Application of pyrrolidone compounds in preparation of myocardial injury protection drugs

Abstract

The invention belongs to the field of medicinal chemistry, and particularly relates to application of specific pyrrolidone chalcone compounds in preparation of drugs for treating myocardial injury related diseases, and the pyrrolidone chalcone compounds can have a good protection effect on myocardial cell injury induced by oxidative stress and high glucose and can be used for preparing drugs for treating myocardial injury related diseases. The compounds can effectively eliminate the generation of active oxygen in oxidative stress injury by activating an Nrf2 / HO-1 antioxidant signal channel, and have a very good protection effect on myocardial cell injury.

Description

Technical field:

[0001] The present invention belongs to the field of pharmaceutical chemistry, and in particular, relates to the application of a specific pyrrolidone chalcone compound in the preparation of therapeutic drugs for myocardial injury-related diseases. Background technology:

[0002] Myocardial ischemia-reperfusion injury, diabetic cardiomyopathy, etc. are heart diseases that seriously endanger human health. There are currently no effective treatments for them. It is of great significance to discover highly effective and low-toxic treatment drugs.

[0003] In the present invention, we introduced a variety of pyrrolidone structural units into the chalcone skeleton, and the target compounds obtained by screening not only have a good protective effect on myocardial cell damage, but their cytotoxicity is also significantly lower than that of the parent chalcone compounds before modification. Summary of the invention:

[0004] The present invention aims to provide the use of 16 pyrrolidone chalcone compounds in the preparation of therapeutic drugs for myocardial injury-related diseases.

[0005] Another object of the present invention is to provide a pharmaceutical composition for treating myocardial disease, which contains a therapeutically effective amount of any one or more of the chalcone compounds described in claim 1 or their pharmaceutically acceptable salts and pharmaceutical excipients as active ingredients.

[0006] Specifically, the 16 pyrrolidone chalcone compounds of the present invention have the following structures:

[0007]

[0008] The molecular formula of C1a is C 26 H 24 N2O5, chemical name: (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, chemical name: (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 23ClN2O5, 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. The 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. The 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. The 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. The 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. The 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. The 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 damaging factor of heart diseases such as myocardial ischemia-reperfusion injury, myocardial infarction, diabetic cardiomyopathy, and myocarditis. Hydrogen peroxide is an important reactive oxygen species and 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 compound. Tert-butylhydroquinone (TBHQ) is a recognized food antioxidant additive that can effectively scavenge free radicals. Therefore, it is used as a positive control substance in this article. Figure 1 As shown in A, all the newly synthesized pyrrolidone compounds have good antioxidant activity. Among them, the activity of some compounds in H9c2 cells is significantly stronger than that of the lead compound C1 or C3. Further cytotoxicity experiments ( Figure 1 В) showed that most compounds were less toxic to cardiomyocytes than 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-induced cardiomyocyte injury models to further study the concentration-dose relationship of compounds Clf and C3a in cardiomyocyte protective activity. Figure 2 A and B show that compounds C1f and C3a have good inhibitory effects 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 could be reversed by compounds C1f and C3a in a concentration-dependent manner and was superior to the positive control TBHQ.

[0011] ROS imbalance leads to oxidative stress, which damages cell components such as proteins, enzymes and lipids, and forms lipid peroxidation product malondialdehyde (MDA). Figure 3As shown in A and B, the MDA content in the H2O2 injury group was significantly higher than that in the DMSO group. After 18 h of drug preincubation, the MDA content was significantly reduced, with a good concentration-dependent inhibitory effect. This indicates that compounds C1f and C3a have an inhibitory effect on the excessive production of cellular ROS induced by H2O2. Dichlorodihydrofluorescein diacetate (DCFH-DA) was used to determine the level of ROS. 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 scavenging effect of this compound on intracellular ROS was dose-dependent and better than the positive control group TBHQ at 5 μM. These findings further confirmed the cytoprotective effect of C1f and C3a.

[0012] To further explore the mechanism of action of the preferred compound C3a, Nrf2 is the main signaling pathway that regulates cellular oxidative damage. Therefore, we used immunofluorescence to detect whether C3a promotes the accumulation of Nrf2 in the cell nucleus. Blue and red staining are for the cell nucleus and Nrf2, such as Figure 4 As shown in A. Compared with the DMSO group, the cell nucleus of the C3a-treated group showed red fluorescence, which was stronger than that of the positive control group (TBHQ). The above results show that C3a can promote the accumulation of Nrf2 in the cell nucleus. During oxidative stress, Nrf2 is activated, inducing the expression of antioxidant enzymes such as heme oxygenase-1 (HO-1), protecting cells through anti-inflammatory and antioxidant mechanisms and regulating cell apoptosis. The effect of C3a on the expression of HO-1 protein in H9c2 cells was further observed. Figure 4 As shown in Figure B, in cells cultured with compound C3a, the expression of HO-1 increased in a concentration-dependent manner. 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 downregulate the expression of Nrf2 protein ( Figure 4 C). The protective effect of C3a on oxidative stress injury in the Nrf2 siRNA group and the control siRNA group was evaluated. Down-regulation of Nrf2 expression inhibited 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 activated the Nrf2 signaling pathway, promoted the translocation of Nrf2 into the nucleus, and upregulated the expression of the antioxidant protein HO-1. However, when Nrf2 expression was silenced by siRNA, its protective antioxidant effect on cells almost disappeared.

[0013] The antioxidant compounds of the present invention can be used in the preparation of antioxidant drugs and therapeutic drugs for myocardial-related diseases, the causes of which are at least partially caused by oxidative stress damage, and the diseases include but are not limited to the following diseases: myocardial ischemia-reperfusion injury, myocardial infarction, diabetic cardiomyopathy, and myocarditis.

[0014] A pharmaceutical composition for treating myocardial diseases, comprising a therapeutically effective amount of any one or more of the 16 pyrrolidone compounds or their pharmaceutically acceptable salts and pharmaceutical excipients as active ingredients. "Pharmaceutical composition" refers to a composition of drugs for preventing and treating myocardial diseases prepared by combining any one or more of the 16 pyrrolidone chalcone compounds or their pharmaceutically acceptable salts with an antioxidant drug currently on the market.

[0015] As used herein, "pharmaceutical excipients" refer to conventional drug carriers in the pharmaceutical field, such as: diluents, excipients such as water, fillers such as starch, sucrose, etc.; binders such as cellulose derivatives, alginates, gelatin and polyvinyl pyrrolidone; wetting agents such as glycerol; disintegrants such as agar, calcium carbonate and sodium bicarbonate; absorption promoters such as quaternary ammonium compounds; surfactants such as cetyl alcohol; adsorption carriers such as kaolin and bentonite; lubricants such as talc, calcium / magnesium stearate, polyethylene glycol, etc. In addition, other adjuvants such as flavoring agents, sweeteners, etc. may be added to the composition.

[0016] The various dosage forms of the pharmaceutical composition of the present invention can be prepared according to conventional production methods in the pharmaceutical field. For example, the active ingredient is mixed with one or more carriers and then made into the desired dosage form. The preparation forms of the drug include injections, tablets, capsules, aerosols, suppositories, films, pills, ointments, controlled release or sustained release agents or nano preparations. The present invention can be administered to patients who need such treatment in the form of a composition by oral, nasal inhalation, rectal or parenteral administration. When used for oral administration, it can be made into conventional solid preparations such as tablets, powders, granules, capsules, etc., and into liquid preparations such as water or oil suspensions or other liquid preparations such as syrups, elixirs, etc.; when used for parenteral administration, it can be made into solutions for injection, water or oil suspensions, etc.

[0017] The present invention will be described in detail below with reference to the embodiments and the accompanying drawings. Description of the drawings:

[0018] Figure 1 . Antioxidant protective activity and cytotoxicity of the compounds on cardiomyocytes H9c2. (A) Antioxidant activity against H2O2-induced oxidative damage in cardiomyocytes. H9c2 cells were pretreated with 5 μM of the compound for 18 h, then stimulated with H2O2 for 24 h, and the survival rate of H9c2 cells was determined by MTT method. The survival rate of untreated cells was 100%. (B) Cytotoxicity screening of compounds on H9c2 cells. The compounds (25 μM) were treated with cells for 42 h, and the cytotoxicity of the compounds was determined by MTT method. 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 oxidative stress injury of H9c2 cells induced by H2O2 and glucose. (A, B) H9c2 cells were pre-incubated with different doses of compounds (1.25μM, 2.5μM, 5μM) and 20μM TBHQ for 18h, and then stimulated with 650μM H2O2 for 24h (A) or 140mM glucose for 48h (A), and the survival rate of cells was measured by MTT. (C) Compounds C1f (1.25μM, 2.5μM, 5μM) and C3a (1.25μM, 2.5μM, 5μM) promoted the colony formation of H9c2 cells after H2O2 treatment. Data are expressed as mean ± SD, n = 3. ###p<0.0001, "p<0.01", p<0.05vs H2O2.

[0020] Figure 3 . Protective effects of C1f and C3a on H9c2 cells induced by H2O2 oxidative stress injury. (A, B) Compounds C1f and C3a reduce the MDA level in H9c2 cells. 200,000 H9c2 cells / well were plated in 6-well plates. After 24 hours, drugs (concentrations of 1.25, 2.5, and 5 μM) were added. The drugs were allowed to act for 18 hours, and H2O2 (900 μM) was added for stimulation. After 2 hours, proteins were collected for MDA experiments. (C) Compounds C1f and C3a reduce the ROS level in H9c2 cells. H9c2 cells were cultured at 1.5×10 5 Cells were plated in 6-well plates at a density of 10 cells / well and incubated for 24 hours. Cells were pretreated with drugs (concentrations of 1.25, 2.5, and 5 μM) for 18 hours and then exposed to 600 μM H2O2 for 2 hours. Cells were incubated with 1 μL of DCFH-DA (10 μM) for 30 minutes. Images were taken under a fluorescence microscope (Nikon). Data are expressed as mean ± SD, n = 3. ####p<0.0001, **p<0.01, *p<0.05 vs H2O2.

[0021] Figure 4. Compound C3a protects H9c2 cells from oxidative stress damage by activating the Nrf2 signaling pathway. (A) Compound C3a induces Nrf2 nuclear translocation in H9c2 cells. After incubation with C3a (10μM) and TBHQ (5μM) for 6h, Nrf2 antibody was incubated and DAPI staining was performed. Nuclear translocation images were taken under a fluorescence microscope. (B) Compound C3a upregulated HO-1 protein expression in H9c2 cells. After incubation with C3a (0.625, 1.25 and 5μM) and TBHQ (5μM) for 18h, HO-1 protein levels were detected by western blot. (C, D) After siRNA downregulated Nrf2 protein levels, the cytoprotective effect of C3a was weakened. H9c2 cells were transfected with Nrf2 siRNA (si-Nrf2) and control siRNA (NC), respectively. C3a (0.625 μM) was pretreated for 18 h, and then exposed to H2O2 (650 μM) for 24 h. Cell viability was detected by MTT method, and protein levels were detected by western blot. Data are expressed as mean + SD, n = 3. p < 0.0001 for DMSO, p < 0.05 for H2O2. Specific implementation method:

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

[0023] Example 1 Synthesis of Compounds

[0024] In a 100mL reaction bottle, add chalcone (0.10g, 0.37mmol / 0.33mmol), sarcosine (0.05g, 0.56mmol), and isatin or acenaphthenequinone containing different substituents in a molar ratio of 1:1.5:1.5. Dissolve with methanol (6-8mL) under ultrasonic action, put into a magnetic heating stirrer oil bath for reaction, the temperature of the whole process is controlled at 67℃, the reaction is about 12h, and the reaction is detected by TCL. After the reaction is completed, use TCL to judge the degree of reaction. If there are few impurities, let it stand in an ice bath, wait for the precipitation to precipitate, filter the precipitate under reduced pressure, rinse with a small amount of dichloromethane, and then recrystallize it with a methanol and water system; if the product is more impure, use petroleum ether: ethyl acetate = 1:1 as the mobile phase to separate and purify it by column chromatography to obtain a pure product, and then characterize the physical and chemical properties.

[0025] (3'S,4'S)-3'-(3,4-dihydroxybenzoyl)-4'-(2-methoxyphenyl)-1'-methylspiro[indoline-3,2'-pyrrolidin]-2-one(C1a).Yellow powder,72.3%yield,mp121.2-123.5℃.1H-NMR(400MHz,DMSO-d6),δ:7.410(d,J=7.6Hz,1H),7.208(t,J=16.0Hz,1H),7.019(t,J=14.8Hz,1H),6.966(s,1H),6.956(s,1H),6.937(t,J=6.0Hz,2H),6.909(d,J=8.4Hz,1H),6.833(t,J=14.8Hz,1H),6.601(d,J=8.0Hz,1H),6.565(d,J=7.6Hz,1H),4.651-4.586(m,1H),4.510(d,J=8.8Hz,1H),3.726(s,3H),3.259(t,J=18.0Hz,2H),2.022(d,J=9.2Hz,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)-1'-methylspiro[indoline-3,2'-pyrrolidin]-2-one.(C1b).Faint yellow powder,70.8%yield,mp 213.5-215.6℃.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℃. 1 H-NMR(400MHz,DMSO-d6),δ:7.367(d,J=6.4Hz,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=8.8Hz,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-bromo-3'-(3,4-dihydroxybenzoyl)-4'-(2-methoxyphenyl)-1'-methylspiro[indoline-3,2'-pyrrolidin]-2-one.(C1d).Faint yellow powder,64.8%yield,mp 126.4-127.2℃. 1H-NMR(400MHz,DMSO-d6),δ:10.622(s,1H),7.372(d,J=7.6Hz,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℃. 1 H-NMR(400MHz,DMSO-d6),δ:10.651(s,1H),9.839(s,1H),9.279(s,1H),7.386(d,J=8.4 Hz,1H),7.228-7.193(m,2H),7.047(d,J=2.0Hz,1H),6.964(d,J=2.8 Hz,1H),6.951(d,J=3.2 Hz,1H),6.933(d,J=3.6 Hz,1H),6.915(d,J=6.0 Hz,1H),6.628(d,J=8.4 Hz,1H),6.542(d,J=8.4 Hz,1H),4.574(d,J=6.4 Hz,1H),4.521(d,J=9.6 Hz,1H),3.718(s,3H),3.381(d,J=8.4 Hz,1H),3.273(t,J=17.2 Hz,1H),2.066(s,3H),1.299-1.174(m,3H).LC-MS m / z:459.2(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℃.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,mp90.6-92.5℃. 1 H-NMR(400MHz,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℃.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℃. 1 H-NMR(400MHz,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=4.8Hz,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 forC 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).Reddishbrown powder,61.1%yield,mp 242.4-245.1℃. 1H-NMR(400MHz,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 forC 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℃. 1 H-NMR(400MHz,DMSO-d6),δ:7.217(d,J=8.0 Hz,1H),7.037(s,1H),6.975(d,J=4.0 Hz,2H),6.898(t,J=18.4 Hz,2H),6.780(d,J=16.1 Hz,1H),6.632(d,J=8.4 Hz,1H),6.549(d,J=8.4 Hz,1H),4.545(t,J=16.4 Hz,1H),4.473(d,J=8.8 Hz,1H),3.682(d,J=8.0 Hz,6H),3.277-3.232(m,2H),2.056(s,3H).LC-MS m / z:553.1(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℃. 1H-NMR(400MHz,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,J1=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℃. 1 H-NMR(400MHz,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,J1=4.4 Hz,J2=7.0 Hz,1H)6.574(dd,J1=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) + ,calcdfor 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(400MHz,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=8.0Hz,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=10.4Hz,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 (400MHz, DMSO-d6), δ: 10.549 (s, 1H), 7.454 (s, 1H), 7.428 (d, J = 6.0Hz, 2H), 7.392 (s, 1H), 7.347 (t, J = 17.2Hz, 3H), 7.289-7.219 (m, 3H), 7.0 06-6.940(m,2H),6.839(t,J=14.8Hz,1H),6.475(d,J=7.6Hz,1H),4.463 (d,J=9.2Hz,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 the Shanghai Cell Bank of the Chinese Academy of Sciences. The cells were placed in a CO2 incubator using the prepared DMEM high-glucose medium. The conditions were set at a temperature of 37°C and a CO2 content of 5%. The cells were grown to the logarithmic growth phase and could be used in the corresponding experiments.

[0042] Example 3 Determining the concentration of H2O2 to create a damage model

[0043] Take H9c2 cells in the logarithmic growth phase and spread them into a 96-well plate, inoculate at a density of 3000 cells / well, and add 100 μL of culture medium to each well. The periphery of the 96-well plate is filled with PBS to prevent the evaporation of water and the occurrence of edge effects. Then place it in a constant temperature cell culture incubator and culture it overnight. After adding H2O2 with different final concentrations (a concentration is set every 20 from 260-580 μM, a total of 17 groups) to the cells, continue to culture in the cell culture incubator for 24 hours, add 20 μL of MTT solution with a concentration of 5 mg / mL, and then aspirate the liquid in the 96-well plate after 4 hours, and then add 120 μL of DMSO solution to each well, shake it in the dark on a micro-oscillator for 10 minutes, and use an enzyme marker to detect the OD value at 490nm. Put PBS as the blank control group A, and calculate the cell survival rate (experimental group A value / control group A value × 100%). The experiment was repeated three times. Finally, the H2O2 concentration corresponding to about 60% cell survival rate was taken as the concentration of H2O2 model damage.

[0044] Example 4 MTT colorimetric assay for oxidative damage

[0045] H9c2 cells were inoculated in a 96-well plate, with about 3000 cells per well. 100 μL of the culture medium was taken and placed in a CO2 incubator with the temperature set to 37°C and the CO2 content set to 5% overnight. A certain concentration of the target compound was added the next day and cultured for 18 hours. A certain concentration of H2O (the concentration of the modeling injury determined in step 1.4.3) was added for stimulation for 24 hours, and finally 20 μL of MTT solution with a concentration of 5 mg / mL was added, and the plate was placed in a CO2 incubator with pre-set conditions for continued culture. After about 4 hours, the liquid in each well was aspirated, and then 120 μL of DMSO solution was added to each well, and the plate was gently shaken for about 1 minute to mix. Then the absorbance was measured, the wavelength of the enzyme-linked immunosorbent assay was set to 490 nm, and DMSO was used as the blank control B. The cell survival rate was calculated (experimental group A value / control group B value × 100%), and the experiment was repeated three times to reduce experimental errors.

[0046] Example 5 MDA assay

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

[0048] Example 6 ROS determination

[0049] H9c2 cells were cultured at 1.5 × 10 5 Cells were plated at a density of 10 cells / well on a 6-well plate and incubated for 24 hours. Cells were pretreated with compounds for 18 hours and then exposed to 600 μM H2O2 for 2 hours. Cells were incubated with 1 μL of 2',7'-dichlorofluorescein diacetate (DCFH-DA; Bio-Tech; Shanghai, China, 10 μM) in the dark at 37°C. Images were taken under a fluorescence microscope to form (Nikon).

[0050] Example 5 Western Blot Analysis

[0051] H9c2 cells in the logarithmic growth phase were seeded in 6-well culture plates, with approximately 1.5 × 10 cells per well. 6Cells were placed in a pre-set cell culture incubator for culture. After about 48 hours, a certain concentration of the synthesized drug and the positive control drug were added, and a blank control group was set. After that, the cells were placed in a cell culture incubator for further culture. After about 18 hours, 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 minutes. The EP tube was marked and the lysed cells were scraped and placed in it. Then centrifugation was performed at 4°C, 12000rpm, and 10 minutes. The protein concentration in the protein supernatant obtained by centrifugation was determined by the Coomassie Brilliant Blue method. After calculation, the required sample was prepared, loading buffer was added, and protein was denatured in a metal bath for 10 minutes, and finally stored at -20°C. Then electrophoresis was performed under polyacrylamide gel, and the membrane was transferred in an ice bath, with the conditions set to 300mA and 90 minutes. Then 5% skim milk powder blocking solution was used for blocking at room temperature for about 90 minutes. After the blocking was completed, the blocking solution was discarded, and then washed with 1×TBST buffer for 7 minutes each time, and repeated 3 times. Then add specific primary antibodies: β-actin (1:2000), HO-1 (1:500), GADPH (1:2000). Incubate on a shaker at 4°C in a refrigerator overnight. The next day, repeat the rinse three times with TBST, each time for about 7 minutes. After rinsing, incubate with secondary antibodies at room temperature for 1 hour, and then rinse three times with 1×TBST solution, each time for about 7 minutes. Finally, use a gel imaging system to develop the color. Use Quantity One gel imaging analysis software to scan the final processed samples, detect the optical density values ​​of the target band and the internal reference, and calculate the ratio of the two for statistical analysis.

[0052] Example 6 Colony cloning experiment

[0053] H9c2 cells were inoculated into 6-well plates at a density of 1000 cells / well and cultured for 24 hours. They were then incubated with a certain concentration of the target compound C3a and DMSO at 37°C and 5% CO2 for 18 hours respectively. In the middle, the original culture medium was replaced with fresh RPMI 1640 culture medium according to the pH change of the culture medium. Finally, the upper culture medium was discarded, and the cells were washed three times with PBS, fixed with paraformaldehyde, and the formaldehyde was discarded after about 10 minutes. The cells were washed three times with the prepared PBS, and finally stained with crystal violet.

Claims

1. Use of any of the following compounds in the preparation of a drug for treating myocardial cell damage:

2. The use according to claim 1, wherein the myocardial cell damage diseases include but are not limited to the following diseases: myocardial ischemia-reperfusion injury, myocardial infarction, diabetic cardiomyopathy, and myocarditis.

3. The use according to claim 1, wherein the compound is preferably selected from any one of the following compounds.

4. A pharmaceutical composition for treating myocardial cell damage, comprising a therapeutically effective amount of the compound according to claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutical excipient.

5. The pharmaceutical composition according to claim 4, wherein the compound according to claim 1 or a pharmaceutically acceptable salt thereof is the sole active ingredient.

6. The pharmaceutical composition according to claim 5, characterized in that: The pharmaceutical composition is in the form of an injection, tablet, capsule, aerosol, suppository, film, pellet, ointment, controlled-release or sustained-release agent and nano-formulation.

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