CN102241628A - (2E)-3-phenyl-N-[2,2,2-trichlorine-1-[[(8-quinolyl amino) thiomethyl]amino]ethyl]-2-acrylamide and medicinal uses thereof - Google Patents
(2E)-3-phenyl-N-[2,2,2-trichlorine-1-[[(8-quinolyl amino) thiomethyl]amino]ethyl]-2-acrylamide and medicinal uses thereof Download PDFInfo
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Abstract
The invention relates to an acrylamide compound with a structural formula I or an isomer thereof, a pharmaceutical salt thereof and a solvate thereof, and also relates to the compound or the isomer thereof or the pharmaceutical salt thereof and the solvate thereof, a composition of a pharmaceutically acceptable vector, an excipient or a diluent, and uses of the compound or the composition in preventing and/or treating diseases or symptoms which relate to cardiomyocyte apoptosis and include but are not limited to the uses of (i) treating hungry myocardial atrophy, (ii) treating myocarditis, (iii) treating cardiac failure, (iv) treating or relieving myocardial damage caused by essential hypertension, (v) treating or relieving myocardial damage caused by early-stage acute myocardial infarction, (vi) treating or relieving myocardial damage caused by acute myocardial infarction reperfusion, (vii) treating or relieving cardiomyocyte pathology caused by heart transplant, (viii) treating or relieving dysplastic cardiomyopathy, cardiomyocyte apoptosis caused by anoxia, or improving cardiovascular system sclerosis.
Description
Technical field
The present invention relates to the medical chemistry field; particularly; the present invention relates to a kind of novel propylene amides (2E)-3-phenyl-N-[2; 2; 2-three chloro-1-[[(8-quinolyl amino) sulphomethyl] amino] ethyl]-2-acrylamide and pharmaceutical composition thereof; the invention still further relates to described compound and pharmaceutical composition thereof and be used for anti-apoptotic; prevention or treatment and apoptosis-related disease or the purposes of symptom are especially for the purposes in protection myocardial cell and prevention or treatment and apoptosis of cardiac muscle diseases associated or the symptom.
Background technology
Apoptosis generally is meant body cell in growth course or under some factor effect, a kind of apoptosis that the regulation and control by gene in the cell and product thereof take place.Apoptosis is prevalent in organic sphere, has both betided under the physiological status, also betides under the pathological state.To fetal development and form take place, defence and immune response, the disease of stable, the body of normal cell populations or cell injury, the generation progress aging, tumour that causes when poisoning play an important role in the tissue, are the focuses of biomedical research always.
Studies show that have the generation of a lot of major diseases all relevant with the cell transition apoptosis, for example in the evolution of acquired immune deficiency syndrome (AIDS), CD4
+The minimizing of T cell number; In the graft-rejection, the necrocytosis of cytotoxic T cell mediation; Ischemic and reperfusion injury, the apoptosis of myocardial cell and neurocyte; Neural system degenerative disorders (as Alzheimer time disease, Parkinson's disease etc.); Be exposed to multiple histocyte apoptosis that ionizing rays causes etc.
Evidence suggests that the generation of apoptosis of cardiac muscle and many heart diseases, development and prognosis have confidential relation.Find that by the research apoptosis of cardiac muscle the myocardium death of infarct is not equal to myocardial necrosis, apoptosis is one of mechanism of myocardial infarction, and be the main mode of the cardiac muscle death due to early stage myocardium death of infarct and the ischemia/reperfusion, a large amount of apoptosis that this moment is myocardium have increased the weight of myocardium destruction.1989, find during observation hunger property myocardial atrophy ultrastructures such as Nepomniashchikh that myocardial cell's structural protein are synthetic to be reduced, cell count reduces, but the companion cell nuclear phase should not reduce pro rata, and tentatively proposing the myocardial atrophy of hunger property thus is by due to the apoptosis.1994, employing Electronic Speculum such as Gottlieb and Kawano obtained the direct evidence of apoptosis of cardiac muscle in conjunction with the dna gel electrophoresis method, and the former discloses reperfusion injury and brings out the rabbit apoptosis of cardiac muscle, the scorching patient of the latter's confirmed myocardial apoptosis of cardiac muscle that occurs together.In the neonatal rat myocardial cell that Tanaka etc. cultivate, also proved the existence of apoptosis.Because methodological progress and Study of apoptosis are deeply, in multiple heart trouble, found the pathological effect of apoptosis of cardiac muscle.Studies show that spontaneously hypertensive mouse (SHR) heart damage is relevant with apoptosis; Turn to heart failure by plump heart late period is due to the apoptosis of cardiac muscle; Acute myocardial infarction is except that necrosis, and the early stage and reperfusion injury of infraction is inducing apoptosis also; Apoptosis of cardiac muscle sees heart and right ventricle's dysplasia myocardosis of transplanting equally, and anoxic is induced apoptosis of cardiac muscle equally.
Apoptosis has recoverability to a certain extent, and the apoptosis in myocardial infarction and the ischemia/reperfusion has its characteristics and rule, utilizes its characteristics can prevent and reduce apoptosis, for the clinical prevention ischemia/reperfusion injury provides enlightenment; In refilling process, the apoptosis that produces contraction bands zone (around the infarct kitchen range) is induced generation by some inducements, can utilize the inhibition factor of apoptosis such as medicine to wait and prevent apoptosis, the corresponding disease that the treatment apoptosis causes.
But at present can be for clinical application be used for anti-apoptotic and the protection medicament categories of cell and quantity also seldom; selectivity and target are not high; therefore constantly research and develop the medicine of new anti-apoptotic safely and effectively and protection cell, the medicine that especially has brand-new mechanism of action has crucial meaning.
Summary of the invention
The objective of the invention is to seek and develop the micromolecular compound that suppresses apoptosis of cardiac muscle, be used for preventing or treating the various pathological changes that apoptosis of cardiac muscle causes.The contriver has found a kind of acrylamides through long-term, a large amount of experimental studies, and it has anti-apoptotic, and protection myocardial cell's effect can be used in prevention or treatment and apoptosis of cardiac muscle diseases associated or symptom.Particularly,
A first aspect of the present invention relates to the compound shown in the formula I, or its isomer, pharmacologically acceptable salt and solvate.
Formula I compound, its chemical name are (2E)-3-phenyl-N-[2,2, and 2-three chloro-1-[[(8-quinolyl amino) sulphomethyl] amino] ethyl]-the 2-acrylamide.
The present invention relates to pharmaceutical composition on the other hand, and it comprises compound shown in the formula I, or its isomer, pharmacologically acceptable salt and solvate, and pharmaceutically acceptable carrier, vehicle or thinner.
The invention still further relates to formula I compound or its isomer, pharmacologically acceptable salt and solvate, be used to prepare anti-apoptotic, the purposes of the medicine of prevention or treatment and apoptosis-related disease or symptom.
The invention still further relates to formula I compound or its isomer, pharmacologically acceptable salt and solvate, be used to prepare the purposes of the medicine of protecting myocardial cell and prevention or treatment and apoptosis of cardiac muscle diseases associated or symptom.
The invention still further relates to a kind of method that prevents and/or treats with apoptosis of cardiac muscle diseases associated or symptom, described method comprises: the aforementioned pharmaceutical compositions that gives therapeutic dose.
The invention still further relates to a kind of myocardial cell's of protection method, described method comprises the aforementioned pharmaceutical compositions that gives therapeutic dose.
Described and apoptosis of cardiac muscle diseases associated or symptom include but not limited to the myocardial atrophy of (i) hunger property, (ii) myocarditis, (iii) in heart failure, the (iv) myocardial damage that causes of essential hypertension, (the v) myocardial damage that causes in early days of acute myocardial infarction, (vi) acute myocardial infarction pours into the myocardial damage that causes again, (vii) myocardial cell's pathology of causing of heart transplantation, (viii) dysplasia myocardosis; Or the apoptosis of cardiac muscle that causes of anoxic, or cardiovascular systems sclerosis.
Compound of the present invention has the effect of treatment chronic heart failure.
Find that by fluidic cell detection and classical TUNEL apoptosis detection method the pre-treatment of use formula I compound can significantly improve tunicamycin inductive apoptosis of cardiac muscle; and, confirm of the provide protection of formula I compound to apoptosis of cardiac muscle along with concentration increases the trend that this anti-apoptosis provide protection has increase.
The present invention selects caspase-12 as the detected object of confirming the apoptosis path, discovery has the expression of caspase-12 in tunicamycin is induced the process of apoptosis of cardiac muscle, and the formula of use I compound can make the caspase-12 expression decreased, this formula I compound intervention can alleviating endocytoplasmic reticulum stress with the expression of subsequently caspase-12, thereby alleviate apoptosis.
In addition, the present invention confirms that formula I compound (during TD50＞100mM), there is no cytotoxic effect, and it does not protected with the irrelevant apoptosis of er stress and stimulates when its maximum cell protection concentration.
The invention still further relates to the method that prevents and/or treats the various diseases that apoptosis of cardiac muscle causes, it comprises needs the above-mentioned patient who prevents and/or treats with preventing and/or treating at least a formula I compound of significant quantity or the composition of its solvate.
Those skilled in the art will recognize that there is chiral centre in compound of Formula I.When the needs compound of Formula I is single enantiomorph, can use the reactant that in all possible step, all is in single enantiomeric forms to prepare, perhaps in the presence of the reagent of single enantiomeric forms or catalyzer, react and prepare, perhaps split stereoisomer mixture and prepare by ordinary method.Some preferable methods comprise uses microorganism to split, the salt of the diastereomer that any spendable acid such as fractionation and chiral acid such as amygdalic acid, camphorsulfonic acid, tartrate, lactic acid forms, the perhaps salt of the diastereomer of formation such as fractionation and chiral base such as brucine (bracine), Peruvian bark alkaloid and derivative thereof.Method commonly used is seen " Enantiomers, Racemates and Resolution " (Wiley Interscience, 1981) that people such as Jaques edits.
It will be appreciated by those skilled in the art that The compounds of this invention also can use with its pharmacologically acceptable salt or solvate forms.Acceptable salt comprises the salt of the routine that is formed by pharmaceutically acceptable mineral acid or organic acid or mineral alkali or organic bases and the acid salt of quaternary ammonium on the physiology of compound of Formula I.The example more specifically of suitable hydrochlorate comprises hydrochloric acid, Hydrogen bromide, sulfuric acid, phosphoric acid, nitric acid, perchloric acid, fumaric acid, acetate, propionic acid, succsinic acid, oxyacetic acid, formic acid, lactic acid, toxilic acid, tartrate, citric acid, pounces on the salt of acid, propanedioic acid, hydroxymaleic acid, toluylic acid, L-glutamic acid, phenylformic acid, Whitfield's ointment, fumaric acid, toluenesulphonic acids, methylsulfonic acid, naphthalene-2-sulfonic acid, Phenylsulfonic acid, hydroxynaphthoic acid, hydroiodic acid HI, oxysuccinic acid, steroic, tannic acid etc.Other acid as oxalic acid, though itself be not pharmaceutically acceptable, can be used to prepare the salt as intermediate, to obtain The compounds of this invention and pharmacologically acceptable salt thereof.The example more specifically of suitable alkali salt comprises sodium, lithium, potassium, magnesium, aluminium, calcium, zinc, N, N '-dibenzyl-ethylenediamin, chloro PROCAINE HCL, PHARMA GRADE, choline, diethanolamine, quadrol, N-methylglucosamine and procaine salt.When after this relating to compound of the present invention, comprise compound of Formula I and pharmacologically acceptable salt thereof and solvate.
The present invention also comprises the prodrug of The compounds of this invention, and this prodrug promptly carries out chemical conversion by metabolic process once administration, becomes afterwards to have active medicine.Usually, this class prodrug is the functional derivatives of The compounds of this invention, and it changes into the compound of required formula (I) in vivo easily.For example, at " Design Of Prodrugs ", H Bund Saard, Elsevier edits, and has described in 1985 and has selected and the ordinary method of the suitable prodrug derivant of preparation.
The present invention also comprises the active metabolite of The compounds of this invention.
Another aspect of the present invention relates to pharmaceutical composition, and it contains raceme or the optically active isomer and at least a pharmaceutically acceptable carrier of The compounds of this invention, and it can be used for interior therapeutic and has biocompatibility.Described pharmaceutical composition can be prepared into various forms according to different way of administration.The mentioned compound of the present invention also can be prepared to various pharmacologically acceptable salts.
Pharmaceutical composition of the present invention comprises compound of Formula I of the present invention or its pharmacologically acceptable salt or hydrate and one or more suitable pharmaceutically acceptable carrier of effective dose.The pharmaceutical carrier here includes but not limited to: ion-exchanger, aluminum oxide, aluminum stearate, Yelkin TTS, serum protein such as human serum albumin, buffer substance such as phosphoric acid salt, glycerine, Sorbic Acid, potassium sorbate, the partial glycerol ester mixture of saturated vegetable fatty acid, water, salt or ionogen, as protamine sulfate, Sodium phosphate dibasic, potassium hydrogen phosphate, sodium-chlor, zinc salt, colloidal silica, Magnesium Trisilicate, polyvinylpyrrolidone, cellulosic material, polyoxyethylene glycol, Xylo-Mucine, polyacrylic ester, beeswax, lanolin.
The pharmaceutical composition of The compounds of this invention can be used with following any-mode: oral, spraying sucks, rectal application, nasal cavity applied medicine, the cheek medication, local application, non-enterally administer, as in subcutaneous, vein, intramuscular, intraperitoneal, the sheath, in the ventricle, in the breastbone and intracranial injection or input, or by the medication of a kind of outer planting reservoir.Wherein preferred oral, intraperitoneal or intravenous administration mode.
When medicine for oral use, The compounds of this invention can be made into oral acceptable dosage form arbitrarily, includes but not limited to tablet, capsule, the aqueous solution or aqeous suspension.Wherein, the carrier that tablet uses generally comprises lactose and W-Gum, also can add lubricant such as Magnesium Stearate in addition.The thinner that capsule preparations uses generally comprises lactose and dried corn starch.Aqueous suspension preparation then normally mixes use with activeconstituents with examples of suitable emulsifiers and suspension agent.If desired, also can add some sweeting agents, perfume compound or tinting material in the above oral preparations form.
When local medication, particularly treat local external application easy to reach and suffer from face or organ, during as eyes, skin or lower intestinal tract nervous system disease, can The compounds of this invention be made different local application's dosage forms, specify as follows according to different trouble faces or organ:
When the eye topical application, The compounds of this invention can be mixed with the dosage form of a kind of micronization suspension or solution, and the carrier that uses is the Sterile Saline of isoosmotic certain pH, wherein can add also not adding preservative agent such as zephiran chloride alkoxide.For eye usefulness, also compound can be made paste form such as vaseline paste.
When topical application, The compounds of this invention can be made into suitable ointment, lotion or creme dosage form, wherein activeconstituents is suspended or is dissolved in one or more carriers.The spendable carrier of ointment formulation includes but not limited to: mineral oil, Albolene, white vaseline, propylene glycol, polyoxyethylene, polyoxytrimethylene, emulsifying wax and water; The spendable carrier of lotion or creme includes but not limited to: mineral oil, and sorbitan monostearate, polysorbate60, the n-Hexadecane ester type waxes, cetene is fragrant and mellow, 2-Standamul G, benzyl alcohol and water.
The all right aseptic injection preparation form medication of The compounds of this invention comprises aseptic injection water or oil suspension or aseptic injectable solution.Wherein, spendable carrier and solvent comprise water, Ringer's solution and isotonic sodium chlorrde solution.In addition, the fixed oil of sterilization also can be used as solvent or suspension medium, as direactive glyceride or two glyceryl ester.
It may be noted that in addition, the using dosage of The compounds of this invention and using method depend on all multifactor, comprise activity intensity, Time of Administration, metabolic rate, the severity of illness and diagnosis and treatment doctor's the subjective judgement of patient's age, body weight, sex, natural health situation, nutritional status, compound.
The beneficial effect of the invention
The invention provides a kind of acrylamides, and prove that it is the potent anti-myocardial apoptosis agent of a class, therefore can be used for but be not limited to the myocardial atrophy of (i) hunger property, (ii) myocarditis, (iii) in heart failure, (iv) treatment or alleviate the myocardial damage that essential hypertension causes, (v) treatment or alleviate the myocardial damage that acute myocardial infarction causes in early days, (vi) treatment or alleviation acute myocardial infarction pour into the myocardial damage that causes again, (vii) treatment or alleviate myocardial cell's pathology that heart transplantation causes, (viii) treatment or alleviate the dysplasia myocardosis; Or improve cardiovascular systems hardened purposes, and be disease or symptom that the treatment apoptosis causes, particularly treat disease or the symptom that apoptosis of cardiac muscle causes new method and approach is provided.
Description of drawings
Fig. 1 different concns formula I compound is to the influence of eIF2 α and P-eIF2 alpha expression
The influence that Fig. 2 formula I compound induces myocardial cell caspase-12 and cleavedcaspase-12 to express to TM
Embodiment
Below in conjunction with embodiment embodiment of the present invention are described in detail, but it will be understood to those of skill in the art that the following example only is used to illustrate the present invention, and should not be considered as limiting scope of the present invention.Unreceipted actual conditions person among the embodiment carries out according to the condition of normal condition or manufacturers's suggestion.The unreceipted person of production firm of agents useful for same or instrument, being can be by the conventional products of commercial acquisition.
Embodiment 1: protection myocardial cell er stress Experimental study on apoptosis
Animal: newborn Wistar rat, in the mouse 24h in age, male and female are not limit
Myocardial cell's separation and cultivation:
Myocardial cell's separation and cultivation are with reference to the adherent isolating method (Kreider of differential, A.Messing, H.Doan, S.U.Kim, R.P.Lisak and D.E.Pleasure, Enrichment of Schwann cell cultures from neonatal rat sciatic nerve by differential adhesion, Brain Res 2 (1981), pp.433444.), get Wistar suckling mouse newborn in the 24h, obtain former generation myocardial cell.
Mtt assay detects the influence of different concns formula I compound to myocardial cell's survival rate
To nourish heart the myocyte according to every hole 10 according to isolating former being commissioned to train that aforesaid method obtains
4Individual cell inoculation is to 96 orifice plates, every pore volume 100ul (marginal pore is filled with aseptic PBS).At 5%CO2, after 37 ℃ of incubators are cultivated 4d, add the formula I compound (0.3 μ M, 1 μ M, 3 μ M, 10 μ M, 30 μ M, 100 μ M) of different concns respectively, each concentration is provided with 3 multiple holes, zeroing hole (substratum, MTT, DMSO) is set simultaneously, control wells (nutrient solution, DMSO).Continue to hatch handle 48h after, every hole adds 20ulMTT solution (5mg/ml is with PBS＜pH=7.4〉join be 0.5%MTT), continues cultivation 4h.Stop cultivating, the careful suction removed nutrient solution in the hole.Every hole adds 150ulDMSO, puts low-speed oscillation 10min on the shaking table, and crystallisate is fully dissolved.Measure each hole absorbancy (OD) value in wavelength 550nm place at enzyme-linked immunosorbent assay instrument, every hole is repeated 5 times and is write down the result.The results are shown in Table 1:
Table 1MTT method detects the influence of formula I compound to myocardial cell's survival rate
| Experimental group | Cell survival rate (* 100%) |
| Control group (DMSO) | 100 |
| Dosing group (0.3 μ M) | 98.8562±6.7316 a |
| Dosing group (1 μ M) | 91.6667±7.6257 a |
| Dosing group (3 μ M) | 94.5261±3.7997 a |
| Dosing group (10 μ M) | 102.7778±3.0645 a |
| Dosing group (30 μ M) | 105.8007±3.2639 a |
| Dosing group (100 μ M) | 104.2484±7.3625 a |
Compare with control group,
aP＞0.05;
Formula I compound is compared myocardial cell's survival rate no difference of science of statistics with control group in 100 μ M concentration.Formula I compound is to normal myocardial cell's not influence of survival rate.
Mtt assay detects different concns formula I compound to tunicamycin (Tunicamycin, TM) influence of inductive apoptosis of cardiac muscle
With isolating myocardial cell according to 104 cell inoculations in every hole to 96 orifice plates, every pore volume 100ul (marginal pore is filled with aseptic PBS).At 5%CO2, after 37 ℃ of incubators are cultivated 4d, after giving formula I compound (5 μ M, 10 μ M, 20 μ M) the processing back 30min of different concns respectively, it is 5 μ g/ml that adding TM makes its final concentration, and it is TM group and the isometric DMSO control group of 5 μ g/ml that final concentration is set simultaneously.Each concentration is provided with 3 multiple holes, zeroing hole (substratum, MTT, DMSO) is set simultaneously, control wells (nutrient solution, DMSO).Continue to cultivate, every hole adds 20ulMTT solution (5mg/ml is with PBS＜pH=7.4〉join be 0.5%MTT), continues cultivation 4h.Stop cultivating, the careful suction removed nutrient solution in the hole.Every hole adds the 150ul dimethyl sulfoxide (DMSO), puts low-speed oscillation 10min on the shaking table, and crystallisate is fully dissolved.Measure each hole absorbancy (OD) value in wavelength 490nm place at enzyme-linked immunosorbent assay instrument OD, every hole is repeated 3 times and is write down the result, press cell survival rate=(A490-TM of control group untreated cell handles the A490 of cell)/A490 * 100% of control group untreated cell and calculate the cell survival rate of each time point, with time is X-coordinate, and light absorption value is that ordinate zou is drawn cell growth curve.
The results are shown in Table 2:
Table 2MTT method detects formula I compound is induced myocardial cell's survival rate to TM influence
Compare #P＜0.05 with the DMSO group; Compare * P＜0.05 with the TM group;
The result shows; the TM intervention group obviously makes myocardial cell's survival rate significantly descend (p＜0.05); compare all significantly increase (p＜0.05) through the pretreated cell survival rate of respectively organizing of each concentration formula I compound with the TM group; show the apoptosis of cardiac muscle that formula I compound can resist tunicamycin (TM) to cause, TM inductive apoptosis of cardiac muscle has been played provide protection.
Embodiment 2:Western Blot detects the apoptosis signal protein and expresses
Will be according to the myocardial cell of the adherent acquisition of embodiment 1 former generation myocardial cell cultural method differential according to every hole 10
6Individual cell inoculation is to 6 orifice plates, every pore volume 2ml, after 37 ℃, 5%CO2 incubator are cultivated 4d, add the intervention of formula I compound respectively by each group of experimental design, after continuing to place 37 ℃, 5%CO2 incubator to be cultured to design time point, with SDS sample loading buffer lysing cell, 100 ℃ of water-bath 10min, 4 ℃ of centrifugal (12000rpm * 10min), collect supernatant.In advance nitrocellulose filter is immersed in 15～20min in the transfering buffering liquid.With sample on 50 μ g albumen/swimming lanes, after 10% polyacrylamide gel SDS-PAGE electrophoretic separation, again gel is tiled in the electrotransfer folder, sponge, filter paper, gel, nitrocellulose filter, filter paper and sponge are housed from the negative pole to the positive pole successively.Constant current 350mA, electricity changes 45min.Electricity changes film to pvdf membrane, take out nitrocellulose filter, after TBST washes film 2min, 5% skim-milk room temperature sealing 1h, add anti-(1: 1000) 4 ℃ of overnight incubation, TBST washes 3 times * 5min of film, adds two of HRP mark again and resists, and hatches 1h under the room temperature, TBST washes 3 times * 5min of film, soak 5min and 10min respectively with TSM1 and TSM2, be dissolved in 1ml TSM2 colour developing with chromogenic substrate 6.6 μ l NBT and 3.3 μ l BCIP, to the clear after washing termination reaction of band.After colour developing stopped, scanning or Taking Pictures recording were preserved.
Detected result:
EIF2 α and P-eIF2 alpha expression
Different concns formula I compound effects 24h Western blotting detects and shows, through 5%CO2,37 ℃ of incubators are cultivated the myocardial cell of 4d, after giving formula I compound (1 μ M, 2 μ M, 5 μ M, 10 μ M, 20 μ M, 40 μ M) the processing 48h of different concns respectively, find that each hole eIF2 protein expression level does not have remarkable change, and P-eIF2 α protein expression level increases (see figure 1) gradually with the increase of formula I compound concentration.
Caspase-12 and Cleaved caspase-12 express and change
Behind the formula I of different concns compound treatment 24h, each is organized Caspase-12 and expresses obviously change of nothing.Cleaved caspase-12 does not have expression in the DMSO group, induce group to express obviously at TM (5 μ g/ml), adding the various I compound group of having used different concns (10 μ M, 20 μ M, 40 μ M), along with the increase Cleaved caspase-12 expression minimizing gradually of formula I compound concentration.Show that Cleaved caspase-12 activates in TM inductive apoptosis of cardiac muscle process, Cleaved caspase-12 protein expression level is dose-dependently decline (see figure 2) with the increase of formula I compound concentration.
Embodiment 3: formula I compound is to the influence of TM inductive apoptosis of cardiac muscle
The former generation myocardial cell 4d that cultivates according to the former generation myocardial cell cultural method of embodiment 1 begins to add tunicamycin (TM) and carries out intervention experiment.Cell is divided into 5 groups at random: solvent control group (DMSO), TM intervention group (5ug/ml), formula I compound+TM intervention group (10 μ M+5 μ g/ml), formula I compound intervention group (20 μ g/ml+5 μ g/ml), formula I compound intervention group (40 μ g/ml+5 μ g/ml).
Flow cytometer detects apoptosis:Behind the drug intervention 24h, press Annexin V-FITC apoptosis kit method and handle cell, detect the apoptosis situation in order to flow cytometer (BDACScalibur, U.S. Becton-Dickinson company) in the 1h.14000 cells of each sample collection are analyzed, and experiment repeats 3 times, results averaged.
TUNEL detects: behind the drug intervention 24h, detect apoptotic cell, press the test kit explanation and handle cell with terminal original position mark (TUNEL) method of the breach of deoxynucleotide terminal enzyme (DNA) mediation.The cell climbing sheet of handling with 0.1mg/ml DNase I is as positive control.The visual field of picked at random more than 5 under the light microscopic, the apoptotic cell in the counting cells (visible brown yellow granule in the karyon) number.Apoptosis rate=(positive staining cell check figure/all cells check figure) * 100%.
Experimental result:Each organizes apoptosis rate relatively:
Pass through cells were tested by flow cytometry, normally cultivating neonatal rat myocardial cell early apoptosis rate is 12.72%; After 5 μ g/ml TM induced 24h, apoptosis reached 29.98%; With the TM group relatively, 10 μ M formula I compound groups, 20 μ M formula I compound groups, 40 μ M formula I compound group apoptosis rate significantly descend (P＜0.05), and it is on a declining curve to increase apoptosis rate with dosage.See Table 3.
Table 3 formula I compound is to the influence of TM inductive apoptosis of cardiac muscle
| Group | Flow cytometer detects apoptosis rate (%) |
| Control group (DMSO) | 12.72±0.96 |
| The TM intervention group | 29.98±1.22 a |
| TM+ formula I 10 μ M | 22.98±1.35 ab |
| TM+ formula I 20 μ M | 20.13±0.87 ab |
| TM+ formula I 40 μ M | 18.64±0.83 ab |
Compare with the DMSO group,
aP＜0.05; Compare with the TM group
bP＜0.05
The TUNEL detected result
DMSO group apoptosis rate is 7.86%, the intervention group apoptosis rate significantly increases (P＜0.05) after 5 μ g/mL TM induce 24h, compare with the TM intervention group, add with formula I compound (10 μ M, 20 μ M, 40 μ M) group, apoptosis rate all obviously reduces (P＜0.05).Formula I compound 40 μ M group cotype I compound 10 μ M, 20 μ M group are compared apoptosis rate decline obviously (P＜0.05)
Table 4 apoptosis rate relatively
Compare with the DMSO group,
aP＜0.05; Compare with the TM group
bP＜0.05; Compare with TM+ formula I 20 μ M group with TM+ formula I10 μ M group,
cP＜0.05
Embodiment 4: formula I compound is to the provide protection of hypoxia inducible apoptosis of cardiac muscle
Animal: newborn Wistar rat, in the mouse 24h in age, male and female are not limit
Cell is prepared: myocardial cell's separation and cultivating with reference to the adherent isolating method of differential, get Wistar suckling mouse newborn in the 24h, through tincture of iodine alcohol disinfecting skin of chest abdomen, core and tiltedly open chest and take out heart and place and ice precooling PBS with scissors median line under xiphoid-process chest of taking back out slightly; Blow and beat heart gently with the PBS of 0.01M and remove blood cell and its hetero-organization, heart is cut into the fragment of 0.5mm3 size, wash 2-3 time repeatedly with 0.01M PBS; Fragment is placed Erlenmeyer flask, add 4ml 0.125% pancreatin, 37 ℃ of water-bath concussions of 1ml 0.1% collagenase II (final concentration is respectively 0.1% and 0.02%) 10min abandons supernatant; Add 4ml 0.125% pancreatin once more, 1ml 0.1% collagenase II, 37 ℃ of water-bath concussion digestion 10min draw supernatant and move to centrifuge tube, supernatant is added the DMEM that contains 10%FBS stop digestion; Repeat water-bath concussion digestion step 3-4 time, till the tissue block complete digestion; With the cell suspension collected with the centrifugal 10min of 1000rpm after, remove supernatant, it is resuspended to add substratum again; With resuspended cell inoculation in Tissue Culture Flask, place 37 ℃ of CO2 incubators to hatch behind the 1.5h with the nutrient solution sucking-off, behind the microscopically counting, adjust cell density with the DMEM nutrient solution that contains 10%FBS, be inoculated into 96 orifice plates by 1 * 104, place 37 ℃ of later half amounts of 5%CO2 incubator 24h to change liquid, add the substratum that contains 0.1%Brdu; Change liquid 1 time behind every afterwards 48h, cultivate and to obtain former generation myocardial cell after 4 days.
Solution is prepared (following reagent can be purchased the company in Invitrogen):
1.1 * wash buffer: the 10 * wash buffer that adds 20ml goes into the 180ml ultrapure water, and total amount is to 200ml, be stored in 4 ℃ 7 days;
2. stationary liquid: 37% formaldehyde solution of 7.3ml is added among 1 * wash buffer of 14.7ml, is preheating to 37 ℃ (this solution needs interim preparation) before the use.
3.1 * infiltration damping fluid: add 10 of 4ml * infiltration damping fluid in the 36ml distilled water, be stored in 4 ℃ 7 days;
4.Mitotracker/Hoechst solution (20 ℃ of preservations): the anhydrous DMSO with 94 μ L dissolves the solution that Mitotracker makes 1mM, and this solution can be preserved 6 months under the condition of-20 ℃ of dry lucifuges.For avoiding repeatedly freezing molten circulation, carry out the packing of single usage quantity.Is 5.5 μ L concentration that the Mitotracker solution of 1mM and the Hoechst dye liquor of 11 μ L are added in the cell culture medium, and obtaining final volume is the application liquid (this solution needs interim preparation) of 5.5mL.
5.Alexa Fluor 488phalloidin Solution: with 140 μ L methyl alcohol AlexaFluor 488phalloidin is dissolved into and makes mother liquor, this solution can be preserved 12 months under the condition of-20 ℃ of dry lucifuges.The Alexa Fluor 488phalloidin mother liquor of 27.5 μ L is added to make among 1 * wash buffer of 5.5ml and uses liquid (needing preparation temporarily).
Experimental procedure:
With isolating myocardial cell according to 104 cell inoculations in every hole to 96 orifice plates, every pore volume 100 μ l (marginal pore is filled with aseptic PBS).At 5%CO2, after 37 ℃ of incubators are cultivated 4d, after giving formula I (5 μ M, 10 μ M, 20 μ M) the processing back 30min of different concns respectively, insert (O2/CO2 in 37 ℃ of airtight anoxic incubators, 5: 95) continue to hatch 16h, the anoxic control wells is set simultaneously, normal control hole 5%CO2,37 ℃ of incubators are cultivated 16h.
2. hatch preceding 30min finishing anoxic, the substratum of 50 μ l and the Mitotracker/Hoechst solution of 50 μ l are added in every hole, continue 37 ℃ of incubated cell 30min.
3. the stationary liquid 100 μ l that will be preheated to 37 ℃ add each culture hole respectively, and do not inhale substratum, ventilation incubated at room 10min.
4. each hole solution (can buckle) that exhausts is washed 1 time with 1 * wash buffer (100 μ l/ hole).Want SC in operation and the washing process, slowly imbibition and liquid feeding are to keep cell attachment and cell integrity.
5. exhaustion wash buffer (can buckle) adds 1 * infiltration damping fluid (100 μ l/ hole) incubated at room 15min.
6. each hole infiltration damping fluid (can buckle) that exhausts is washed 1 time with 1 * wash buffer (100 μ l/ hole).
7. each the hole wash buffer (can buckle) that exhausts, every hole adds 50 μ l AlexaFluor 488phalloidin solution, and the room temperature lucifuge is hatched 30min.
8. each the hole Alexa Fluor 488phalloidin solution (can buckle) that exhausts is washed 2 times with 1 * wash buffer (100 μ l/ hole), adds 1 * wash buffer (100 μ l/ hole) again.
9. board edge is wrapped (preventing drying) with sealing film, on HCS Reader, detected.
High intension screening analytical method:
During cell generation apoptosis, the remarkable change of morphocytology can appear usually, and a series of variations such as biochemical and molecular marker.A lot of about apoptotic detection method, but can only single index detect at the apoptosis of apoptotic cell mostly.It is emerging in recent years a kind of apoptosis detection method that high intension screening is analyzed, and by specific fluorescent dye apoptosis is carried out multiplicity simultaneously.Main analyze three parameters relevant with apoptotic process: comprise the karyomorphology change, mitochondrial swelling with or mitochondrial membrane potential, F-Actin muscle content.The height of specificity and reliability have to(for) complete cell.
Apoptotic cells takes place show two types nuclear alteration usually, nuclear fragmentationization or nuclear concentrate, and in the process of nuclear fragmentationization, circular or oval-shaped nuclear becomes lobulated, and finally is cracked into a plurality of subnucleus structures.Because the destruction of structure components such as endonuclear euchromosome, kernel increases nuclear density.HCS Reader can observe by the dye morphology of pair cell nuclear of Hoechst, and pair cell nuclear area and nuclear intensity are carried out quantitative comparison.
Intramitochondrial variation is the important morphological change of apoptosis, and plastosome discharges the apoptogene factor by adventitia, simultaneously, because mitochondrial permeability increases, the electrochemistry composition of mitochondrial inner membrane is changed, and causes the decline or the disappearance of membrane potential.Under the stimulation of apoptosis, plastosome is also expanded and is become big, causes the increase of plastosome volume.The increase of the decline of mitochondrial membrane potential and plastosome volume has been acknowledged as the early stage mark of apoptosis, can pass through the plastosome tracer agent
Red carries out quantitatively.
The variation of actin cytoskeleton has been in the news and has changed relevant important parameter as one with apoptosis, and during apoptosis, F-Actin muscle content increases in early days.By the special staining agent Alexa of F-Actin muscle
The dyeing of 488Phalloidin (phalloidin) can be determined F-Actin muscle content, and the degree of pair cell apoptosis quantizes comparison.
Formula I compound is to the provide protection detected result of hypoxia inducible apoptosis of cardiac muscle:
1) nuclear area detected result:
| Group | The nuclear area |
| Control group | 113.86±3.06 |
| The anoxic group | 98.26±1.00 |
| Formula I compound 5 μ M group | 109.90±3.97 |
| Formula I compound 10 μ M group | 107.43±7.77 |
| Formula I compound 20 μ M group | 111.90±10.76 |
2) nuclear intensity detection result:
| Group | Nuclear intensity |
| Control group | 122.70±2.38 |
| The anoxic group | 118.23±1.33 |
| Formula I compound 5 μ M group | 124.90±2.25 |
| Formula I compound 10 μ M group | 123.21±2.71 |
| Formula I compound 20 μ M group | 121.40±2.24 |
3) light flux detected result:
| Group | light?flux |
| Control group | 1247.52±48.059 |
| The anoxic group | 1044.91±16.53 |
| Formula I compound 5 μ M group | 1201.92±29.95 |
| Formula I compound 10 μ M group | 1166.12±91.06 |
| Formula I compound 20 μ M group | 1214.09±107.59 |
4) Actin muscle fiber detected result:
| Group | Fibre strength | The short major axis ratio of fiber |
| Control group | 461.77±8.68 | 0.74±0.00 |
| The anoxic group | 541.01±41.14 | 0.77±0.01 |
| Formula I compound 5 μ M group | 498.03±76.67 | 0.75±0.02 |
| Formula I compound 10 μ M group | 503.64±49.23 | 0.74±0.01 |
| Formula I compound 20 μ M group | 471.83±69.38 | 0.74±0.02 |
5). the mitochondrial membrane potential detected result:
| Group | Membrane |
| Control group | |
| 1±0.01 | |
| The anoxic group | 96.04％±3.61％ |
| Formula I compound 5 μ M group | 96.69％±5.06％ |
| Formula I compound 10 μ M group | 97.19％±2.82 |
| Formula I compound 20 μ M group | 99.40％±2.80％ |
6) cell counting detected result:
| Group | Cell counting |
| |
1±0.11 |
| The anoxic group | 71.51％±5.73％ |
| Formula I compound 5 μ M group | 88.57％±7.64％ |
| Formula I compound 10 μ M group | 98.03％±16.24 |
| Formula I compound 20 μ M group | 101.73％±22.42％ |
Compare with control group, the cell counting of anoxic group has descended 28.49%, and the formula I compound (5 μ M, 10 μ M, 20 μ M) that has used different concns afterwards, compares cell counting raise respectively (17.06%, 26.52% and 30.22%) with the anoxic group.
Compare with control group, the anoxic group causes declines such as nuclear area, nuclear intensity, intensity of illumination, Actin muscle fiber, mitochondrial membrane potential and cell counting, formula I compound can obviously improve above-mentioned each desired value of myocardial cell under the anoxic condition, and high intension The selection result shows that formula I compound has the obvious apoptosis of cardiac muscle effect that anoxic causes that improves.
Although the specific embodiment of the present invention has obtained detailed description, it will be understood to those of skill in the art that.According to disclosed all instructions, can carry out various modifications and replacement to those details, these change all within protection scope of the present invention.Four corner of the present invention is provided by claims and any equivalent thereof.
Claims (7)
1. the compound of formula I, or its isomer, pharmacologically acceptable salt and solvate.
2. pharmaceutical composition, it comprises the compound of formula I, or its isomer, pharmacologically acceptable salt and solvate, and pharmaceutically acceptable carrier, vehicle or thinner.
3. the described compound of claim 1 or its isomer, pharmacologically acceptable salt and solvate are used to prepare anti-apoptotic, the purposes of the medicine of prevention or treatment and apoptosis-related disease or symptom.
4. the described compound of claim 1 or its isomer, pharmacologically acceptable salt and solvate are used to prepare the purposes of the medicine of protection myocardial cell and prevention or treatment and apoptosis of cardiac muscle diseases associated or symptom.
5. method that prevents and/or treats with apoptosis of cardiac muscle diseases associated or symptom, described method comprises: the described pharmaceutical composition of claim 2 that gives therapeutic dose.
6. method of protecting the myocardial cell, described method comprises the described pharmaceutical composition of the claim 2 that gives therapeutic dose.
7. each described and apoptosis of cardiac muscle diseases associated or symptom of claim 3-5, comprise: the myocardial atrophy of hunger property, myocarditis, in heart failure, the myocardial damage that causes is poured in the myocardial damage that the myocardial damage that essential hypertension causes, acute myocardial infarction cause in early days, acute myocardial infarction again, myocardial cell's pathology that heart transplantation causes, the dysplasia myocardosis; Apoptosis of cardiac muscle that anoxic causes or cardiovascular systems sclerosis.
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| CN103387551A (en) * | 2012-05-11 | 2013-11-13 | 中国人民解放军军事医学科学院毒物药物研究所 | Thiazole compounds and uses thereof |
| WO2015090209A1 (en) * | 2013-12-20 | 2015-06-25 | 中国人民解放军军事医学科学院毒物药物研究所 | New urea compound, manufacturing method and application thereof |
| CN112007156A (en) * | 2019-05-30 | 2020-12-01 | 复旦大学 | Application of cannabinoid receptor drug in preparation of drug for treating myocardial cell necrotic apoptosis |
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| Publication number | Priority date | Publication date | Assignee | Title |
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| CN103387551A (en) * | 2012-05-11 | 2013-11-13 | 中国人民解放军军事医学科学院毒物药物研究所 | Thiazole compounds and uses thereof |
| WO2013166989A1 (en) * | 2012-05-11 | 2013-11-14 | 中国人民解放军军事医学科学院毒物药物研究所 | Thiazole compound and use thereof |
| CN103387551B (en) * | 2012-05-11 | 2015-05-20 | 中国人民解放军军事医学科学院毒物药物研究所 | Thiazole compounds and uses thereof |
| US9139543B2 (en) | 2012-05-11 | 2015-09-22 | Institute Of Pharmacology And Toxicology Academy Of Military Medical Sciences P.L.A. China | Thiazole compounds and uses thereof |
| WO2015090209A1 (en) * | 2013-12-20 | 2015-06-25 | 中国人民解放军军事医学科学院毒物药物研究所 | New urea compound, manufacturing method and application thereof |
| CN105814018A (en) * | 2013-12-20 | 2016-07-27 | 中国人民解放军军事医学科学院毒物药物研究所 | New urea compound, manufacturing method and application thereof |
| US9718770B2 (en) | 2013-12-20 | 2017-08-01 | Institute Of Pharmacology And Toxicology Academy Of Military Medical Sciences P.L.A. China | Substituted thioureas as heat shock protein 70 inhibitors |
| CN112007156A (en) * | 2019-05-30 | 2020-12-01 | 复旦大学 | Application of cannabinoid receptor drug in preparation of drug for treating myocardial cell necrotic apoptosis |
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