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Malic acid phosphate ester and applications of malic acid phosphate ester in inhibition of calcium ion deposition diseases

A technology of phosphate malate and dibenzyl malate, which is applied in the field of chemical biology, can solve the problems of poor fat solubility and low stability, and achieve the effect of reducing the concentration of calcium ions

Inactive Publication Date: 2015-07-22
NANCHANG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, due to the disadvantages of poor fat solubility and low stability of citrate phosphate, it is one of the hot spots in drug research and development in recent years to search for and discover new derivatives with higher physiological activity based on citrate phosphate. one

Method used

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  • Malic acid phosphate ester and applications of malic acid phosphate ester in inhibition of calcium ion deposition diseases
  • Malic acid phosphate ester and applications of malic acid phosphate ester in inhibition of calcium ion deposition diseases
  • Malic acid phosphate ester and applications of malic acid phosphate ester in inhibition of calcium ion deposition diseases

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0017] Example 1. Preparation of malic acid phosphate.

[0018] Weigh 1g of D-L malic acid, add 0.01g of p-toluenesulfonic acid under the protection of nitrogen, put it into an oil bath and stir. Raise the temperature to about 130°C until the solid becomes molten. Continue to stir for about 10 minutes. After the malic acid is completely liquid, put it into an oil bath at 80°C. 1.6 g of benzyl alcohol was added. The temperature was slowly raised to 120°C, and the reaction progress was tracked by thin-layer chromatography, and the reaction was terminated after 6 hours. After cooling to room temperature, a saturated sodium bicarbonate solution was slowly added dropwise to the reaction liquid until neutral. Add anhydrous diethyl ether for extraction until the white substance in the solution disappears completely. The extract was washed three times with saturated brine, and dried over anhydrous magnesium sulfate overnight. Add 8 mL of re-distilled dichloromethane to dissolve,...

Embodiment 2

[0019] Example 2. Inhibitory effect of malate phosphate on C2C12 calcification in mouse myoblasts.

[0020] Establish cell model, its method is as follows: First, mouse myoblast C 2 C 12 Take 5×10 5 cells / ml were inoculated in 24-well plates, and the next day, serum-free medium was added and cultured in an incubator for 24 hours. On the third day, use 1mCi / ml45Ca 2+ The labeled medium was exchanged for cells, and then ATP was added at a final concentration of 1 mM. Negative controls were performed with cells not treated with ATP or β-glycerophosphate. After 48 or 72 hours, the medium was discarded, the cells were washed 5 times with low-temperature Hank's equilibrium solution, and 0.1N NaOH was added. Finally, the amount of radioactive substance in the cell lysate was determined, and the experiment was carried out three times, and the average value was calculated. The results are shown in Table 1.

[0021] Table 1: Malate phosphate inhibits C 2 C 12 Study on Cell Cal...

Embodiment 3

[0023] Example 3. Reducing effect of malate phosphate on calcium ion concentration in HSK of human skeletal muscle cells.

[0024] Human skeletal muscle cells HSK were seeded in a 96-well plate at 10,000 / well, cultured in a medium containing malate phosphate or calcium channel inhibitor Diltiazem for 48 hours, and then added Fluo-4 calcium ion fluorescent indicator, And placed in a fluorescence plate reader to measure the fluorescence intensity. The intracellular calcium ion concentration is directly proportional to the fluorescence intensity. Three experiments were carried out in this way, and the average value was calculated at the end. The results are shown in Table 2.

[0025] Table 2: Study on reduction of calcium ion concentration in HSK cells by malate phosphate

[0026]

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Abstract

The invention relates to malic acid phosphate ester and applications of the malic acid phosphate ester in inhibition of calcium ion deposition diseases, wherein the structure of the malic acid phosphate ester is represented by the following formula. The synthesis steps comprise: carrying out a stirring reaction of malic acid and benzyl alcohol for 6 h at a temperature of 80-130 DEG C, carrying out pressure reducing distillation at a temperature of 175-185 DEG C to remove the unreacted benzyl alcohol, carrying out recrystallization with ethanol to obtain dibenzyl malate, dissolving the dibenzyl malate in dichloromethane under N2 protection, adding triethylamine, adopting 4-dimethylaminopyridine as a catalyst, stirring for 0.5 h in a 0 DEG C ice bath, slowly adding diphenyl chlorophosphate in a dropwise manner, carrying out a stirring reaction for 0.5 h, carrying out a room temperature for 12 h, carrying out pressure reducing distillation, carrying out recrystallization with ethanol to obtain dibenzyl phosphate ester dibenzyl malate, adding the dibenzyl phosphate ester dibenzyl malate and platinum oxide to distilled water, carrying out a catalysis reaction for 20-24 h, extracting, and carrying out low temperature freeze-drying to obtain the product. According to the present invention, with the malic acid phosphate ester, the mouse myoblast C2C12 calcification can be well inhibited, and the concentration of the calcium ions in the human skeletal muscle cell HSK can be effectively reduced.

Description

technical field [0001] The invention belongs to the technical field of chemical biology. technical background [0002] Calcium-containing crystals such as monosodium urate, calcium pyrophosphate CPPD, basic calcium phosphate BCP crystals, and calcium oxalate polymers are ubiquitous in the human body. Studies have shown that the level of calcium crystals in human cells can regulate gene expression and affect many life processes, from cell growth to apoptosis. The deposition of calcium crystals in bone, articular cartilage and joint tissue can lead to joint disease. For example, monosodium urate is associated with gout, calcium pyrophosphate CPPD is associated with pseudogout, basic calcium phosphate BCP crystals are associated with cartilage degeneration and acute calcific periarthritis, calculi, and c-fos and c-jun proto-oncogene expression. [0003] The diversity of structures and biological activities of natural products makes them occupy a very important position in the...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07F9/09A61P3/14
Inventor 陆豫郑楠余勃潘栋梁胡川
Owner NANCHANG UNIV
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