CN102241628B - (2E)-3-phenyl-N-[the chloro-1-of 2,2,2-tri-[[(8-quinolinyl-amino) sulphomethyl] is amino] ethyl]-2-acrylamide and medicinal use thereof - Google Patents
(2E)-3-phenyl-N-[the chloro-1-of 2,2,2-tri-[[(8-quinolinyl-amino) sulphomethyl] is amino] ethyl]-2-acrylamide and medicinal use thereof Download PDFInfo
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Abstract
The present invention relates to acrylamides or its isomer of formula I, pharmacologically acceptable salt and solvate, comprise this compound or its isomer, pharmacologically acceptable salt and solvate, and pharmaceutically acceptable carrier, the composition of vehicle or thinner, and described compound or composition are for preventing and/or treating the purposes in the disease relevant with apoptosis of cardiac muscle or symptom, including, but not limited to (i) inanition myocardial atrophy, (ii) myocarditis, (iii) in heart failure, (iv) myocardial damage that essential hypertension causes is treated or alleviates, v myocardial damage that () treatment or alleviation acute myocardial infarction cause in early days, (vi) myocardial damage that acute myocardial infarction Reperfu-sion causes is treated or alleviates, (vii) myocardial cell's pathology that heart transplantation causes is treated or alleviates, (viii) treatment or alleviation dysplasia myocardosis, or the apoptosis of cardiac muscle that anoxic causes, or improve the purposes of cardiovascular systems sclerosis.
Description
Technical field
The present invention relates to medicinal chemistry arts; particularly; the present invention relates to a kind of novel propylene amides (2E)-3-phenyl-N-[2; 2; the chloro-1-of 2-tri-[[(8-quinolinyl-amino) sulphomethyl] amino] ethyl]-2-acrylamide and pharmaceutical composition thereof; the invention still further relates to described compound and pharmaceutical composition thereof for anti-apoptotic; the purposes of prevention or treatment and apoptosis-related disease or symptom, especially for protection myocardial cell and the purposes in prevention or the treatment disease relevant with apoptosis of cardiac muscle or symptom.
Background technology
Apoptosis generally refers to that body cell is in growth course or under some factor effect, by the regulation and control of genes within cells and product thereof and a kind of apoptosis occurred.Apoptosis is prevalent in organic sphere, under both betiding physiological status, under also betiding pathological state.Fetal development and form are occurred, the defence of stable, the body of normal cell populations and immune response in tissue, disease or poisoning time cause cell injury, generation progress that is aging, tumour plays an important role, and is the focus of biomedical research always.
Research shows, has the generation of a lot of major disease all relevant with cell transition apoptosis, such as, in the evolution of acquired immune deficiency syndrome (AIDS), and CD4
+the minimizing of T cell number; In graft-rejection, the necrocytosis of cytotoxic T cell mediation; Reperfusion iujurt, the apoptosis of myocardial cell and neurocyte; Neuro-degenerative disorders (as Alzheimer time disease, Parkinson's disease etc.); Be exposed to the Various Tissues apoptosis etc. that ionizing rays causes.
Evidence suggests, the generation of apoptosis of cardiac muscle and many heart diseases, development and prognosis have close relationship.Find that the Myocardial death of infarct is not equal to myocardial necrosis by research apoptosis of cardiac muscle, apoptosis is one of mechanism of myocardial infarction, and be the major way of the early stage Myocardial death of infarct and the Myocardial death caused by ischemia/reperfusion, now a large amount of apoptosis of cardiac muscle, have increased the weight of myocardium destruction.1989, Nepomniashchikh etc. find when observing inanition myocardial atrophy ultrastructure, and the synthesis of myocardial ultramicrostructure albumen reduces, and cell count reduces, but companion cell nuclear phase should not reduce pro rata, the inanition myocardial atrophy of preliminary proposition is caused by apoptosis thus.1994, Gottlieb and Kawano etc. adopted Electronic Speculum to achieve the direct evidence of apoptosis of cardiac muscle in conjunction with DNA gel electrophoresis method, and the former discloses reperfusion injury and brings out Rabbit cardiomyocyte apoptosis, and the scorching patient of the latter's confirmed myocardial is occurred together apoptosis of cardiac muscle.In the neonatal rat myocardial cell that Tanaka etc. cultivate, also demonstrate the existence of apoptosis.Because the research of methodological progress and apoptosis is goed deep into, in multiple heart trouble, find the pathological effect of apoptosis of cardiac muscle.Research shows, spontaneously hypertensive mouse (SHR) heart damage is relevant with apoptosis; Late period turns to heart failure caused by apoptosis of cardiac muscle by Hypertrophic Heart; Acute myocardial infarction, except necrosis, blocks early stage and reperfusion injury also inducing apoptosis; Apoptosis of cardiac muscle sees heart and the right ventricular dysplasia myocardosis of transplanting equally, the same inducing cardiomyocytes apoptosis of anoxic.
Apoptosis has recoverability to a certain extent, and the apoptosis in myocardial infarction and ischemia/reperfusion has its feature and rule, utilizes its feature can prevent and reduce apoptosis, for clinical prevention ischemia/reperfusion injury provides enlightenment; In refilling process, the apoptosis producing contraction bands region (around infarct) induces generation by some inducements, and the restraining factors of apoptosis such as medicine etc. can be utilized to prevent apoptosis, the corresponding disease that treatment apoptosis causes.
But at present can for the medicament categories for anti-apoptotic and Cell protection of clinical application and quantity also little; selectivity and targeting are not high; therefore constantly research and develop the medicine of new anti-apoptotic safely and effectively and Cell protection, the medicine tool especially with brand-new mechanism of action is of great significance.
Summary of the invention
The object of the invention is to find and exploitation suppresses the micromolecular compound of apoptosis of cardiac muscle, be used for the various pathological changes prevented or treatment apoptosis of cardiac muscle causes.Contriver is through long-term, a large amount of experimental studies, and found a kind of acrylamides, it has anti-apoptotic, the effect of protection myocardial cell, can be used in prevention or treats the disease relevant with apoptosis of cardiac muscle or symptom.Particularly,
A first aspect of the present invention relates to the compound shown in formula I, or its isomer, pharmacologically acceptable salt and solvate.
Formula I, its chemical name is (2E)-3-phenyl-N-[the chloro-1-of 2,2,2-tri-[[(8-quinolinyl-amino) sulphomethyl] is amino] ethyl]-2-acrylamide.
The present invention relates to pharmaceutical composition, compound shown in its contained I, or its isomer, pharmacologically acceptable salt and solvate, and pharmaceutically acceptable carrier, vehicle or thinner on the other hand.
The invention still further relates to formula I or its isomer, pharmacologically acceptable salt and solvate, for the preparation of 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 or its isomer, pharmacologically acceptable salt and solvate, for the preparation of the purposes of the medicine of protection myocardial cell and prevention or the treatment disease relevant with apoptosis of cardiac muscle or symptom.
The invention still further relates to and a kind ofly prevent and/or treat the disease relevant with apoptosis of cardiac muscle or the method for symptom, described method comprises: the aforementioned pharmaceutical compositions giving therapeutic dose.
The invention still further relates to a kind of method protecting myocardial cell, described method comprises the aforementioned pharmaceutical compositions giving therapeutic dose.
The described disease relevant with apoptosis of cardiac muscle or symptom include but not limited to (i) inanition myocardial atrophy, (ii) myocarditis, (iii) in heart failure, (iv) myocardial damage that causes of essential hypertension, v myocardial damage that () acute myocardial infarction causes in early days, (vi) myocardial damage that causes of acute myocardial infarction Reperfu-sion, (vii) myocardial cell's pathology of causing of heart transplantation, (viii) dysplasia myocardosis; Or the apoptosis of cardiac muscle that anoxic causes, or cardiovascular systems sclerosis.
Compound of the present invention has the effect for the treatment of chronic heart failure.
Find that the pre-treatment of use formula I significantly can improve the apoptosis of cardiac muscle of tunicamycin induction by FCM analysis and classical TUNEL apoptosis detection method; and increase the trend that this anti-apoptotic provide protection has increase along with concentration, confirm that formula I is to the provide protection of apoptosis of cardiac muscle.
The present invention selects caspase-12 as the detected object confirming apoptosis path, find the expression having caspase-12 in the process of tunicamycin inducing cardiomyocytes apoptosis, and the formula I of use can make caspase-12 express minimizing, this formula I intervention can alleviate endocytoplasmic reticulum stress with the expression of caspase-12 subsequently, thus alleviate apoptosis.
In addition, the present invention confirms formula I when its maximum cell protection concentration and, time (TD50 > 100mM), there is no cytotoxic effect, and it does not protect the apoptosis irrelevant with er stress to stimulate.
The invention still further relates to the method preventing and/or treating the various diseases that apoptosis of cardiac muscle causes, it comprises and gives needs the above-mentioned patient prevented and/or treated the composition of at least one formula I or its solvate that prevent and/or treat significant quantity.
Those skilled in the art will recognize that compound of Formula I exists chiral centre.When being single enantiomorph when needing compound of Formula I, the reactant being all in single enantiomeric forms in all possible step can be used in prepare, or carry out reacting preparing under the reagent of single enantiomeric forms or the existence of catalyzer, or prepared by ordinary method fractionation stereoisomer mixture.Some preferred methods comprise use microorganism and split, split the salt of the diastereomer formed as any spendable acid such as amygdalic acid, camphorsulfonic acid, tartrate, lactic acid with chiral acid, or the salt of diastereomer that fractionation and chiral base are formed as brucine (bracine), cinchona alkaloid and derivative thereof etc.Conventional method is shown in " Enantiomers, RacematesandResolution " (WileyInterscience, 1981) that the people such as Jaques edit.
It will be appreciated by those skilled in the art that the compounds of this invention also can use with the form of its pharmacologically acceptable salt or solvate.On the physiology of compound of Formula I, acceptable salt comprises the salt of routine and the acid salt of quaternary ammonium that are formed by pharmaceutically acceptable mineral acid or organic acid or mineral alkali or organic bases.The example more specifically of suitable hydrochlorate comprises hydrochloric acid, Hydrogen bromide, sulfuric acid, phosphoric acid, nitric acid, perchloric acid, fumaric acid, acetic acid, propionic acid, succsinic acid, oxyacetic acid, formic acid, lactic acid, toxilic acid, tartrate, citric acid, flutters 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, although itself is not pharmaceutically acceptable, may be used for preparing the salt being used 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, chloroprocaine, choline, diethanolamine, quadrol, N-METHYL-ALPHA-L-GLUCOSAMINE 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, once administration, namely carries out chemical conversion by metabolic process, becomes the activated medicine of tool afterwards.Usually, this kind of prodrug is the functional derivatives of the compounds of this invention, and it easily changes into the compound of required formula (I) in vivo.Such as, at " DesignOfProdrugs ", HBundSaard, Elsevier edit, and describe the ordinary method selecting and prepare suitable prodrug derivatives in 1985.
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 the raceme of the compounds of this invention or optically active isomer and the pharmaceutically acceptable carrier of at least one, 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.Compound mentioned by the present invention also can be prepared to various pharmacologically acceptable salts.
Pharmaceutical composition of the present invention comprises the compound of Formula I of the present invention of effective dose or its pharmacologically acceptable salt or hydrate and one or more suitable pharmaceutically acceptable carrier.Here pharmaceutical carrier includes but not limited to: ion-exchanger, aluminum oxide, aluminum stearate, Yelkin TTS, serum protein is as human serum albumin, and buffer substance is as phosphoric acid salt, glycerine, Sorbic Acid, potassium sorbate, the partial glyceride mixtures 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 any-mode below: oral, spraying sucks, rectal application, nasal cavity applied medicine, cheek medication, local application, non-bowel medication, as in subcutaneous, vein, intramuscular, intraperitoneal, sheath, in ventricle, in 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 oral medication, the compounds of this invention can be made into any oral acceptable dosage form, 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 in addition as Magnesium Stearate.The thinner that capsule preparations uses generally comprises lactose and dried corn starch.Aqueous suspension preparation then normally by activeconstituents and suitable emulsifying agent and suspension agent used in combination.If needed, in above oral dosage form, also some sweeting agents, perfume compound or tinting material can be added.
When local application, particularly treat Local out dressing easy to reach and suffer from face or organ, during as eyes, skin or lower intestinal tract nervous system disease, according to different trouble faces or organ, the compounds of this invention can be made different using topical preparations forms, be described as follows:
When eye topical application, the compounds of this invention can be mixed with the dosage form of a kind of micronized suspension or solution, use carrier to be the Sterile Saline of isotonic certain pH, wherein can add also can not adding preservative agent as zephiran chloride alkoxide.For eye use, also compound can be made ointment as vaseline paste.
When topical application, the compounds of this invention can be made into suitable ointment, lotion or cream formulation form, is wherein suspended by activeconstituents 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; Lotion or the spendable carrier of creme include but not limited to: mineral oil, sorbitan monostearate, polysorbate60, cetyl ester wax, and cetene is fragrant and mellow, 2-Standamul G, benzyl alcohol and water.
The compounds of this invention can also the medication of aseptic injection preparation form, 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 sterilizing also can be used as solvent or suspension medium, as direactive glyceride or two glyceryl ester.
It may be noted that in addition, using dosage and the using method of the compounds of this invention depend on factors, comprise the age of patient, body weight, sex, natural health situation, nutritional status, the activity intensity of compound, Time of Administration, metabolic rate, the severity of illness and the subjective judgement of diagnosis and treatment doctor.
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 may be used for but be not limited to (i) inanition myocardial atrophy, (ii) myocarditis, (iii) in heart failure, (iv) myocardial damage that essential hypertension causes is treated or alleviates, v myocardial damage that () treatment or alleviation acute myocardial infarction cause in early days, (vi) myocardial damage that acute myocardial infarction Reperfu-sion causes is treated or alleviates, , (vii) myocardial cell's pathology that heart transplantation causes is treated or alleviates, (viii) treatment or alleviation dysplasia myocardosis, or improve the purposes of cardiovascular systems sclerosis, be the treatment disease that causes of apoptosis or symptom, particularly treat disease that apoptosis of cardiac muscle causes or symptom provides new method and access.
Accompanying drawing explanation
Fig. 1 different concns formula I is on the impact of eIF2 α and P-eIF2 alpha expression
The impact that Fig. 2 formula I is expressed TM inducing cardiomyocytes caspase-12 and cleavedcaspase-12
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 for illustration of the present invention, and should not be considered as limiting scope of the present invention.Unreceipted actual conditions person in embodiment, the condition of conveniently conditioned disjunction manufacturers suggestion is carried out.Agents useful for same or the unreceipted production firm person of instrument, being can by the conventional products of commercial acquisition.
embodiment 1: the experimental study of protection myocardial cell er stress apoptosis
Animal: newborn Wistar rats, in mouse 24h in age, male and female are not limit
the separation of myocardial cell and cultivation:
The separation of myocardial cell with cultivate the method (Kreider be separated with reference to differential velocity adherent, A.Messing, H.Doan, S.U.Kim, R.P.LisakandD.E.Pleasure, EnrichmentofSchwanncellculturesfromneonatalratsciaticner vebydifferentialadhesion, BrainRes2 (1981), pp.433444.), get Wistar suckling mouse newborn in 24h, obtain primary cardiomyocytes.
mtt assay detects different concns formula I to the impact of myocyte survival rate
By the Primary cultured myocardial cells of the separation according to aforesaid method acquisition according to every hole 10
4individual cell is inoculated into 96 orifice plates, every pore volume 100ul (the aseptic PBS of marginal pore fills).At 5%CO2, after 37 DEG C of incubators cultivate 4d, add the formula I (0.3 μM, 1 μM, 3 μMs, 10 μMs, 30 μMs, 100 μMs) of different concns respectively, each concentration arranges 3 multiple holes, zeroing hole (substratum, MTT, DMSO) is set simultaneously, control wells (nutrient solution, DMSO).After continuing to hatch process 48h, every hole adds 20ulMTT solution (5mg/ml joins i.e. 0.5%MTT with PBS<pH=7.4>), continues to cultivate 4h.Stop cultivating, carefully suck nutrient solution in hole.Every hole adds 150ulDMSO, puts low-speed oscillation 10min on shaking table, crystallisate is fully dissolved.Measure each hole absorbancy (OD) value at enzyme-linked immunosorbent assay instrument in wavelength 550nm place, every hole is repeated 5 times and is recorded result.The results are shown in Table 1:
Table 1MTT method detects formula I to the impact of myocyte survival rate
| Experimental group | Cell survival rate (× 100%) |
| Control group (DMSO) | 100 |
| Dosing group (0.3 μM) | 98.8562±6.7316 a |
| Dosing group (1 μM) | 91.6667±7.6257 a |
| Dosing group (3 μMs) | 94.5261±3.7997 a |
| Dosing group (10 μMs) | 102.7778±3.0645 a |
| Dosing group (30 μMs) | 105.8007±3.2639 a |
| Dosing group (100 μMs) | 104.2484±7.3625 a |
Compared with control group,
ap > 0.05;
Formula I, in 100 μMs of concentration, compares myocyte survival rate no difference of science of statistics with control group.The survival rate of formula I on normal myocardial cells does not affect.
mtt assay detects different concns formula I to the impact of the apoptosis of cardiac muscle that tunicamycin (Tunicamycin, TM) is induced
The myocardial cell of separation is inoculated into 96 orifice plates according to 104, every hole cell, every pore volume 100ul (the aseptic PBS of marginal pore fills).At 5%CO2, after 37 DEG C of incubators cultivate 4d, after giving the rear 30min of formula I (5 μMs, 10 μMs, 20 μMs) process of different concns respectively, add TM and make its final concentration be 5 μ g/ml, TM group and isometric DMSO control group that final concentration is 5 μ g/ml are set simultaneously.Each concentration arranges 3 multiple holes, arranges zeroing hole (substratum, MTT, DMSO), control wells (nutrient solution, DMSO) simultaneously.Continue to cultivate, every hole adds 20ulMTT solution (5mg/ml joins i.e. 0.5%MTT with PBS<pH=7.4>), continues to cultivate 4h.Stop cultivating, carefully suck nutrient solution in hole.Every hole adds 150ul dimethyl sulfoxide (DMSO), puts low-speed oscillation 10min on shaking table, crystallisate is fully dissolved.Each hole absorbancy (OD) value is measured in wavelength 490nm place at enzyme-linked immunosorbent assay instrument OD, every hole is repeated 3 times and is recorded result, the cell survival rate of each time point is calculated by A490 × 100% of cell survival rate=(A490 of the A490-TM process cell of control group untreated cell)/control group untreated cell, take time as X-coordinate, light absorption value is that ordinate zou draws cell growth curve.
The results are shown in Table 2:
Table 2MTT method detects formula I to the impact of TM inducing cardiomyocytes survival rate
Compare with DMSO group, #P < 0.05; Compare with TM group, * P < 0.05;
Result shows; TM intervention group obviously makes myocyte survival rate significantly decline (p < 0.05); all significantly increase (p < 0.05) is compared with TM group through the pretreated each group of cell survival rate of each concentration formula I; show the apoptosis of cardiac muscle that formula I can resist tunicamycin (TM) and causes, provide protection is served to the apoptosis of cardiac muscle of TM induction.
embodiment 2:westernBlot detects antiapoptotic signals protein expression
By the myocardial cell that obtains according to embodiment 1 primary myocardial cell culture method differential velocity adherent according to every hole 10
6individual cell is inoculated into 6 orifice plates, every pore volume 2ml, 37 DEG C, 5%CO2 incubator cultivates after 4d, empirically design the intervention of each component Bie Jiashi I, continue to be placed in 37 DEG C, after 5%CO2 incubator is cultured to design time point, with SDS sample loading buffer lysing cell, 100 DEG C of water-bath 10min, 4 DEG C centrifugal (12000rpm × 10min), collects supernatant.In advance nitrocellulose filter is immersed in 15 ~ 20min in transfering buffering liquid.With 50 μ g albumen/swimming lane loadings, after 10% polyacrylamide gel SDS-PAGE electrophoretic separation, then gel is laid in electrotransfer folder, from negative pole to positive pole, sponge, filter paper, gel, nitrocellulose filter, filter paper and sponge is housed successively.Constant current 350mA, electricity turns 45min.Electricity transferring film is to pvdf membrane, take out nitrocellulose filter, after TBST washes film 2min, 5% skim-milk room temperature closes 1h, add primary antibodie (1: 1000) 4 DEG C of overnight incubation, TBST washes film 3 times × 5min, then add HRP mark two resist, incubated at room temperature 1h, TBST washes film 3 times × 5min, soak 5min and 10min respectively with TSM1 and TSM2, be dissolved in 1mlTSM2 colour developing, to the clear after washing termination reaction of band with chromogenic substrate 6.6 μ lNBT and 3.3 μ lBCIP.After colour developing stops, scanning or Taking Pictures recording are preserved.
Detected result:
eIF2 α and P-eIF2 alpha expression
Different concns formula I effect 24hWestern blotting detection display, through 5%CO2,37 DEG C of incubators cultivate the myocardial cell of 4d, after giving formula I (1 μM, 2 μMs, 5 μMs, 10 μMs, 20 μMs, 40 μMs) the process 48h of different concns respectively, find that each hole eIF2 protein expression level is without remarkable change, and P-eIF2 α protein expression level increases with formula I concentration and increases gradually (see Fig. 1).
caspase-12 and Cleavedcaspase-12 expresses change
After the formula I process 24h of different concns, each group Caspase-12 expresses without obviously changing.Cleavedcaspase-12 expresses in DMSO group nothing, express obviously in TM (5 μ g/ml) induction group, adding the respective compounds of formula I group having used different concns (10 μMs, 20 μMs, 40 μMs), the increase Cleavedcaspase-12 along with formula I concentration expresses and reduces gradually.Show to activate in the apoptosis of cardiac muscle process that Cleavedcaspase-12 induces at TM, Cleavedcaspase-12 protein expression level increases with formula I concentration and declines (see Fig. 2) in dose-dependently.
embodiment 3: formula I is on the impact of the apoptosis of cardiac muscle that TM induces
The primary cardiomyocytes 4d cultivated according to embodiment 1 primary myocardial cell culture method starts 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+TM intervention group (10 μM of+5 μ g/ml), formula I intervention group (20 μ g/ml+5 μ g/ml), formula I intervention group (40 μ g/ml+5 μ g/ml).
flow cytomery apoptosis:after drug intervention 24h, by AnnexinV-FITC apoptosis kit method process cell, in 1h, detect apoptosis situation in order to flow cytometer (BDACScalibur, Becton-Dickinson company of the U.S.).Each sample collection 14000 cells are analyzed, experiment repetition 3 times, results averaged.
tUNEL detects: after drug intervention 24h, detect apoptotic cell by Nick End labeled in situ (TUNEL) method of deoxynucleotide terminal enzyme (DNA) mediation, by test kit, process cell is described.Using the cell climbing sheet of 0.1mg/mlDNaseI process as positive control.The visual field of random selecting more than 5 under light microscopic, apoptotic cell (in the karyon visible brown yellow granule) number in counting cells.Apoptosis rate=(positive stained cells check figure/all cells check figure) × 100%.
experimental result:each group of apoptosis rate compares:
pass through cells were tested by flow cytometry, normal cultured neonatal rat heart cells early apoptosis rate is 12.72%; After 5 μ g/mlTM induce 24h, apoptosis reaches 29.98%; Compare with TM group, 10 μMs of formula I groups, 20 μMs of formula I groups, 40 μMs of formula I group apoptosis rate significantly decline (P < 0.05), and it is on a declining curve to increase apoptosis rate with dosage.In table 3.
Table 3 formula I is on the impact of the apoptosis of cardiac muscle that TM induces
| Group | Flow cytomery apoptosis rate (%) |
| Control group (DMSO) | 12.72±0.96 |
| TM intervention group | 29.98±1.22 a |
| TM+ formula I 10 μMs | 22.98±1.35 ab |
| TM+ formula I 20 μMs | 20.13±0.87 ab |
| TM+ formula I 40 μMs | 18.64±0.83 ab |
Compared with DMSO group,
ap < 0.05; Compared with TM group
bp < 0.05
tUNEL detected result
DMSO group apoptosis rate is 7.86%, intervention group apoptosis rate after 24h is induced significantly to increase (P < 0.05) through 5 μ g/mLTM, compare with TM intervention group, add by formula I (10 μMs, 20 μMs, 40 μMs) group, apoptosis rate all obviously reduces (P < 0.05).Formula I 40 μMs group Formula I compounds 10 μMs, 20 μMs of groups are compared apoptosis rate and are declined obviously (P < 0.05)
Table 4 apoptosis rate compares
Compared with DMSO group,
ap < 0.05; Compared with TM group
bp < 0.05; Organize compare with a TM+ formula I20 μM group with TM+ formula I10 μM,
cp < 0.05
embodiment 4: formula I is to the provide protection of hypoxia inducible apoptosis of cardiac muscle
animal: newborn Wistar rats, in mouse 24h in age, male and female are not limit
cell prepares: the separation of myocardial cell with cultivate the method is separated with reference to differential velocity adherent, get Wistar suckling mouse newborn in 24h, through tincture of iodine alcohol disinfecting skin of chest abdomen, core and tiltedly open chest taking-up heart with scissors median line under xiphoid-process chest of opening slightly to the left and be placed in ice precooling PBS; 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, repeatedly rinses 2-3 time with 0.01MPBS; Fragment is placed in Erlenmeyer flask, adds 4ml0.125% pancreatin, 1ml0.1% collagenase II (final concentration is respectively 0.1% and 0.02%) 37 DEG C of water-bath concussion 10min, abandon supernatant; Again add 4ml0.125% pancreatin, 1ml0.1% collagenase II, 37 DEG C of water-bath concussion digestion 10min, draw supernatant and move to centrifuge tube, the DMEM added by supernatant containing 10%FBS stops digestion; Repeat water-bath concussion digestion step 3-4 time, till tissue block complete digestion; By the cell suspension of collection with after the centrifugal 10min of 1000rpm, remove supernatant, then it is resuspended to add substratum; Resuspended cell is inoculated in Tissue Culture Flask, be placed in 37 DEG C of CO2 incubators to hatch nutrient solution sucking-off after 1.5h, under the microscope after counting, with the DMEM nutrient solution adjustment cell density containing 10%FBS, 96 orifice plates are inoculated into by 1 × 104, be placed in 37 DEG C of later half amounts of 5%CO2 incubator 24h and change liquid, add the substratum containing 0.1%Brdu; Change liquid 1 time after every 48h afterwards, cultivate and can obtain primary cardiomyocytes after 4 days.
Solution prepares (following reagent can be purchased from Invitrogen company):
1.1 × washbuffer: the 10 × washbuffer adding 20ml enters 180ml ultrapure water, total amount to 200ml, be stored in 4 DEG C 7 days;
2. stationary liquid: 37% formaldehyde solution of 7.3ml is added in the 1 × washbuffer of 14.7ml, is preheating to before using 37 DEG C (this solution needs Extemporaneous).
3.1 × permeabilization buffer: the 10 × permeabilization buffer adding 4ml in 36ml distilled water, be stored in 4 DEG C 7 days;
4.Mitotracker/Hoechst solution (-20 DEG C of preservations): dissolve with the anhydrous DMSO of 94 μ L the solution that Mitotracker makes 1mM, this solution can be preserved 6 months under the condition of-20 DEG C of dry lucifuges.For avoiding repeatedly freeze-thaw to circulate, carry out the packing of single usage quantity.Be that the Mitotracker solution of 1mM and the Hoechst dye liquor of 11 μ L are added in cell culture medium 5.5 μ L concentration, obtain the application liquid (this solution needs Extemporaneous) that final volume is 5.5mL.
5.AlexaFluor488phalloidinSolution: with 140 μ L methyl alcohol AlexaFluor488phalloidin is dissolved into and makes mother liquor, this solution can be preserved 12 months under the condition of-20 DEG C of dry lucifuges.The AlexaFluor488phalloidin mother liquor of 27.5 μ L is added in the 1 × washbuffer of 5.5ml and makes application liquid (needing Extemporaneous).
experimental procedure:
1. the myocardial cell of separation is inoculated into 96 orifice plates according to 104, every hole cell, every pore volume 100 μ l (the aseptic PBS of marginal pore fills).At 5%CO2, after 37 DEG C of incubators cultivate 4d, after giving the rear 30min of formula I (5 μMs, 10 μMs, 20 μMs) process of different concns respectively, insert (O2/CO2 in 37 DEG C of airtight anoxic incubators, 5: 95) continue to hatch 16h, arrange anoxic control wells, normal control hole 5%CO2,37 DEG C of incubators cultivate 16h simultaneously.
2. 30min before completing anoxic and hatching, the substratum of 50 μ l and the Mitotracker/Hoechst solution of 50 μ l are added in every hole, continue 37 DEG C of incubated cell 30min.
3. the stationary liquid 100 μ l being preheated to 37 DEG C is added each culture hole respectively, and do not suck substratum, ventilation incubated at room 10min.
4. exhaust each hole solution (can buckle), washes 1 time with 1 × washbuffer (100 μ l/ hole).Want SC in operation and washing process, slow imbibition and liquid feeding are to keep the integrity of cell attachment and cell.
5. exhaust washbuffer (can buckle), adds 1 × permeabilization buffer (100 μ l/ hole) incubated at room 15min.
6. exhaust each hole permeabilization buffer (can buckle), washes 1 time with 1 × washbuffer (100 μ l/ hole).
7. exhaust each hole washbuffer (can buckle), and every hole adds 50 μ lAlexaFluor488phalloidin solution, and room temperature lucifuge hatches 30min.
8. exhaust each hole AlexaFluor488phalloidin solution (can buckle), washes 2 times, then add 1 × washbuffer (100 μ l/ hole) with 1 × washbuffer (100 μ l/ hole).
9. with sealed membrane, board edge is wrapped (preventing drying), HCSReader detects.
High content screening analytical method:
During apoptosis, usually there will be the remarkable change of morphocytology, and a series of change such as biochemistry and molecular marker.A lot of about apoptotic detection method, but mostly can only detect for the single index of the apoptosis of apoptotic cell.High content screening analysis is emerging a kind of apoptosis detection method in recent years, carries out multiplicity by specific fluorescent dye to apoptosis simultaneously.Three parameters that Main Analysis is relevant to apoptotic process: comprise karyomorphology change, mitochondrial swelling and or mitochondrial membrane potential, F-actin content.Complete cell is had to specificity and the reliability of height.
The cell that apoptosis occurs shows the nuclear alteration of two types usually, and nuclear fragmentation or core concentrate, and in the process of nuclear fragmentation, circular or oval core becomes lobulated, and are finally cracked into multiple subnucleus structure.Because the destruction of the structure components such as endonuclear euchromosome, kernel makes nuclear density increase.HCSReader observes nuclear morphology by Hoechst dyeing, and carries out quantitative comparison to nuclear area and core intensity.
Intramitochondrial change is the important morphological change of apoptosis, and plastosome, by adventitia release apoptosis gene factor, meanwhile, because mitochondrial permeability increases, also can make the electrochemistry ingredient change of mitochondrial inner membrane, cause decline or the disappearance of membrane potential.Under the stimulation of apoptosis, plastosome is also expanded and is become large, causes the increase of plastosome volume.The decline of mitochondrial membrane potential and the increase of plastosome volume, be acknowledged as the mark that apoptosis is early stage, by plastosome tracer agent
red carries out quantitatively.
The change of actin cytoskeleton has been in the news and has changed relevant important parameter as one with apoptosis, and in early days during apoptosis, F-actin content increases.By the staining agent Alexa that F-Actin muscle is special
the dyeing of 488Phalloidin (phalloidin), can determine F-actin content, carries out quantification compare apoptotic degree.
Formula I is to the provide protection detected result of hypoxia inducible apoptosis of cardiac muscle:
1) long term voyage detected result:
| Group | Long term voyage |
| Control group | 113.86±3.06 |
| Anoxic group | 98.26±1.00 |
| Formula I 5 μMs of groups | 109.90±3.97 |
| Formula I 10 μMs of groups | 107.43±7.77 |
| Formula I 20 μMs of groups | 111.90±10.76 |
2) core detected intensities:
| Group | Core intensity |
| Control group | 122.70±2.38 |
| Anoxic group | 118.23±1.33 |
| Formula I 5 μMs of groups | 124.90±2.25 |
| Formula I 10 μMs of groups | 123.21±2.71 |
| Formula I 20 μMs of groups | 121.40±2.24 |
3) lightflux detected result:
| Group | light flux |
| Control group | 1247.52±48.059 |
| Anoxic group | 1044.91±16.53 |
| Formula I 5 μMs of groups | 1201.92±29.95 |
| Formula I 10 μMs of groups | 1166.12±91.06 |
| Formula I 20 μMs of groups | 1214.09±107.59 |
4) actin fiber detected result:
| Group | Fibre strength | The short major axis ratio of fiber |
| Control group | 461.77±8.68 | 0.74±0.00 |
| Anoxic group | 541.01±41.14 | 0.77±0.01 |
| Formula I 5 μMs of groups | 498.03±76.67 | 0.75±0.02 |
| Formula I 10 μMs of groups | 503.64±49.23 | 0.74±0.01 |
| Formula I 20 μMs of groups | 471.83±69.38 | 0.74±0.02 |
5). mitochondrial membrane potential detected result:
| Group | Membrane potential |
| Control group | 1±0.01 |
| Anoxic group | 96.04％±3.61％ |
| Formula I 5 μMs of groups | 96.69％±5.06％ |
| Formula I 10 μMs of groups | 97.19％±2.82 |
| Formula I 20 μMs of groups | 99.40％±2.80％ |
6) cell counting detected result:
| Group | Cell counting |
| Control group | 1±0.11 |
| Anoxic group | 71.51％±5.73％ |
| Formula I 5 μMs of groups | 88.57％±7.64％ |
| Formula I 10 μMs of groups | 98.03％±16.24 |
| Formula I 20 μMs of groups | 101.73％±22.42％ |
Compare with control group, the cell counting of anoxic group have dropped 28.49%, and after employing the formula I (5 μMs, 10 μMs, 20 μMs) of different concns, compare cell counting with anoxic group and increase respectively (17.06%, 26.52% and 30.22%).
Compare with control group, anoxic group causes the declines such as nuclear area, core intensity, intensity of illumination, actin fiber, mitochondrial membrane potential and cell counting, formula I can significantly improve the above-mentioned each desired value of myocardial cell under anoxic condition, the apoptosis of cardiac muscle effect that High content screening result shows to have clear improvement formula I tool anoxic causes.
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 amendment 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 (3)
1. the compound or pharmaceutically acceptable salt thereof of formula I is for the preparation of the purposes of the medicine of protection myocardial cell and prevention or the treatment disease relevant with apoptosis of cardiac muscle or symptom
2. the method protecting myocardial cell in vitro of non-treatment object, described method comprises the compound or pharmaceutically acceptable salt thereof of the contained I giving therapeutic dose, and the pharmaceutical composition of pharmaceutically acceptable carrier, vehicle or thinner
3. purposes according to claim 1, wherein relevant with apoptosis of cardiac muscle disease or symptom comprise: inanition myocardial atrophy, myocarditis, in heart failure, the myocardial damage that essential hypertension causes, the myocardial damage that acute myocardial infarction causes in early days, the myocardial damage that acute myocardial infarction Reperfu-sion causes, myocardial cell's pathology that heart transplantation causes, dysplasia myocardosis; The apoptosis of cardiac muscle that anoxic causes or cardiovascular systems sclerosis.
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