CN104337809B - The purposes of N- (4,5- dihydro -2- thiazoles) -2- (4- methylenedioxy phenoxies methyl) thiazole -4-carboxamide - Google Patents

Abstract

The invention discloses the purposes of 4 formamide (formula (I)) of compound N (4,5 dihydro, 2 thiazole) 2 (4 methylenedioxy phenoxy methyl) thiazole, belong to pharmaceutical field.The compound indicated by formula (I) is to be adjusted on ABCA1, and can be used for adjusting blood fat and for treating or preventing atherosclerosis.The present disclosure additionally applies for the compounds to be used to raise ABCA1 expression activities, adjusts blood fat and treatment and/or the pharmaceutical composition for preventing the purposes, the active constituent being selected from containing therapeutically effective amount and optional pharmaceutically acceptable carrier.

Description

N- (4,5- dihydro -2- thiazoles) -2- (4- methylenedioxy phenoxies methyl) thiazole -4-carboxamide Purposes

Technical field

The present invention relates to compound N-(4,5- dihydro -2- thiazoles) -2- (4- methylenedioxy phenoxies methyl) thiazole -4-carboxamide, Applied to the treatment and/or prevention of Atherosclerotic cardiovascular disease, belong to pharmaceutical field；Compound of the present invention is used for Prepare the purposes of adjustment, prevention and/or the treatment of lipid lowering agent and antiatherosclerosis on ABCA1；The present invention also relates to And the pharmaceutical composition of the compound.

Background technology

Angiocardiopathy is the primary killers for endangering human health in developed country and most developing countries, in recent years With the raising of people's living standard, the incidence of cardiovascular and cerebrovascular disease is in apparent ascendant trend, and cardiovascular and cerebrovascular disease is dead The composition for accounting for population in the world death is died up to 1/3.Atherosclerosis (Atherosclerosis) is a kind of chronic inflammatory disease Disease is the pathologic basis of a variety of severe cardiovascular diseases (such as coronary heart diseases and angina pectoris, myocardial infarction, cerebral apoplexy).China at present High incidence, rejuvenation trend is presented in atherosclerosis.

Atherosclerosis drug therapy includes vasodilator drug, adjustment hypolipidemic medicine, antiplatelet drug etc. at present. Though existing method can slow down the disease process, cannot still cure.Current clinically widely applied statins are mainly logical It crosses the biosynthesis for inhibiting cholesterol and enhancing peripheral cells and access is adjusted to the ldl receptor that cholesterol absorbs to realize anti-artery Atherosis, but can only reduce by 20%~40% cardiovascular event【1】, and statins find many livers at present Dirty, heart etc. toxicity.The harm for further decreasing angiocardiopathy, while reducing low density lipoprotein cholesterol The drug with novel mechanism must be found from the new therapy target of prevention and/or reversal of atherosclerosis.

Peripheral cells inner cholesterol dysbolism is the important pathogenesis of atherosclerosis, and excess cholesterol is outside liver Removing in tissue is to prevent and treat the committed step of atherosclerosis【2】.High-density lipoprotein (HDL) is by peripheral tissues In cholesterol transport be metabolized to liver or drained in the form of cholic acid again, this process is referred to as reverse cholesterol transport (RCT)【3】.It is to treat the effective means of atherosclerosis that RCT and cholesterol, which exclude approach,.People's ATP binding cassette transporter bodies A1 (ABCA1, (ATP-binding cassette transporter (ABC) A1) play a significant role in RCT【4】.

ABCA1 gene mutations cause Tangier disease (TD) diseases, tissue macrophages inner cholesterol to be built up, blood plasma HDL cholesterol reduces (HDL cholesterol, HDL-C), increases the risk of angiocardiopathy【5】.Height expression ABCA1's turns DNA murine can increase the level of blood plasma HDL, apoA-I, and Macrophage cholesterol outflow is made to obviously increase, to drop The danger of low atherosclerosis.The major function of ABCA1 is that the cholesterol of endocellular liberation and phosphatide are transported to poor fat Or the apolipoprotein A-1 (apoA-I) without fat, this is first speed limit link that reverse cholesterol transport (RCT) and HDL are generated, because This, ABCA1 has great influence to generation, the development of lipid-metabolism and atherosclerosis；More and more clinical test tables Bright raising HDL levels are beneficial to angiocardiopathy, and do not depend on reduction LDL【6】.Studies have shown that tables of the ABCA1 in liver It is all critically important up to for being metabolized HDL down to the homeostasis of entire cholesterol in vivo【7,8】, for internal macrophage In RCT also function to the effect just regulated and controled【9】.ABCA1 is considered as the potential new target drone for finding Novel cardiovascular drug 【10-13】。

In short, there is also heavier side effects for the Statins medicine clinically used, still lack treatment artery at present The specific medicament of atherosis.For obtain tissue specificity it is stronger, by adjust RCT key receptors to promote outside cholesterol Row reduces lipid accumulation, to play prevention and/or treatment atherosclerosis newtype drug, utilizes the Chinese Academy of Medical Sciences Screening model of being adjusted on the ABCA1 of country of Institute of Medicinal Biological Technique new drug (microorganism) screening experiment room structure carries out extensive Screening finds that compound N-(4,5- dihydro -2- thiazoles) -2- (4- methylenedioxy phenoxies methyl) thiazole -4-carboxamide has on apparent The activity of ABCA1 is adjusted, and determines confirm that it has antiatherosclerosis angiocardiopathy active in vitro.N- (4,5- bis- Hydrogen -2- thiazoles) -2- (4- methylenedioxy phenoxies methyl) thiazole -4-carboxamide there are no document in terms of study of anti-atherogenic effect Report, is the discovery for the first time of present patent application.This group of statins amine formyl benzene sulfonyl class compound and clinically used Object is compared, and structure does not have similitude, and plays study of anti-atherogenic effect mechanism difference, and being expected to, which becomes specificity, is adjusted RCT key receptors ABCA1 expression reduces lipid, reduces cholesterol, to cardiovascular as antiatherosclerosis or even protection The medicine of effect.Therefore, which is likely to be the antiatherosclerosis compound with novel mechanism, has Wide development prospect.

Invention content

Present invention discover that being related to compound N-(4,5- dihydro -2- thiazoles) -2- (4- methylenedioxy phenoxies methyl) thiazole -4- formyls Amine, the application in the drug for preparing antiatherosclerosis angiocardiopathy, the compound have structure novel, mechanism of action The characteristics of unique, good anti-atherosclerotic effect, shown in structure such as formula (I)：

It should be appreciated that the compound of the present invention carries out the method that structure of modification is taken, pharmaceutical field crowd institute can be passed through Known any method is prepared into required structure.

The application of compound of the present invention, it is characterised in that the drug is that can raise ATP binding cassette transporter (ABCA1) drug of expression activity.

The application of compound of the present invention, it is characterised in that the drug is the drug for adjusting blood fat.

A kind of application of pharmaceutical composition in the drug for preparing antiatherosclerosis angiocardiopathy, it is characterised in that institute The compound that pharmaceutical composition contains therapeutically effective amount is stated as active constituent and one or more pharmaceutically acceptable Carrier.

The application of pharmaceutical composition of the present invention, it is characterised in that it is 0.1%- that described pharmaceutical composition, which contains weight ratio, 99.5% active constituent preferably comprises the active constituent that weight ratio is 0.5%-99.5%.

The application of pharmaceutical composition of the present invention, it is characterised in that the drug is that can raise ATP binding cassette transporter (ABCA1) drug of expression activity, adjusting blood fat.

The application of pharmaceutical composition of the present invention, it is characterised in that the drug is the drug of antiatherosclerosis.

Pharmaceutical composition of the present invention, various dosage forms can be prepared according to the conventional production process of pharmaceutical field, such as So that active constituent is mixed with one or more carriers, be then made into required dosage form, is suitable for passing through any suitable approach Treat the form (such as the approach such as oral, subcutaneous, intramuscular, intravenous and intradermal) of application.

Compound of the present invention can be prepared according to the synthetic method of pharmaceutical field.

It should be appreciated that the suitable dosage of the compound of the present invention and combinations thereof is likely to be dependent on the type, serious of disease Degree and stage, and it is different with patient.Determining that optimal dose generally comprises makes treatment advantages level be controlled with of the invention Any dangerous or harmful side effect treated balances each other.

The people ABCA1 screening models that the present inventor is established using this laboratory evaluate the activity of patents, Selection 0.1%DMSO is negative control, and 9CRA is positive control, and measurement compound is under 10 μ g/ml concentration in ABCA1 models Up-regulation rate, multiple determination of activity the results show, the compounds of this invention (number T61) has good up-regulation to ABCA1 Effect, and its application in terms for the treatment of atherosclerosis is provided, develop into novel anti-atherogenic for the compound Hardening drug is laid a good foundation.As a result compound effects show that it can be on dose dependent in adjusting screening model on people ABCA1 It is 0.04 μ g/ml (Fig. 1) to adjust the expression of ABCA1, EC50.Compound T61 is acted at (0.05,0.24,1.2,6.0 μ g/ml) In HepG2 cells 18-24h, western blot the results show that compound can be thin in the horizontal apparent up-regulation HepG2 of albumen (Fig. 2) The expression of ABCA1 in born of the same parents.Thus it proves, compound or composition of the present invention can be used as up-regulation ATP binding cassette transporter The drug of ABCA1 expression activities.

apoE^-/-Mouse aorta en face carry out oil red dyeing, as a result such as Fig. 3.As can be seen that drink high in fat from Fig. 3 A There is apparent arteriosclerosis plaque in the entire aorta for eating model group (Fig. 3 b), illustrates the success of artery sclerosis model construction.With High fat diet model group is compared, and the apparent less, reduction for the treatment of group (Fig. 3 c) mouse aorta patch illustrates that T61 can be hard to artery Positive effect is played in the treatment of change.Negative control group (Fig. 3 a) in aorta scalp it can also be seen that do existing smaller arterial plaque Block, may be due to apoE^-/-The spontaneous generation of mouse.

By apoE^-/-Mouse liver frozen section oil red coloration result such as Fig. 4.High fat diet group (Fig. 4 b) liver utilizes oil The pathological section of red O dyeing, oil red dyeing is very deep, and fat deposition area is very big.There is minimal amount in administration group (Fig. 4 c) liver cell Fat drips exist, it is similar to the effect of negative control group.

Adjusting level such as tables 1 of the T61 to blood fat.Table 1 is shown, compared with high fat diet group, total cholesterol TC in administration group, Low density lipoprotein cholesterol LDL-C, triglycerides TG are significantly reduced, and illustrate that compound has the function of adjusting blood fat.

ApoEs of the table 1IMB-T61 to high fat diet^-/-The influence of lipid of mice

Group	Only	TC(mg/dL)	LDL-C(mg/dL)	HDL-C(mg/dL)	TG(mg/dL)
						Blank	7	414.73±58.02	224.28+59.94	54.02±8.15	97.59±22.52
Model	7	810.64±88.78	527.33±41.35	78.04±12.28	165.87±23.14
						IMB-T61	7	706.25±86.08	418.32±62.31	91.77±5.59	116.23±12.72

Based on the above results, illustrate that T61 has good study of anti-atherogenic effect.

Advantageous effect of the invention

1) novelty of action target spot：Current study show that ABCA1 is the novel targets of antiatherosclerosis, in artery congee Generation, the development of sample hardening have great influence, are dedicated to finding upper adjustment or the agonist of ABCA1 at present both at home and abroad, but There has been no the drug appearance for acting on this target spot；

2) novelty of compound：The present invention elaborates compound N-(4,5- dihydro -2- thiazoles) -2- (4- first for the first time Base Phenoxymethyl) definite effect of the thiazole -4-carboxamide in terms of artereosclerotic cardiovascular, and explained from for the first time from molecular mechanism The reason of stating its anti arteriosclerosis effect, this at home and abroad belongs to for the first time, this is anti-with independent intellectual property right for China's exploitation Atherosclerosis drug has great importance.

3) prevention and treatment of artery sclerosis：Cardiovascular and cerebrovascular disease especially atherosclerosis has seriously threatened the mankind Life and health, existing drug early has been unable to meet clinical demand, cannot fundamentally treat.Present invention discover that chemical combination Object has good anti-atherosclerotic effect in animal body, and good guide is provided for exploitation anti arteriosclerosis drug Compound has broad application prospects.

Description of the drawings

Fig. 1 is amount effect relation curves of the T61 in ABCA1 up-regulated expression agent screening models；

Fig. 2 is the protein expression that T61 (0.05,0.24,1.20,6.0 μ g/ml) increases ABCA1 in HepG2 cells；

Fig. 3 is the apoE that T61 reduces high fat diet^-/-Mouse aorta patch；

Fig. 4 is the apoE that T61 reduces high fat diet^-/-Lipid content in mouse liver；

Specific implementation mode

Following embodiment can make professional technician that the present invention be more completely understood, but not limit this hair in any way It is bright.

One the compounds of this invention of embodiment measures the EC50 of adjustment screening model on ABCA1

Compound is dissolved in the mother liquor that DMSO is made into 10mg/ml.By ABCA1-LUC HepG2 cells with 5 × 10⁴A/hole It is inoculated in 96 porocyte culture plates, about 6h removes the culture medium containing serum after cell is adherent, and cell is gently rinsed with PBS Once.With the diluted compound of serum-free RPMI1640 or MEM at a series of concentration, 0.001-100 μm of ol/L, 200 holes μ l/. The hole of final concentration 0.1%DMSO culture mediums is as blank control.37 DEG C are continued at, after cultivating 18-24h under the conditions of 5%CO2, PBS (200 holes μ l/) board-washing 2 times, abandons PBS.Cell pyrolysis liquid (20 holes μ l/) (Promega) is added, after 15-30min, under microscope After observation cell cracking is complete, luciferase (60 holes μ l/) is added, measures uciferase activity (microplate reader reading), meter immediately Calculate rate of change of the sample to be tested to uciferase activity.EC is calculated using Origin8.5₅₀。

The measurement that two the compounds of this invention of embodiment influences ABCA1 protein expression levels

Influence method using western blot methods detection compounds to ABCA1 protein expressions is the same.HepG2 cells are spread In six orifice plates (Costar), sets up dosing group T61 (0.05,0.24,1.20,6.0 μ g/ml) and negative control group (is added without Sample to be tested).RIPA cell pyrolysis liquids extract total protein of cell, and BCA kits (Pierce) carry out protein quantification, then carry out 10%SDS-PAGE electrophoresis, 30 μ g albumen are per hole.Each protein antibodies use concentration：ABCA1(NB400-105；1： 500dilution；Novus Biologicals Inc., San Jose, CA)；β-actin(1∶5000；Invitrogen)； peroxidase (HRP)-conjugated goat anti-rabbit IgG antibody(1∶3000dilution； Santa Cruz)；peroxidase(HRP)-conjugated rabbit anti-mouse IgG antibody(1∶ 3000dilution；Santa Cruz).It is detected with chemoluminescence method (ECL, Millipore) kit, photograph.

Embodiment three is in apoE^-/-Pharmacodynamic evaluation in Mice Body

apoE^-/-The structure of rat aorta hardening model

apoE^-/-Mouse, 7 week old, normal diet are fed one week；

By apoE^-/-Mouse is weighed, random to be grouped, and is divided into 4 groups (model group, administration group, negative control groups), every group 6-8 is only；

Since 8 week old, model group and administration group feed high lipid food, and negative control group continues to feed normal diet；

T61 (20mg/Kg) gastric infusion；Negative control group and model group are to carboxymethylcellulose sodium solution, gastric infusion； Administration 8 weeks；

By 8 weeks apoE of administration^-/-Mouse fasting 6h；It plucks eyeball and takes blood, collect blood with the EP pipes of heparin rinse, up and down It is reverse, it is placed on ice, 4000rpm/min, supernatant is transferred in new EP pipes and (is divided to two parts) by 4 DEG C of centrifugation 3min, be placed in- 20 DEG C of preservations, using Zhong Shengbeikong companies lipid determination kit measurement blood lipid level.

Fixed mouse cuts off skin until abdomen, cuts off abdominal cavity, first leave and take fresh with operating scissors along the median line of neck Liver organization, be put into EP pipes (point three parts), be immediately placed in liquid nitrogen, stay and be RNA and Protein Assav；

After having taken liver, cut off thoracic cavity, cut off breastbone, exposure heart carries out cardiac perfusion immediately, first with 4% poly Formaldehyde pours into about 1ml, then changes PBS into and pours into about 4ml, stops after liver bleaches；

Liver and small intestine are taken out successively；Aorta is isolated to the artery overall length of the total branch of ilium, takes out heart and artery. After dissection, liver, heart and aorta are put into 4% paraformaldehyde, 37 DEG C of fixed 2h, be then placed in 20% sucrose it is molten In liquid, 4 DEG C overnight.

B) the production method of frozen section

1.5ml EP pipes are cut off from middle part, part with cover is left, lid is covered, mark；

OCT embedding mediums are added in EP pipes, bubble has been careful not to；

The tissue impregnated in 20% sucrose is put into OCT, when heart embeds, about 1/3 heart is stayed, cuts flat with, by the heart Nose part upward, is slowly put into OCT；When liver embeds, when section being kept to be put into OCT in one plane；

Organized EP pipes will be wrapped slowly to be put into liquid nitrogen；

Wrap the EP pipes freezed immediately with tinfoil, -20 DEG C are kept in dark place, and long-term preservation is placed on -80 DEG C.

C) aorta oil red O stain

Aorta is taken out from 20% sucrose, PBS is washed once；

Under a dissecting microscope, aorta is longitudinally splitted；

By the aorta cut off distillation washing 3 times；

60% isopropanol impregnates 10min, synchronizing；

(oil red storing liquid configures, and is used in filtering 1-2h) is put into the oil red working solution newly prepared by what is synchronized, 30min；

It is put into 60% isopropanol color separation 1min；

Distilled water is washed 3 times；

The aorta that solution is splitted is laid on black wax, camera is taken a picture immediately.

As a result it shows：See attached drawing

D) frozen section oil red O stain (heart efferent tract and liver organization)

Slice is placed into 30min at room temperature, dries up frozen section；

Slice is put into 4% paraformaldehyde, fixed 10min；

Paraformaldehyde solution is abandoned, distilled water is added, washes 3 times, each 3min；

60% isopropanol impregnates slice 3min, synchronizing；

Then the slice synchronized is put into the oil red working solution newly prepared (configuration of oil red storing liquid, filtering 1-2h Interior use), 30min；

Slice is put into 60% isopropanol color separation, is observed under the microscope at any time；

Distilled water is washed 3 times；

Hematoxylin contaminates 2-3min, redyes nucleus；

After distilled water washes 3 times, slice is placed in distilled water；

Aqueous mountant mounting (+1 part of distilled water of 9 parts of medical glycerines)；

The slice, thin piece surrounding sealed is applied into nail polish, shady place dries in the shade, takes a picture immediately.

Bibliography

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Claims (9)

1. compound N-(4,5- dihydro -2- thiazoles) -2- (4- methylenedioxy phenoxies methyl) thiazole -4-carboxamides are preparing anti-artery congee Application in the drug of sample hardening, shown in the structure such as formula (I) of the compound：

2. application according to claim 1, which is characterized in that the drug is that can raise ATP binding cassette transporter (ABCA1) drug of expression activity.

3. application according to claim 1, which is characterized in that the drug is the drug for adjusting blood fat.

4. a kind of pharmaceutical composition of antiatherosclerosis, which is characterized in that described pharmaceutical composition contains therapeutically effective amount If formula (I) compound represented is as active constituent and one or more pharmaceutically acceptable carriers.

5. pharmaceutical composition according to claim 4, which is characterized in that described pharmaceutical composition contains weight ratio and is The active constituent of 0.1%-99.5%.

6. pharmaceutical composition according to claim 5, which is characterized in that described pharmaceutical composition contains weight ratio and is The active constituent of 0.5%-99.5%.

7. pharmaceutical composition according to claim 4, which is characterized in that the pharmaceutical composition is that up-regulation ATP is combined The pharmaceutical composition of cassette transporter (ABCA1) expression activity.

8. pharmaceutical composition according to claim 4, which is characterized in that the pharmaceutical composition is to adjust the medicine of blood fat Compositions.

9. the preparation method of pharmaceutical composition according to claim 4, which is characterized in that given birth to according to the routine of pharmaceutical field Production method, makes active constituent be mixed with one or more carriers, is then made into required dosage form.