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Preparation method and application of protein degradation targeting chimera

A technology of protein degradation and chimera, applied in the field of medicine

Pending Publication Date: 2022-03-01
SHANGHAI UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Theoretically, the use of PROTACs to achieve double knockout of BRD4 and HDACs will overcome the problems of dual target inhibitors, and there is no literature report on the simultaneous double knockout of two target proteins using rationally designed PROTACs

Method used

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  • Preparation method and application of protein degradation targeting chimera
  • Preparation method and application of protein degradation targeting chimera
  • Preparation method and application of protein degradation targeting chimera

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0055] 8-(2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazole [4,3-a][1,4]diazepin-1-yl)-N-(2-(2-(2-(2-((2-(2,6-dioxopiperidine- Synthesis of 3-yl) 1-oxoisoindol-4-yl) amino)-2-oxoethoxy) ethoxy) ethoxy) ethyl) acetamido)-N-hydroxyoctylamide ( M10)

[0056] Step a: tert-butyl (2-(2-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)1-oxoisoindolyl-4 Synthesis of -yl)amino)-2-oxoethoxy)ethoxy)ethoxy)urethane (M2)

[0057] Step a: Compound M0 (1.0g, 3.1mmol) H, ATU (2.4g, 6.2mmol) D, IPEA (1.0 mL, 7.8mmol) were dissolved in dry DMF, reacted at room temperature for 0.5h and then added Nalidomide M1 (0.80 g, 3.1 mmol) was reacted for 4 h, and the reaction was complete as detected by TLC. The reaction solution was slowly poured into a mixture of ice water (1.5L), extracted with EA (30mL×3), and the organic phases were combined, and the solvent was evaporated under reduced pressure, and the residue was separated by silica gel column chromatography, and the mobile phase ...

Embodiment 2

[0073] Example 2: Test of the inhibitory activity (Ki) of the compounds of the present invention on BRD4 and HDAC1.

[0074] Add 5 μL of the compound to be tested (each diluted concentration), BRD4 (20 nM) and PMDM6-F (20 nM) (buffer: 100 mM tripotassium phosphate, pH = 7.5; 100 μg / mL BGG; 0.02% sodium azide) into 96-well black The flat-bottom microplate plate was raised to a final volume of 115 μL, and after incubation at room temperature for 1 hour, the fluorescence polarization value was read with a Biotek-Synergy microplate reader (excitation light: 485nM, emission light: 528nM).

[0075] According to the fluorescence polarization value obtained by the above method, the curve was drawn with Origin 9.0 software, and the protein binding inhibition constant (Ki) was calculated. HDAC1 test method is the same as BRD4.

[0076] Experimental results: First, the inhibitory activity of all target compounds on BRD4 and HDAC1 proteins was tested, and (+)-JQ1 and SAHA were selected as ...

Embodiment 3

[0079] Embodiment 3: the in vitro antitumor activity test (IC of compound of the present invention) 50 ).

[0080] The compound of the present invention is tested for three kinds of tumor cell proliferation inhibitory ability, and the test method adopts the conventional CCK8 method. For the logarithmic growth phase tumor cells (MCF-7 (human breast cancer cells)), A549 (human lung cancer cells), HepG2 (human liver cancer cells) were digested with trypsin, and then culture medium (DMEM+10%FBS or PRMI1640+10% FBS) to dilute and suspend the cells into a single cell suspension, adjust the cell density to 5×10 4 cells / mL, add 100 μL to each well and inoculate in a 96-well plate, place at 37°C, 5% CO 2 Cultivate in the incubator for 24 hours, and then add different concentrations of compounds, each concentration of three parallel wells, and set the experimental group and control group, continue to incubate for 72 hours, add 10 μL of CCK8 solution to each well, and then at 37 ° C A...

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PUM

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Abstract

The invention discloses a preparation method and application of a protein degradation targeting chimera. The compound has a structure as shown in a general formula (I). The compound is reasonably designed through a BET inhibitor ((+)-JQ1), an HDAC inhibitor and E3 ubiquitin ligase. Pharmacological experiments show that the compounds have relatively strong binding inhibition activity on BET protein and HDAC protein and in-vitro anti-tumor proliferation activity. Mechanism experiments show that the compound BET / HDAC double-target PROTAC can obviously induce degradation of the BRD4 protein and the HDACs protein, and can be applied to tumor diseases with pathological characteristics mediated by the BRD4 protein and the HDACs protein. As a BET / HDAC double-target PROTAC molecule reported for the first time, the compound has further development and research values.

Description

technical field [0001] The invention relates to the field of medical technology, in particular to a preparation method and application of a protein degradation targeting chimera, and the application of this type of small molecular compound in the treatment of malignant tumor diseases. Background technique [0002] As an emerging technology, PROTAC enables the target protein to be recognized by E3 ubiquitin ligase and labeled with ubiquitin through a bifunctional small molecule, and then uses the cell's own ubiquitin-proteasome system to degrade the target protein. Currently, most studies on PROTACs focus on degrading a single target protein. Although some literatures have published that the downregulation of two or more proteins can be achieved, most of them are caused by the off-target effect of PROTAC, or the result of the downregulation of multiple proteins downstream due to the inhibition of upstream signals, or due to the effect on The ligand pocket of the same type of...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D493/14A61P35/00A61K31/551
CPCC07D493/14A61P35/00
Inventor 何世鹏马俊辉余自强刘莹马浩骞陈宝宝纪雅静
Owner SHANGHAI UNIV
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