid oil pump was drained to obtain 80 mg of a white solid.

The characterization data for this compound 17 is: ¹ H NMR (400 MHz, CD ₃ OD, δ _ppm ): 7.51 (d, J = 6.4 Hz, 2H), 7.42 (d, J = 6.4 Hz, 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, 5.6 Hz, 2H), 3.34 -3.12 (m, 1H), 1.24 (t, J = 6.0 Hz, 3H), 1.08 (d, J = 5.6 Hz, 6H). ¹³ C NMR (125 MHz, CD ₃ OD, 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. MS m / z: 522.1 (M + H) ⁺ , 520.1 (MH) ^- .

Example 18

Compound 18: 5-(2,4-dihydroxy-5-isopropylphenyl)-N-methyl-4-(4-(morpholin-4-carbonyl)phenyl)isoxazole-3-methyl For the preparation of the amide, refer to the method of Example 17.

The characterization data for this compound 18 is: ¹ H NMR (400 MHz, CD ₃ OD, 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, 7.2 Hz, 2H), 3.14 - 3.07 (m, 1H), 1.21 (t, J = 7.2 Hz, 3H), 1.05 (d, J = 6.8 Hz, 6H). ¹³ C NMR (125MHz, CD ₃ OD, 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 (MH) ^- .

The structure of the compound 18 is:

Example 19

Follow the above steps, including the following steps:

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

Take a 100mL single-mouth bottle, add 3g (s)-(+)-mandelic acid, dissolve with ethanol (30mL), add concentrated sulfuric acid (0.3mL), reflux at 80 °C, stir for 3h, TLC tracking shows the raw material reaction Finished. The saturated NaHCO ₃ solution was added until no bubbles were formed, and most of the ethanol solution was added, and the mixture was extracted with EA (30 mL), and brine was evaporated. 98.5%).

The characterization data for this compound IV-1 is: ¹ H NMR (400 MHz, Chloroform-d, δ _ppm ): 7.42 - 7.27 (m, 5H), 5.15 (d, J = 4.0 Hz, 1H), 4.27 - 4.10 (m) , 2H), 3.68 (d, J = 4.0 Hz, 1H), 1.20 (t, J = 8.0 Hz, 3H)

(2) Preparation of (S)-ethyl-2-hydroxy-2-(4-iodophenyl)acetate (Compound IV-2).

Take a 100mL single-mouth bottle, add compound IV-1 (3.2g, 17.8mmol), dissolve in anhydrous DCM (60mL), wrap the outer wall of the bottle with tin foil, avoid iodine, add iodine (4.5g, 17.7mmol) and trifluoromethane Silver acid (4.56 g, 17.7 mmol) was stirred at room temperature for 4 h. The solution was quenched by the addition of sodium thiosulfate solution, the precipitate was filtered, washed with DCM, and the mixture was washed with saturated brine. The organic phase was collected, dried over anhydrous sodium sulfate, dried, and then passed to the column (PE: EA=8:1) to give brown oil liquid.

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

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

Take a 50 ml two-necked bottle and add compound IV-2 (0.55 g, 1.8 mmol), boranoic acid pinacol ester (0.55 g, 2.16 mmol), [1,1'-bis(diisopropylphosphine) ferrocene Iron] dichloropalladium (54 mg, 0.09 mmol), potassium acetate (529 mg, 5.4 mmol), argon exchange, plus The mixture was dissolved in DMF, placed at 90 ° C, stirred for 1 h, and TLC tracking showed that the reaction of the starting material was completed. Rotate most of the DMF, add EA, filter the precipitate, rinse with EA, spin dry, and pass through the column (PE: EA = 8:1). An anhydrous oily liquid (390 mg, 70.9%) was obtained.

The characterization data for this compound IV-3 is: ¹ H NMR (400 MHz, Chloroform-d, δ _ppm ): 7.81 (d, J = 7.6 Hz, 2H), 7.44 (d, J = 7.2 Hz, 2H), 5.16 ( s, 1H), 4.23-4.09 (m, 2H), 3.56 (s, 1H), 1.33 (s, 12H), 1.22 (t, J = 7.2 Hz, 3H).

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

A 100 mL single-mouth bottle was added, and compound 3 (1 g, 3.27 mmol) was added, dissolved in anhydrous DCM (10 mL), dry triethylamine (1.4 mL, 9.8 mmol, 3 eq.) was added, and TsCl (1.86 g, 9.8 mmol) was slowly added. 3 equivalents), placed at 50 ° C, stirred overnight. TLC tracking showed that the reaction of the starting material was completed. Water and DCM were added, and the mixture was washed with saturated brine, dried over anhydrous sodium sulfate, dried, and then passed to the column (PE: EA = 10:1). An anhydrous oily liquid (1 g, 66.7%) was obtained.

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

A 50 mL single-mouth flask was taken, and compound IV-4 (0.5 g, 1.09 mmol) was added, and toluene (8 mL) was dissolved, and dried triethylamine (0.3 mL, 2.17 mmol, 2 eq.), morpholine (0.14 mL, 1.64 mmol, 1.5 equivalents), placed in an oil bath and refluxed. After stirring for 2 h, TLC tracking showed that the reaction of the starting material was completed. Most of the toluene was taken up, and EA and saturated brine were added, extracted, dried over anhydrous sodium sulfate and dried (260 mg, 64%).

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

Take a 50mL single-mouth bottle, add compound IV-5 (250mg, 0.67mmol), add 10mL of HCl solution (adjusted to pH=3), add n-hexane (10mL), stir, add phenylboronic acid (81mg, 0.67mmol, 1 equivalent ), stirring vigorously until all the phenylboronic acid was dissolved, and stirring for another 0.5 h. TLC tracking showed that the reaction of the starting material was completed. Extract, retain the aqueous phase, add n-hexane and wash once, add EA to wash away the incomplete phenylboric acid, retain the aqueous phase, add saturated sodium bicarbonate solution, adjust the aqueous phase to alkaline, then add EA*2 extraction Dry with anhydrous sodium sulfate, spin dry and use directly in the next step. The product was obtained as a colorless viscous product, 160 mg, 82%.

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

A 25 mL vial was added and compound I-10 (102 mg, 0.17 mmol), compound IV-6 (60 mg, 0.2 mmol, 1.2 eq.), Pd(PPh ₃ ) ₂ Cl ₂ (12 mg, 0.02 mmol, 10 mol%), Sodium bicarbonate (43 mg, 0.51 mmol, 3 eq.), argon exchange, DMF: H ₂ O = 10 mL: 2 mL solution, placed at 80 ° C and stirred for 3 h. TLC tracking monitoring. Most of the solvent was spun off and passed through a column (PE: EA = 4:1) to give a brown-yellow product of 75 mg. ESI-MS m/z: 718.1 (M+H) ⁺ .

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

Take a 25 mL two-necked flask, add the crude compound IV-7 (68 mg), argon-protected, add anhydrous DCM (10 mL) to dissolve, place at 0 ° C, stir for 15 min, add dropwise a solution of boron tribromide in dichloromethane ( 1 M, 0.28 mL, 3 eq.), stirred for 10 min, placed at room temperature and stirred for 1 h. TLC tracking showed that the reaction of the starting material was completed. Dilute with DCM (10 mL) at 0 ° C, quench with saturated NaHCO ₃ solution, extract and collect organic phase. EA was added to the aqueous phase, and the resulting flocculent solid was dissolved, and the organic phase was collected, mixed, dried over anhydrous sodium sulfate, and concentrated to a column (DCM: acetone = 1:1). The obtained yellow viscous product was beaten, petroleum ether was added, and the mixture was allowed to stand at a low temperature to precipitate a white solid, and the yellow liquid was taken up, and the obtained solid oil pump was drained to obtain 25 mg of a white solid (S configuration).

The characterization data for this compound 19 are: ¹ H NMR (400 MHz, CD ₃ OD, δ _ppm ): 7.44 (d, J = 6 Hz, 2H), 7.33 (d, J = 5.6 Hz, 2H), 6.79 (s, 1H) ), 6.39 (s, 1H), 4.22 (d, J = 8 Hz, 1H), 4.19 (d, J = 5.6 Hz, 1H), 4.12 (d, J = 5.6 Hz, 1H), 4.02 (s, 4H) , 3.40 (d, J = 5.6 Hz, 2H), 3.10 (t, J = 5.2 Hz, 1H), 2.47 (s, 4H), 1.22 (d, J = 5.6 Hz, 6H), 0.98 (d, J = 5.2 Hz, 6H). ¹³ C NMR (125 MHz, CD ₃ OD, 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 (MH) ^- .

Example 20

Compound 20: (S)-ethyl-2-(4-(5-(2,4-dihydroxy-5-isopropylphenyl)-3-(ethylcarbamoyl)isoxazole-4- Benzene Preparation of ethyl 2-(4AR,7AS)-tetrahydro-2H-[1,4]dioxane[2,3-c]pyrrole-6-(3H)-yl)acetate The method of Example 19.

The characterization data for this compound 20 is: ¹ H NMR (400 MHz, CD ₃ OD, δ _ppm ): 7.44 (d, J = 6.4 Hz, 2H), 7.33 (d, J = 6.4 Hz, 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, 6.0 Hz, 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, 5.6 Hz, 6H), 0.99 (t, J = 2.4 Hz, 6H). ¹³ C NMR (125 MHz, CD ₃ OD, 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 (MH) ^- .

The structural formula of the compound 20 is:

Example 21

Compound 21: (S)-isopropyl-2-(4-(5-(2,4-dihydroxy-5-isopropylphenyl)-3-(ethylcarbamoyl)isoxazole-4 -yl)phenyl)-2-(4AR,7AS)-tetrahydro-2H-[1,4]dioxane[2,3-c]pyrrole-6-(3H)-yl)acetate For the preparation, refer to the method of Example 19.

The characterization data for this compound 21 are: ¹ H NMR (400 MHz, CD ₃ OD, δ _ppm ): 7.44 (d, J = 8.4 Hz, 2H), 7.33 (d, J = 8.4 Hz, 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, 5.6 Hz, 2H), 3.11-3.04 (m, 2H), 2.86-2.78 (m, 2H), 2.75-2.72 (m, 1H), 1.27 (d, J = 4.8 Hz, 3H), 1.22 (t, J = 5.6 Hz, 3H), 1.14 (d, J = 4.8 Hz, 3H), 1.00 (t, J = 3.2 Hz, 6H). ¹³ C NMR (125 MHz, CD ₃ OD, 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 (MH) ^- .

The structural formula of the compound 21 is:

Example 22

Compound 22: (S)-ethyl 2-(4-(5-(2,4-dihydroxy-5-isopropylphenyl)-3-(ethylcarbamoyl)isoxazole-4-yl For the preparation of phenyl)-2-((2S,6R)-2,6-dimethylmorpholine) acetate, the method of Example 19 was followed.

The characterization data for this compound 22 are: ¹ H NMR (400 MHz, CD ₃ OD, δ _ppm ): 7.39 (d, J = 8.4 Hz, 2H), 7.31 (d, J = 8.0 Hz, 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, 7.2 Hz, 2H) , 3.09-3.02 (m, 1H), 2.86 (d, J = 6.8 Hz, 1H), 2.57 (d, J = 11.2 Hz, 1H), 1.94 (t, J = 6.8 Hz, 1H), 1.66 (t, J = 10.8 Hz, 1H), 1.20 - 1.16 (s, 6H), 1.22 (d, J = 6.4 Hz, 3H), 1.03 (d, J = 6.4 Hz, 3H), 0.96 (d, J = 6.4 Hz, 6H). ¹³ C NMR (125 MHz, CD ₃ OD, 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 (MH) ^- .

The structural formula of the compound 22 is:

Example 23

Compound 23: (R)-ethyl-2-(4-(5-(2,4-dihydroxy-5-isopropylphenyl)-3-(ethylcarbamoyl)isoxazole-4- For the preparation of phenyl)-2-morpholinoacetate, reference is made to the method of Example 19.

The characterization data for this compound 23 are: ¹ H NMR (400 MHz, CD ₃ OD, δ _ppm ): 7.40 (d, J = 6.4 Hz, 2H), 7.30 (d, J = 6.4 Hz, 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, 5.6 Hz, 2H), 3.08-3.03 (m, 1H), 2.45 (d, J = 3.6 Hz, 4H), 1.20, 1.17 (dd, J = 5.6, 6.0 Hz, 6H ), 0.95 (d, J = 5.6 Hz, 6H). ¹³ C NMR (125 MHz, CD ₃ OD, 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 (MH) ^- .

The structural formula of the compound 23 is:

Example 24

Compound 24: (R)-ethyl-2-(4-(5-(2,4-dihydroxy-5-isopropylphenyl)-3-(ethylcarbamoyl)isoxazole-4- Preparation of ethyl phenyl)-2-(4AR,AS)-tetrahydro-2H-[1,4]dioxane[2,3-c]pyrrole-6-(3H)-yl)acetate Refer to the method of Example 19.

The characterization data for this compound 24 are: ¹ H NMR (400 MHz, CD ₃ OD, δ _ppm ): 7.41 (d, J = 6.4 Hz, 2H), 7.30 (d, J = 6.4 Hz, 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, 5.6 Hz, 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, 5.6 Hz, 6H), 0.97 (dd, J = 2.8, 2.8 Hz, 6H). ¹³ C NMR (125 MHz, CD ₃ OD, 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 (MH) ^- .

The structural formula of the compound 24 is:

Example 25

Follow the above steps, including the following steps:

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

Take a 100 mL single-mouth bottle, add 4-bromobenzyl alcohol (5 g, 26.9 mmol), imidazole (2.2 g, 1.2 equivalents, 32.3 mmol) and 4-dimethylaminopyridine (DMAP) (catalytic amount), dissolved in DCM (40 mL) tert-Butyldimethylchlorosilane (TBSCl) (4.87 g, 1.2 eq., 32.3 mmol) was slowly added and stirred overnight. After the reaction was completed by TLC, water was added, and the resulting solid was dissolved, extracted, and washed with saturated brine. The organic phase was collected, dried over anhydrous sodium sulfate and evaporated to remove organic solvent to afford 7.7 g of colorless oil. ESI-MS m/z: 301.0 (M+H) ⁺ .

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

A 50 mL two-necked flask was taken and compound 2 (4 g, 13.3 mmol) and tetrakistriphenylphosphine palladium (0.77 g, 5 mmol%, 0.67 mmol) were added. Argon exchange, dissolved in DMF, added tributyl(1-ethoxyethylene)tin (5.4 mL, 1.2 eq, 16 mmol), placed at 100 ° C, stirred for 2 h, and the reaction was monitored by TLC. Depressurize with oil pump, vacuum to remove most of DMF, add EA and 1N hydrochloric acid solution, stir, TLC monitoring reaction, extract, add saturated brine, collect organic phase, dry anhydrous sodium sulfate The sample was mixed with a column (PE: DCM = 8:1) to obtain 2.5 g of a colorless liquid, yield 70%.

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

Take a 50 mL single-mouth bottle, add compound 3 (1 g, 3.8 mmol), dissolve in toluene, add diethyl carbonate (0.91 mL, 2 equivalents, 7.6 mmol), slowly add NaH (0.3 g, 2 equivalents, 7.6 mmol), set Stir at reflux at 100 ° C overnight. The reaction was monitored by TLC. The mixture was placed at 0 ° C, quenched with water, and the organic solvent was removed by rotary evaporation. 1). 650 mg of a colorless oil were obtained in a 50% yield. The characterization data of the compound VII-3 is: ¹ H NMR (400 MHz, CD ₃ Cl, δ _ppm ): 7.93 (d, J = 8.1 Hz, 2H), 7.44 (d, J = 8.1 Hz, 2H), 4.80 ( s, 2H), 4.24 (dd, J = 7.2, 7.2 Hz, 2H), 3.98 (s, 2H), 0.95 (s, 9H), 0.11 (s, 6H).

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

Take a 50 mL single-mouth bottle and add compound VII-3 (600 mg, 1.8 mmol) and (((4-azido-6-isopropyl-1,3-phenylenebis(oxy))))) Diphenyl (1 g, 1.5 eq., 2.7 mmol, mixture), EtOAc was evaporated, EtOAc EtOAc EtOAc EtOAc EtOAc After adding water and extracting twice, the organic phase was collected, dried over anhydrous sodium sulfate, and dried over a column of PE: EA = 8:1 to obtain 800 mg of oil, yield 64%.

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

A 50 mL single-mouth bottle was taken, and compound VII-4 (800 mg, 1.16 mmol) was added thereto, dissolved in ethanol, and an aqueous solution of ethylamine (10 mL) was added thereto, and the mixture was placed at 80 ° C, refluxed for 1 d, and the reaction was monitored by TLC. Most of the organic solvent was removed by rotary evaporation, EA was added, and the mixture was washed twice.

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

A 50 mL vial was added, and compound VII-5 (600 mg, 0.87 mmol) was added, dissolved in tetrahydrofuranamine (TBAF) in THF (10 mL), and stirred at room temperature for 2 h. The reaction was monitored by TLC. Most of the organic solvent was removed by rotary evaporation, EA was added, and extracted twice.

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

Take a 50 mL single-mouth bottle, add compound VII-6 (500 mg, 0.87 mmol), dissolve in DCM, add 2,2,6,6-tetramethylpiperidine oxide (TEMPO) and tetrabutylammonium bromide (TBAB). (Catalytic amount), a sodium hypochlorite (NaOCl) solution (5 mL) and a NaHCO ₃ solution (2 mL) were added and stirred at room temperature for 1 h. The solution turned from reddish brown to light yellow. The reaction was monitored by TLC. After adding DCM, the mixture was extracted three times, washed with saturated brine, dried over anhydrous sodium sulfate, and dried, and then passed to the column (PE: EA = 5:1) to obtain 250 mg of viscous product, yield 50%.

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

(8) Ethyl-2-(4-(1-(2,4-bis(benzyloxy)-5-isopropylphenyl)-4-(ethylcarbamoyl)-1H-1, Preparation of 2,3-triazol-5-yl)phenyl)-2-hydroxyacetic acid (Compound VII-8).

Take a 50 mL single-mouth bottle, add compound VII-7 (250 mg, 0.44 mmol), dissolve in DCM, add I ₂ (catalytic amount), add trimethylcyanosilane (TMSCN) (0.11 mL, 2 equivalents, 0.87 mmol), room temperature Stir for 30 min and the reaction was monitored by TLC. It was quenched by the addition of water, extracted, washed with saturated brine, dried and dried over anhydrous sodium sulfate. A solution of hydrogen chloride in ethanol (10 mL) was added and stirred at room temperature overnight. The reaction was monitored by TLC. Quenched by the addition of saturated sodium bicarbonate solution, the most organic solvent was removed by rotary evaporation, EA was added, extracted twice, washed with saturated brine, dried over anhydrous sodium sulfate, and dried over a column of PE: EA = 4:1 to yield 140 mg. The rate was 49.1%. ESI-MS m/z: 649.0 (M+H) ⁺ .

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

A 25 mL vial was added, and compound VII-8 (140 mg, 0.22 mmol) was added, dissolved in DCM, and then added with TEA (92 μL, 3 equivalents, 0.66 mmol) and TsCl (126 mg, 3 equivalents, 0.66 mmol), placed at 40 ° C, Stir for 4 h. The reaction was monitored by TLC. The mixture was extracted with saturated brine, and the organic layer was dried, dried over anhydrous sodium sulfate, and evaporated to dryness.

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

A 25 mL single-mouth bottle was taken, compound 11 (50 mg, 0.06 mmol) was added, and toluene was dissolved. Morpholine (20 μL, 4 equivalents, 0.24 mmol) and TEA (33 μL, 4 equivalents, 0.24 mmol) were added, and the mixture was placed at 100 ° C and refluxed. 2h. The reaction was monitored by TLC. Most of the organic solvent was removed by rotary evaporation, and extracted with saturated brine. The organic phase was collected, dried over anhydrous sodium sulfate and evaporated

ESI-MS m/z: 718.0 (M+H) ⁺ .

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

Take a 25mL single-mouth bottle, add compound VII-10 (30mg, 0.04mmol), argon exchange, add anhydrous DCM to dissolve, set at -5 ° C, stir for 15min, add dropwise boron tribromide dichloromethane solution (0.12 mL, 3 equivalents, 0.12 mmol), stirred for 15 min, stirred at room temperature for 2 h. After being added to a saturated aqueous solution of sodium hydrogencarbonate, the mixture was evaporated, evaporated, evaporated, evaporated, evaporated, evaporated.

The characterization data for this compound 25 is: ¹ H NMR (400 MHz, CD ₃ OH, δ _ppm ): 7.43 (d, J = 8.4 Hz, 2H), 7.39 (d, J = 8.4 Hz, 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, 6.4 Hz, 2H) , 3.16-3.09 (m, 1H), 2.48-2.41 (m, 4H), 1.27 (t, J = 7.2 Hz, 3H), 1.18 (t, J = 7.2 Hz, 3H), 1.09 (d, J = 6.8) Hz,6H). ¹³ C NMR (125MHz, CD ₃ OD, 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 (MH) ^- .

Experimental example

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

Principle: FITC-labeled geldanamycin binds to HSP90 and produces fluorescence polarization. If the compound can compete with geldanamycin for inhibition of HSP90 enzyme, no fluorescence polarization is detected.

Reagents and instruments: fluorescently labeled geldanamycin (Sigma), Hsp90α or Hsp90β enzyme solution (Stressgen Bioreagents Corp, cat.no.SPP-776), DTT (Promega), bovine serum albumin (BSA) (Hyclone), DMSO (Sigma). Envision 2104 Fluorometer (Perkin Elmer, USA), sampler (Eppendorf), microplate (corning).

Each compound was set up in a plurality of different dose groups. The high dose group was configured as a mother liquor, and the remaining dose group was diluted three times to the lowest dose group. All samples were dissolved in DMSO and stored at -20 ° C for use. The experimental buffer contained 20 mmol/l HEPES (K), 50 mmol/l KCl, 5 mmol/l MgCl ₂ , 20 mmol/l Na ₂ MoO ₄ and 0.01% NP40, and the pH was 7.3. 5 μl of a reaction buffer containing 40 mM DTT and 2 mg/ml BSA was added before each experiment, and then 2.5 μl of fluorescently-labeled geldanamycin (reaction concentration: 5 nM) was added. Then, 10 nl of a 1000× gradient-concentrated compound was added to the reaction solution using a compound transfer apparatus Liquid Handler Echo520, and finally 2.5 μl of Hsp90βor Hsp90α enzyme solution (reaction concentration: 35 ng/μl) was added. The reaction was gently shaken at room temperature for 2 hours, and finally the reading was measured with a microplate reader, the excitation light was 485 nm, the emission light was 530 nm, and the data was processed by Graphpad Prism 5 software.

(2) A screening method for anti-cancer activity of a target compound.

CCK kit works: Cell Counting Kit is called CCK kit, which is based on WST-8 (chemical name: 2-(2-methoxy) Base 4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H-tetrazole monosodium salt) for cell proliferation and cytotoxicity Fast and highly sensitive detection kit. WST-8 is an upgraded product of MTT. Its working principle is: in the presence of electron coupling reagent, it can be reduced by dehydrogenase in mitochondria to produce highly water-soluble orange-yellow formazan product (formazan) and precipitated in cells. Medium, while dead cells do not have this function. The depth of color is directly proportional to the proliferation of cells and inversely proportional to cytotoxicity. The absorbance was measured at a wavelength of 540/720 nm using an enzyme-linked detector to indirectly reflect the number of viable cells.

Reagents and instruments: various cancer cell lines (ATCC, USA), penicillin, streptomycin, DMSO (Sigma), CCK8 (CK04, Japan Tongren Chemical), RPMI1640 (GIBCO), BSA (Hyclone), trypsin (Gino Corporation), 384-well cell culture plate (Corning).

Each cancer cell was suspended in the corresponding culture medium to prepare a suitable concentration, and the volume was 50 μl/well implanted in a 384-well plate, and cultured at 37 ° C in a 5% CO ₂ incubator for 24 hours. The compound was dissolved in DMSO into 10 mM mother liquor, and diluted with DMSO 1:3 to 10 concentration gradients of 1000× compound series concentration stock solution, and the 1000× series concentration compound storage solution was transferred to the cell 384 well using the compound transfer instrument Liquid Handler Echo520. An appropriate volume of vehicle DMSO was added to the corresponding wells of the plate at 50 nL per well and blank control wells. Gently mix and continue to culture at 37 °C. After 72 h, the corresponding medium was replaced again, and 3 μl of CCK8 cell proliferation-toxicity detecting reagent was added to each well. The plates were incubated for 2 h in a 37 ° C incubator and the absorbance was measured at 540/720 nm using an enzyme-linked detector.

In this experiment, the inhibitory activities of the target compounds on HSP90α and HSP90β of HSP90 protein were determined by the above methods. Human lung cancer cell line A549, human breast cancer cell line MCF-7, chronic bone marrow with high expression of HSP90 The inhibitory activity of the leukemia cell line K562, the prostate cancer cell line DU145, and the test proliferation epidermal cancer cell line Hela. The results are shown in Table 1.

Table 1 Inhibitory activity of target compound against HSP90 protein and tumor cell line IC ₅₀ (unit: nM)

According to Table 1, from the enzyme activity level, the above compounds have inhibitory effects on both HSP90 isoforms, and most of the compounds have stronger binding ability to HSP90α than HSP90β. Compared to the positive control compound NVP/AUY-922 (CAS: 747412-49-3), most of the compounds showed stronger inhibitory activity, further demonstrating that the target backbone is more suitable for the active pocket of the target. At the cellular level, the above compounds have strong inhibitory activity against five types of cancer cell lines, and most of the compounds can reach several or several nanomolar levels, especially for the epidermal cancer cell line Hela. effect.

The technical features of the above-described embodiments may be arbitrarily combined. For the sake of brevity of description, all possible combinations of the technical features in the above embodiments are not described. However, as long as there is no contradiction between the combinations of these technical features, All should be considered as the scope of this manual.

The above-described embodiments are merely illustrative of several embodiments of the present invention, and the description thereof is more specific and detailed, but is not to be construed as limiting the scope of the invention. It should be noted that a number of variations and modifications may be made by those skilled in the art without departing from the spirit and scope of the invention. Therefore, the scope of the invention should be determined by the appended claims.

Claims

A heat shock protein inhibitor having the structural features of Formula I, or a pharmaceutically acceptable salt thereof:

among them:

R 1 , R 7 and R 8 are each independently selected from: H, C 1 -C 6 alkyl, C 2 -C 6 unsaturated alkyl, halogen, hydroxy, C 1 -C 6 alkoxy, NHCOOR, SO 2 NHR, NHSO 2 R, CN, NHCOR, CONHR;

R is selected from the group consisting of: H, C 1 -C 6 alkyl, C 3 -C 6 unsaturated alkyl, C 3 -C 8 cycloalkyl;

R 2 and R 3 are each independently selected from the group consisting of: H, D, C 1 -C 6 alkyl, C 3 -C 6 unsaturated alkyl, C 3 -C 8 cycloalkyl, phenyl, substituted phenyl, hetero Aryl group, acyl group;

R 4 is selected from the group consisting of: an amino group, a C 1 -C 6 saturated alkylamine, a C 2 -C 6 saturated heterocyclic amine, a C 1 -C 6 alkylamido group, an arylamide group, a substituted arylamide group, a hetero Arylamide group, substituted heteroarylamide group;

R 5 and R 6 are the same oxygen atom or are each independently selected from: H, D, C 1 -C 6 alkyl, C 3 -C 8 cycloalkyl, carbonyl, cyano, C 1 -C 6 alkoxy ,COOR',NHCOR',CONHR';

R' is selected from the group consisting of: H, C 1 -C 6 alkyl, C 3 -C 6 unsaturated alkyl;

X, Y, Z, and W are each independently selected from: C, NH, CH, N, O, S.
The heat shock protein inhibitor or a pharmaceutically acceptable salt thereof according to claim 1, wherein the five-membered aromatic heterocyclic ring containing X, Y, Z, W is selected from the following structures:
The heat shock protein inhibitor of claim 1 or a pharmaceutically acceptable salt thereof, wherein the heat shock protein inhibitor is selected from the group consisting of the following compounds of formula II:

among them:

R 7 and R 8 are selected from the group consisting of: H, C 1 -C 6 alkyl, C 2 -C 6 unsaturated alkyl, halogen, hydroxy, C 1 -C 6 alkoxy, CN;

R 1 is selected from the group consisting of C 1 -C 6 alkyl, C 2 -C 6 unsaturated alkyl, C 1 -C 6 alkoxy.
The heat shock protein inhibitor of claim 1 or a pharmaceutically acceptable salt thereof, wherein said R 2 and R 3 are each independently selected from the group consisting of: H, C 3 -C 8 cycloalkyl, C 1 -C 6 alkyl, C 3 -C 6 unsaturated alkyl.
The heat shock protein inhibitor according to any one of claims 1 to 4, wherein R 4 is selected from the group consisting of amino groups, C 1 -C 6 saturated alkylamines, C 2 -, or a pharmaceutically acceptable salt thereof. C 6 saturated heterocyclic amine, C 1 -C 6 alkylamide group, aryl amide group, substituted aryl amide group, heteroaryl amide group, substituted heteroaryl amide group;

R 5 is selected from the group consisting of: H or the same oxygen atom as R 6 ;

R 6 is selected from the group consisting of CN, COOR', CONHR', or the same oxygen atom as R 5 ;

Wherein R' is selected from the group consisting of H, C 1 -C 6 alkyl, C 3 -C 6 unsaturated alkyl, C 3 -C 8 cycloalkyl.
The heat shock protein inhibitor according to claim 5 or a pharmaceutically acceptable salt thereof, wherein R 4 is selected from the group consisting of:
The heat shock protein inhibitor of claim 1 or a pharmaceutically acceptable salt thereof, wherein the heat shock protein inhibitor is selected from the group consisting of compounds of formula III:

among them:

R 1 is selected from the group consisting of: C 1 -C 6 alkyl;

R 7 and R 8 are selected from the group consisting of: a hydroxyl group;

R 2 is selected from: H;

R 3 is selected from the group consisting of: C 1 -C 6 alkyl;

R 4 is selected from the group consisting of:

R 5 is selected from the group consisting of: H or the same oxygen atom as R 6 ;

R 6 is selected from the group consisting of CN, COOR', CONHR', or the same oxygen atom as R 5 ;

Wherein R' is selected from the group consisting of H, C 1 -C 6 alkyl, C 3 -C 6 unsaturated alkyl, C 3 -C 8 cycloalkyl.
The heat shock protein inhibitor of claim 1 or a pharmaceutically acceptable salt thereof, which is selected from the group consisting of:
A method for producing a heat shock protein inhibitor according to any one of claims 1 to 8, or a pharmaceutically acceptable salt thereof, which comprises the following route synthesis:

Wherein: R 10 is a hydroxy protecting group.
Use of the heat shock protein inhibitor of any one of claims 1-8, or a pharmaceutically acceptable salt thereof, for the manufacture of a medicament for the prevention and treatment of a disease having an increased pathological characteristic of heat shock protein 90 expression.
The use according to claim 10, wherein the disease having an increased pathological characteristic of heat shock protein 90 expression is: cancer, metabolic disease, myelodysplastic syndrome, systemic mastocytosis, Hippe At least one of Lindau syndrome, multicenter Castleman's disease, and psoriasis.