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

Abstract

The present invention relates to an acrylamide compound of formula I or its isomer, pharmaceutically acceptable salt and solvate, including the compound or its isomer, pharmaceutically acceptable salt and solvate, and pharmaceutically acceptable carrier, excipient Formulation or diluent composition, and the use of said compound or composition for preventing and/or treating diseases or symptoms related to cardiomyocyte apoptosis, including but not limited to (i) starvation myocardial atrophy, (ii) myocarditis, (iii) heart failure, (iv) treatment or alleviation of myocardial injury caused by essential hypertension, (v) treatment or alleviation of myocardial injury caused by early acute myocardial infarction, (vi) treatment or alleviation of acute myocardial infarction Myocardial injury caused by infarct reperfusion, (vii) treatment or alleviation of cardiomyocyte lesion caused by heart transplantation, (viii) treatment or alleviation of dysplastic cardiomyopathy; or cardiomyocyte apoptosis caused by hypoxia, or improvement of cardiovascular system sclerosis the use of.

Description

(2E)-3-phenyl-N-[2,2,2-trichloro-1-[[(8-quinolylamino)thiomethyl]amino]ethyl]-2-acrylamide and its medical use

technical field

The present invention relates to the field of medicinal chemistry, specifically, the present invention relates to a novel acrylamide compound (2E)-3-phenyl-N-[2,2,2-trichloro-1-[[(8-quinoline Base amino) thiomethyl] amino] ethyl] -2-acrylamide and its pharmaceutical composition, the present invention also relates to said compound and its pharmaceutical composition for anti-apoptosis, prevention or treatment and apoptosis Use for related diseases or symptoms, especially for protecting cardiomyocytes and preventing or treating diseases or symptoms related to cardiomyocyte apoptosis.

Background technique

Apoptosis generally refers to a kind of programmed cell death that occurs during the development of cells in the body or under the action of certain factors through the regulation of intracellular genes and their products. Apoptosis is ubiquitous in the biological world, and occurs in both physiological and pathological conditions. It plays an important role in embryonic development and morphogenesis, the stability of normal cell populations in tissues, the defense and immune response of the body, cell damage caused by disease or poisoning, aging, and the occurrence and progression of tumors. It has always been a hot spot in biomedical research.

Studies have shown that the occurrence of many major diseases is related to excessive apoptosis, such as the decrease in the number of CD4 ⁺ T cells in the development of AIDS; cell death mediated by cytotoxic T cells in transplant rejection; ischemia And reperfusion injury, apoptosis of cardiomyocytes and nerve cells; degenerative diseases of the nervous system (such as Alzheimer's disease, Parkinson's disease, etc.); apoptosis of various tissue cells caused by exposure to ionizing radiation, etc.

Evidence shows that cardiomyocyte apoptosis is closely related to the occurrence, development and prognosis of many heart diseases. Through the study of myocardial cell apoptosis, it is found that myocardial death in infarction is not equal to myocardial necrosis. Apoptosis is one of the mechanisms of myocardial infarction, and it is the main way of myocardial death in the early stage of infarction and myocardial death caused by ischemia/reperfusion. At this time, myocardial A large number of apoptosis, aggravated the destruction of myocardium. In 1989, when Nepomniashchikh et al. observed the ultrastructure of starvation myocardial atrophy, they found that the synthesis of myocardial cell structure protein decreased, and the number of cells decreased, but there was no corresponding decrease in the nucleus, which preliminarily proposed that starvation myocardial atrophy was caused by apoptosis. due to. In 1994, Gottlieb and Kawano obtained direct evidence of cardiomyocyte apoptosis by electron microscopy combined with DNA gel electrophoresis. The former revealed that reperfusion injury induced apoptosis of rabbit cardiomyocytes, and the latter confirmed that myocarditis patients were accompanied by cardiomyocyte apoptosis. The existence of apoptosis was also proved in the neonatal rat cardiomyocytes cultured by Tanaka et al. Due to the advancement of methodology and the in-depth study of apoptosis, the pathological role of cardiomyocyte apoptosis has been found in many heart diseases. Studies have shown that heart damage in spontaneously hypertensive mice (SHR) is related to apoptosis; late hypertrophy to heart failure is caused by cardiomyocyte apoptosis; in addition to necrosis in acute myocardial infarction, early infarction and reperfusion injury also induce apoptosis. Cardiomyocyte apoptosis is also seen in transplanted hearts and right ventricular dysplastic cardiomyopathy, and hypoxia also induces cardiomyocyte apoptosis.

Apoptosis is reversible to a certain extent. Apoptosis in myocardial infarction and ischemia/reperfusion has its characteristics and rules. Using its characteristics can prevent and reduce apoptosis, which provides a basis for clinical prevention of ischemia/reperfusion. Injury provides enlightenment; in the process of reperfusion, the apoptosis in the contractile zone area (around the infarct) is induced by some inducements, and the inhibitory factors of apoptosis, such as drugs, can be used to prevent apoptosis and treat apoptosis-induced corresponding diseases.

However, the types and quantities of drugs for anti-apoptosis and cell protection available for clinical application are still very small, and their selectivity and targeting are not high. Drugs for cells, especially those with new mechanisms of action, are of great significance.

Contents of the invention

The purpose of the present invention is to search for and develop small molecule compounds that inhibit cardiomyocyte apoptosis, so as to prevent or treat various pathological changes caused by cardiomyocyte apoptosis. After long-term and extensive experimental research, the inventors have discovered an acrylamide compound, which can resist apoptosis and protect cardiomyocytes, and can be used to prevent or treat diseases or symptoms related to cardiomyocyte apoptosis. specifically,

The first aspect of the present invention relates to the compound represented by formula I, or its isomer, pharmaceutically acceptable salt and solvate.

Formula I compound, its chemical name is (2E)-3-phenyl-N-[2,2,2-trichloro-1-[[(8-quinolylamino)thiomethyl]amino]ethyl ]-2-acrylamide.

Another aspect of the present invention relates to a pharmaceutical composition, which comprises the compound represented by formula I, or its isomer, pharmaceutically acceptable salt and solvate, and a pharmaceutically acceptable carrier, excipient or diluent.

The present invention also relates to the use of the compound of formula I or its isomers, pharmaceutically acceptable salts and solvates for preparing anti-apoptosis, preventing or treating diseases or symptoms related to apoptosis.

The present invention also relates to the use of the compound of formula I or its isomers, pharmaceutically acceptable salts and solvates for preparing medicines for protecting cardiomyocytes and preventing or treating diseases or symptoms related to cardiomyocyte apoptosis.

The present invention also relates to a method for preventing and/or treating diseases or symptoms related to cardiomyocyte apoptosis, the method comprising: administering a therapeutic amount of the above-mentioned pharmaceutical composition.

The present invention also relates to a method for protecting cardiomyocytes, the method comprising administering a therapeutic amount of the above-mentioned pharmaceutical composition.

The diseases or symptoms related to cardiomyocyte apoptosis include but are not limited to (i) starvation myocardial atrophy, (ii) myocarditis, (iii) heart failure, (iv) myocardial injury caused by essential hypertension, (v ) Myocardial injury caused by early acute myocardial infarction, (vi) Myocardial injury caused by reperfusion after acute myocardial infarction, (vii) Myocyte lesion caused by heart transplantation, (viii) Dysplastic cardiomyopathy; or Myocyte caused by hypoxia Apoptosis, or hardening of the cardiovascular system.

The compound of the present invention has the effect of treating chronic heart failure.

Through flow cytometry and the classic TUNEL apoptosis detection method, it was found that pretreatment with the compound of formula I can significantly improve the cardiomyocyte apoptosis induced by tunicamycin, and the anti-apoptotic protective effect tends to increase as the concentration increases. The protective effect of the compound of formula I on cardiomyocyte apoptosis was confirmed.

The present invention selects caspase-12 as the detection object for confirming the apoptosis pathway, and finds that there is caspase-12 expression in the process of tunicamycin-induced cardiomyocyte apoptosis, and the use of the compound of formula I can reduce the expression of caspase-12, which means It shows that the intervention of the compound of formula I can reduce the stress of endoplasmic reticulum and the subsequent expression of caspase-12, thereby reducing cell apoptosis.

In addition, the present invention proves that the compound of formula I has no cytotoxic effect at its maximum cytoprotective concentration (TD50>100mM), and it does not protect against apoptotic stimuli unrelated to endoplasmic reticulum stress.

The present invention also relates to a method for preventing and/or treating various diseases caused by cardiomyocyte apoptosis, which comprises administering a preventive and/or therapeutically effective amount of at least one compound of formula I or a solvate thereof to those in need of the aforementioned prevention and/or treated patients.

Those skilled in the art will recognize the presence of chiral centers in compounds of general formula I. When a compound of general formula I is desired as a single enantiomer, it can be prepared using reactants that are in single enantiomeric form in all possible steps, or reagents or catalysts that are in single enantiomeric form The reaction is carried out in the presence of , or it is prepared by resolution of a mixture of stereoisomers by conventional methods. Some preferred methods include resolution using microorganisms, resolution of diastereomeric salts with chiral acids such as mandelic acid, camphorsulfonic acid, tartaric acid, lactic acid, etc. Diastereoisomer salts formed by sex bases such as bracine, cinchona alkaloids and their derivatives. Commonly used methods are found in "Enantiomers, Racemates and Resolution" edited by Jaques et al. (Wiley Interscience, 1981).

Those skilled in the art will appreciate that the compounds of the present invention may also be used in the form of their pharmaceutically acceptable salts or solvates. The physiologically acceptable salts of the compounds of general formula I include conventional salts formed from pharmaceutically acceptable inorganic or organic acids or bases and acid addition salts of quaternary ammoniums. More specific examples of suitable acid salts include hydrochloric acid, hydrobromic acid, sulfuric acid, phosphoric acid, nitric acid, perchloric acid, fumaric acid, acetic acid, propionic acid, succinic acid, glycolic acid, formic acid, lactic acid, maleic acid, tartaric acid , citric acid, pamoic acid, malonic acid, hydroxymaleic acid, phenylacetic acid, glutamic acid, benzoic acid, salicylic acid, fumaric acid, toluenesulfonic acid, methanesulfonic acid, naphthalene-2-sulfonic acid, benzene Salts of sulfonic acid, hydroxynaphthoic acid, hydroiodic acid, malic acid, steroic acid, tannic acid, etc. Other acids, such as oxalic acid, although not pharmaceutically acceptable in themselves, can be used in the preparation of salts used as intermediates to obtain the compounds of the invention and their pharmaceutically acceptable salts. More specific examples of suitable base salts include sodium, lithium, potassium, magnesium, aluminum, calcium, zinc, N,N'-dibenzylethylenediamine, chloroprocaine, choline, diethanolamine, ethyl Diamine, N-methylglucamine and procaine salts. Hereinafter, references to compounds of the present invention include compounds of general formula I and pharmaceutically acceptable salts and solvates thereof.

The present invention also includes prodrugs of the compounds of the present invention, which, upon administration, are chemically transformed by metabolic processes and then become active drugs. Typically, such prodrugs are functional derivatives of the compounds of the invention which are readily converted in vivo to the desired compound of formula (I). General methods for selecting and preparing suitable prodrug derivatives are described, for example, in "Design Of Prodrugs", H Bund Saard, Elsevier ed., 1985.

The present invention also includes active metabolites of the compounds of the present invention.

Another aspect of the present invention relates to a pharmaceutical composition, which contains the racemate or optical isomer of the compound of the present invention and at least one pharmaceutically acceptable carrier, which can be used for in vivo therapy and has biocompatibility. The pharmaceutical composition can be prepared in various forms according to different administration routes. The compounds mentioned in the present invention can also be prepared into various pharmaceutically acceptable salts.

The pharmaceutical composition of the present invention comprises an effective dose of the compound of general formula I of the present invention or a pharmaceutically acceptable salt or hydrate thereof and one or more suitable pharmaceutically acceptable carriers. The pharmaceutical carriers here include but are not limited to: ion exchangers, alumina, aluminum stearate, lecithin, serum proteins such as human albumin, buffer substances such as phosphate, glycerol, sorbic acid, potassium sorbate, saturated vegetable Partial glyceride mixtures of fatty acids, water, salts or electrolytes such as protamine sulfate, disodium hydrogen phosphate, potassium hydrogen phosphate, sodium chloride, zinc salts, colloidal silicon dioxide, magnesium trisilicate, polyvinylpyrrolidone, Cellulosic substances, macrogol, sodium carboxymethylcellulose, polyacrylates, beeswax, lanolin.

The pharmaceutical compositions of the compounds of this invention may be administered in any of the following ways: oral, inhalation spray, rectal, nasal, buccal, topical, parenteral, e.g. subcutaneous, intravenous, intramuscular, intraperitoneal, sheath Intravenous, intraventricular, intrasternal and intracranial injection or infusion, or with the aid of an explanted reservoir. Among them, oral, intraperitoneal or intravenous administration is preferred.

When administered orally, the compounds of the present invention may be prepared in any orally acceptable preparation form, including but not limited to tablets, capsules, aqueous solutions or suspensions. Wherein, the carrier that tablet uses generally comprises lactose and cornstarch, also can add lubricating agent such as magnesium stearate in addition. Diluents used in capsule formulations generally include lactose and dried cornstarch. Aqueous suspensions are usually prepared by mixing the active ingredient with suitable emulsifying and suspending agents. If desired, some sweetening, flavoring or coloring agents may also be added to the above oral preparation forms.

When using locally, especially when treating the affected areas or organs that are easily accessible by local external application, such as eyes, skin or lower intestinal nerve diseases, the compound of the present invention can be made into different topical preparations according to different affected areas or organs form, as follows:

When administered topically to the eye, the compounds of the present invention may be formulated as a micronized suspension or solution in the form of isotonic sterile saline at a pH with or without the addition of a preservative such as benzyl chloride. alkyl alkoxides. For ophthalmic use, the compounds may also be formulated in ointments such as petrolatum.

When applied topically to the skin, the compounds of the invention may be formulated in suitable ointments, lotions or creams, wherein the active ingredients are suspended or dissolved in one or more carriers. Carriers that can be used in ointment formulations include, but are not limited to: mineral oil, liquid petrolatum, white petrolatum, propylene glycol, polyethylene oxide, polypropylene oxide, emulsifying wax, and water; carriers that can be used in lotions or creams include, but are not limited to: mineral oil Oil, sorbitan monostearate, Tween 60, cetyl esters wax, cetyl aryl alcohol, 2-octyldodecanol, benzyl alcohol and water.

The compounds of this invention can also be administered in the form of sterile injectable preparations, including sterile injectable aqueous or oily suspensions or sterile injectable solutions. Among them, usable vehicles and solvents include water, Ringer's solution and isotonic sodium chloride solution. In addition, sterile, fixed oils, such as mono- or diglycerides, can be employed as a solvent or suspending medium.

In addition, it should be pointed out that the dose and method of use of the compounds of the present invention depend on many factors, including the patient's age, body weight, sex, natural health status, nutritional status, activity intensity of the compound, time of administration, metabolic rate, severity of the disease, and The subjective judgment of the treating physician.

Beneficial Effects of the Invention

The present invention provides an acrylamide compound, and proves that it is a kind of potent anti-cardiomyocyte apoptosis agent, so it can be used for but not limited to (i) starvation myocardial atrophy, (ii) myocarditis, (iii) Heart failure, (iv) treatment or alleviation of myocardial injury caused by essential hypertension, (v) treatment or alleviation of myocardial injury caused by acute myocardial infarction, (vi) treatment or alleviation of myocardial injury caused by acute myocardial infarction reperfusion, , (vii) treating or alleviating cardiomyocyte lesions caused by heart transplantation, (viii) treating or alleviating dysplastic cardiomyopathy; or improving cardiovascular system sclerosis, for treating diseases or symptoms caused by apoptosis, especially for treating Diseases or symptoms caused by cardiomyocyte apoptosis provide new methods and approaches.

Description of drawings

Figure 1 Effects of different concentrations of formula I compounds on the expression of eIF2α and P-eIF2α

Effect of Fig. 2 formula I compound on TM-induced cardiomyocyte caspase-12 and cleavedcaspase-12 expression

Detailed ways

Embodiments of the present invention will be described in detail below in conjunction with examples, but those skilled in the art will understand that the following examples are only used to illustrate the present invention, and should not be considered as limiting the scope of the present invention. Those who do not indicate the specific conditions in the examples are carried out according to the conventional conditions or the conditions suggested by the manufacturer. The reagents or instruments used were not indicated by the manufacturer, and they were all commercially available conventional products.

Embodiment 1 : Experimental research on protecting cardiomyocyte endoplasmic reticulum stress apoptosis

Animals: Newborn Wistar rats, within 24 hours of age, male or female

Isolation and culture of cardiomyocytes :

Isolation and culture of cardiomyocytes refer to the method of differential adhesion separation (Kreider, A.Messing, H.Doan, S.U.Kim, R.P.Lisakand D.E.Pleasure, Enrichment of Schwanncellcultures from neonatal ratsciatic nerve by differential adhesion, BrainRes2 (1981), pp.433444.), take 24h Primary cardiomyocytes were obtained from newborn Wistar suckling mice.

Effects of Different Concentrations of Compounds of Formula I on the Survival Rate of Cardiomyocytes by MTT Assay

The isolated primary cultured cardiomyocytes obtained by the above method were inoculated into a 96-well plate at 10 ⁴ cells per well, with a volume of 100 ul per well (edge wells were filled with sterile PBS). After incubating in a 5% CO2, 37°C incubator for 4 days, add different concentrations of the compound of formula I (0.3 μM, 1 μM, 3 μM, 10 μM, 30 μM, 100 μM), set 3 replicate wells for each concentration, and set the zero adjustment well (medium, MTT, DMSO), control wells (medium, DMSO). After continued incubation for 48 hours, 20ul of MTT solution (5 mg/ml, 0.5% MTT in PBS<pH=7.4>) was added to each well, and the culture was continued for 4 hours. Terminate the culture, and carefully aspirate the culture medium in the well. Add 150ul of DMSO to each well, shake on a shaker at low speed for 10min, and fully dissolve the crystals. The absorbance (OD) value of each well was measured at a wavelength of 550 nm by an enzyme-linked immunosorbent assay instrument, and each well was repeated 5 times and the results were recorded. The results are shown in Table 1:

Table 1 MTT method detects the impact of formula I compound on cardiomyocyte viability

test group Cell viability (×100%) Control group (DMSO) 100 Drug-dosed group (0.3μM) 98.8562±6.7316 ^a Drug-dosed group (1μM) 91.6667±7.6257 ^a Drug-dosed group (3μM) 94.5261±3.7997 ^a Drug-dosed group (10μM) 102.7778±3.0645 ^a Drug-dosed group (30μM) 105.8007±3.2639 ^a Drug-dosed group (100μM) 104.2484±7.3625 ^a

Compared with the control group, ^a P>0.05;

Compared with the control group, the compound of formula I has no statistical difference in the survival rate of cardiomyocytes within the concentration of 100 μM. It shows that the compound of formula I has no effect on the survival rate of normal cardiomyocytes.

Effects of different concentrations of compounds of formula I on cardiomyocyte apoptosis induced by tunicamycin (TM) by MTT assay

The isolated cardiomyocytes were inoculated into a 96-well plate according to 104 cells per well, and the volume of each well was 100ul (edge wells were filled with sterile PBS). After culturing in an incubator at 5% CO2 at 37°C for 4 days, give different concentrations of compounds of formula I (5 μM, 10 μM, 20 μM) and treat them for 30 minutes, then add TM to make the final concentration 5 μg/ml, and set the final concentration to 5 μg /ml of TM group and the same volume of DMSO control group. Three replicate wells were set for each concentration, and zero-setting wells (medium, MTT, DMSO) and control wells (culture solution, DMSO) were set at the same time. To continue culturing, add 20ul of MTT solution (5mg/ml, mixed with PBS<pH=7.4>, that is, 0.5% MTT) into each well, and continue culturing for 4h. Terminate the culture, and carefully aspirate the culture medium in the well. Add 150ul of dimethyl sulfoxide to each well, shake on a shaker at low speed for 10min, and fully dissolve the crystals. Measure the absorbance (OD) value of each well at the wavelength 490nm at the OD of the enzyme-linked immunosorbent detector, repeat 3 times in each well and record the results, according to the cell survival rate=(the A490 of the A490-TM treated cells of the control group untreated cells)/ The A490×100% of untreated cells in the control group was used to calculate the cell survival rate at each time point, and the cell growth curve was drawn with time as the abscissa and absorbance value as the ordinate.

The results are shown in Table 2:

Table 2 MTT method detects the effect of formula I compound on TM-induced cardiomyocyte survival rate

Compared with DMSO group, #P<0.05; compared with TM group, *P<0.05;

The results showed that the TM intervention group significantly decreased the survival rate of cardiomyocytes (p<0.05), and the cell survival rates of each group pretreated with the compound of formula I at various concentrations were significantly increased compared with the TM group (p<0.05), indicating that the formula Compound I can resist the apoptosis of cardiomyocytes induced by tunicamycin (TM), and has a protective effect on the apoptosis of cardiomyocytes induced by TM.

Example 2: Detection of apoptosis signal protein expression by WesternBlot

The cardiomyocytes obtained by differential adherence according to the primary cardiomyocyte culture method in Example 1 were inoculated into a ⁶ -well plate at 10 cells per well, with a volume of 2 ml per well. According to the experimental design, each group was intervened with the compound of formula I respectively, and continued to be placed in a 37°C, 5% CO2 incubator to cultivate until the designed time point, and the cells were lysed with SDS loading buffer, 100°C water bath for 10min, and centrifuged at 4°C (12000rpm× 10min), and the supernatant was collected. Soak the nitrocellulose membrane in transfer buffer for 15-20 minutes in advance. Load 50 μg protein/lane, separate by 10% polyacrylamide gel SDS-PAGE electrophoresis, then spread the gel in the electrotransfer holder, and install sponge, filter paper, gel, nitric acid in order from the negative electrode to the positive electrode Cellulose membranes, filter papers and sponges. Constant current 350mA, electric transfer 45min. Electrotransfer the membrane to PVDF membrane, take out the nitrocellulose membrane, wash the membrane with TBST for 2 minutes, block with 5% skimmed milk powder at room temperature for 1 hour, add primary antibody (1:1000) and incubate overnight at 4°C, wash the membrane 3 times × 5 minutes with TBST, and then add HRP Labeled secondary antibody, incubated at room temperature for 1h, washed with TBST for 3 times×5min, soaked in TSM1 and TSM2 for 5min and 10min respectively, dissolved in 1mlTSM2 with 6.6μl NBT and 3.3μlBCIP for color development, and washed with water until the bands were clear Stop the reaction. After the color development is terminated, the scanning or photographing records are saved.

Test results:

eIF2α and P-eIF2α expression

24h Western blot detection of different concentrations of compounds of formula I showed that after 5% CO2, cardiomyocytes cultured in a 37° C. Afterwards, it was found that the expression level of eIF2 protein in each well had no significant change, while the expression level of P-eIF2α protein gradually increased with the increase of the concentration of the compound of formula I (see Figure 1).

Expression changes of caspase-12 and Cleavedcaspase-12

After being treated with different concentrations of the compound of formula I for 24 hours, the expression of Caspase-12 in each group had no obvious change. Cleavedcaspase-12 was not expressed in the DMSO group, but was significantly expressed in the TM (5 μg/ml) induction group. In the groups of various formula I compounds with different concentrations (10 μM, 20 μM, 40 μM), with the increase of the concentration of the formula I compound, Cleavedcaspase -12 expression gradually decreased. It shows that Cleavedcaspase-12 is activated during TM-induced cardiomyocyte apoptosis, and the protein expression level of Cleavedcaspase-12 decreases in a dose-dependent manner as the concentration of the compound of formula I increases (see Figure 2).

Embodiment 3 : the effect of the compound of formula I on the cardiomyocyte apoptosis induced by TM

The primary cardiomyocytes cultured according to the method for culturing primary cardiomyocytes in Example 1 began to be added with tunicamycin (TM) for the intervention experiment at 4 days. The cells were randomly divided into 5 groups: solvent control group (DMSO), TM intervention group (5ug/ml), formula I compound+TM intervention group (10μM+5μg/ml), formula I compound intervention group (20μg/ml+5μg/ml ), the compound intervention group of formula I (40 μg/ml+5 μg/ml).

Cell apoptosis detected by flow cytometry: After 24 hours of drug intervention, the cells were treated according to the method of AnnexinV-FITC cell apoptosis kit, and the cell apoptosis was detected by flow cytometry (BDACScalibur, Becton-Dickinson Company, USA) within 1 hour. 14,000 cells were collected from each sample for analysis, the experiment was repeated 3 times, and the results were averaged.

TUNEL detection : After 24 hours of drug intervention, the apoptotic cells were detected by deoxynucleotidyl terminal transferase-mediated nick end in situ labeling (TUNEL) method, and the cells were treated according to the instructions of the kit. Cell slides treated with 0.1 mg/ml DNaseI were used as a positive control. Under the light microscope, more than 5 visual fields were randomly selected, and the number of apoptotic cells (brown yellow particles were visible in the nucleus) in the cells was counted. Apoptosis rate=(Number of positively stained nuclei/Number of all nuclei)×100%.

Experimental results: comparison of apoptosis rate in each group:

As determined by flow cytometry , the early apoptosis rate of normal cultured neonatal rat cardiomyocytes was 12.72%; after 24 hours of induction with 5 μg/ml TM, the apoptosis rate reached 29.98%; group and the 40 μM formula I compound group significantly decreased the apoptosis rate of cardiomyocytes (P<0.05), and the apoptosis rate showed a downward trend with the dose increasing. See Table 3.

The effect of table 3 formula I compound on the cardiomyocyte apoptosis induced by TM

group Cardiomyocyte apoptosis rate detected by flow cytometry (%) Control group (DMSO) 12.72±0.96 TM intervention group 29.98±1.22 ^a TM + Formula I 10 μM 22.98± ^1.35ab TM + Formula I 20 μM 20.13± ^0.87ab TM + Formula I 40 μM 18.64± ^0.83ab

Compared with DMSO group, ^a P<0.05; compared with TM group, ^b P<0.05

TUNEL test results

The apoptosis rate in the DMSO group was 7.86%. After being induced by 5 μg/mLTM for 24 hours, the apoptosis rate in the intervention group increased significantly (P<0.05). group, the apoptosis rate was significantly reduced (P<0.05). Compared with the 10 μ M and 20 μ M groups of formula I compound 40 μ M, the apoptosis rate decreased significantly (P < 0.05)

Table 4 Comparison of cardiomyocyte apoptosis rate

Compared with DMSO group, ^a P<0.05; compared with TM group, ^b P<0.05; compared with TM+Formula I10μM group and TM+Formula I20μM group, ^c P<0.05

Embodiment 4 : Formula I compound is to hypoxia-induced cardiomyocyte apoptosis protective effect

Animals : Newborn Wistar rats, within 24 hours of age, male or female

Cell preparation : Isolation and culture of cardiomyocytes refer to the method of differential adherence separation. Newborn Wistar suckling mice were taken within 24 hours, and the skin of the chest and abdomen was disinfected with iodine alcohol. Take out the heart by oblique thoracotomy and place it in ice-cold PBS; blow the heart gently with 0.01M PBS to remove blood cells and other tissues, cut the heart into 0.5mm3 pieces, and rinse repeatedly with 0.01MPBS 2-3 times Place the fragments in the Erlenmeyer flask, add 4ml0.125% trypsin, 1ml0.1% collagenase II (final concentration is 0.1% and 0.02% respectively) 37 ℃ water bath and shake for 10min, discard the supernatant; add 4ml0 again .125% trypsin, 1ml 0.1% collagenase II, 37 ℃ water bath shaking digestion for 10min, absorb the supernatant and transfer to a centrifuge tube, add the supernatant to DMEM containing 10% FBS to stop the digestion; repeat the water bath shaking digestion steps 3-4 centrifuge the collected cell suspension at 1000rpm for 10min, remove the supernatant, and resuspend in the medium; inoculate the resuspended cells into cell culture flasks and incubate at 37°C with CO2 After incubating in the box for 1.5h, suck out the culture solution, count under the microscope, adjust the cell density with DMEM culture solution containing 10% FBS, inoculate 1×104 into a 96-well plate, and place it in a 5% CO2 incubator at 37°C After 24 hours, half of the medium was changed, and medium containing 0.1% Brdu was added; after that, the medium was changed once every 48 hours, and primary cardiomyocytes were obtained after 4 days of culture.

Solution preparation (the following reagents can be purchased from Invitrogen):

1.1×washbuffer: Add 20ml of 10×washbuffer into 180ml of ultrapure water, the total amount reaches 200ml, store at 4°C for 7 days;

2. Fixative solution: add 7.3ml of 37% formaldehyde solution to 14.7ml of 1×washbuffer, and preheat to 37°C before use (this solution needs to be prepared temporarily).

3. 1× permeation buffer: add 4ml of 10× permeation buffer to 36ml double distilled water, store at 4°C for 7 days;

4. Mitotracker/Hoechst solution (stored at -20°C): Dissolve Mitotracker in 94 μL of anhydrous DMSO to make a 1 mM solution. This solution can be stored at -20°C in a dry and dark condition for 6 months. To avoid multiple freeze-thaw cycles, aliquots are made for single use. Add 5.5 μL of 1 mM Mitotracker solution and 11 μL of Hoechst staining solution to the cell culture medium to obtain an application solution with a final volume of 5.5 mL (this solution needs to be prepared temporarily).

5. AlexaFluor488phalloidin Solution: Dissolve AlexaFluor488phalloidin in 140 μL of methanol to make a mother solution. This solution can be stored at -20°C in a dry and dark condition for 12 months. Add 27.5μL of AlexaFluor488phalloidin master solution to 5.5ml of 1×washbuffer to make application solution (temporary preparation is required).

Experimental steps :

1. The isolated cardiomyocytes were inoculated into a 96-well plate according to 104 cells per well, with a volume of 100 μl per well (edge wells were filled with sterile PBS). After culturing in a 5% CO2, 37°C incubator for 4 days, they were treated with different concentrations of formula I (5 μM, 10 μM, 20 μM) for 30 minutes, and then placed in a closed 37°C hypoxic incubator (O2/CO2, 5: 95) Continue to incubate for 16 hours, and at the same time set hypoxia control wells, normal control wells with 5% CO2, and incubate in a 37°C incubator for 16 hours.

2. 30 minutes before the completion of the hypoxic incubation, add 50 μl of medium and 50 μl of Mitotracker/Hoechst solution to each well, and continue to incubate the cells at 37° C. for 30 minutes.

3. Add 100 μl of fixative solution preheated to 37°C to each culture well without aspirating the medium, and incubate at room temperature for 10 minutes in a ventilated place.

4. Aspirate the solution in each well (you can pinch the plate), and wash once with 1×washbuffer (100μl/well). Take special care during handling and washing, and slowly aspirate and add liquid to maintain cell attachment and cell integrity.

5. Aspirate the washbuffer (you can pinch the plate), add 1× permeation buffer (100 μl/well) and incubate at room temperature for 15 minutes.

6. Absorb the permeation buffer in each well (you can pinch the plate), and wash once with 1×washbuffer (100 μl/well).

7. Aspirate the washbuffer of each well (you can pinch the plate), add 50 μl AlexaFluor488phalloidin solution to each well, and incubate at room temperature for 30 minutes in the dark.

8. Aspirate the AlexaFluor488phalloidin solution in each well (the plate can be pinched), wash twice with 1×washbuffer (100μl/well), and then add 1×washbuffer (100μl/well).

9. Wrap the edge of the board with parafilm (to prevent drying), and detect it on the HCSReader.

High-content screening assays:

When cells undergo apoptosis, there are usually significant changes in cell morphology, as well as a series of changes in biochemical and molecular markers. There are many methods for detecting apoptosis, but most of them can only detect a single indicator of apoptosis in apoptotic cells. High-content screening analysis is a new method for apoptosis detection that has emerged in recent years. It uses specific fluorescent staining to simultaneously perform multi-factor analysis on apoptosis. Three parameters related to the apoptosis process were mainly analyzed: including nuclear morphological changes, mitochondrial swelling and/or mitochondrial membrane potential, and F-actin content. Highly specific and reliable for intact cells.

Apoptotic cells usually exhibit two types of nuclear changes, nuclear fragmentation or nuclear condensation, in which round or oval nuclei become lobulated and eventually split into multiple subunits. nuclear structure. Due to the destruction of structural components such as autosomes and nucleoli in the nucleus, the nuclear density increases. HCSReader can observe the morphology of cell nuclei through Hoechst staining, and quantitatively compare the area and intensity of nuclei.

Changes in mitochondria are important morphological changes in apoptosis. Mitochondria release apoptosis gene factors through the outer membrane. At the same time, because of the increase in mitochondrial permeability, the electrochemical composition of the inner mitochondrial membrane can also be changed, resulting in a decrease in membrane potential or disappear. Under the stimulation of apoptosis, mitochondria also expand and enlarge, causing an increase in mitochondrial volume. The decline of mitochondrial membrane potential and the increase of mitochondrial volume have been recognized as early markers of apoptosis, which can be detected by mitochondrial tracer Red for quantification.

Changes in the actin cytoskeleton have been reported as an important parameter associated with altered apoptosis, with increased F-actin content during early apoptosis. Via the F-actin-specific stain Alexa The staining of 488 Phalloidin (submuscarine) can determine the content of F-actin and quantify the degree of apoptosis.

The protective effect detection result of the compound of formula I to cardiomyocyte apoptosis induced by hypoxia:

1) Nuclear area test results:

group nuclear area control group 113.86±3.06 hypoxia group 98.26±1.00 Formula I compound 5μM group 109.90±3.97 Formula I compound 10μM group 107.43±7.77 Formula I compound 20μM group 111.90±10.76

2) Nuclear intensity test results:

group nuclear strength control group 122.70±2.38 hypoxia group 118.23±1.33 Formula I compound 5μM group 124.90±2.25 Formula I compound 10μM group 123.21±2.71 Formula I compound 20μM group 121.40±2.24

3) lightflux detection results:

group light flux control group 1247.52±48.059 hypoxia group 1044.91±16.53 Formula I compound 5μM group 1201.92±29.95 Formula I compound 10μM group 1166.12±91.06 Formula I compound 20μM group 1214.09±107.59

4) Actin fiber detection results:

group Fiber Strength Fiber short to long axis ratio control group 461.77±8.68 0.74±0.00 hypoxia group 541.01±41.14 0.77±0.01 Formula I compound 5μM group 498.03±76.67 0.75±0.02 Formula I compound 10μM group 503.64±49.23 0.74±0.01 Formula I compound 20μM group 471.83±69.38 0.74±0.02

5). Mitochondrial membrane potential detection results:

group membrane potential control group 1±0.01 hypoxia group 96.04%±3.61% Formula I compound 5μM group 96.69%±5.06% Formula I compound 10μM group 97.19%±2.82 Formula I compound 20μM group 99.40%±2.80%

6) Cell count test results:

group cell counts control group 1±0.11 hypoxia group 71.51%±5.73% Formula I compound 5μM group 88.57%±7.64% Formula I compound 10μM group 98.03%±16.24 Formula I compound 20μM group 101.73%±22.42%

Compared with the control group, the cell counts in the hypoxia group decreased by 28.49%, and after using different concentrations of the formula I compound (5 μM, 10 μM, 20 μM), compared with the hypoxia group, the cell counts increased respectively (17.06%, 26.52% and 30.22%).

Compared with the control group, the hypoxia group caused the decrease of nucleus area, nucleus intensity, light intensity, actin fibers, mitochondrial membrane potential and cell count, etc., and the compound of formula I could obviously improve the above-mentioned index values of cardiomyocytes under the hypoxia state, The results of high-content screening show that the compound of formula I can significantly improve cardiomyocyte apoptosis caused by hypoxia.

Although specific embodiments of the present invention have been described in detail, those skilled in the art will understand. Based on all the teachings that have been disclosed, various modifications and substitutions can be made to those details, and these changes are all within the scope of the invention. The full scope of the invention is given by the appended claims and any equivalents 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.