Use of a class of pyrrolidinone compounds for preparing myocardial injury protective drugs

Abstract

The application belongs to the field of pharmaceutical chemistry and particularly relates to application of specific pyrrolidone chalcone compounds in preparation of therapeutic drugs for diseases related to myocardial injury, the pyrrolidone chalcone compounds can have good protective effect on oxidative stress and high glucose induced myocardial cell injury, they can effectively eliminate generation of active oxygen in oxidative stress injury by activating Nrf2 / HO-1 antioxidant signal pathway, and have good protective effect on myocardial cell injury.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of pharmaceutical chemistry, and in particular, the present application relates to the use of specific pyrrolidinone chalcone compounds in the preparation of a medicament for treating myocardial injury related diseases. BACKGROUND

[0002] Myocardial ischemia reperfusion injury, diabetic cardiomyopathy and other diseases are serious heart diseases that endanger human health, and there is currently no effective treatment drug, so it is of great significance to find a highly effective and low-toxicity treatment drug.

[0003] In the present application, various pyrrolidinone structural units are introduced into the chalcone skeleton, and the target compounds screened have not only good protective effect on myocardial cell injury, but also their cytotoxicity is significantly lower than that of the parent chalcone compound before modification. SUMMARY

[0004] The present application aims to provide the use of 16 pyrrolidinone chalcone compounds in the preparation of a medicament for treating myocardial injury related diseases.

[0005] Another object of the present application is to provide a pharmaceutical composition for treating myocardial diseases, which contains a therapeutically effective amount of any one or more of the chalcone compounds of claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient and a pharmaceutical adjuvant.

[0006] Specifically, the 16 pyrrolidinone chalcone compounds of the present application have the following structures:

[0007]

[0008] The molecular formula of C1a is C 26 H 24 N2O5, the chemical name is: (3'S, 4'S)-3'-(3, 4-dihydroxybenzoyl)-4'-(2-methoxyphenyl)-1'-methylspiro[indoline-3, 2'-pyrrolidin]-2-one. The molecular formula of C1b is C 27 H 25 FN2O6, the chemical name is: (3'S, 4'S)-3'-(3, 4-dihydroxybenzoyl)-5-fluoro-4'-(2-methoxyphenyl)-1'-methylspiro[indoline-3, 2'-pyrrolidin]-2-one. The molecular formula of C1c is C 26 H 23CIN2O5, chemical name: (3'S,4'S)-5-chloro-3'-(3,4-dihydroxybenzoyl)-4'-(2- methoxyphenyl)-1 '-methylspiro[indoline-3,2'-pyrrolidin]-2-one. The molecular formula of C1d is C 26 H 23 BrN2O5, chemical name: (3'S,4'S)-5-bromo-3'-(3,4-dihydroxybenzoyl)-4'-(2- methoxyphenyl)-1 '-methylspiro[indoline-3,2'-pyrrolidin]-2-one. The molecular formula of C1e is C 27 H 22 N2O5, chemical name: (3'S,4'S)-3'-(3,4-dihydroxybenzoyl)-4'-(2-methoxyphenyl)-1',5- dimethylspiro[indoline-3,2'-pyrrolidin]-2-one. The molecular formula of C1f is C 27 H 26 N2O6, chemical name: (3'S,4'S)-3'-(3,4-dihydroxybenzoyl)-5-methoxy-4'-(2- methoxyphenyl)-1 '-methylspiro[indoline-3,2'-pyrrolidin]-2-one. The molecular formula of C1g is C 31 H 27 NO5, chemical name: (1R,3'S,4'S)-3'-(3,4-dihydroxybenzoyl)-4'-(2-methoxyphenyl)-1'- methyl-2H-spiro[acenaphthylene-1,2'-pyrrolidin]-2-one. The molecular formula of C3a is C 27 H 26 N2O6, chemical name: (3'S,4'S)-3'-(3,4-dihydroxybenzoyl)-4'-(2,5-dimethoxyphenyl)-1'- methylspiro[indoline-3,2'-pyrrolidin]-2-one. The molecular formula of C3b is C 27 H 25FN2O6, chemical name: (3'S,4'S)-3'-(3,4-dihydroxybenzoyl)-4'-(2,5-dimethoxyphenyl)-5-fluoro-1'-methylspiro[indoline-3,2'-pyrrolidin]-2-one. Molecular formula of C3c is C 27 H 25 ClN2O6, chemical name: (3'S,4'S)-5-chloro-3'-(3,4-dihydroxybenzoyl)-4'-(2,5-dimethoxyphenyl)-1'-methylspiro[indoline-3,2'-pyrrolidin]-2-one. Molecular formula of C3d is C 27 H 25 BrN2O6, chemical name: (3'S,4'S)-5-bromo-3'-(3,4-dihydroxybenzoyl)-4'-(2,5-dimethoxyphenyl)-1'-methylspiro[indoline-3,2'-pyrrolidin]-2-one. Molecular formula of C3e is C 28 H 28 N2O6, chemical name: (3'S,4'S)-3'-(3,4-dihydroxybenzoyl)-4'-(2,5-dimethoxyphenyl)-1',5-dimethylspiro[indoline-3,2'-pyrrolidin]-2-one. Molecular formula of C3f is C 28 H 28 N2O7, chemical name: (3'S,4'S)-3'-(3,4-dihydroxybenzoyl)-4'-(2,5-dimethoxyphenyl)-5-methoxy-1'-methylspiro[indoline-3,2'-pyrrolidin]-2-one. Molecular formula of C3g is C 32 H 29 NO6, chemical name: (1R,3'S,4'S)-3'-(3,4-dihydroxybenzoyl)-4'-(2,5-dimethoxyphenyl)-1'-methyl-2H-spiro[acenaphthylene-1,2'-pyrrolidin]-2-one. Molecular formula of CS1 is C 25 H 22N2O2, chemical name: (3'S,4'S)-3'-benzoyl-1'-methyl-4'-phenylspiro[indoline-3,2'-pyrrolidin]-2-one. The molecular formula of CS2 is C 30 H 25 NO2, chemical name: (1S,3'S,4'R)-3'-benzoyl-1',3'-dimethyl-4'-phenyl-2H-spiro[acenaphthylene-1,2'-pyrrolidin]-2-one.

[0009] Oxidative stress injury is the main injury factor of myocardial ischemia-reperfusion injury, myocardial infarction, diabetic cardiomyopathy, myocarditis and other heart diseases. Hydrogen peroxide is an important reactive oxygen species, which is often used to induce oxidative damage to cardiomyocytes in vitro. Therefore, we used the hydrogen peroxide-induced oxidative damage model to evaluate the cell protective activity of the compounds. Tert-butyl hydroquinone (TBHQ) is a recognized food antioxidant additive that can effectively scavenge free radicals. Therefore, it was used as a positive control substance in this paper. As shown in Figure 1 A, all newly synthesized pyrrolidinones have good antioxidant activity. Among them, the activity of some compounds in H9c2 cells is significantly stronger than that of the lead compounds Cl or C3. Further cytotoxicity experiments Figure 1 В) showed that the toxicity of most compounds to cardiomyocytes was lower than that of the lead compounds Cl or C3.

[0010] In diabetic cardiomyopathy, high glucose is the main factor inducing myocardial injury. Therefore, we selected hydrogen peroxide and high glucose to induce myocardial cell injury models, respectively, to further study the concentration-dose relationship of compounds Clf and C3a in myocardial cell protective activity. Figure 2 A, B show that compounds C1f and C3a have good inhibitory effect on both injury models, and are concentration-dependent. In the colony formation experiment Figure 2 C), H2O2-induced injury severely inhibited the colony formation of H9c2 cells, but this inhibitory effect can be reversed by compounds C1f and C3a in a concentration-dependent manner, and is better than the positive control TBHQ.

[0011] ROS imbalance leads to oxidative stress, which damages cellular components such as proteins, enzymes and lipids, forming lipid peroxidation product malondialdehyde (MDA). The results of MDA detection are as follows Figure 3A, B showed that the MDA content of H2O2 damage group was significantly higher than that of DMSO group. After 18h pre-incubation of the drug, the MDA content was significantly reduced, with good concentration-dependent inhibition. This indicates that compounds C1f and C3a have inhibitory effects on H2O2-induced excessive production of ROS in cells. The level of ROS was determined by using dichlorofluorescein diacetate (DCFH-DA). Compared with the DMSO group, the ROS level of H9c2 cells under the action of H2O2 was significantly increased, and the fluorescence intensity was stronger. The ROS scavenging effect of the compound on the cells showed a dose-dependent manner, and was better than that of the positive control group TBHQ at 5μM. These findings further confirmed the cell protection effect of C1f and C3a.

[0012] Further explore the mechanism of the preferred compound C3a, Nrf2 is the main signaling pathway to regulate cell oxidative damage. Therefore, we detected whether C3a promotes the accumulation of Nrf2 in the nucleus by immunofluorescence. Blue and red staining is the nucleus and Nrf2, as shown in Figure 4 A. Compared with the DMSO group, the nucleus of the C3a treatment group showed red fluorescence, which was stronger than the positive control group (TBHQ). The above results show that C3a can promote the accumulation of Nrf2 in the nucleus. During oxidative stress, Nrf2 is activated to induce the expression of antioxidant enzymes such as heme oxygenase-1 (HO-1), which protects cells through anti-inflammatory and antioxidant mechanisms and regulates apoptosis. Further observe the effect of C3a on the expression of HO-1 protein in H9c2 cells. As shown in Figure 4 B, the expression of HO-1 in cells cultured with compound C3a increased in a concentration-dependent manner. In order to further confirm the protective effect of compound C3a on H2O2-induced oxidative stress damaged cells, we transfected H9c2 cells with Nrf2 small interfering (si) RNA to down-regulate the expression of Nrf2 protein Figure 4 C). Evaluate the protective effect of C3a on oxidative stress damage in Nrf2 siRNA group and control siRNA group. Down-regulation of Nrf2 expression inhibits the protective effect of C3a Figure 4 D). Therefore, C3a can protect H9c2 cells from oxidative damage by targeting Nrf2 to enhance the expression of HO-1. In addition, C3a significantly activates the Nrf2 signaling pathway, promotes the translocation of Nrf2 into the nucleus, and up-regulates the expression of antioxidant protein HO-1, while when Nrf2 expression is silenced by siRNA, its protective antioxidant effect on cells almost disappears.

[0013] The antioxidant compounds described in the present application can be applied to the preparation of antioxidant drugs and drugs for the treatment of myocardial related diseases, the etiology of which is at least partially caused by oxidative stress damage, including but not limited to the following diseases: myocardial ischemia-reperfusion injury, myocardial infarction, diabetic cardiomyopathy, myocarditis.

[0014] A pharmaceutical composition for treating myocardial disease, comprising a therapeutically effective amount of any one or more of the above-mentioned 16 pyrrolizidine compounds or pharmaceutically acceptable salts thereof as an active ingredient, and a pharmaceutical adjuvant. The "pharmaceutical composition" refers to a combination of any one or more of the 16 pyrrolizidine compounds or pharmaceutically acceptable salts thereof with an existing antioxidant drug, which is used to prepare a composition for preventing and treating myocardial disease.

[0015] The "pharmaceutical adjuvant" used herein refers to a conventional pharmaceutical carrier in the field of pharmacy, such as a diluent, an excipient, e.g., water, a filler, e.g., starch, sucrose, etc.; a binder, e.g., cellulose derivatives, alginate, gelatin, and polyvinylpyrrolidone; a humectant, e.g., glycerol; a disintegrant, e.g., agar, calcium carbonate, and sodium bicarbonate; an absorption enhancer, e.g., a quaternary ammonium compound; a surfactant, e.g., cetyl alcohol; an adsorptive carrier, e.g., kaolin and bentonite; a lubricant, e.g., talc, calcium / magnesium stearate, polyethylene glycol, etc. In addition, other adjuvants such as a flavoring agent, a sweetening agent, etc. can be further added to the composition.

[0016] The pharmaceutical composition of the present application can be prepared according to the conventional production method in the field of pharmacy. For example, the active ingredient is mixed with one or more carriers, and then it is prepared into a desired dosage form. The preparation form of the pharmaceutical includes an injection, a tablet, a capsule, an aerosol, a suppository, a film, a dripping pill, an ointment, a controlled or sustained release agent, or a nano preparation. The present application can be administered to a patient in need of such treatment in the form of a composition by oral, nasal inhalation, rectal, or parenteral administration. For oral administration, it can be prepared into a conventional solid preparation such as a tablet, a powder, a granule, a capsule, etc., a liquid preparation such as an aqueous or oily suspension, or other liquid preparations such as a syrup, an elixir, etc.; for parenteral administration, it can be prepared into an injection solution, an aqueous or oily suspension, etc.

[0017] The present application will be described in detail below with reference to examples and the accompanying drawings. BRIEF DESCRIPTION OF DRAWINGS

[0018] Figure 1 . Antioxidant protective activity and cytotoxicity of the compounds on H9c2 myocardial cells. (A) Antioxidant activity against H2O2-induced oxidative damage of myocardial cells. H9c2 cells were pretreated with 5 μM of the compounds for 18 h, and then stimulated with H2O2 for 24 h. The survival rate of H9c2 cells was determined by MTT assay. The survival rate of untreated cells was 100%. (B) Cytotoxicity screening of the compounds on H9c2 cells. The compounds (25 μM) were applied to the cells for 42 h, and the cytotoxicity of the compounds was determined by MTT assay. The data are expressed as mean ± SD, n = 3. Compared with DMSO, ****P < 0.0001, ***P < 0.001, **P < 0.01, *P < 0.05.

[0019] Figure 2 Protective effects of C1f and C3a on H2O2 and glucose-induced oxidative stress injury in H9c2 cells. (A, B) H9c2 cells were pre-incubated with different doses of compounds (1.25, 2.5, 5 μΜ) and 20 μΜ of TBHQ for 18 h, then stimulated with 650 μΜ of H2O2 for 24 h (A) or 140 mM of glucose for 48 h (A), respectively, and cell viability was determined by MTT assay. (C) Compounds C1f (1.25, 2.5, 5 μΜ) and C3a (1.25, 2.5, 5 μΜ) promoted H9c2 cell colony formation after H2O2 treatment. Data are presented as mean ± SD, n = 3. ###p<0.0001, ##p<0.01, *p<0.05 vs H2O2.

[0020] Figure 3 Protective effects of C1f and C3a on H2O2-induced oxidative stress injury in H9c2 cells. (A, B) Compounds C1f and C3a reduced MDA levels in H9c2 cells. H9c2 cells were plated at 2 × 105 cells / well in 6-well plates, and after 24 h, drugs were added (concentration 1.25, 2.5, 5 μΜ) for 18 h, and then H2O2 (900 μΜ) was added for stimulation. After 2 h, the protein was collected for MDA experiments. (C) Compounds C1f and C3a reduced ROS levels in H9c2 cells. H9c2 cells were plated at 1.5 × 105 cells / well in 6-well plates, incubated for 24 h, and then drugs were added (concentration 1.25, 2.5, 5 μΜ) for 18 h of pre-treatment, and then exposed to 600 μΜ of H2O2 for 2 h. Cells were incubated with 1 μL of DCFH-DA (10 μΜ) for 30 min. Images were taken under a fluorescence microscope (Nikon). Data are presented as mean ± SD, n = 3. ###p<0.0001, ##p<0.01, *p<0.05 vs H2O2. 5

[0021] Figure 4 ​Compound C3a protects H9c2 cells from oxidative stress injury by activating Nrf2 signaling pathway. (A) Compound C3a induces Nrf2 nuclear translocation in H9c2 cells. After incubation with C3a (10 μΜ) and TBHQ (5 μΜ) for 6 h, Nrf2 antibody was incubated, and DAPI staining was performed. Nuclear translocation images were taken under fluorescence microscope. (B) Compound C3a up-regulates HO-1 protein expression in H9c2 cells. After incubation with C3a (0.625, 1.25 and 5 μΜ) and TBHQ (5 μΜ) for 18 h, western blot was performed to detect HO-1 protein level. (C, D) The cell protection effect of C3a is weakened after down-regulation of Nrf2 protein level by siRNA. H9c2 cells were transfected with Nrf2 siRNA (si-Nrf2) and control siRNA (NC), respectively. C3a (0.625 μΜ) was pre-incubated for 18 h, and then exposed to H2O2 (650 μΜ) for 24 h. Cell viability was detected by MTT method, and protein level was detected by western blot. Data were expressed as mean + SD, n = 3. p < 0.0001 vs. DMSO, p < 0.05 vs. H2O2. DETAILED DESCRIPTION

[0022] The present application is further illustrated in the following examples. These examples are for illustrative purposes only and are not intended to limit the scope of the present application.

[0023] Synthesis of compounds in Example 1

[0024] In a 100 mL reaction bottle, chalcone (0.10 g, 0.37 mmol / 0.33 mmol), sarcosine (0.05 g, 0.56 mmol), and indigo or acenaphthenequinone containing different substituents were added in a molar ratio of 1:1.5:1.5. Methanol (6-8 mL) was used to dissolve under the action of ultrasonic waves, and the reaction was placed in a magnetic heating stirrer oil bath. The temperature of the whole process was controlled at 67°C, and the reaction was carried out for about 12 h. The reaction was detected by TCL. After the reaction was completed, the degree of reaction was judged by TCL. If the impurities were less, the ice bath was placed, and the precipitate was precipitated. The precipitate was reduced pressure filtration, washed with a small amount of dichloromethane, and then recrystallized with a methanol and water system; if the product was more impure, column chromatography was used for separation and purification with petroleum ether: ethyl acetate = 1:1 as the mobile phase to obtain pure product, and then physical and chemical property characterization was performed.

[0025] (3'S,4'S)-3'-(3,4-dihydroxybenzoyl)-4'-(2-methoxyphenyl)-l'-methylspiro[indoline-3,2'-pyrrolidin]-2-one (C1a). Yellow powder, 72.3% yield, mp 121.2-123.5 °C.1H-NMR (400 MHz, DMSO-d6), δ: 7.410 (d, J = 7.6 Hz, 1H), 7.208 (t, J = 16.0 Hz, 1H), 7.019 (t, J = 14.8 Hz, 1H), 6.966 (s, 1H), 6.956 (s, 1H), 6.937 (t, J = 6.0 Hz, 2H), 6.909 (d, J = 8.4 Hz, 1H), 6.833 (t, J = 14.8 Hz, 1H), 6.601 (d, J = 8.0 Hz, 1H), 6.565 (d, J = 7.6 Hz, 1H), 4.651-4.586 (m, 1H), 4.510 (d, J = 8.8 Hz, 1H), 3.726 (s, 3H), 3.259 (t, J = 18.0 Hz, 2H), 2.022 (d, J = 9.2 Hz, 3H). LC-MS m / z: 445.2 (M+H) + , calcd for C 26 H 24 N2O5: 444.17.

[0026] (3'S,4'S)-3'-(3,4-dihydroxybenzoyl)-5-fluoro-4'-(2-methoxyphenyl)-l'-methylspiro[indoline-3,2'-pyrrolidin]-2-one. (C1b). Faint yellow powder, 70.8% yield, mp 213.5-215.6 °C. LC-MS m / z: 463.2. (M+H) + , calcd for C 26 H 23 FN2O5: 462.16.

[0027] (3'S,4'S)-5-chloro-3'-(3,4-dihydroxybenzoyl)-4'-(2-methoxyphenyl)-1'- methylspiro[indoline-3,2'-pyrrolidin]-2-one. (C1c). Faint yellow powder, 69.8% yield, mp 176.4-178.2 °C. 1 H-NMR (400 MHz, DMSO-d6), δ: 7.367 (d, J = 6.4 Hz, 1H), 7.1893 (d, J = 16.8 Hz, 1H), 7.070 (d, J = 6.8 Hz, 1H), 6.958 (t, J = 11.6 Hz, 2H), 6.900 (d, J = 8.8 Hz, 1H), 6.871 (s, 1H), 6.783 (s, 1H), 6.599 (d, J = 8.4 Hz, 1H), 6.260 (d, J = 8.4 Hz, 1H), 4.553 (t, J = 24.4 Hz, 3H), 3.906 (s, 1H), 3.792 (s, 1H), 2.050 (s, 2H). LC-MS m / z: 479.2 (M+H) + , calcd for C 26 H 23 ClN2O5: 478.13.

[0028] (3'S,4'S)-5-chloro-3'-(3,4-dihydroxybenzoyl)-4'-(2-methoxyphenyl)-1'- methylspiro[indoline-3,2'-pyrrolidin]-2-one. (C1c). Faint yellow powder, 69.8% yield, mp 176.4-178.2 °C. 1H-NMR (400 MHz, DMSO-d6), δ: 10.622 (s, 1H), 7.372 (d, J = 7.6 Hz, 1H), 7.209-7.176 (m, 2H), 7.025 (d, J = 1.6 Hz, 1H), 6.943 (d, J = 2.4 Hz, 2H), 6.929-6.891 (m, 2H), 6.611 (d, J = 8.4 Hz, 1H), 6.522 (d, J = 8.0 Hz, 1H), 4.568 (t, J = 16.4 Hz, 1H), 4.510 (t, J = 13.6 Hz, 1H), 3.701 (s, 3H), 3.249 (t, J = 17.6 Hz, 2H), 2.043 (s, 3H). LC-MS m / z: 523.1 (M+H) + , calcd for C 26 H 23 BrN2O5: 522.08.

[0029] (3'S,4'S)-3'-(3,4-dihydroxybenzoyl)-4'-(2-methoxyphenyl)-1',5-dimethylspiro[indoline-3,2'-pyrrolidin]-2-one (C1e). Faint yellow powder, 68.8% yield, mp 104.3-107.5 °C. 1 H-NMR (400 MHz, DMSO-d6), δ: 10.622 (s, 1H), 7.372 (d, J = 7.6 Hz, 1H), 7.209-7.176 (m, 2H), 7.025 (d, J = 1.6 Hz, 1H), 6.943 (d, J = 2.4 Hz, 2H), 6.929-6.891 (m, 2H), 6.611 (d, J = 8.4 Hz, 1H), 6.522 (d, J = 8.0 Hz, 1H), 4.568 (t, J = 16.4 Hz, 1H), 4.510 (t, J = 13.6 Hz, 1H), 3.701 (s, 3H), 3.249 (t, J = 17.6 Hz, 2H), 2.043 (s, 3H). LC-MS m / z: 523.1 (M+H) + , calcd for C 27 H 26N2O5: 458.18.

[0030] (3'S,4'S)-3'-(3,4-dihydroxybenzoyl)-5-methoxy-4'-(2-methoxyphenyl)-1'- methylspiro[indoline-3,2'-pyrrolidin]-2-one (C1f). Faint yellow powder, 69.3% yield, mp 96.5-99.4 °C. LC-MS m / z: 475.2. (M+H) + , calcd for C 27 H 26 N2O6: 474.18.

[0031] (1R,3'S,4'S)-3'-(3,4-dihydroxybenzoyl)-4'-(2-methoxyphenyl)-1'-methyl-2H- spiro[acenaphthylene-1,2'-pyrrolidin]-2-one. (C1g). White powder, 69.1% yield, mp 90.6-92.5 °C. 1 H-NMR (400 MHz, DMSO-d6), δ: 8.067 (d, J = 6.4 Hz, 1H), 7.832 (d, J = 5.6 Hz, 1H), 7.770 (d, J = 6.8 Hz, 1H), 7.678 (s, 1H), 7.591 (s, 1H), 7.498 (s, 2H), 7.368 (s, 3H), 7.248 (s, 1H), 7.074 (d, J = 4.8 Hz, 1H), 7.009 (s, 2H), 6.862 (s, 2H), 4.579-4.505 (m, 2H), 3.543 (d, J = 6.4 Hz, 2H), 1.994 (s, 3H). LC-MS m / z: 480.2 (M+H) + , calcd for C 30 H 25 NO5: 479.17.

[0032] (3'S,4'S)-3'-(3,4-dihydroxybenzoyl)-4'-(2,5-dimethoxyphenyl)-1'-methylspiro[indoline-3,2'-pyrrolidin]-2-one. (C3a). Claybank powder, 59.8% yield, mp 229.1-230.5 °C. LC-MS m / z: 475.2 (M+H) + , calcd for C 27 H 26 N2O6: 474.18.

[0033] (3'S,4'S)-3'-(3,4-dihydroxybenzoyl)-4'-(2,5-dimethoxyphenyl)-5-fluoro-1'-methylspiro[indoline-3,2'-pyrrolidin]-2-one. (C3b). Claybank powder, 69.1% yield, mp 248.3-377.5 °C. 1 H-NMR (400 MHz, DMSO-d6), δ: 6.987 (d, J = 3.2 Hz, 1H), 6.885 (s, 1H), 6.839 (d, J = 14.4 Hz, 3H), 6.759 (d, J = 9.2 Hz, 2H), 6.591 (d, J = 4.8 Hz, 1H), 6.351 (s, 1H), 4.560 (t, 1H), 4.482 (d, J = 9.6 Hz, 1H), 3.700 (s, 3H), 3.670 (s, 3H), 3.366 (t, J = 6.6 Hz, 1H), 3.179 (s, 1H), 2.046 (s, 3H). LC-MS m / z: 493.2 (M+H) + , calcd for C 27 H 25 FN2O6: 492.17.

[0034] (3'S,4'S)-5-chloro-3'-(3,4-dihydroxybenzoyl)-4'-(2,5-dimethoxyphenyl)-1'-methylspiro[indoline-3,2'-pyrrolidin]-2-one. (C3c). Reddish brown powder, 61.1% yield, mp 242.4-245.1 °C. 1H-NMR (400 MHz, DMSO-d6), δ: 7.062 (s, 1H), 6.947 (s, 2H), 6.852 (s, 2H), 6.744 (s, 1H), 6.596 (s, 1H), 4.527 (s, 2H), 3.681 (s, 8H), 3.347 (s, 1H), 3.167 (s, 1H), 2.029 (s, 3H). LC-MS m / z: 509.2 (M+H) + , calcd for C 27 H 25 ClN2O6: 508.14.

[0035] (3'S,4'S)-5-bromo-3'-(3,4-dihydroxybenzoyl)-4'-(2,5-dimethoxyphenyl)-1'- methylspiro[indoline-3,2'-pyrrolidin]-2-one. (C3d). Claybank powder, 61.1% yield, mp 269.3-271.6 °C. 1 H-NMR (400 MHz, DMSO-d6), δ: 7.062 (s, 1H), 6.947 (s, 2H), 6.852 (s, 2H), 6.744 (s, 1H), 6.596 (s, 1H), 4.527 (s, 2H), 3.681 (s, 8H), 3.347 (s, 1H), 3.167 (s, 1H), 2.029 (s, 3H). LC-MS m / z: 509.2 (M+H) + , calcd for C 27 H 25 BrN2O6: 552.09.

[0036] (3'S,4'S)-3'-(3,4-dihydroxybenzoyl)-4'-(2,5-dimethoxyphenyl)-1',5-dimethylspiro[indoline-3,2'-pyrrolidin]-2-one. (C3e). Reddish brown powder, 69.8% yield, mp 271.2-272.5 °C. 1H-NMR (400 MHz, DMSO-d6), δ: 6.994 (d, J = 2.8 Hz, 1H), 6.939 (d, J = 2.0 Hz, 1H), 6.889 (t, J = 14.8 Hz, 2H), 6.818 (d, J = 7.6 Hz, 1H), 6.766 (dd, Ji = 10.2, J2= 4.8 Hz, 2H), 6.606 (d, J = 8.4 Hz, 1H), 6.452 (d, J = 8.0 Hz, 1H), 4.621 - 4.558 (m, 1H), 4.454 (d, J = 9.6 Hz, 1H), 3.684 (s, 6H), 3.485 - 3.432 (m, 1H), 3.216 (t, J = 18.4 Hz, 1H), 2.174 (s, 3H), 2.019 (s, 3H). LC-MS m / z: 489.2 (M+H) + , calcd for C 28 H 28 N2O6: 488.19.

[0037] (3'S,4'S)-3'-(3,4-dihydroxybenzoyl)-4'-(2,5-dimethoxyphenyl)-5-methoxy-1'- methylspiro[indoline-3,2'-pyrrolidin]-2-one (C3f). Claybank powder, 63.1% yield, mp 228.1-234.5 °C. 1 H-NMR (400 MHz, DMSO-d6), δ: 7.001 (d, J = 2.4 Hz, 1H), 6.924 (s, 1H), 6.869 (d, J = 3.2 Hz, 1H), 6.744 (dd, Ji = 4.4 Hz, J2= 7.0 Hz, 1H) 6.574 (dd, Ji = 4.0 Hz, J2= 13.6 Hz, 1H), 6.533 (d, J = 2.0 Hz, 1H), 6.476 (d, J = 6.8 Hz, 1H), 4.597 - 4.546 (m, 1H), 4.425 (d, J = 7.2 Hz, 1H), 3.661 (d, J = 4.4 Hz, 7H), 3.629 (s, 3H), 3.337 (t, J = 12.4 Hz, 1H), 3.219 (t, J = 14.4 Hz, 1H), 2.016 (s, 3H). LC-MS m / z: 505.2 (M+H) + , calcd for C 28 H 28 N2O7: 504.19.

[0038] 1R,3'S,4'S)-3'-(3,4-dihydroxybenzoyl)-4'-(2,5-dimethoxyphenyl)-1'-methyl-2H-spiro[acenaphthylene-1,2'-pyrrolidin]-2-one.(C3g).White powder,64.8%yield,mp 195.4-199.3℃. 1 H-NMR (400 MHz, DMSO-d6), δ: 8.141 (d, J = 8.0 Hz, 1H), 7.879 (d, J = 6.8 Hz, 1H), 7.816 (d, J = 8.0 Hz, 1H), 7.729 (t, J = 15.2 Hz, 1H), 7.600 (t, J = 15.6 Hz, 1H), 7.386 (d, J = 6.8 Hz, 1H), 7.087 (d, J = 2.8 Hz, 1H), 6.905 (d, J = 8.8 Hz, 1H), 6.793 (dd, J1= 4.2 Hz, J2= 13.8 Hz, 1H), 6.695 (d, J = 2.0 Hz, 1H), 6.571 (d, J = 8.0 Hz, 1H), 6.246 (d, J = 8.4 Hz, 1H), 4.762-4.699 (m, 1H), 4.586 (d, J = 9.2 Hz, 1H), 3.726 (s, 3H), 3.698 (s, 3H), 3.522 (t, J = 20.8 Hz, 1H), 3.371 (s, 1H), 1.940 (d, J = 10.4 Hz, 3H). LC-MS m / z: 510.2 (M+H) + , calcd for C 31 H 27 NO6: 509.18

[0039] (3'S,4'S)-3'-benzoyl-1'-methyl-4'-phenylspiro[indoline-3,2'-pyrrolidin]-2-one.(CS1). White powder, 59.8%yield, mp 200.8-203.0℃. 1H-NMR (400 MHz, DMSO-d6), δ: 10.549 (s, 1H), 7.454 (s, 1H), 7.428 (d, J = 6.0 Hz, 2H), 7.392 (s, 1H), 7.347 (t, J = 17.2 Hz, 3H), 7.289-7.219 (m, 3H), 7.006-6.940 (m, 2H), 6.839 (t, J = 14.8 Hz, 1H), 6.475 (d, J = 7.6 Hz, 1H), 4.463 (d, J = 9.2 Hz, 1H), 4.406-4.341 (m, 1H), 3.434 (m, 2H), 2.078 (s, 3H). LC-MS m / z: 383.1 (M+H) + , calcd for C 25 H 22 N2O2: 382.17.

[0040] Example 2 Cell culture

[0041] H9c2 cells were purchased from Shanghai Cell Bank of Chinese Academy of Sciences, and were placed in a CO2 incubator with a temperature of 37°C and a CO2 content of 5% using a prepared DMEM high-sugar medium. The cells were grown to the logarithmic growth phase and were used for the corresponding experiments.

[0042] Example 3 Determination of the concentration of H2O2 damage model

[0043] H9c2 cells in the logarithmic growth phase were placed in a 96-well plate at a density of 3000 cells per well, with 100 μL of medium per well. The periphery of the 96-well plate was filled with PBS to prevent edge effects caused by water evaporation. Then, the plate was placed in a constant-temperature cell incubator and incubated overnight. Different final concentrations (from 260-580 μM, with a concentration interval of 20, for a total of 17 groups) of H2O2 were added to the cells, which were then incubated in the cell incubator for 24 h. Then, 20 μL of MTT solution with a concentration of 5 mg / mL was added, and after 4 h, the liquid in the 96-well plate was removed, and 120 μL of DMSO solution was added to each well. The plate was shaken for 10 min on a micro-vibrator in the dark, and the OD value at 490 nm was detected using an enzyme-labeled instrument. PBS was used as a blank control group A, and the cell survival rate was calculated (experimental group A value / blank control group A value x 100%). The experiment was repeated three times. Finally, the concentration of H2O2 corresponding to about 60% cell survival rate was taken as the concentration of the H2O2 damage model.

[0044] Example 4 Determination of oxidative damage by MTT colorimetry

[0045] H9c2 cells were seeded in 96-well plates, approximately 3000 cells per well, with 100 μL of culture medium. The plates were placed in a CO2 incubator at 37°C with 5% CO2 concentration and incubated overnight. The next day, a specific concentration of the target compound was added and the cells were cultured for 18 hours. Then, a specific concentration of H2O (determined in step 1.4.3 for modeling damage) was added for 24 hours of stimulation. Finally, 20 μL of 5 mg / mL MTT solution was added, and the plates were placed in a CO2 incubator under pre-set conditions for further culture. After approximately 4 hours, the liquid in each well was aspirated, and 120 μL of DMSO solution was added to each well, gently swirling for about 1 minute to mix. Absorbance was then measured using an ELISA reader set to 490 nm, with DMSO used as a blank control (B). Cell viability was calculated as (experimental group A value / control group B value × 100%). The experiment was repeated three times to minimize experimental error.

[0046] Example 5: MDA Determination

[0047] H9c2 cells were seeded at a density of 2 × 10⁵ cells / well in 6-well plates and allowed to adhere for 48 hours. The compound was added and the cells were incubated for 18 hours. Then, H₂O₂ (900 μM) was added for stimulation, and protein was collected after 2 hours. MDA was detected using an MDA kit (beyotimebiotech, China).

[0048] Example 6: ROS Measurement

[0049] H9c2 cells were fed at a concentration of 1.5 × 10⁻⁶. 5 Cells were seeded at a density of 100 cells / well in 6-well plates and incubated for 24 hours. Cells were pretreated with a compound for 18 hours, followed by exposure to 600 μM H₂O₂ for 2 hours. Cells were then incubated in the dark at 37°C with 1 μL of 2',7'-dichlorofluorescein diacetate (DCFH-DA; Beyotime; Shanghai, China, 10 μM). Images were taken under a fluorescence microscope for formation (Nikon).

[0050] Example 5: Western Blot Analysis

[0051] H9c2 cells in logarithmic growth phase were seeded in 6-well plates, approximately 1.5 × 10⁶ cells per well. 6The cells were placed in a pre-set cell incubator and cultured for about 48 h, after which a certain concentration of the synthesized drug and a positive control drug were added, and a blank control group was set. Then they were placed in the cell incubator for continuous culture, and after about 18 h, each well was washed 3 times with cold PBS, placed on ice, and then 60 μL of lysis solution was added for lysis for about 10 min. The EP tube was labeled, and the lysed cells were scraped into it. Then centrifugal stratification was performed at 4°C, 12000 rpm, and 10 min. The protein concentration in the protein supernatant obtained by centrifugation was determined by the Coomassie brilliant blue method, and the required sample was prepared after calculation, loading buffer was added, and protein denaturation was performed in a metal bath for 10 min. Finally, it was stored in -20°C. Then electrophoresis separation was performed in a polyacrylamide gel, and the membrane was transferred in an ice bath, with the condition set to 300 mA for 90 min. Then 5% skimmed milk powder blocking solution was used to block at room temperature for about 90 min. After blocking, the blocking solution was discarded, and then 1x TBST buffer was used for washing, each time for 7 min, and repeated 3 times. Then specific primary antibodies were added: β-actin (1:2000), HO-1 (1:500), GADPH (1:2000). Incubate overnight in the refrigerator at 4°C on a shaking table. The next day, rinse with TBST for three times, each time for about 7 min. After rinsing, incubate the secondary antibody at room temperature for 1 h, and then rinse with 1x TBST solution for three times, each time for about 7 min. Finally, develop the color with a gel imaging system. The processed samples were scanned using Quantity One gel imaging analysis software, the optical density values of the target band and the internal reference were detected, and the ratio of the two was calculated for statistical analysis.

[0052] Example 6 Colony Cloning Experiment

[0053] H9c2 cells were seeded in a 6-well plate at a density of 1000 cells / well and cultured for 24 h, and then incubated with a certain concentration of target compounds C3a and DMSO at 37°C, 5% CO2 incubator for 18 h. The original culture medium was replaced with fresh RPMI 1640 culture medium according to the change of pH. Finally, the upper culture medium was discarded, washed with PBS for 3 times, fixed with paraformaldehyde, and after about 10 min, the paraformaldehyde was discarded, and then the prepared PBS was washed for 3 times, and finally stained with crystal violet.

Claims

1. The use of any of the following compounds in the preparation of therapeutic drugs for cardiomyocyte injury diseases, wherein the cardiomyocyte injury diseases are myocardial ischemia-reperfusion injury, myocardial infarction, diabetic cardiomyopathy, and myocarditis, and the structural formulas of the compounds are as follows: 。 2. The application according to claim 1, wherein the compound is selected from any of the following compounds: 。 3. A pharmaceutical composition for treating myocardial cell injury, comprising a therapeutically effective amount of the following compounds or their pharmaceutically acceptable salts and excipients: 。 4. The pharmaceutical composition according to claim 3, characterized in that, The compound or its pharmaceutically acceptable salt is the sole active ingredient.

5. The pharmaceutical composition according to claim 4, characterized in that: The pharmaceutical composition is formulated in the form of injections, tablets, capsules, aerosols, suppositories, films, pellets, ointments, controlled-release or sustained-release formulations, and nanoformulations.

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