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Quinazoline fluorescent probe as well as preparation method and application thereof

A technology of fluorescent probes and quinazolines, which is applied in the field of quinazoline fluorescent probes and their preparation, can solve the problems of reduced drug efficacy, and achieve the effects of simple operation, fast analysis, and high inhibitory activity

Inactive Publication Date: 2014-12-17
无锡艾德美特生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Patients with tumors with high expression of EGFR are sensitive to Gefitinib (gefitinib) targeted drugs, on the contrary, the drug effect is significantly reduced

Method used

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  • Quinazoline fluorescent probe as well as preparation method and application thereof
  • Quinazoline fluorescent probe as well as preparation method and application thereof
  • Quinazoline fluorescent probe as well as preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0040] Preparation of fluorescent probes

[0041] (1) In a 100mL single-necked flask, 1.1g (about 6.59mmol) of ethyl bromoacetate, 2.0g (about 6.27mmol) of raw material a: (4-(3'-chloro-4'-fluorophenylamino)- 7-methoxy-6-hydroxyquinazoline), 2.6g (about 18.8mmol) of potassium carbonate were dispersed in 20mL of N,N-dimethylformamide solvent, stirred at 40°C, followed by TLC to observe the reaction process, the reaction is complete in about 0.5 hours, stop the reaction, filter the reaction solution, collect the filtrate, drop the filtrate into 80mL of distilled water, and immediately precipitate a large amount of yellow precipitate, after standing for 2 hours, filter, collect the filter cake, and vacuum dry to obtain The crude product was recrystallized with a mixed solvent of V (dichloromethane): V (methanol) = 20: 1 to obtain a light yellow precipitate pure product b, which is an acetate derivative intermediate, and the yield was 75%; Melting point, and carry out mass spectr...

Embodiment 2

[0048] Probe biological activity detection

[0049] (1) ELISA measures the IC of quinazoline fluorescent probes of the present invention inhibiting EGFR protein kinase activity 50

[0050] The kinase inhibitory activity of the synthesized compound d was determined by enzyme-linked immunosorbent assay (Enzyme Immunosorbent Assay, ELISA). Using the protein kinase detection kit of CST company: PTP1B (Tyr66) Biotinylated Peptide as the substrate of the kinase EGFR (Epidermal Growth Factor Receptor), the inhibition degree of compound d to the phosphorylation reaction of the kinase substrate was investigated by spectrophotometry, and determined IC 50 value.

[0051] In the ELISA experiment, positive control and negative control are used to control the experimental conditions. In order to improve the test accuracy, three groups of parallel experiments were performed for each concentration. A known EGFR inhibitor (4-(3'-chloro-4'-fluoroanilino)-7-methoxy-6-hydroxyquinazoline) was...

Embodiment 3

[0056] Determining the localization distribution of probes on the cell membrane

[0057] (1) Cell culture

[0058] Five cell lines (all purchased from the Cell Resource Center, Institute of Basic Medical Sciences, Chinese Academy of Medical Sciences): A431 epidermal squamous cell line, A549 human lung adenocarcinoma cell line, HeLa human cervical cancer cell line, MCF-7 human breast cancer cell line strain, HBE human bronchial epithelial cell line. Medium composition: DMEM with antibiotics (100U mL -1 Penicillin and 100mg mL -1 streptomycin) and 10% fetal bovine serum. Cell seeding density: 5×10 cells per 100 mm culture dish 6 , at 37°C with 5% CO 2 Culture in a cell culture incubator until the cells cover 80-90%, and replace the medium every two days or so.

[0059] (2) Fluorescence confocal imaging

[0060] Before the experiment, use 1×10 per dish 4 Cells were inoculated into confocal glass small dishes, each dish was added with 1.5mL medium, at 37°C with 5% CO 2 Cu...

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Abstract

The invention discloses a quinazoline fluorescent probe and a preparation method thereof. The fluorescent probe structure is fluorescein-labeled Gefitinib, and has relatively strong inhibitory activity on EGFR protein kinase and potential cancer treatment effect; according to confocal fluorescence imaging results, the fluorescent probe is uniformly distributed on a cell membrane after being acted with a plurality of cell strains, and can be used for cancer cell imaging. According to tested results of flow cytometry, the intensification factor of the cell fluorescence intensity has positive correlation property with the EGFR content in the cell. By virtue of a flow cytometry technology, the fluorescent probe can be applied to relative quantification detection of a biomarker EGFR in the cell, so that relatively direct information is provided for cancer diagnosis. The fluorescent probe has functions of diagnosing and treating cancers, can be used for directly detecting living cells, is quick to analyze, simple and convenient to operate and low in cost, and has a development and application value.

Description

technical field [0001] The invention relates to the field of cancer diagnosis and treatment in the field of biotechnology, in particular to a class of quinazoline fluorescent probes with functions of cancer diagnosis and treatment, and a preparation method and application thereof. Background technique [0002] Malignant tumor (cancer) is one of the most serious diseases that endanger human health. It is a multi-stage complex disease involving multiple genes. In the complex process of cell signal transduction, the overexpression of certain key proteins and regulatory factors can lead to the loss of normal growth regulation of cells, resulting in excessive proliferation and the formation of tumors. [0003] Epidermal Growth Factor Receptor (EGFR) is a transmembrane glycoprotein with tyrosine protein kinase activity. As an important regulatory factor in the process of tumor formation and development, EGFR has become one of the main targets of tumor targeted therapy as a tumor ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C09K11/06C07D405/12A61K31/517A61P35/00G01N21/64
Inventor 汪福意罗群郑伟
Owner 无锡艾德美特生物科技有限公司
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