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Application of 7-hydroxycoumarin pyrazoline derivative in preparing GRP94 inhibitors

A kind of inhibitor and pyrazole technology, applied in the field of coumarin pyrazoline fluorescent compounds

Active Publication Date: 2017-08-22
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] After searching and searching by authoritative organizations, using the above-mentioned coumarin pyrazoline derivative 3-(1,5-diphenyl-4,5-dihydro-1H-pyrazol-3-yl)-7-hydroxyl-2H The research and application of -chromen-2-one as a GRP94 inhibitor has not been reported at home and abroad

Method used

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  • Application of 7-hydroxycoumarin pyrazoline derivative in preparing GRP94 inhibitors
  • Application of 7-hydroxycoumarin pyrazoline derivative in preparing GRP94 inhibitors
  • Application of 7-hydroxycoumarin pyrazoline derivative in preparing GRP94 inhibitors

Examples

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Effect test

Embodiment 1

[0042] Example 1: Preparation of 3-(1,5-diphenyl-4,5-dihydro-1H-pyrazol-3-yl)-7-hydroxy-2H-chromen-2-one

[0043] 2,4-Dihydroxybenzaldehyde (20mmol) and ethyl acetoacetate (24mmol) were dissolved in 30ml of ethanol solution, and 1ml of piperidine was added dropwise as a catalyst. The reaction solution was heated to reflux for 3 hours to the end of the reaction, and a large amount of grass green solid was precipitated after cooling to room temperature. The crude product is obtained by suction filtration, and then recrystallized with ethanol to obtain the green product 7-hydroxy-3-acetyl coumarin.

[0044] Dissolve 7-hydroxy-3-acetylcoumarin, benzaldehyde and a catalytic amount of piperidine (about 1 ml) in 20 ml of absolute ethanol, and reflux the reaction solution for 4 hours to cool to room temperature. Because this reaction has a low conversion rate under weak alkaline conditions, 10 equivalents of benzaldehyde are added to the reaction, and the reaction is carried out thoroughl...

Embodiment 2

[0046] Example 2: HCP1 interacts directly with GRP94

[0047] Surface plasmon resonance analysis was performed by Biacore T200 to detect the binding of HCP1 to the full-length GRP94 protein. The full-length GRP94 protein was immobilized on the surface of the CM5 chip by amino coupling. 2.74μM, 1.83μM, 1.22μM, 0.81μM and 0.54μM HCP1 were used for injection. The injection time of HCP1 is 120s, the flow rate is 10ul / min, and the dissociation time is 600s. The injection buffer is PBS (10mM phosphate, 137mM NaCl, 2.68mM KCl, 0.1% dimethyl sulfoxide [v / v], pH 8.0) at a temperature of 25°C. After each injection, 10mM NaOH was used for chip regeneration. The combined response value is the RU value, the blank control is subtracted from the data processing, and the Biacore T200 evaluation software is used for 1:1 combined simulation analysis.

[0048] The results show that the response value obtained by Biacore T200 corresponds to the concentration of HCP1, and the equilibrium dissociati...

Embodiment 3

[0049] Example 3: Co-localization of HCP1 and GRP94

[0050] Inoculate A549 cells in a glass petri dish with a diameter of 3.5cm at 37℃, CO 2 After 24 hours of incubation in the incubator, add HCP1 (10μM) for 3 hours, remove the culture medium, wash twice with 0.1M PBS buffer, add 1640 stock solution, and place it under a laser confocal microscope with different wavelength excitation light (405nm, 488nm, 546nm, 633nm and 647nm) take pictures.

[0051] Inoculate A549 cells in a glass dish with a diameter of 3.5cm, 37℃, CO 2 After 24 hours of incubation in the incubator, HCP1 (10 μM) was added for treatment for 3 hours, the culture medium was removed, the cells were washed twice with 0.1M PBS, and the cells were fixed with 4% paraformaldehyde for 15 minutes. Discard 4% paraformaldehyde, wash 3 times with 0.1M PBS buffer, 5 min each time; discard 0.1M PBS buffer, add normal serum blocking solution, and block for 20 min at room temperature; discard blocking solution and add GRP94 prima...

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Abstract

The invention discloses application of 3-(1,5-diphenyl-4,5-dihydro-1H-pyrazole-3-yl)-7-hydroxy-2H-chroman-2-one in preparing GRP94 inhibitors. The concentration of the 3-(1,5-diphenyl-4,5-dihydro-1H-pyrazole-3-yl)-7-hydroxy-2H-chroman-2-one which effectively inhibits the biological functions of GRP94 is 1-10 micromole. According to the application, a foundation is laid for researching and developing the inhibitors for the GRP94, and the compound can also be used as an effective chemical tool to provide a powerful means for research of the biological functions of the GRP94.

Description

Technical field [0001] The present invention relates to an application of a coumarin pyrazoline fluorescent compound, in particular to a 7-hydroxycoumarin pyrazoline derivative, namely 3-(1,5-diphenyl-4,5-dihydro-1H- Application of pyrazol-3-yl)-7-hydroxy-2H-chromen-2-one in the preparation of GRP94 inhibitors. Background technique [0002] GRP94 (glucose regulatory protein 94) is encoded by the HSP90B1 gene, which is a subtype of HSP90 (heat shock protein 90) in the endoplasmic reticulum, and is structurally highly conserved with HSP90. GRP94 is mainly involved in the folding and assembly of secretory and membrane proteins in cells. Clinical studies have shown that GRP94 is involved in the development and metastasis of a series of cancers such as melanoma, ovarian cancer, multiple myeloma, and lung cancer. GRP94 has become a new target for tumor treatment. In addition, GRP94 is also involved in autoimmunity and inflammation-related diseases. The molecular chaperone function o...

Claims

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Application Information

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IPC IPC(8): C07D405/04A61K31/4155A61P35/00
CPCC07D405/04
Inventor 苗俊英赵宝祥张尚立苏乐魏群
Owner SHANDONG UNIV
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