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23-hydroxybetulinic acid fluorescent probe and its preparation method and use in cellular localization and uptake

A fluorescent probe, the technology of betulinic acid, which is applied in the detection of the application of 23-hydroxy betulinic acid in the localization and uptake of target cells, the preparation of a fluorescent probe of 23-hydroxy betulinic acid, a natural product of anti-tumor activity, in the field of fluorescent probes, It can solve the problems that the molecular mechanism of action remains to be explored, the anti-tumor molecular mechanism of 23-HBA has not been fully elucidated, and the cellular target and mode of action are unclear, and achieve the effect of good effect, mild conditions and anti-tumor effect.

Inactive Publication Date: 2016-05-04
CHINA PHARM UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0004] However, the anti-tumor molecular mechanism of 23-HBA has not been fully elucidated, and the intracellular target and mode of action are also unclear, and the relevant molecular mechanism of action remains to be explored

Method used

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  • 23-hydroxybetulinic acid fluorescent probe and its preparation method and use in cellular localization and uptake
  • 23-hydroxybetulinic acid fluorescent probe and its preparation method and use in cellular localization and uptake
  • 23-hydroxybetulinic acid fluorescent probe and its preparation method and use in cellular localization and uptake

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0041] Synthesis of 23-HBA fluorescent probe

[0042] Compound a (2g, 7.7mmol), N-hydroxysuccinimide (1.3g, 11.5mmol) and EDCI (1.8g, 9.2mmol) were dissolved in dichloromethane (20.0mL), stirred at room temperature for 4h, concentrated Add water, extract with dichloromethane (30mL×3), combine the organic layers, wash with water, wash with saturated brine, anhydrous Na 2 SO 4 After drying and concentration, column chromatography (CH 2 Cl 2 / CH 3 OH 100:1, v / v) yielded the target compound b (2 g, 72.9%). 1 H-NMR (CDCl 3 , 300MHz) δ: ppm1.26 (6H, t, J = 7.1Hz), 2.90 (4H, s), 3.48 (4H, q, J = 7.1Hz), 6.45 (1H, d, J = 1.9Hz, Ar -H), 6.65 (1H, dd, J=9.0Hz, J=2.2Hz, Ar-H), 7.38 (1H, d, J=9.0Hz, Ar-H), 8.59 (1H, s, Ar-H ).

[0043] Dissolve compound b (700mg, 1.95mmol), N-Boc-ethylenediamine (0.4mL) and DIPEA (1.4mL) in dichloromethane (20.0mL), stir at room temperature for 3h, add water after concentration, dichloromethane Extract (30mL×3), combine the organic layers, wash w...

Embodiment 2

[0049] Anti-tumor Proliferation Activity of 23-HBA Fluorescent Probe

[0050] The tumor cells in the logarithmic growth phase were digested, counted, and prepared at a concentration of 5×10 4 cells / mL of cell suspension, 100 μL of cell suspension was added to each well of a 96-well plate (5×10 3 cells). Place the 96-well plate at 37°C, 5% CO 2 After culturing in the incubator for 24 hours, the drug was diluted to the desired concentration with complete medium, and 100 μL of the corresponding drug-containing medium was added to each well. Place the 96-well plate at 37°C, 5% CO 2 After culturing in the incubator for 72 hours, stain the 96-well plate with MTT; add 20 μL MTT (5 mg / mL) to each well, and continue culturing in the incubator for 4 hours; discard the medium, add 150 μL DMSO to each well to dissolve, and shake gently for 10 minutes. Mix well; λ=490nm, read the OD value of each well with a microplate reader, and calculate the inhibition rate. Experimental results (s...

Embodiment 3

[0052] 23-HBA fluorescent probe staining of B16F10 cells

[0053] B16F10 cells in the logarithmic growth phase were selected and inoculated on glass coverslips placed in a 6-well plate, 5×10 cells per well 4 After 24 hours of attachment, compound g (2 μM, 4 μM, 10 μM and 20 μM InDMSO) was added to the complete medium and incubated at different time points (15min, 30min, 1h, 2h, 4h, 24h). After incubation for the corresponding time, the cells were fixed with 4% paraformaldehyde (in 1×PBS) for 10 minutes, and after washing with PBS, the glass coverslips were taken out for fluorescence imaging. Under the fluorescent microscope, observe the coloring part of the cells, the distribution of fluorescence and the change of brightness, etc., the results are shown in figure 2 a.

[0054] B16F10 cells in the logarithmic growth phase were selected and inoculated on glass coverslips placed in a 6-well plate, 5×10 cells per well 5 After 24 hours of attachment, 10 μM compound g was added ...

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Abstract

The invention relates to the field of novel 23-hydroxybetulinic acid fluorescent derivative organic synthesis and pharmaceutical chemistry and concretely relates to a 23-hydroxybetulinic acid fluorescent probe obtained through connection of a coumarin fluorophore to a 23-hydroxybetulinic acid skeleton. The invention discloses a use of the 23-hydroxybetulinic acid fluorescent derivative in antineoplastic mechanism research and a potential use of the 23-hydroxybetulinic acid fluorescent derivative in cancer treatment and especially relates to a use of the 23-hydroxybetulinic acid fluorescent probe in detection of target cell localization and uptake under action of 23-hydroxybetulinic acid.

Description

technical field [0001] The present invention relates to the fields of organic synthesis and medicinal chemistry, in particular to the preparation of a new type of anti-tumor active natural product 23-hydroxybetulinic acid fluorescent probe, in particular to the use of this type of fluorescent probe to detect the presence of 23-hydroxybetulinic acid in target cells Applications in Positioning and Uptake. Background technique [0002] With the development of economy, tumor has become the number one killer threatening human health. Scientists have been working on tumor research for centuries, and drugs for various tumor diseases have been developed accordingly. Clinical data show that 80% of antineoplastic drugs are derived from natural products. Natural products have played an important role in the development of anticancer drugs. Although natural products have achieved success in tumor therapy, the anti-tumor mechanisms of most natural products are still unclear, which lim...

Claims

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Application Information

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IPC IPC(8): C07J63/00C09K11/06A61K31/585A61K49/00A61P35/00G01N21/64
CPCA61K31/585A61K49/0039A61K49/0052C07J63/008C09K11/06C09K2211/1011C09K2211/1088G01N21/6402G01N21/6486
Inventor 徐进宜魏国湘徐盛涛王艺玮张恒源陈红宁刘剑吴晓明叶文才姚和权谢唯佳
Owner CHINA PHARM UNIV
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