NO donor compound, method for producing the same, pharmaceutical composition, and use

Abstract

The present invention discloses a NO donor compound and its preparation method, pharmaceutical composition and use. The structure of this compound is as shown in Formula I. This NO donor compound and its pharmaceutical composition can be prepared as a drug for treating myocardial hypoxic injury disease as a hypoxia-activated NO donor, and the prepared drug can exert its medicinal effect at both cell and animal levels, and the synthesis of this compound is simple and easy to operate.

Description

Technical Field

[0001] The present invention relates to an NO donor compound, a method for producing the same, a pharmaceutical composition and use thereof, and particularly relates to an NO donor compound that can be produced as a drug for treating myocardial hypoxic injury-related diseases, a method for producing the same, a pharmaceutical composition and use 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 occlusion of the coronary artery, and is also called ischemic heart disease. According to the data report released by the Chinese Cardiovascular Disease Center in 2020, the number of people suffering from cardiovascular diseases such as coronary heart disease in China has already reached 290 million. Currently, the high incidence and fatality rate of coronary heart disease pose a serious threat to the health of the public. Nitric oxide (NO) donor drugs are often used for the treatment of coronary heart disease caused by ischemia because they release exogenous NO molecules in the body to promote vasodilation. However, conventional drugs lack selectivity for hypoxia and cannot release a sufficient amount of NO concentrated at the hypoxic site, resulting in serious side effects.

[0003] In recent years, hypoxia-activated prodrugs have attracted wide attention, as they can reduce off-target toxicity by promoting the selective release of active drug groups in the hypoxic microenvironment. In view of the fact that hypoxia is an important factor causing myocardial injury, using this design concept in the structural design of NO donor drugs to obtain hypoxia-activated prodrugs is an effective way to improve the therapeutic effect of such drugs and reduce side effects. Nitroreductase (NTR) is present in mammalian heart, liver, kidney, lung, and brain tissues, and it can catalyze the reduction of nitroaromatic compounds by providing hydrogen from NADPH and NADH under anaerobic conditions. Therefore, nitroaromatic compounds are recognized hypoxia-activating groups and may be used in the design of hypoxia-activated prodrugs. This is because NTR in hypoxic cells selectively reduces the nitro group on the aromatic ring and further promotes the release of the active drug group from the prodrug through intramolecular electron transfer. Since NTR is highly expressed in hypoxic cells and can effectively promote the concentrated release of the active drug group in the hypoxic microenvironment of the prodrug, using the hypoxia-activating properties of nitroaromatic compounds, binding it to an NO donor to obtain a prodrug, and releasing a sufficient amount of NO under hypoxia activation has important significance for the treatment of cardiovascular diseases such as coronary heart disease.

Summary of the Invention

Problems to be Solved by the Invention

[0004] Objective of the Invention: In order to solve problems such as the poor therapeutic effect of conventional NO donor drugs in treating myocardial hypoxia injury and the large side effects, the present invention aims to provide an NO donor compound that can effectively improve myocardial hypoxia injury in a hypoxic microenvironment, its manufacturing method, pharmaceutical composition, and use.

Means for Solving the Problems

[0005] Technical Solution: As a first aspect of the present invention, the NO donor compound of the present invention has the structure of Formula I, JPEG0007683872000001.jpg55135 Here, R 1 when R is a nitro group, R 2 is hydrogen, halogen or C 1 ~C 4 alkyl group, and R 3 is hydrogen, halogen or C 1 ~C 4 alkyl group, R 2 when R is a nitro group, R 1 is hydrogen, halogen or C 1 ~C 4 alkyl group, and R 3 is hydrogen, halogen or C 1 ~C 4 alkyl group.

[0006] The present invention binds a hypoxia-activated group nitroaromatic compound to a NO donor molecule via a linking group to obtain a hypoxia-activated type NO donor compound.

[0007] Preferably, in the above structure, R 1 is a nitro group, R 2 is hydrogen, halogen or a methyl group, and R 3 is hydrogen.

[0008] More specifically, the above NO donor compound is any one compound selected from JPEG0007683872000002.jpg109170.

[0009] As a second aspect of the present invention, the method for producing the above NO donor compound is to subject Compound 1 and Compound 2 to an acylation reaction to obtain Compound I, and the specific synthesis steps are as follows.

[0010] (1) 4-Hydroxy(N-methyl)aniline hemisulfate and NaNO 2React in the presence of acetic acid to obtain Compound 1. In step (i), set the reaction temperature to 0 °C, use acetic acid (AcOH) as the solvent, and set the reaction time to 3 h. (2) React a benzyl alcohol derivative with bis(trichloromethyl) carbonate (triphosgene, BTC) in the presence of N,N-diisopropylethylamine (DIPEA) to obtain Compound 2. In step (ii), set the reaction temperature to room temperature, use tetrahydrofuran (THF) as the solvent, and set the reaction time to 5 h. (3) React Compound 1 and Compound 2 in the presence of N,N-diisopropylethylamine to obtain Product I. In step (iii), set the reaction temperature to room temperature, use dichloromethane (DCM) as the solvent, and set the reaction time to 5 h.

[0011] JPEG0007683872000003.jpg51170 Here, R 1 、R 2 、R 3 are defined as described above.

[0012] As a third aspect of the present invention, the pharmaceutical composition of the present invention contains the NO donor compound and a pharmaceutically acceptable carrier.

[0013] The NO donor compound can be used to produce a general pharmaceutical preparation by adding a pharmaceutically acceptable carrier, for example, tablets, capsules, syrups, suspensions or injections. Commonly used pharmaceutical adjuvants such as flavors, sweeteners, liquid / solid fillers, diluents, etc. can be added to the preparation.

[0014] As a fourth aspect of the present invention, the NO donor compound and pharmaceutical composition of the present invention can be manufactured as a drug for treating myocardial hypoxic injury diseases. The above compound is a hypoxia-activated NO donor compound and is used to improve the survival rate of cardiomyocytes under hypoxic conditions. Specifically, it is used to treat coronary heart disease. The above compound can release NO to cells in a hypoxic microenvironment and effectively improve myocardial hypoxic injury.

Advantages of the Invention

[0015] Beneficial effects: Compared with the prior art, the present invention has the following remarkable advantages.

[0016] (1) Such NO donor compounds and pharmaceutical compositions can effectively release NO under hypoxic conditions, and the release amount can reach up to more than 40% at most, and can improve the survival rate of cardiomyocytes, and can function as a hypoxia-activated NO donor both in vivo and in vitro. (2) Such NO donor compounds and pharmaceutical compositions are widely used and can be manufactured as drugs for treating myocardial hypoxia injury diseases. The said drugs can exert medicinal effects both at the cellular level and the animal level, and the therapeutic effect is better. (3) The manufacturing method of the compound is simple and easy to operate.

Brief Description of the Drawings

[0017]

Fig. 1a

Fig. 1b

Fig. 2

Fig. 3

Modes for Carrying Out the Invention

[0018] Hereinafter, the technical solution of the present invention will be further described in conjunction with examples.

[0019] Example 1: Preparation of N-methyl-N-nitroso-p-phenol (Compound 1) JPEG0007683872000004.jpg47130

[0020] Under light - shielding conditions, 4 - hydroxy(N - methyl)aniline hemisulfate (1.722 g, 10.0 mmol) was added to 50 mL of acetic acid and dissolved by ultrasonic treatment. A sodium nitrite (1.380 g, 20.0 mmol) solution dissolved in 10 ml of water was cooled in an ice - water bath and added dropwise to the acetic acid solution. After the addition was complete, the reaction was continued for 3 h in an ice - water bath. After the reaction was completed, the reaction solution was transferred to a beaker, 50 mL of water was added, the pH was adjusted to 7 with a saturated aqueous sodium hydrogen carbonate solution, and this aqueous solution was extracted with ethyl acetate. The organic phases were combined, dried over anhydrous sodium sulfate, and then spin - dried to obtain 1.46 g of the product, with a yield of 96%.

[0021] 1 H NMR(600MHz, DMSO - d 6 ):δ9.79(s, 1H), 7.41(d, J = 8.8Hz, 2H), 6.89(d, J = 8.8Hz, 2H), 3.38(s, 3H)ppm.

[0022] Example 2: Preparation of p - nitrobenzyl chloroformate (Compound 2a) JPEG0007683872000005.jpg40148

[0023] p - Nitrobenzyl alcohol (0.168 g, 1.1 mmol) and BTC (0.296 g, 1.0 mmol) were dissolved in 20 mL of anhydrous tetrahydrofuran, cooled in an ice - water bath, and N,N - diisopropylethylamine (0.142 g, 1.1 mmol) was added. The reaction was carried out for 30 min under nitrogen gas protection and then for 5 h at room temperature. After the reaction was completed, the reaction solution was concentrated, excess phosgene was removed with a small amount of anhydrous tetrahydrofuran, and after adding anhydrous dichloromethane, it was directly used in the next reaction.

[0024] Example 3: Preparation of 4 - ((methylnitroso)amino)phenyl(4 - nitrobenzyl)carbonate (Compound I1) JPEG0007683872000006.jpg42142

[0025] 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, cooled in an ice - water bath, and a solution of anhydrous dichloromethane containing compound 2a obtained in Example 2 was slowly added dropwise to the above solution. After the addition was complete, the ice - water bath was removed and the reaction was carried out at room temperature for 5 h. The reaction solution was washed three times each with 0.5 M dilute hydrochloric acid, water, and saturated brine, the organic phase was separated, dried over anhydrous sodium sulfate overnight, and then concentrated by rotary evaporation to obtain a yellow solid. The solid was recrystallized from a mixed solvent of ethyl acetate and petroleum ether to obtain 285 mg of pale - yellow crystals, with a yield of 86%.

[0026] 1H NMR (600 MHz, DMSO - d6): δ 8.28 - 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. 13C NMR (150 MHz, DMSO - d6): δ 153.15, 150.02, 147.87, 143.07, 140.37, 129.32, 124.15, 122.79, 121.20, 69.03, 32.14 ppm. HRMS (m / z) (ESI): calcd for C15H14N3O6[M + H]+: 332.08, found: 332.11.

[0027] Example 4: Preparation of 4 - ((methylnitroso)amino)phenyl (2 - chloro - 4 - nitrobenzyl) carbonate (Compound I2) JPEG0007683872000007.jpg41143

[0028] Referring to the methods described in Examples 2 and 3, 2 - chloro - 4 - nitrobenzyl alcohol was used as the raw material to obtain Compound I2, which was pale - yellow crystals with a yield of 23%.

[0029] 1H NMR (600 MHz, DMSO-d6): δ 8.38 - 8.39 (d, J = 2.3 Hz, 1H), 8.28 - 8.30 (dd, 1H), 7.88 - 7.90 (d, J = 8.5 Hz, 1H), 7.71 - 7.72 (m, 2H), 7.46 - 7.48 (m, 2H), 5.49 (s, 2H), 3.44 (s, 3H) ppm. 13C NMR (150 MHz, DMSO-d6): δ 152.99, 149.95, 148.49, 140.41, 140.18, 133.62, 131.17, 124.78, 122.96, 122.77, 121.25, 66.92, 32.20 ppm. HRMS (m / z) (ESI): calcd for C15H13ClN3O6Na [M+Na]+: 388.03, found: 388.10.

[0030] Example 5: Preparation of 4-((methylnitroso)amino)phenyl (2-methyl-4-nitrobenzyl) carbonate (Compound I3) JPEG0007683872000008.jpg45162

[0031] Referring to the methods described in Examples 2 and 3, 2-methyl-4-nitrobenzyl alcohol was used as the raw material to obtain Compound I3, which was a pale yellow crystal with a yield of 45%.

[0032] 1H NMR (600 MHz, DMSO-d6): δ 8.15 - 8.16 (d, J = 2.2 Hz, 1H), 8.10 - 8.14 (dd, J = 8.4, 2.4 Hz, 1H), 7.71 - 7.73 (d, J = 2.2 Hz, 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. 13C NMR (150 MHz, DMSO-d6): δ 153.11, 150.01, 147.78, 141.17, 140.37, 139.16, 129.71, 125.06, 122.80, 121.49, 121.21, 67.66, 32.17, 18.77 ppm. HRMS (m / z) (ESI): calcd for C16H15N3O6 [M+H]+: 346.10, found: 346.10.

[0033] Example 6: Preparation of 4-((Methylnitroso)amino)phenyl(2-nitrobenzyl)carbonate (Compound I4) JPEG0007683872000009.jpg43136

[0034] Referring to the methods described in Examples 2 and 3, 2-nitrobenzyl alcohol was used as the raw material to obtain Compound I4, which is a pale yellow crystal with a yield of 73%.

[0035] 1H NMR (600 MHz, DMSO-d6): δ 8.17 - 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. 13C NMR (150 MHz, DMSO-d6): δ 153.04, 149.98, 147.82, 140.40, 134.85, 130.81, 130.25, 130.06, 125.49, 122.77, 121.24, 67.09, 32.16 ppm. HRMS (m / z) (ESI): calcd for C15H14N3O6 [M+H]+: 332.09, found: 332.02..

[0036] Example 7: Preparation of 4-((Methylnitroso)amino)phenyl(2-nitro-4-chlorobenzyl)carbonate (Compound I5) JPEG0007683872000010.jpg43146

[0037] Referring to the methods described in Examples 2 and 3, 2-nitro-4-chlorobenzyl alcohol was used as the raw material to obtain Compound I5, which is a pale yellow crystal with a yield of 46%.

[0038] 1H NMR (600 MHz, DMSO-d6): δ 8.26 - 8.27 (d, J = 2.2 Hz, 1H), 7.94 - 7.96 (dd, 1H), 7.81 - 7.83 (d, J = 8.4 Hz, 1H), 7.70 - 7.72 (m, 2H), 7.45 - 7.46 (m, 2H), 5.62 (s, 2H), 3.44 (s, 3H) ppm. 13C NMR (150 MHz, DMSO-d6): δ 152.94, 149.96, 148.47, 140.42, 134.51, 134.21, 131.84, 129.81, 125.32, 122.75, 121.24, 66.53, 32.15 ppm. HRMS (m / z) (ESI): calcd for C15H13ClN3O6Na [M+Na]+: 388.03, found: 388.00.

[0039] Example 8: Preparation of 4-((methylnitroso)amino)phenyl (2-nitro-4-bromobenzyl) carbonate (Compound I6) JPEG0007683872000011.jpg44135

[0040] Referring to the methods described in Examples 2 and 3, 2-nitro-4-bromobenzyl alcohol was used as the raw material to obtain Compound I6, which is a pale yellow crystal with a yield of 33%.

[0041] 1H NMR (600 MHz, DMSO-d6): δ 8.36 - 8.37 (d, J = 2.1 Hz, 1H), 8.07 - 8.09 (dd, 1H), 7.73 - 7.75 (d, J = 8.3 Hz, 1H), 7.70 - 7.72 (m, 2H), 7.44 - 7.46 (m, 2H), 5.60 (s, 2H), 3.44 (s, 3H) ppm. 13C NMR (150 MHz, DMSO-d6): δ 152.94, 149.96, 148.49, 140.42, 137.45, 131.94, 130.19, 128.03, 122.75, 122.16, 121.24, 66.58, 32.15. HRMS (m / z) (ESI): calcd for C15H13BrN3O6 [M+H]+: 431.98, found: 431.90.

[0042] Example 9: Preparation of 4-((methylnitroso)amino)phenyl(2-nitro-4-methylbenzyl)carbonate (Compound I7) JPEG0007683872000012.jpg38129 Referring to the methods described in Examples 2 and 3, 2-nitro-4-methylbenzyl alcohol was used as the raw material to obtain Compound I7, which is a pale yellow crystal with a yield of 51%.

[0043] 1H NMR (600 MHz, DMSO-d6): δ8.00 - 8.01 (m, 1H), 7.70 - 7.72 (m, 2H), 7.66 - 7.67 (d, J = 0.9 Hz, 2H), 7.43 - 7.46 (m, 2H), 5.60 (s, 2H), 3.44 (s, 3H), 2.44 (s, 3H) ppm. 13C NMR (150 MHz, 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, 20.74 ppm. HRMS (m / z) (ESI): calcd for C16H16N3O6 [M+H]+: 346.10, found: 345.90.

[0044] Example 10: Preparation of 4-((methylnitroso)amino)phenyl(2-nitro-5-chlorobenzyl)carbonate (Compound I8) JPEG0007683872000013.jpg48129

[0045] Referring to the methods described in Examples 2 and 3, 2-nitro-5-chlorobenzyl alcohol was used as the raw material to obtain Compound I8, which is a pale yellow crystal with a yield of 37%.

[0046] 1H NMR (600 MHz, DMSO-d6): δ 8.21 - 8.23 (d, J = 8.8 Hz, 1H), 7.83 - 7.84 (d, J = 2.3 Hz, 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. 13C NMR (150 MHz, DMSO-d6): δ 152.82, 149.98, 146.36, 140.43, 139.48, 133.27, 130.05, 129.67, 127.63, 122.76, 121.23, 66.56, 32.14 ppm. HRMS (m / z) (ESI): calcd for C15H13ClN3O6Na [M+Na]+: 388.03, found: 388.00.

[0047] Example 11: Preparation of 4-((methylnitroso)amino)phenyl (2-nitro-5-methylbenzyl) carbonate (Compound I9) JPEG0007683872000014.jpg49128

[0048] Referring to the methods described in Examples 2 and 3, 2-nitro-5-methylbenzyl alcohol was used as the raw material to obtain Compound I9, which was a pale yellow crystal with a yield of 48%.

[0049] 1H NMR (600 MHz, DMSO-d6): δ 8.09 - 8.11 (d, J = 8.4 Hz, 1H), 7.72 - 7.73 (d, J = 2.2 Hz, 1H), 7.71 - 7.72 (d, J = 2.3 Hz, 1H), 7.57 - 7.58 (m, 1H), 7.48 - 7.49 (m, 1H), 7.46 - 7.47 (d, J = 2.3 Hz, 1H), 7.45 - 7.46 (d, J = 2.2 Hz, 1H), 5.63 (s, 2H), 3.44 (s, 3H), 2.47 (s, 3H) ppm. 13C NMR (150 MHz, DMSO-d6): δ 153.00, 150.01, 145.93, 145.49, 140.40, 130.95, 130.47, 130.26, 125.70, 122.78, 121.24, 67.21, 32.15, 21.53 ppm. HRMS (m / z) (ESI): calcd for C16H15N3O6Na [M+Na]+: 368.09, found: 368.10.

[0050] Example 12: Detection of NO release levels after the compound acts on cardiomyocytes H9c2 under normoxia or hypoxia by the Griess method Cardiomyocytes H9c2 were resuscitated, cultured using DMEM medium, placed in a carbon dioxide incubator, and cultured to adhere to the wall and grow. After the cells reached an appropriate density, they were adhered to a porous plate, placed in a carbon dioxide incubator, and cultured at 37 °C, 5% CO 2 and saturated humidity conditions for 24 h until the cells adhered to the wall. The supernatant was discarded, 2 mL of fresh DMEM medium was added, and compounds at certain concentrations (5, 10, 15 μM) and the control drug isosorbide dinitrate were added. Normoxia incubator (37 °C, 21% O 2 ) or hypoxia incubator (37 °C, 1% O 2) After culturing for 4 h, the supernatant was removed, and the cells in the plate were collected into 1.5 mL centrifuge tubes respectively, centrifuged at 1000 rpm for 5 min, the supernatant was discarded, 1 mL of PBS was added to resuspend the cells, centrifuged again, the PBS was discarded, 100 μL of cell lysis solution (for NO measurement only) was added to the centrifuge tube, left on ice for half a minute, and then centrifuged at 13300 rpm for 5 min at 4 °C, and the supernatant was collected. 50 μL of the supernatant was taken into a 96-well plate, three parallel groups were set for each sample, and 50 μL of Griess reagent I and 50 μL of Griess reagent II were added sequentially. The OD value of each well was measured using a full wavelength microplate reader at a wavelength of 540 nm, the NO concentration was calculated from the standard curve, and the results are as shown in Table 1 and Table 2.

[0051] Table 1 NO release amount after 4 h of treatment of cardiomyocyte H9c2 with compounds under normoxia JPEG0007683872000015.jpg100170*. NO release amount = measured NO concentration / calculated NO concentration, data are represented as the mean ± SD of three independent experiments: *p < 0.05, **p < 0.01, a. Represents unmeasured, b. Calculated at 1 / 2 of the release amount.

[0052] Table 2 NO release amount after 4 h of treatment of cardiomyocyte H9c2 with compounds under hypoxia JPEG0007683872000016.jpg99170*. NO release amount = measured NO concentration / calculated NO concentration, data are represented as the mean ± SD of three independent experiments: *p < 0.05, **p < 0.01, a. Represents unmeasured, b. Calculated at 1 / 2 of the release amount.

[0053] According to the data in Table 1 and Table 2, after treating cardiomyocytes with compounds at different concentrations under normoxia, the amount of NO released in cells became very small, and all were lower than the maximum release amount of Compound I2 (<3.34%, 10 μM). However, under hypoxia, in addition to Compounds I5 - I9, treating cardiomyocytes with different concentrations of Compounds I1 - I4 could effectively cause the release of NO in cells, and the release amount was between 20.15% and 45.45%, showing as a hypoxia-activated NO donor. The control drug, isosorbide dinitrate (ISDN), released a certain amount of NO after treating cardiomyocytes regardless of normoxia or hypoxia. The amount of NO released in cells under normoxia was relatively large, reaching 41.63% (10 μM), and it could also cause the release of NO in cells under hypoxia, but the release amount was smaller than that under normoxia and lower than the release levels of Compounds I2 and I3.

[0054] Example 13: Improvement effect of compounds on the survival rate of cardiomyocytes H9c2 under normoxia or hypoxia Wash the cells twice with 5 mL of PBS, add 1 mL of trypsin to digest monolayer-cultured cardiomyocytes H9c2, prepare a single-cell suspension with a culture medium containing serum, inoculate 5000 - 10000 cells per well into a 96-well culture plate, and fill each well with a medium volume of 100 μL and the edge wells with sterilized pure water. Transfer the culture plate to a carbon dioxide incubator and culture for 24 h under the conditions of 37 °C, 5% CO 2 and saturated humidity until the cells adhere to the wall. Aspirate 100 μL of the medium in the well, add 50 μL of fresh medium, and give different concentrations of the compound and the control drug isosorbide dinitrate. Add 50 μL of the drug per well, and set up three parallel groups and one normoxia non-drug administration group as controls. (The concentration gradient is 10, 1, 0.1, 0.01, 0.001 μM). The normoxia culture group was incubated in a normal incubator at 5% CO 2 , 37 °C for 4 h, and the hypoxia group was incubated in a hypoxia incubator (O 2Incubate at 37 °C for 4 h at a concentration ≤ 1%), and after the completion of the culture, add 10 μL of CCK8 reagent per well and continue the culture in a normal oxygen incubator for 4 h. After the culture was terminated, the absorbance value of each well was measured at 450 nm with a microplate reader, and the cell viability of cardiomyocytes at each concentration was calculated. The results are as shown in Figures 1a and 1b.

[0055] Under normal oxygen conditions (see Figure 1a), for compounds I1-I9, as the concentration increased, except that compound I4 slightly improved the cell viability below 1 μM, the other compounds showed no improvement in cell viability. On the contrary, they also affected the cell viability at a high concentration of 10 μM and showed relatively low toxicity. Under hypoxic conditions (see Figure 1b), compounds I1-I4 significantly restored the viability of hypoxic cells within the concentration range of 0.001-10 μM, and it improved with the increase in compound concentration. However, compounds I5-I9 had no obvious effect on restoring the viability of hypoxic cells. The control drug isosorbide dinitrate showed no improvement in cell viability under normal oxygen conditions and showed a certain ability only at high concentrations under hypoxic conditions, but it was inferior to compounds I1-I4. Combining the data in Table 1 and Table 2, such hypoxia-activated NO donor compounds I1-I4 can effectively release NO under hypoxic conditions and can improve the viability of cardiomyocytes in a certain concentration range, and their activity was superior to that of the conventional drug isosorbide dinitrate. This beneficial effect was also verified in animal-level experiments.

[0056] Example 14: Detection of the NO release level in the heart of hypoxia-modeling mice of compound I1 using a NO detection kit (a) Drugs, reagents and experimental animals NO detection kit (Biyuntian Biotechnology Co., Ltd., Shanghai), hypoxia-modeling and administered mice, heart tissue homogenates of blank control group mice, male Kunming mice aged 6-8 weeks.

[0057] (b) Experimental principle NO has chemical activity, and in the body, NO 3- And NO 2 - Converted quickly to NO 2 - No further 3 - Using the Griess reagent, NO is converted to 3 - NO 2 - Reduced to NO 2 - has a characteristic absorption at 540 nm, and the absorbance at 540 nm was detected using a microplate reader, and the NO concentration was calculated using a standard curve.

[0058] (c) Experimental procedure (1) Hypoxia model (85 mg / kg isoproterenol), the treatment group was intraperitoneally injected with 20 mg / kg compound I1 before hypoxia modeling; (2) Centrifuge at 12,000 rpm for 15 min to collect cardiac tissue homogenate proteins. (3) The working solution for the NO detection kit was prepared, and the working solution was added to the proteins of each group. After incubation, the NO content of each group was analyzed by detecting it with a microplate reader. The results are shown in Figure 2.

[0059] As shown in FIG. 2, after hypoxia modeling and administration to mice, the intracardiac NO level in the compound I1 group was significantly higher than that in the other two groups.

[0060] Example 15: Study of the regulatory effect of compound I1 on the expression level of myocardial injury marker protein in myocardial hypoxic mice by Western blot analysis (a) Drugs and reagents and laboratory animals 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 antibodies (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), male Kunming mice at 6 - 8 weeks of age.

[0061] (b) Experimental principle Based on the Western Blot bands, the changes in the expression levels of proteins were analyzed.

[0062] (c) Experimental operation (1) Extract mouse heart tissue proteins, centrifuge at 12,000 rpm for 15 min to collect the proteins, quantify the proteins with the BCA kit, add to boiling water with loading buffer and boil for 5 min as a preparation. (2) Prepare SDS-PAGE gel, load the samples to separate the protein bands. (3) After transferring the protein bands to the PVDF membrane, block with milk, incubate with primary antibody and secondary antibody in sequence, and analyze the changes in the expression of proteins in each group by visualizing the protein bands using a chemiluminescence imager. The results are as shown in Figure 3.

[0063] As shown in Fig. 3, protein samples were extracted from the hearts of blank group mice, hypoxia model mice, and administration + hypoxia modeling group mice, respectively, and Western blot experiments were conducted. Compared with the blank group, the decrease in TSC2-P expression in hypoxia model group mice caused a significant increase in the expression of mTORC1 protein, indicating that obvious myocardial damage appeared in the model group. The mTORC1 expression in the hearts of mice pretreated with compound I at 120 mg / kg recovered to a level close to that of the normal group, indicating that this compound has an excellent effect on protecting against myocardial hypoxia injury.

Claims

Claim 1 An NO donor compound having a structure of Formula I, wherein, R １ When R is a nitro group, ２ R is hydrogen, halogen or C １ to C ４ alkyl group, and R ３ is hydrogen, halogen or C １ to C ４ alkyl group, and R ２ When R is a nitro group, １ R is hydrogen, halogen or C １ ~C ４ alkyl group, and R ３ is hydrogen, halogen or C １ ~C ４ alkyl group, and the NO donor compound is characterized by this. Claim 2 In the above structure, R １ is a nitro group, R ２ is hydrogen, a halogen or a methyl group, R ３ is hydrogen, and the NO donor compound according to claim 1, characterized in that. The NO donor compound according to claim 1, which is any one compound selected from [

3. ]. Claim 4 A method for producing the NO donor compound according to any one of claims 1 to 3, comprising subjecting Compound 1 and Compound 2 to an acylation reaction to obtain Compound I, Here, R １ , R ２ , R ３ is defined as described in any one of claims 1 to 3, and a method characterized by this. Claim 5 A pharmaceutical composition comprising the NO donor compound according to any one of claims 1 to 3 and a pharmaceutically acceptable carrier. Claim 6 Use of the NO donor compound according to any one of claims 1 to 3 in the manufacture of a medicament for treating myocardial hypoxic injury-related diseases. Claim 7 Use of the pharmaceutical composition according to claim 5 in the manufacture of a medicament for treating myocardial hypoxic injury-related diseases. Claim 8 The use according to claim 6, wherein the compound is a hypoxia-activated NO donor compound. Claim 9 The use according to claim 6, wherein the medicament is used to improve the survival rate of cardiomyocytes under hypoxic conditions. Claim 10 The use according to claim 6, wherein the disease is coronary heart disease.

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