NO donor compound and preparation method, pharmaceutical composition and application thereof

Abstract

The present invention discloses a class of NO donor compounds and their preparation methods, pharmaceutical compositions and applications. The compound structure is shown in Formula I. The NO donor compounds and their pharmaceutical compositions can be prepared as drugs for treating myocardial hypoxia injury diseases as hypoxia-activated NO donors. The prepared drugs can exert their efficacy at both the cellular level and the animal level, and the synthesis method of the compounds is simple and easy to operate.

Description

Technical Field

[0001] The present invention relates to a class of NO donor compounds and a preparation method, a pharmaceutical composition and application thereof, and in particular to a class of NO donor compounds which can be prepared as medicines for treating myocardial hypoxia injury diseases and a preparation method, a pharmaceutical composition and application thereof. Background Art

[0002] Coronary heart disease is myocardial damage caused by myocardial tissue ischemia (hypoxia at the cellular level) due to organic stenosis or obstruction of the coronary arteries, also known as ischemic heart disease. According to a data report released by my country's Cardiovascular Disease Center in 2020, the number of people suffering from cardiovascular diseases such as coronary heart disease in China has reached 290 million. The current high incidence and mortality of coronary heart disease pose a serious threat to people's health. Since nitric oxide (NO) donor drugs can release exogenous NO molecules in the body to promote vasodilation, they are often used to treat coronary heart disease caused by ischemia. However, existing drugs lack selectivity for hypoxia and cannot release sufficient NO in the hypoxic area, which brings serious side effects.

[0003] In recent years, hypoxia-activated prodrugs have attracted extensive attention. They can promote the selective release of active drug groups in hypoxic microenvironments, thereby reducing off-target toxicity. Given that hypoxia is an important factor causing myocardial injury, applying this design concept to the structural design of NO donor drugs to obtain hypoxia-activated prodrugs is an effective method to improve the efficacy of such drugs and reduce side effects. Nitroreductase (NTR) exists in the heart, liver, kidney, lung, and brain tissues of mammals. It can catalyze the reduction of nitroaromatic compounds by providing hydrogen with NADPH and NADH under anaerobic conditions. Therefore, nitroaromatic compounds are a recognized hypoxia-activated group and can be used in the design of hypoxia-activated prodrugs. This is because NTR in hypoxic cells can selectively reduce the nitro group on the aromatic ring, thereby promoting the release of active drug groups from prodrugs through intramolecular electron transfer. Since NTR is highly expressed in hypoxic cells, it can effectively promote the concentrated release of active drug groups in prodrugs in the hypoxic microenvironment. Therefore, the hypoxia-activated properties of nitroaromatic compounds are utilized to combine them with NO donors to obtain prodrugs, which can release sufficient amounts of NO under hypoxia activation, which is of great significance for the treatment of cardiovascular diseases such as coronary heart disease. Summary of the invention

[0004] Purpose of the invention: In order to solve the problems of poor efficacy and large side effects of existing NO donor drugs in treating myocardial hypoxic injury, the present invention aims to provide a class of NO donor compounds that can effectively improve myocardial hypoxic injury in a hypoxic microenvironment, as well as their preparation method, pharmaceutical composition and application.

[0005] Technical solution: As the first aspect of the present invention, the NO donor compound of the present invention has a structure of formula I:

[0006]

[0007] in:

[0008] R 1 When it is nitro, R 2 is hydrogen, halogen or C1-C4 alkyl, R 3 is hydrogen, halogen or C1-C4 alkyl;

[0009] R 2 When it is nitro, R 1 is hydrogen, halogen or C1-C4 alkyl, R 3 It is hydrogen, halogen or C1-C4 alkyl.

[0010] The invention bonds a nitro aromatic compound with a hypoxia activated group to an NO donor molecule through a connecting group to obtain a hypoxia activated NO donor compound.

[0011] Preferably, in the structure:

[0012] R 1 For nitro, R 2 is hydrogen, halogen or methyl, R 3 For hydrogen.

[0013] More specifically, the NO providing compound is selected from any of the following compounds:

[0014]

[0015] As the second aspect of the present invention, the preparation method of the NO donor compound is to obtain compound I by acylation reaction of compound 1 and compound 2. The specific synthesis steps are as follows:

[0016] (1) 4-Hydroxy(N-methyl)aniline hemisulfate reacts with NaNO2 in the presence of acetic acid to obtain compound 1; in step (i), the reaction temperature is 0°C, the solvent is acetic acid (AcOH), and the reaction time is 3h;

[0017] (2) a benzyl alcohol derivative is reacted with bis(trichloromethyl) carbonate (triphosgene, BTC) in the presence of N,N-diisopropylethylamine (DIPEA) to obtain compound 2; in step (ii), the reaction temperature is room temperature, the solvent is tetrahydrofuran (THF), and the reaction time is 5 h;

[0018] (3) Compound 1 and Compound 2 react in the presence of N,N-diisopropylethylamine to obtain product I; in step (iii), the reaction temperature is room temperature, the solvent is dichloromethane (DCM), and the reaction time is 5 h.

[0019]

[0020] Among them, R 1 , R 2 , R 3 The definition of is as mentioned above.

[0021] As the third aspect of the present invention, the pharmaceutical composition of the present invention comprises the NO donor compound and a pharmaceutically acceptable carrier.

[0022] The NO donor compound can be added with a pharmaceutically acceptable carrier to prepare common pharmaceutical preparations, such as tablets, capsules, syrups, suspensions or injections. The preparations can be added with common pharmaceutical excipients such as flavors, sweeteners, liquid / solid fillers, diluents, etc.

[0023] As the fourth aspect of the present invention, the NO donor compound and the pharmaceutical composition of the present invention can be prepared as a drug for treating myocardial hypoxic injury diseases. The above compound is a hypoxia-activated NO donor compound, which is used to improve the viability of myocardial cells under hypoxic conditions, and is specifically used to treat coronary heart disease. The above compound can release NO in cells in the hypoxic microenvironment, effectively improving myocardial hypoxic injury.

[0024] Beneficial effects: Compared with the prior art, the present invention has the following significant advantages:

[0025] (1) Such NO donor compounds and pharmaceutical compositions can effectively release NO under hypoxic conditions, with the release amount reaching more than 40%, and can improve the vitality of cardiomyocytes, and can serve as hypoxia-activated NO donors both in vivo and in vitro;

[0026] (2) Such NO donor compounds and pharmaceutical compositions are widely used and can be prepared as drugs for treating myocardial hypoxia and injury-related diseases; the drugs can exert their efficacy at both the cellular level and the animal level, and have a more excellent therapeutic effect;

[0027] (3) The preparation method of the compound is simple and easy to operate. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] Figure 1a The compound improves the vitality of myocardial cells H9c2 under normoxic conditions;

[0029] Figure 1b The compound improves the vitality of cardiomyocytes H9c2 under hypoxic conditions;

[0030] Figure 2 is the NO release level of compound I1 in the heart of hypoxia model mice;

[0031] Figure 3 is the expression level of mTORC1 and TSC2-P proteins in mouse heart. DETAILED DESCRIPTION

[0032] The technical solution of the present invention is further described below in conjunction with embodiments.

[0033] Example 1: Preparation of N-methyl-N-nitroso-p-phenol (Compound 1)

[0034]

[0035] Add 4-hydroxy(N-methyl)aniline hemisulfate (1.722 g, 10.0 mmol) to 50 mL of acetic acid under light-proof conditions and dissolve it by ultrasound. Add sodium nitrite (1.380 g, 20.0 mmol) dissolved in 10 ml of water to the acetic acid solution under ice-water bath cooling, and continue to react for 3 h under ice-water bath after the addition. After the reaction is completed, transfer the reaction solution to a beaker, add 50 mL of water, adjust the pH to 7 with saturated sodium bicarbonate aqueous solution, then extract the aqueous solution with ethyl acetate, combine the organic phases, dry over anhydrous sodium sulfate, and spin dry to obtain 1.46 g of product with a yield of 96%.

[0036] 1 H NMR (600MHz, DMSO-d6): δ9.79 (s, 1H), 7.41 (d, J = 8.8Hz, 2H), 6.89 (d, J = 8.8Hz, 2H), 3.38 (s, 3H) ppm.

[0037] Example 2: Preparation of p-nitrobenzyl chloroformate (Compound 2a)

[0038]

[0039] Dissolve p-nitrobenzyl alcohol (0.168 g, 1.1 mmol) and BTC (0.296 g, 1.0 mmol) in 20 mL of anhydrous tetrahydrofuran, add N,N-diisopropylethylamine (0.142 g, 1.1 mmol) under ice-water bath cooling, and react for 30 min under nitrogen protection. Then react at room temperature for 5 h. After the reaction is completed, concentrate the reaction solution, remove excess phosgene with a small amount of anhydrous tetrahydrofuran, and add anhydrous dichloromethane and use it directly in the next step reaction.

[0040] Example 3: Preparation of 4-((methylnitroso)amino)phenyl(4-nitrobenzyl)carbonate (Compound I1)

[0041]

[0042] Compound 1 (0.152 g, 1.0 mmol) and N,N-diisopropylethylamine (0.258 g, 2.0 mmol) were dissolved in 20 mL of anhydrous dichloromethane. The anhydrous dichloromethane solution containing compound 2a obtained in Example 2 was slowly dripped into the above solution under ice-water bath cooling. After the dripping was completed, the ice-water bath was removed and the reaction was carried out at room temperature for 5 h. The reaction solution was washed with 0.5 M dilute hydrochloric acid, water, and saturated brine for 3 times respectively, and the organic phase was separated, dried over anhydrous sodium sulfate overnight, and then concentrated by rotary evaporation to obtain a yellow solid, which was recrystallized with a mixed solvent of ethyl acetate and petroleum ether to obtain 285 mg of light yellow crystals with a yield of 86%.

[0043] 1 H NMR (600MHz, DMSO-d6): δ8.28–8.30(m,2H),7.74–7.76(m,2H),7.70–7.72(m,2H),7.45–7.48(m,2H),5.46(s,2H),3.44(s,3H)ppm. 13 C NMR (150MHz, DMSO-d6): δ153.15,150.02,147.87,143.07,140.37,129.32,124.15,122.79,121.20,69.03,32.14ppm.HRMS(m / z)(ESI):calcd for C 15 H 14 N3O6[M+H] + :332.08,found:332.11.

[0044] Example 4: Preparation of 4-((methylnitroso)amino)phenyl(2-chloro-4-nitrobenzyl)carbonate (Compound I2)

[0045]

[0046] Referring to the method described in Examples 2 and 3, using 2-chloro-4-nitrobenzyl alcohol as raw material, compound I2 was obtained as light yellow crystals with a yield of 23%.

[0047] 1 H NMR (600MHz, DMSO-d6): δ8.38-8.39(d,J=2.3Hz,1H),8.28-8.30(dd,1H),7.88-7.90 (d,J=8.5Hz,1H),7.71–7.72(m,2H),7.46-7.48(m,2H),5.49(s,2H),3.44(s,3H)ppm. 13C NMR (150MHz, DMSO-d6): δ152.99,149.95,148.49,140.41,140.18,133.62,131 .17,124.78,122.96,122.77,121.25,66.92,32.20ppm.HRMS(m / z)(ESI):calcd forC 15 H 13 ClN3O6Na[M+Na] + :388.03,found:388.10.

[0048] Example 5: Preparation of 4-((methylnitroso)amino)phenyl(2-methyl-4-nitrobenzyl)carbonate (Compound I3)

[0049]

[0050] Referring to the method described in Examples 2 and 3, using 2-methyl-4-nitrobenzyl alcohol as raw material, compound I3 was obtained as light yellow crystals with a yield of 45%.

[0051] 1 H NMR (600MHz, DMSO-d6): δ8.15-8.16 (d, J=2.2Hz, 1H), 8.10-8.14 (dd, J=8.4, 2.4Hz, 1H), 7.71-7.73 (d,J=2.2Hz,1H),7.68–7.70(m,2H),7.45–7.48(m,2H),5.44(s,2H),3.44(s,3H),2.47(s,3H)ppm. 13 C NMR (150MHz, DMSO-d6): δ153.11,150.01,147.78,141.17,140.37,139.16,129.71 ,125.06,122.80,121.49,121.21,67.66,32.17,18.77ppm.HRMS(m / z)(ESI):calcd for C 16 H 15 N3O6[M+H] + :346.10,found:346.10.

[0052] Example 6: Preparation of 4-((methylnitroso)amino)phenyl(2-nitrobenzyl)carbonate (Compound I4)

[0053]

[0054] Referring to the method described in Examples 2 and 3, 2-nitrobenzyl alcohol was used as raw material to obtain compound I4 as pale yellow crystals with a yield of 73%.

[0055] 1 H NMR (600MHz, DMSO-d6): δ8.17-8.19(dd,1H),7.85-7.88(m,1H),7.79–7.80(m,1H),7 .70–7.73(m,2H),7.68–7.69(m,1H),7.45–7.47(m,2H),5.65(s,2H),3.44(s,3H)ppm. 13 C NMR (150MHz, DMSO-d6): δ153.04,149.98,147.82,140.40,134.85,130.81,130 .25,130.06,125.49,122.77,121.24,67.09,32.16ppm.HRMS(m / z)(ESI):calcd for C 15 H 14 N3O6[M+H] + :332.09,found:332.02..

[0056] Example 7: Preparation of 4-((methylnitroso)amino)phenyl(2-nitro-4-chlorobenzyl)carbonate (Compound I5)

[0057]

[0058] Referring to the method described in Examples 2 and 3, using 2-nitro-4-chlorobenzyl alcohol as raw material, compound I5 was obtained as light yellow crystals with a yield of 46%.

[0059] 1 H NMR (600MHz, DMSO-d6): δ8.26-8.27(d,J=2.2Hz,1H),7.94-7.96(dd,1H),7.81-7.83 (d,J=8.4Hz,1H),7.70–7.72(m,2H),7.45–7.46(m,2H),5.62(s,2H),3.44(s,3H)ppm. 13 C NMR (150MHz, DMSO-d6): δ152.94,149.96,148.47,140.42,134.51,134.21,131 .84,129.81,125.32,122.75,121.24,66.53,32.15ppm.HRMS(m / z)(ESI):calcd forC 15 H 13ClN3O6Na[M+Na] + :388.03,found:388.00.

[0060] Example 8: Preparation of 4-((methylnitroso)amino)phenyl(2-nitro-4-bromobenzyl)carbonate (Compound I6)

[0061]

[0062] Referring to the method described in Examples 2 and 3, using 2-nitro-4-bromobenzyl alcohol as raw material, compound I6 was obtained as light yellow crystals with a yield of 33%.

[0063] 1 H NMR (600MHz, DMSO-d6): δ8.36-8.37(d,J=2.1Hz,1H),8.07-8.09(dd,1H),7.73-7.75 (d,J=8.3Hz,1H),7.70–7.72(m,2H),7.44–7.46(m,2H),5.60(s,2H),3.44(s,3H)ppm. 13 C NMR (150MHz, DMSO-d6): δ152.94,149.96,148.49,140.42,137.45,131.94,13 0.19,128.03,122.75,122.16,121.24,66.58,32.15.HRMS(m / z)(ESI):calcd forC 15 H 13 BrN3O6[M+H] + :431.98,found:431.90.

[0064] Example 9: Preparation of 4-((methylnitroso)amino)phenyl(2-nitro-4-methylbenzyl)carbonate (Compound I7)

[0065]

[0066] Referring to the method described in Examples 2 and 3, using 2-nitro-4-methylbenzyl alcohol as raw material, compound I7 was obtained as light yellow crystals with a yield of 51%.

[0067] 1 H NMR (600MHz, DMSO-d6): δ8.00–8.01(m,1H),7.70–7.72(m,2H),7.66-7.67(d ,J=0.9Hz,2H),7.43–7.46(m,2H),5.60(s,2H),3.44(s,3H),2.44(s,3H)ppm.13 C NMR (150MHz, DMSO-d6): δ153.04,149.99,147.77,140.57,140.38,135.29,130.22 ,127.75,125.56,122.74,121.20,67.02,32.12,20.74ppm.HRMS(m / z)(ESI):calcd for C 16 H 16 N3O6[M+H] + :346.10,found:345.90.

[0068] Example 10: Preparation of 4-((methylnitroso)amino)phenyl(2-nitro-5-chlorobenzyl)carbonate (Compound I8)

[0069]

[0070] Referring to the method described in Examples 2 and 3, using 2-nitro-5-chlorobenzyl alcohol as raw material, compound I8 was obtained as light yellow crystals with a yield of 37%.

[0071] 1 H NMR (600MHz, DMSO-d6): δ8.21-8.23(d,J=8.8Hz,1H),7.83-7.84(d,J=2.3Hz,1H),7. 76-7.79(dd,1H),7.71–7.73(m,2H),7.46–7.48(m,2H),5.65(s,2H),3.44(s,3H)ppm. 13 C NMR (150MHz, DMSO-d6): δ152.82,149.98,146.36,140.43,139.48,133.27,130 .05,129.67,127.63,122.76,121.23,66.56,32.14ppm.HRMS(m / z)(ESI):calcd forC 15 H 13 ClN3O6Na[M+Na] + :388.03,found:388.00.

[0072] Example 11: Preparation of 4-((methylnitroso)amino)phenyl(2-nitro-5-methylbenzyl)carbonate (Compound I9)

[0073]

[0074] Referring to the method described in Examples 2 and 3, using 2-nitro-5-methylbenzyl alcohol as raw material, compound I9 was obtained as light yellow crystals with a yield of 48%.

[0075] 1 H NMR (600MHz, DMSO-d6): δ8.09-8.11(d,J=8.4Hz,1H),7.72-7.73(d,J=2.2Hz,1H),7.71-7.72(d,J=2.3Hz,1H),7.57–7.58(m ,1H),7.48–7.49(m,1H),7.46-7.47(d,J=2.3Hz,1H),7.45-7.46(d,J=2.2Hz,1H),5.63(s,2H),3.44(s,3H),2.47(s,3H)ppm. 13 C NMR (150MHz, DMSO-d6): δ153.00,150.01,145.93,145.49,140.40,130.95,130.47 ,130.26,125.70,122.78,121.24,67.21,32.15,21.53ppm.HRMS(m / z)(ESI):calcd for C 16 H 15 N3O6Na[M+Na] + :368.09,found:368.10.

[0076] Example 12: Griess method for detecting NO release level after the compound interacts with myocardial cells H9c2 under normoxia or hypoxia

[0077] Cardiomyocyte H9c2 is revived, DMEM culture medium is used to cultivate, and is placed in a carbon dioxide incubator and cultivated to make it adherent growth. After the cell reaches suitable density, it is inoculated in a multi-well plate, placed in a carbon dioxide incubator, and cultivated for 24h to cell adhesion at 37°C, 5%CO2 and saturated humidity conditions, and supernatant is discarded, fresh DMEM culture medium 2mL is added, and then the compound and reference substance medicine isosorbide dinitrate of certain concentration (5,10,15 μM) are added. After cultivating 4h in a normoxic incubator (37°C, 21%O2) or anoxic incubator (37°C, 1%O2), supernatant is removed, and then the cells in the plate are collected in 1.5mL centrifuge tubes respectively, 1000rpm, centrifugal 5min, supernatant is discarded, 1mL PBS is added to resuspend cells, centrifugal again, PBS is discarded, 100 μL cell lysates (NO is measured exclusively) are added in the centrifuge tube, half a minute is placed on ice, and then 4°C of lower 13300rpm centrifugal 5min, supernatant is collected. Take 50 μL of supernatant in a 96-well plate, set up three parallel groups for each sample, and add 50 μL of Griess reagent I and 50 μL of Griess reagent II successively. Measure the OD value of each well with a full-wavelength microplate reader at a wavelength of 540 nm, and calculate the NO concentration using the standard curve. The results are shown in Tables 1 and 2.

[0078] Table 1. NO release in H9c2 cardiomyocytes treated with compounds for 4 h under normoxia

[0079]

[0080] *. NO release amount = measured NO concentration / calculated NO concentration, data are expressed as the average SD of three independent experiments: *p<0.05,

[0081] **p<0.01;

[0082] a. Indicates not measured, b. Calculated based on 1 / 2 of the release amount.

[0083] Table 2. NO release in myocardial cells H9c2 after treatment with compounds for 4 hours under hypoxia

[0084]

[0085] *. NO release amount = measured NO concentration / calculated NO concentration, data are expressed as the average SD of three independent experiments: *p<0.05,

[0086] **p<0.01;

[0087] a. Indicates not measured, b. Calculated based on 1 / 2 of the release amount.

[0088] The data in Table 1 and Table 2 show that the amount of NO released in the cardiomyocytes after the treatment of the compounds at different concentrations under normoxia is very small, which is lower than the maximum release of compound I2 (<3.34%, 10 μM); but under hypoxia, except for compounds I5-I9, the treatment of cardiomyocytes with compounds I1-I4 at different concentrations can effectively lead to the release of NO in the cells, with the release amount ranging from 20.15% to 45.45%, showing a hypoxia-activated NO donor. The reference substance isosorbide dinitrate (ISDN) releases a certain amount of NO after treating cardiomyocytes under normoxia or hypoxia; it leads to a large amount of NO released in the cells under normoxia, up to 41.63% (10 μM), and can also lead to the release of NO in the cells under hypoxia, but the release amount is less than that under normoxia, and lower than the release level of compounds I2 and I3.

[0089] Example 13: Effect of the compound on the vitality of myocardial cells H9c2 under normoxia or hypoxia

[0090] Wash the cells twice with 5mL PBS, add 1mL trypsin to digest the monolayer cultured cardiomyocytes H9c2, prepare a single cell suspension with a serum-containing culture solution, and inoculate 5000-10000 cells per well in a 96-well culture plate, with a culture medium volume of 100μL per well, and fill the edge wells with sterilized pure water. Move the culture plate into a carbon dioxide incubator, culture for 24h at 37°C, 5% CO2 and saturated humidity until the cells adhere to the wall, aspirate the 100μL culture medium in the well, add 50μL fresh culture medium, and give different concentrations of compounds and reference drug isosorbide dinitrate respectively. Add 50μL of drug to each well, set 3 parallel groups and 1 normoxic non-drug group as controls. (Concentration gradient is 10, 1, 0.1, 0.01, 0.001μM). The normoxic culture group was incubated in a normal incubator at 5% CO2 and 37°C for 4 hours; the hypoxic group was incubated in a hypoxic incubator (O2 concentration ≤ 1%) at 37°C for 4 hours. After the culture was completed, 10 μL of CCK8 reagent was added to each well and the culture was continued in the normoxic incubator for 4 hours. The culture was terminated, and the absorbance of each well was measured at 450nm on an ELISA reader. The cell survival rate of cardiomyocytes at each concentration was calculated. The results are shown in Figure 1a and Figure 1b .

[0091] Under normoxic conditions (see Figure 1a ), compounds I1-I9 showed no improvement on cell viability except compound I4 at 1 μM, as the concentration increased. On the contrary, they had some effect on cell viability at a high concentration of 10 μM, showing less toxicity. Under hypoxic conditions (see Figure 1b), compounds I1-I4 can significantly restore the vitality of hypoxic cells in the concentration range of 0.001 to 10 μM, and the activity increases with the increase of compound concentration, while compounds I5-I9 have no obvious effect on restoring the vitality of hypoxic cells. The control drug isosorbide dinitrate has no improvement on the vitality of cells under normoxic conditions. Under hypoxic conditions, it only shows a certain ability at high concentrations, but not as good as compounds I1-I4. Combining the data in Tables 1 and 2, this type of hypoxia-activated NO donor compound I1-I4 can effectively release NO under hypoxic conditions, and can improve the vitality of cardiomyocytes in a certain concentration range. Its activity is better than that of the existing drug isosorbide dinitrate. This beneficial effect has also been verified in animal level experiments.

[0092] Example 14: NO detection kit to detect the NO release level of compound I1 in the heart of hypoxia model mice

[0093] (a) Drugs, reagents and experimental animals

[0094] NO detection kit (Biyuntian Biotechnology Co., Ltd., Shanghai), heart tissue homogenate of hypoxia modeling and drug-treated mice and blank control group mice, and 6-8 week old male Kunming mice.

[0095] (b) Experimental principle

[0096] NO is chemically active and is rapidly converted into NO3 in the body - and NO2 - , NO2 - Further converted into NO3 - . Use Griess reagent to convert NO3 - Reduction to NO2 - , NO2 - There is characteristic absorption at 540nm. The absorbance at 540nm is detected by an enzyme reader, and the NO concentration is converted using a standard curve.

[0097] (c) Experimental operation

[0098] (1) Hypoxia model (85 mg / kg isoproterenol), the drug-treated group was intraperitoneally injected with 20 mg / kg compound I1 before hypoxia modeling;

[0099] (2) Centrifuge at 12,000 rpm for 15 min to collect cardiac tissue homogenate protein;

[0100] (3) Prepare the working solution in the NO detection kit, add the working solution to each group of proteins, incubate and detect with an ELISA instrument to analyze the NO content in each group. The results are shown in Figure 2 .

[0101] like Figure 2As shown, after hypoxia modeling and drug administration to mice, the NO level in the heart of the compound I1 group was significantly higher than that of the other two groups.

[0102] Example 15: Western blot study on the regulatory effect of compound I1 on the expression level of myocardial injury marker proteins in myocardial hypoxia mice

[0103] (a) Drugs, reagents and experimental animals

[0104] Western Blot gel preparation kit (Biyuntian Biotechnology Co., Ltd., Shanghai), BCA detection kit for protein quantification (Biyuntian Biotechnology Co., Ltd., Shanghai), ionization buffer, wet transfer solution, primary antibody (anti-TSC2-P, Abgent, 1:1000, A-AP3825a; anti-mTORC1, Abcam, 1:1000, ab120224; anti-GAPDH, Bioworld Technology, 1:500, AP0063), secondary antibody (Goat Anti-Rabbit IgG (H+L) HRP, 1:3000, Bioworld Technology, BS13278), 6-8 week old male Kunming mice.

[0105] (b) Experimental principle

[0106] The changes in protein expression levels were analyzed based on Western Blot bands.

[0107] (c) Experimental operation

[0108] (1) Extract mouse heart tissue protein, collect the protein by centrifugation at 12,000 rpm for 15 min, quantify the protein using a BCA kit, add the protein to boiling loading buffer for 5 min, and set aside;

[0109] (2) preparing SDS-PAGE gel and loading the sample to separate protein bands;

[0110] (3) After the protein bands were transferred to the PVDF membrane, they were blocked with milk, incubated with primary and secondary antibodies in sequence, and the protein bands were visualized using a chemiluminescence developer to analyze the changes in protein expression in each group. The results are shown in Figure 3 .

[0111] like Figure 3As shown, protein samples were extracted from the hearts of blank group mice, hypoxia model mice, and drug-treated and hypoxia modeling mice for Western blot experiments. Compared with the blank group, the down-regulation of TSC2-P expression in the hypoxia model group mice caused a significant up-regulation of mTORC1 protein expression, indicating that the model group had obvious myocardial damage. The expression of mTORC1 in the hearts of mice pretreated with 20 mg / kg compound I1 was restored to a level close to that of the normal group, indicating that the compound has excellent anti-myocardial hypoxia injury effect.

Claims

1. A NO donor compound, characterized in that Having the structure of Formula I: ， in: R 1 When it is nitro, R 2 is hydrogen, halogen or C1-C4 alkyl, R 3 is hydrogen; R 2 When it is nitro, R 1 is hydrogen, R 3 For hydrogen.

2. The NO donor compound according to claim 1, characterized in that In the structure: R 1 For nitro, R 2 is hydrogen, halogen or methyl, R 3 For hydrogen.

3. The NO providing compound according to claim 1, characterized in that Select any of the following compounds: 。 4. A method for preparing the NO donor compound according to any one of claims 1 to 3, characterized in that: Compound 1 and compound 2 are subjected to acylation reaction to obtain compound I, ； Among them, R 1 , R 2 , R 3 The definition is as described in any one of claims 1 to 3.

5. A pharmaceutical composition, characterized in that The pharmaceutical composition comprises the NO donor compound according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier.

6. Use of the NO donor compound according to any one of claims 1 to 3 in the preparation of a drug for treating myocardial hypoxia-damage diseases.

7. Use of the pharmaceutical composition according to claim 5 in preparing drugs for treating myocardial hypoxia and injury diseases.

8. The use according to claim 6 or 7, characterized in that: The compound is a hypoxia activated NO donor compound.

9. The use according to claim 6 or 7, characterized in that: The drug is used to improve the viability of myocardial cells under hypoxic conditions.

10. The use according to claim 6 or 7, characterized in that: The disease is coronary heart disease.

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