CN103936608B

CN103936608B - The activity of fighting against senium of a kind of novel chalcone compound

Info

Publication number: CN103936608B
Application number: CN201410157929.5A
Authority: CN
Inventors: 王艳艳; 于爽; 牛雯颖
Original assignee: Heilongjiang University of Chinese Medicine
Current assignee: Heilongjiang University of Chinese Medicine
Priority date: 2014-04-17
Filing date: 2014-04-17
Publication date: 2016-06-15
Anticipated expiration: 2034-04-17
Also published as: CN103936608A

Abstract

The present invention provides a kind of novel chalcone compound with anti-aging effects. Can be used as preparation various formulation have delay senility or with immunomodulatory, promote brain function related drugs bulk drug, be used for the treatment of the medicinal use with diseases associated with senescence.

Description

The activity of fighting against senium of a kind of novel chalcone compound

Technical field

The invention belongs to medicinal chemistry art, specifically, the present invention relates to the chemical structure of the novel chalcone compound with anti-aging effects. In addition, the present invention also relates to anti-aging effects mechanism and the purposes etc. of this kind of compound.

Background technology

The degeneration change that aging is that body is respectively organized, organ dysfunction increases with the age and occurs is the general performance of the various biochemical reaction of body, is the result of the many factors in the inside and outside acting in conjunction such as (environmental pollution, spirit nervous, hereditary). Along with, within the scope of the world, the particularly development of China's aging population trend, this worldwide medical science problem anti-ageing is also paid close attention to day by day in China. In recent years about aging grind high and according to aging mechanism searching efficient antiaging agent research become the hot issue in current medicine drug development field. Antiaging agent be a class to improve the medicine of life efficiency as final purpose, it plays its adjustment function, delay senility, it is to increase quality of life from multisystem, multi-level and multistage.

Antiaging agent can be divided into by physics and chemistry attribute: 1. chemicals, has antioxidant, antiaging hormone, nutrient substance, oxidase inhibitor, immunomodulator, biochemical preparation, brain function to promote medicine etc.; 2. Chinese medicine, has single medicinal material to comprise anti-ageing plant amedica, anti-ageing animal drugs, anti-ageing mineral drug and compound preparation etc. Although there is a large amount of antiaging agent on market, but these medicines are old medicine newly use a bit, such as vitamins; Some is then the new drug developed for other medical treatment objects (such as hypertension, reducing blood-fat, antitumor etc.) in recent years, simple is used as anti-ageing medicine at present also seldom. So, the active drug finding delaying sanility process is not only the expectation of whole world people, the responsibilities and obligations of pharmaceutical researchers especially.

Chalcone compounds is with 1,3-diphenylprop ketenes is the organic compound of basic skeleton structure, extensively it is present in the multiple medicinal plant such as Radix Glycyrrhizae, safflower, it it is the precursor of synthesis flavones in plant materials, it also it is simultaneously the important organic medicinal intermediate of a class, owing to its molecule has bigger flexibility, can from different receptors bind, show many-sided biologic activity, particularly significantly active in anti-ageing, antitumor, parasiticide, inflammation antiviral, antibacterial, anti-, anti-platelet aggregation etc. Therefore, the research and development of chalcone compounds are become a pharmaceutical chemical research focus.

Cultured cell in vitro premature senescence is the recurrence of organism life cycle at cell levels, Dimri etc. propose a kind of biology mark beta-galactosidase enzymes that can differentiate senile cell in body first in nineteen ninety-five, report propose in body or external senescent fibroblast and keratinocyte all express this enzyme, rest cell and terminally differentiated cells then lack, also expression without this enzyme in immortalized cells, betagalactosidase activity detection is the premature senescence detection technique generally acknowledged at present. Telomere is a kind of special construction of cell chromosome end, and it is in chromosomal localization, duplication, protection and has vital role in the growth of control cell, life-span etc. Research shows, the aging of telomerase activation and cell is closely related. Klotho genetic expression, in distal renal tubular and brain choroid plexus, by the regulation and control of Regular Insulin/IGF-1 signal path and p53 gene are affected senescence process, is the popular target spot of old and feeble Mechanism Study. Not yet have at present and play its adjustment function antiaging agent and enter Clinical practice from multisystem, multi-level and multistage, therefore develop the new small molecule antiaging agent tool with China's independent intellectual property right and be of great significance.

At present also not from the research report of the multi-level chalcones medicine playing anti-aging effects, the present inventor is through long-term and arduous research experiment, design, synthesize and final obtained a series of chalcone compound compounds, and find that they have good activity of fighting against senium through pharmacologically active detection.

Summary of the invention

The technical problem to be solved in the present invention is to provide new anti-senescence compounds type and medicine thereof. Specifically, the present invention provides the compound shown in formula (A322) or its pharmacy acceptable salt:

Compound (A322) is:

(E)-1-(3-(Dimethylamino)phenyl)-3-[4-hydroxy-3-(3-methylbut-2-en-1-yl)phenyl]prop-2-en-1-one

(E)-[3-(dimethylin) phenyl]-3-[4-hydroxyl-3-(3-methyl-2-butene base) phenyl]-3-amylene-2-ketone

¹HNMR(CDCl₃, 300MHz): 67.74 (d, 1H, J=15.6Hz, H β), 7.32-7.44 (m, 3H, H2, H5 ', H6 '), 7.38 (s, 1H, H2 '), 7.37 (d, 1H, J=15.6Hz, H α), 7.33 (s, 1H, H6), 6.92-6.96 (m, 1H, H4 '), 6.84 (d, 1H, J=7.8Hz, H3), 5.30-5.36 [m, 1H, ArCH₂CHC(CH₃)₂], 3.38 [d, 2H, J=7.2Hz, ArCH ₂CHC(CH₃)₂], 3.02 [s, 6H, N (CH ₃)₂], 1.79 [s, 6H, ArCH₂CHC(CH ₃)₂].

An object of the present invention is to provide a kind of novel chalcone compound with activity of fighting against senium.

The two of the object of the present invention are to provide preparation method and the activity of fighting against senium of a kind of novel chalcone compound.

In the present invention, first we utilize chemical process to synthesize novel chalcone compound A322. Application H₂O₂Mouse cardiac muscle cell (H9c2 (2-1)) and Human umbilical vein endothelial cells (HUVEC-12) aging model of induction confirm that compound A-13 22 has significant anti-aging effects (details are shown in embodiment 1), simultaneously, utilize HUVEC-12 cell that this compound carries out the research of anti-aging effects mechanism (details are shown in embodiment 2), mtt assay measures cell survival rate, cell aging is detected with telomerase activation (TRAP-argentation), the generation of ROS in cell is detected by active oxygen Fluorescence kit, the expression of klothomRNA in real-time quantitative PCR detection cell. compound involved in the present invention is at H₂O₂H9c2 (2-1) cell of induction and HUVEC-12 cell aging model can make betagalactosidase activity significantly decline, show that this compound has good activity of fighting against senium. In the research of anti-aging effects mechanism, normal Endothelial Cell Survival rate is had no significant effect by this compound in Valid concentration, it is possible to significantly suppress H₂O₂The telomerase activation of induction reduces, and raises the expression of anti-ageing gene klotho simultaneously, has significant anti-aging effects.

Accompanying drawing illustrates:

Here cited compound is compounds category and the structure formation in order to the present invention is described better, and unrestricted the present invention.

Fig. 1 illustrates the compound structure of the compound A-13 22 of synthesis;

Fig. 2 illustrates the activity of fighting against senium checking of compound on cell levels;

Fig. 3 illustrates that compound is on the impact of normal Endothelial Cell Survival rate;

Fig. 4 illustrates that compound can effectively suppress H₂O₂The endotheliocyte telomerase activation of induction;

Fig. 5 illustrates that compound can raise the expression of klothomRNA.

The activity of fighting against senium checking of compound on embodiment 1 cell levels

Compound A-13 22 is configured to DMSO respectively the solution of 3,10 and 30 μMs of concentration, after the rat myocardial cell H9c2 (2-1) merged Asia respectively and Human umbilical vein endothelial cells HUVEC-12 is inoculated in Tissue Culture Plate 48h, the active ingredient matter sample adding above-mentioned different concns intervenes 2h, and H9c2 (2-1) cell adds the H of 160 μMs subsequently₂O₂Process 2h is also replaced with normal nutrient solution and continues to cultivate 72h; HUVEC-12 cell is added the H of 100 μMs simultaneously₂O₂Process 48h, is also replaced with normal nutrient solution and continues to cultivate 10 days, within every 2 days, change a nutrient solution. Cell aging state is evaluated by observing betagalactosidase activity, absorb the nutrient solution of above-mentioned two kinds of cells, PBS washs, beta-galactosidase enzymes dyeing stationary liquid is fixed, after washing stationary liquid, beta-galactosidase enzymes dyeing working fluid dyeing, observes random counter 200 cells, records the transfect cell number that dyes/refuse under ordinary optical microscope. The inhibiting rate of medicine=(model group staining cell number-medicine group staining cell number)/model group staining cell number × 100%.

Experimental result shows, normal blank group cell almost has no blue transfect cell (H9c2 (2-1) cell 0.53 ± 0.31/200 cells, HUVEC-12 cell 0.59 ± 0.27/200 cells), H₂O₂After process, blue transfect cell significantly increases, and reaches 69.47 ± 2.27 and 71.47 ± 3.36/200 cells respectively), compare with blank group and have pole significant difference (P < 0.001). Compound A-13 22 can significantly reduce H under 30 μMs of concentration₂O₂H9c2 (2-1) cell (see Fig. 2 A) of induction and the betagalactosidase activity of HUVEC-12 cell (see Fig. 2 B), effect, with positive control resveratrol (30 μMs), shows that this compound has good activity of fighting against senium.

Table 1A322 compound is to H₂O₂The impact (X ± SD, n=3) of the cell aging of induction

Embodiment 2 compound A-13 22 mechanism of resisting senility is studied

1, mtt assay measures cell survival rate: the HUVEC-12 cell in vegetative period of taking the logarithm, is inoculated in 96 well culture plates, is placed in containing 5%CO₂37 DEG C of constant incubators in cultivate 24h, after cell pastes wall completely, every hole adds the testing sample solution of 100 μ l different concns (3,10 and 30 μMs) respectively, establish the blank group and the positive controls (resveratrol that only add perfect medium and do not add cell simultaneously, 30 μMs), often organize and parallel establish four multiple holes. After cultivating 48h, every hole adds MTT liquid 20 μ l (5mg/mL), continues to cultivate 4h, abandons nutrient solution, and every hole adds 100 μ lDMSO, after dissolving completely to be crystallized, surveys absorbancy by microplate reader in 570nm place, calculates comparative survival rate of cells. Comparative survival rate of cells={ [(mean value of the mean value of medicine group OD value-blank group OD value)]/(mean value of the mean value of control group OD value-blank group OD value) } × 100%.

Experimental result shows, normal Endothelial Cell Survival rate is had no significant effect (see Fig. 3) by compound A-13 22 (3-30 μM) and resveratrol (30 μMs).

2, TRAP-argentation measures telomerase activation: telomere enzyme extraction: cell PBS is washed twice, 2000rpm, centrifugal 5min (4 DEG C), collecting cell. Adding the Washbuffer that 1ml is ice-cold, put 5min, 4000rpm on ice, centrifugal 5min (4 DEG C), abandons supernatant. Adding ice-cold Lysisbuffer and 0.5 μ l β-coloured glaze base ethanol, vibration 10s, puts 30min on ice, 4 DEG C, centrifugal (13000rpm, 30min), removes cell debris, Aspirate supernatant, illustrates by BCA test kit and surveys protein concentration. Pcr amplification: get endotheliocyte telomere zyme extract 2 μ g and add following reaction system: 10 × TRAP reaction solution 5 μ l, dNTPs1 μ l, Taq-DNA polysaccharase 1 μ l, TS primer 1 μ l, add DEPC and process water to 50 μ l, through 23 DEG C of 10min, add CX primer 1 μ l, mixed even, carry out pcr amplification: 94 DEG C of 30s, 50 DEG C of 30s, 72 DEG C of 90s, 27 circulations, 72 DEG C of 5min. Polyacrylamine gel electrophoresis: prepare 12% non denatured polyacrylamine gel, 180V prerunning 1h. Get PCR primer 10 μ l and carry out 12% Root of Medicinal Indian mulberry activity of fighting against senium composition and study on mechanism non denatured polyacrylamine gel, 180V electrophoresis 1h. Silver dye: take out gel, soaks 1min with fixing 30min and the 0.2g/l Sulfothiorine of 10% acetic acid, respectively with distilled water rinsing 3 times. Dye 30min and colour developing liquid colour developing 10min through Silver Nitrate dye liquor, and slight concussion, until ladder shape DNA band occurs, adds 10% acetic acid termination reaction. Use gel imaging system analytical results.

Experimental result shows, through H₂O₂The endotheliocyte that (100 μMs) process, telomerase activation significantly declines (P < 0.001). Compound A-13 22 (10,30 μMs) or the positive control drug resveratrol (30 μMs) of pre-treatment different concns can significantly suppress H₂O₂The telomerase activation of induction reduces (see Fig. 4).

3, the mensuration of oxyradical in cell: fluorescence dye H2DCF-DA is hydrolyzed to H in cell₂DCF, through peroxidation formed green fluorescence material DCF, therefore, can according to DCF fluorescence number carry out ROS level in reacting cells. By endotheliocyte by 5 × 10⁵Individual/ml is inoculated in 6 orifice plates, adds different treatment factor and test after growth 48h. After process, take out cell, with anteserum-less substrate rinsing 3 times under room temperature, add 10 μMs of H₂DCF-DA fluorescence dye 37 DEG C of lucifuges hatch 20min, after anteserum-less substrate rinsing 3 times, by multi-functional reading plate instrument fluorescence intensity.

Oxidative stress is its vital role in cellular senescence process, and experimental result shows, H₂O₂(100 μMs) hatch HUVEC-12 cell 48h, can obviously strengthen fluorescence intensity (reflection reactive oxygen species). But, compound A-13 22 (3,10,30 μMs) or the positive control drug resveratrol (30 μMs) of pre-treatment different concns can significantly suppress H₂O₂The reactive oxygen species of induction increases.

Table 1A322 compound is to H₂O₂Endotheliocyte oxyradical change (X ± SD, n=3) of induction

4, Real-TimePCR detects the expression of klothomRNA: 0.25% trypsin digestion and cell, by 5 × 10⁵Individual/ml is inoculated in 6 orifice plates to cultivate. After cytogamy, use the DMEM nutrient solution of 1% calf serum instead, make cell synchronization, then can divide into groups to test. After experiment terminates, detecting the expression of mRNA by Real-TimePCR method, step is as follows: the extraction of (1) cell total rna: adding 1mlTrizol in every porocyte, room temperature places 5min, collects in 1.5mlEP pipe;Then adding chloroform 200 μ l, violent jolting 15s, room temperature places 3min; 12000rpm4 DEG C of centrifugal 15min, gets upper strata aqueous phase and puts in another EP pipe; Adding Virahol 200 μ l, repeatedly put upside down mixed even, room temperature places 10min; 12000rpm4 DEG C of centrifugal 15min, it is seen that the RNA precipitate of white is at the bottom of pipe; Remove supernatant, add 75% ethanol 1ml, repeatedly put upside down several times, washing precipitation; 7000rpm4 DEG C of centrifugal 15min, removes supernatant, the air-dry 10min of room temperature; Add DEPC water 20 μ l and dissolve RNA, for reverse transcription after 4 DEG C of placement 30min. (2) reverse transcription synthesis: DNA: quantitative analyzing RNA concentration (OD₂₆₀/OD₂₈₀For 1.8-2.0). EP pipe adds following reacted constituent: TotalRNA0.1-5 μ g (2 μ l), Oligo (dt)₁₈1 μ l, deionized water adds to 12 μ l. Hatch 5min, cooled on ice for 70 DEG C, of short duration centrifugal, collect liquid pearl; Each composition is added in the following order: 5 × reactionbuffer4 μ l in Tube; RnaseInhibitor (200U/ μ l) 1 μ l, 10mMdNTPmix2 μ l. Mixed even gently, of short duration centrifugal collection liquid pearl. Hatch 5min for 37 DEG C, add reversed transcriptive enzyme (200U/ μ l) 1 μ l. Hatching 60min for 42 DEG C, 70 DEG C of heating 10min, termination reaction, the cDNA that cooled on ice is extracted can directly carry out PCR reaction. (3) Real-TimePCR measures: in the Real-timePCR reaction system containing primer, cDNA, makes inherent genetic contrast with phosphoglyceraldehy-de dehydrogenase (GAPDH). Gene primer sequence: GAPDHF+:5 '-CCAGCAAGAGCACAAGAGGAA-3 ', GAPDHF-:5 '-ATGGTACATGACAAGGTGCGG-3 '; KlothoF+:5 '-TGGCAAGAGGTTACAGCATC-3 '; KlothoF-:5 '-CAGGTCCAGACGCAGGATGGC-3 '. Reaction conditions: 94 DEG C of 10s, 95 DEG C of 5s, 60 DEG C of 30s, totally 35 circulations. Reaction confirms amplification curve and melt curve analysis after terminating, carry out PCR quantitative time production standard curve etc.

Experimental result shows, through H₂O₂The endotheliocyte that (100 μMs) process, klothomRNA expresses without significantly decline (P < 0.001). The compound A-13 22 (10,30 μMs) of pre-treatment different concns can significantly increase klothomRNA and express (see Fig. 5).

Claims

1. there is the compound or pharmaceutically acceptable salt thereof of the following chemical structure:

。

2. compound or pharmaceutically acceptable salt thereof according to claim 1, is characterized in that: it is for the relevant disease that delays senility.

3. compound or pharmaceutically acceptable salt thereof according to claim 1, is characterized in that: for the pharmaceutical composition of the relevant disease that delays senility, it comprises compound or pharmaceutically acceptable salt thereof according to claim 1, and pharmaceutically acceptable carrier.

4. the compound or pharmaceutically acceptable salt thereof stated according to claim 3, is characterized in that: described pharmaceutical composition, and it is made into tablet, capsule, oral liquid, injection, pulvis, paste or external medicinal liquid.

5. compound or pharmaceutically acceptable salt thereof according to claim 1, is characterized in that: the application of described compound or pharmaceutically acceptable salt thereof in the medicine for the preparation of the relevant disease that delays senility.