Preparation method and application of a class of sponge-derived 2-pyrrolidone alkaloids

Abstract

The application discloses a preparation method and application of 2-pyrrolidone alkaloids from sponges. P. suberitoides Six 2-pyrrolidone alkaloid compounds (compounds 1-6) are separated from sponges in the application, and the pharmacological activity of the 2-pyrrolidone alkaloids in heart protection is disclosed. In the prior art, the application of 2-pyrrolidone alkaloids from marine sponges in the field of heart protection has not been reported, and the application fills the technical gap. The compounds provided by the application can effectively reduce myocardial cell damage under oxygen-glucose deprivation conditions, can improve heart function after myocardial infarction, and can effectively inhibit pathological ventricular remodeling after myocardial infarction, and thus can play a dual antioxidant role by removing mitochondrial reactive oxygen species and up-regulating endogenous antioxidant enzymes, thereby providing a new scheme for preparation of heart protection drugs.

Description

Technical Field

[0001] This invention belongs to the field of marine natural product drug development technology, specifically relating to the preparation method and application of a class of sponge-derived 2-pyrrolidone alkaloids. Background Technology

[0002] The statements herein provide only background information in relation to this invention and do not necessarily constitute prior art.

[0003] Oxidative stress induced by myocardial infarction and reperfusion is the core mechanism of myocardial injury and pathological remodeling. During ischemia, hypoxia and reperfusion, cardiomyocyte mitochondria produce a large amount of reactive oxygen species (ROS), exceeding the clearance capacity of the endogenous antioxidant defense system, leading to lipid peroxidation, protein denaturation and DNA damage, ultimately causing cardiomyocyte apoptosis and necrosis, which in turn leads to ventricular dilation and progressive decline in cardiac function.

[0004] Currently used ACE inhibitors in clinical practice β While postoperative secondary prevention drugs such as beta-blockers can delay ventricular remodeling to some extent, they cannot fundamentally reverse existing myocardial damage and have adverse effects such as hypotension, bradycardia, and electrolyte disturbances. Therefore, developing cardioprotective drugs that can directly scavenge excess reactive oxygen species and enhance endogenous antioxidant defense capabilities is of significant clinical importance for reducing myocardial ischemia-induced damage and inhibiting pathological remodeling after myocardial infarction.

[0005] In recent years, upregulating the expression of endogenous antioxidant enzymes (such as SOD, CAT, and HO-1) by activating the Nrf2 / ARE signaling pathway has become an important strategy in the development of cardioprotective drugs. Several natural products have been shown to exert cardioprotective effects through this pathway, such as ursolic acid from Prunella vulgaris, astaxanthin and fucoxanthin from seaweed, and neoagartetrasaccharides from red algae. However, these compounds are mostly polysaccharides, terpenes, or phenols, with relatively concentrated chemical skeletons, highlighting the urgent need to discover novel cardioprotective active molecules.

[0006] Marine sponges are an important source of naturally bioactive compounds, and the alkaloids they produce possess various pharmacological activities, including antitumor, antibacterial, and anti-inflammatory effects. Previous studies have reported that polyketides (such as gigantdioxin) from sponge-derived fungal metabolites exhibit cardioprotective effects in an OGD / R cardiomyocyte injury model; however, their mechanism of action is mainly through targets such as the adenosine A2a receptor (ADORA2A), and their association with antioxidant pathways remains unclear. Other literature reports on sponge-derived... Stylissa massa and Pseudospongosorites suberitoidesThree pyrrolidone steroid analogs with AKR1B1-targeting inhibitory activity have been identified, suggesting their potential use in treating chronic diabetic syndromes. Furthermore, while marine-derived pyrrolidone alkaloids have been reported to possess antitumor activities, their cardioprotective applications are currently unknown. Therefore, the discovery of novel cardioprotective drugs from marine sponges with antioxidant activity holds significant scientific importance and promising application prospects. Summary of the Invention

[0007] To address the shortcomings of existing technologies, the purpose of this invention is to provide a method for preparing and applying a class of sponge-derived 2-pyrrolidone alkaloids. This invention utilizes sponges... P. suberitoides ( Pseudospongosorites suberitoides Six 2-pyrrolidone alkaloids were isolated from [the sample / organism / organism], of which compounds 1-3 were novel compounds, and compounds 4-6 were known compounds. Furthermore, the cardioprotective activities of the isolated 2-pyrrolidone alkaloids were screened under an oxygen-glucose deprivation (OGD) model. The results showed that all tested compounds could alleviate excessive mitochondrial ROS production and moderately upregulate the expression of antioxidant genes (SOD1, SOD2, CAT, HO-1). Compounds 1, 2, and 6 significantly reduced cardiomyocyte viability loss. In addition, compound 1 exhibited the strongest protective effect and antioxidant activity against cardiomyocytes. In vivo cardioprotective activity evaluation showed that compound 1, through an antioxidant mechanism, could effectively improve cardiac function after myocardial infarction, inhibit pathological ventricular remodeling (including ventricular dilation and fibrosis), and reduce cardiomyocyte loss.

[0008] To achieve the above objectives, the present invention is implemented through the following technical solution: In a first aspect, the present invention provides a class of sponge-derived 2-pyrrolidone alkaloids, selected from at least one of the following compounds: .

[0009] In a second aspect, the present invention provides a method for preparing sponge-derived 2-pyrrolidone alkaloids as described in the first aspect, comprising the following steps: sponge P. suberitoides After pulverization, it is extracted with methanol and concentrated to obtain a crude extract; The crude extract was dissolved in water, extracted with ethyl acetate, and concentrated under reduced pressure to obtain the aqueous primary fraction containing the target 2-pyrrolidone alkaloids. The primary aqueous fraction was subjected to gradient elution using a forward silica gel column to obtain the secondary aqueous fraction containing the target 2-pyrrolidone alkaloids; The aqueous secondary fraction was eluted by medium-pressure liquid chromatography to obtain the aqueous tertiary fraction containing the target 2-pyrrolidone alkaloids; The aqueous tertiary fraction was eluted by gel column chromatography and then by secondary medium-pressure liquid chromatography to obtain the aqueous quaternary fraction containing the target 2-pyrrolidone alkaloids. The target 2-pyrrolidone alkaloids were obtained by high performance liquid chromatography separation of the tertiary and quaternary fractions in the aqueous phase.

[0010] In some embodiments, the aqueous tertiary fraction is purified by high performance liquid chromatography to obtain compound 3, compound 4, compound 5, or compound 6; In some embodiments, the aqueous quaternary fraction is purified by high performance liquid chromatography to obtain compound 1 or compound 2.

[0011] In some embodiments, the gradient elution via a forward silica column is performed using a dichloromethane / methanol mixture as the eluent, with the ratio changing from 10:1 to 0:1 (v / v), followed by elution with methanol / trifluoroacetic acid, the methanol / trifluoroacetic acid gradient ratio being 1000:1 to 1000:2 (v / v).

[0012] In some embodiments, the elution is performed by medium-pressure liquid chromatography and secondary medium-pressure liquid chromatography, with the eluent being a methanol / water mixture containing 0.09%-0.11% trifluoroacetic acid; during elution, the gradient is from 5:95 to 100:0 (v / v), and the flow rate is 16.0-20.0 mL / min.

[0013] In some embodiments of the present invention, the elution by gel column chromatography uses pure methanol as the eluent and is separated by Sephadex LH-20 gel column chromatography.

[0014] In some embodiments, the high-performance liquid chromatography separation uses a mobile phase of methanol / water containing 0.09-0.11% trifluoroacetic acid, an Agilent XDB-C18 column, a flow rate of 1.9-2.1 mL / min, a detection wavelength of 254±2 nm, and a column temperature of 24.5-25.5℃.

[0015] Thirdly, the present invention provides the application of the sponge-derived 2-pyrrolidone alkaloids described in the first aspect in the preparation of cardioprotective drugs.

[0016] Preferably, in the OGD cardiomyocyte injury model, compounds 1, 2, and 6 can alleviate OGD-induced loss of cardiomyocyte viability; Preferably, compounds 1-6 can all reduce mitochondrial ROS levels in cardiomyocytes and inhibit oxidative stress; Preferably, compounds 1-6 can upregulate SOD1, SOD2, CAT, and HO. 1. Increased expression of antioxidant gene mRNA enhances myocardial endogenous antioxidant capacity; More preferably, in this series, compound 1 exhibits the best myocardial protective and antioxidant activity.

[0017] Preferably, a myocardial infarction model was established by ligation of the left anterior descending coronary artery in mice. Compound 1 was injected intraperitoneally 3 days after the operation at a dose of 10 mg / kg / d for 7 consecutive days. Preferably, compound 1 can increase the left ventricular ejection fraction and the left ventricular fractional shortening rate in a myocardial infarction model mouse, thereby improving cardiac pumping function. Preferably, compound 1 can increase the thickness of the left ventricular anterior wall at end-systole and end-diastole, and decrease the left ventricular diameter at end-systole and end-diastole. Preferably, compound 1 can reduce the ratio of heart weight to body weight, reduce the area of ​​myocardial infarction, reduce the area of ​​myocardial fibrosis scar, increase the number of surviving myocardial cells in the infarct boundary area, and inhibit pathological cardiac remodeling after myocardial infarction. Preferably, compound 1 can upregulate the expression of antioxidant-related genes in myocardial tissue and exert antioxidant effects in vivo.

[0018] Fourthly, the present invention provides a cardioprotective pharmaceutical composition comprising a therapeutically effective amount of the sponge-derived 2-pyrrolidone alkaloids described in the first aspect and a pharmaceutically acceptable carrier or excipient.

[0019] Preferably, the dosage form of the pharmaceutical composition is selected from tablets, capsules, injections, oral liquid preparations, or lyophilized powder injections.

[0020] The beneficial effects achieved by one or more embodiments of the present invention described above are as follows: (1) This invention uses sponge P. suberitoides Six 2-pyrrolidone alkaloids were isolated from sponges, of which compounds 1, 2, and 3 are new compounds, while compounds 4, 5, and 6 are known compounds, enriching the library of sponge-derived pyrrolidone alkaloids.

[0021] (2) The present invention screened the efficacy of six compounds in vitro. The results of the OGD model showed that compounds 1, 2 and 6 could alleviate the decline in cardiomyocyte viability. All tested compounds could reduce the level of mitochondrial ROS, upregulate the mRNA expression of antioxidant genes SOD1, SOD2, CAT and HO-1, and enhance the endogenous antioxidant capacity of myocardium. Among them, compound 1 had the best cardioprotective and antioxidant activity and was a candidate active monomer with excellent performance.

[0022] (3) The efficacy of compound 1 in vivo was verified using a mouse model of left anterior descending artery ligation myocardial infarction, and the in vivo and in vitro experimental results corroborated each other. At the functional level, compound 1 can activate the myocardial endogenous antioxidant system, significantly increase the left ventricular ejection fraction and short-axis shortening rate, and improve cardiac pumping function. At the structural level, compound 1 can effectively alleviate the thinning of the left ventricular anterior wall in the infarct area, inhibit the pathological dilation of the left ventricular cavity, reduce the heart weight-to-body weight ratio, reduce the myocardial infarction area and reduce the formation of fibrotic scars, and increase the number of surviving cardiomyocytes in the infarct boundary area, thereby comprehensively inhibiting pathological cardiac remodeling after myocardial infarction.

[0023] (4) This invention confirms that 2-pyrrolidone alkaloids derived from marine sponge-like organisms have the potential for the treatment of myocardial infarction, providing a new direction for the development of drugs for the treatment of myocardial infarction. Attached Figure Description

[0024] The accompanying drawings, which form part of this invention, are used to provide a further understanding of the invention. The illustrative embodiments of the invention and their descriptions are used to explain the invention and do not constitute an improper limitation of the invention.

[0025] Figure 1 These are the structural formulas of compounds 1-6 provided by this invention; Figure 2 It is compound 1 6. Oxygen-glucose deprivation injury H9c2(2) 1) Protective effect on myoblasts of the cardiac muscle; Figure 3 This refers to the in vivo cardioprotective effect of compound 1 on a mouse model of myocardial infarction. Figure 4 It is compound 1 1 H-NMR spectrum; Figure 5 It is compound 1 13 C-NMR spectrum; Figure 6 It is compound 1 1 H- 1 H COSY spectrum; Figure 7 This is the HSQC spectrum of compound 1; Figure 8 This is the HMBC spectrum of compound 1; Figure 9 This is the NOESY spectrum of compound 1; Figure 10 This is the HR-ESI-MS spectrum of compound 1; Figure 11 It is compound 2. 1 H-NMR spectrum; Figure 12It is compound 2. 13 C-NMR spectrum; Figure 13 It is compound 2. 1 H- 1 H COSY spectrum; Figure 14 This is the HSQC spectrum of compound 2; Figure 15 This is the HMBC spectrum of compound 2; Figure 16 This is the NOESY spectrum of compound 2; Figure 17 This is the HR-ESI-MS spectrum of compound 2; Figure 18 It is compound 3. 1 H-NMR spectrum; Figure 19 It is compound 3. 13 C-NMR spectrum; Figure 20 These are the DEPT135 and DEPT90 spectra of compound 3; Figure 21 It is compound 3. 1 H- 1 H COSY spectrum; Figure 22 This is the HSQC spectrum of compound 3; Figure 23 This is the HMBC spectrum of compound 3; Figure 24 This is the NOESY spectrum of compound 3; Figure 25 This is the HR-ESI-MS spectrum of compound 3; Figure 26 It is compound 4. 1 H-NMR spectrum; Figure 27 This is the DEPTQ spectrum of compound 4; Figure 28 It is compound 5. 1 H-NMR spectrum; Figure 29 This is the DEPTQ spectrum of compound 5; Figure 30 It is compound 6. 1 H-NMR spectrum; Figure 31 It is compound 6. 13 C-NMR spectrum; Figure 32 These are the DEPTQ, DEPT135, and DEPT90 spectra of compound 6; Figure 33 It is compound 6. 1 H- 1 H COSY spectrum; Figure 34 This is the HSQC spectrum of compound 6; Figure 35 This is the HMBC spectrum of compound 6; Figure 36 This is the NOESY spectrum of compound 6; Figure 37 This is the HR-ESI-MS spectrum of compound 6. Detailed Implementation

[0026] It should be noted that the following detailed description is illustrative and intended to provide further explanation of the invention. Unless otherwise specified, all technical and scientific terms used in this invention have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0027] The present invention will be further described below with reference to the embodiments.

[0028] The sponge used in this research P. suberitoides The samples were collected in July 2018 from the Xisha Islands in the South China Sea. They were immediately frozen after collection to maintain their biological activity and chemical stability. Species identification was performed by Dr. Nicole J. de Voogd of the Leiden Centre for Natural Biodiversity Research in the Netherlands, ensuring accurate taxonomic identification. The voucher specimen (No. XS-2018-70) is preserved in Laboratory 453 of the School of Pharmacy, Nanchang University, for verification in subsequent research.

[0029] Example 1: Sponge P. suberitoides Extraction, separation and purification methods of active compounds I. Raw material pretreatment and preparation of crude extract Take freshly collected sponges P. suberitoides The sample, with a wet weight of 10.7 kg, was crushed and then extracted four times with methanol at room temperature, each time until the solvent color no longer deepened. The four extracts were combined and concentrated under reduced pressure to remove the solvent, yielding 219.5 g of a brown crude extract.

[0030] II. Liquid-Liquid Extraction Separation The crude extract residue was dissolved in 2 L of distilled water and extracted five times consecutively with an equal volume of ethyl acetate. The ethyl acetate layers were combined and concentrated under reduced pressure to obtain 79.1 g of the ethyl acetate-soluble fraction; the aqueous layer was concentrated under reduced pressure to obtain 138.0 g of the aqueous fraction.

[0031] III. Separation of the Aqueous Phase Aqueous phase samples were separated by normal-phase silica gel column chromatography, eluted with a dichloromethane / methanol gradient solvent system at a gradient ratio of 10:1 to 0:1 (v / v), followed by elution with methanol / trifluoroacetic acid (1000:1 to 1000:2, v / v), and 10 subfractions PS-1 to PS-10 were collected.

[0032] IV. Isolation and purification of PS-3 subfractions Medium-pressure liquid chromatography (LC) for rapid preparative separation: PS-3 subfraction was taken and eluted using LC. The mobile phase was methanol / water containing 0.1% trifluoroacetic acid, with a gradient from 5:95 to 100:0 (v / v), a flow rate of 18.0 mL / min, and a run time of 110 min. A total of 11 subfractions, from PS-3-1 to PS-3-11, were collected.

[0033] Sephadex LH-20 gel column chromatography separation: PS-3-5, PS-3-6 and PS-3-7 were combined and separated by Sephadex LH-20 gel column chromatography using pure methanol as eluent. 32 subfractions were collected and numbered from PS-3-5-1 to PS-3-5-32.

[0034] Secondary medium-pressure liquid chromatography rapid preparative separation: PS-3-5-10 to PS-3-5-19 were combined and eluted again by medium-pressure liquid chromatography rapid preparative chromatography. The mobile phase was methanol / water containing 0.1% trifluoroacetic acid, with a gradient from 5:95 to 100:0 (v / v), a flow rate of 18.0 mL / min, and a run time of 100 min, yielding two subfractions, PS-3-5-10-1 and PS-3-5-10-2.

[0035] High performance liquid chromatography purification: PS-3-5-10-1 was purified by high-performance liquid chromatography (HPLC) using an XDB-C18 column. The mobile phase was methanol / water containing 0.1% trifluoroacetic acid (15:85, v / v), the flow rate was 2.0 mL / min, the detection wavelength was 254 nm, and the collection retention time was [not specified]. R = 43.3 min major chromatographic peak, yielding compound 2 (colorless oil, 6.9 mg).

[0036] PS-3-5-10-2 was purified by high-performance liquid chromatography (HPLC) under the same chromatographic conditions as above, and the retention time t was collected. R = 12.1 min major chromatographic peak, yielding compound 1 (colorless oil, 6.9 mg).

[0037] V. Isolation and purification of PS-3-8 subfractions The PS-3-8 subfraction was purified by high-performance liquid chromatography (HPLC) using an XDB-C18 column. The mobile phase was methanol / water containing 0.1% trifluoroacetic acid (22:78, v / v), the flow rate was 2.0 mL / min, the detection wavelength was 254 nm, and the collection retention time was [not specified]. R = 36.0 min major chromatographic peak, yielding compound 3 (colorless oil, 1.2 mg).

[0038] VI. Isolation and purification of PS-1 subfractions Medium-pressure liquid chromatography (LC) for rapid preparative separation: The PS-1 subfraction was eluted by LC with the mobile phase being methanol / water containing 0.1% trifluoroacetic acid, with a gradient from 5:95 to 100:0 (v / v), a flow rate of 18.0 mL / min, and a run time of 110 min. Seven subfractions, from PS-1-1 to PS-1-7, were collected.

[0039] High-performance liquid chromatography (HPLC) purification: The PS-1-5 subfraction was purified by HPLC using an XDB-C18 column. The mobile phase was methanol / water containing 0.1% trifluoroacetic acid (25:75, v / v), the flow rate was 2.0 mL / min, the detection wavelength was 254 nm, and the collection retention time was [not specified]. R = 18.3 min main chromatographic peak, yielding subfraction PS-1-5-2 and compound 4 (colorless oil, 6.69 mg).

[0040] Secondary high-performance liquid chromatography (HPLC) purification: PS-1-5-2 was further purified by HPLC using an XDB-C18 column. The mobile phase was methanol / water containing 0.1% trifluoroacetic acid (15:85, v / v), the flow rate was 2.0 mL / min, and the detection wavelength was 254 nm. Retention times t were collected separately. R = 39.3 min and t R = 45.0 min chromatographic peak, yielding compound 5 (colorless oil, 5.61 mg) and compound 6 (colorless oil, 2.63 mg).

[0041] The obtained six compounds were analyzed by NMR and HR-ESI-MS to determine their planar and stereo structures. Compounds 1-3 were identified as novel 2-pyrrolidone alkaloids, while compounds 4-6 were identified as known 2-pyrrolidone alkaloids.

[0042] Compound 1 dissolves in DMSO- d 6 1 See the H-NMR spectrum (600 MHz). Figure 4 Compound 1 is soluble in DMSO-d 6 13 C-NMR spectrum (600 MHz) see Figure 5 Compound 1 is soluble in DMSO- d 6 1 H- 1 The H COSY spectrum (600 MHz) can be found here. Figure 6 Compound 1 is soluble in DMSO- d The HSQC spectrum (600 MHz) in section 6 is shown below. Figure 7 Compound 1 is soluble in DMSO- d The HMBC spectrum (600MHz) in section 6 is shown below. Figure 8 Compound 1 is soluble in DMSO- d The NOESY spectrum (600 MHz) in section 6 is shown below. Figure 9 The HR-ESI-MS spectrum of compound 1 is shown below. Figure 10 .

[0043] Compound 2 dissolves in DMSO- d 6 1 See the H-NMR spectrum (600 MHz). Figure 11 Compound 2 is soluble in DMSO- d 6 13 C-NMR spectrum (600 MHz) see Figure 12 Compound 2 is soluble in DMSO- d 6 1 H- 1 The H COSY spectrum (600 MHz) can be found here. Figure 13 Compound 2 is soluble in DMSO- d The HSQC spectrum (600 MHz) in section 6 is shown below. Figure 14 Compound 2 is soluble in DMSO- d The HMBC spectrum (600 MHz) in section 6 is shown below. Figure 15 Compound 2 is soluble in DMSO- d The NOESY spectrum (600 MHz) in section 6 is shown below. Figure 16 The HR-ESI-MS spectrum of compound 2 is shown below. Figure 17 .

[0044] Compound 3 is soluble in DMSO- d 6 1 See the H-NMR spectrum (600 MHz). Figure 18 Compound 3 is soluble in DMSO- d 6 13 C-NMR spectrum (600 MHz) see Figure 19 Compound 3 is soluble in DMSO- dThe DEPT135 and DEPT90 spectra (150MHz) in section 6 are shown below. Figure 20 Compound 3 is soluble in DMSO- d 6 1 H- 1 The H COSY spectrum (600 MHz) can be found here. Figure 21 Compound 3 is soluble in DMSO- d The HSQC spectrum (600 MHz) in section 6 is shown below. Figure 22 Compound 3 is soluble in DMSO- d The HMBC spectrum (600 MHz) in section 6 is shown below. Figure 23 Compound 3 is soluble in DMSO- d The NOESY spectrum (600 MHz) in section 6 is shown below. Figure 24 The HR-ESI-MS spectrum of compound 3 is shown in [reference needed]. Figure 25 .

[0045] Compound 4 is soluble in DMSO- d 6 1 See the H-NMR spectrum (600 MHz). Figure 26 Compound 4 is soluble in DMSO- d The DEPTQ spectrum (150 MHz) in section 6 is shown below. Figure 27 .

[0046] Compound 5 is soluble in DMSO- d 6 1 See the H-NMR spectrum (600 MHz). Figure 28 Compound 5 is soluble in DMSO- d The DEPTQ spectrum (150 MHz) in section 6 is shown below. Figure 29 .

[0047] Compound 6 is soluble in DMSO- d 6 1 See the H-NMR spectrum (600 MHz). Figure 30 Compound 6 is soluble in DMSO- d 6 13 C-NMR spectrum (150 MHz) see Figure 31 Compound 6 is soluble in DMSO- d The DEPTQ, DEPT135, and DEPT90 spectra (150 MHz) in section 6 are shown below. Figure 32 Compound 6 is soluble in DMSO- d 6 1 H- 1 The H COSY spectrum (600 MHz) can be found here. Figure 33 Compound 6 is soluble in DMSO- dThe HSQC spectrum (600 MHz) in section 6 is shown below. Figure 34 Compound 6 is soluble in DMSO- d The HMBC spectrum (600 MHz) in section 6 is shown below. Figure 35 Compound 6 is soluble in DMSO- d The NOESY spectrum (600 MHz) in section 6 is shown below. Figure 36 The HR-ESI-MS spectrum of compound 6 is shown below. Figure 37 .

[0048] For each compound 1 H NMR and 13 Characterization was performed using C NMR signals. The characterization of compounds 1-3 is shown in Tables 1 and 2. The assignment of compound 4 is shown in Table 3. The characterization of compound 5 is shown in Table 4. The characterization of compound 6 is shown in Table 5.

[0049] Table 1. Compounds 1-3 1 H NMR data (600 MHz, DMSO- d 6)

[0050] Table 2. Compounds 1-3 13 C NMR data (150 MHz, DMSO- d 6)

[0051] Table 3. Compound 4 1 H NMR (600 MHz) and 13 C NMR (150 MHz) data (DMSO- d 6)

[0052] Table 4. Compound 5 1 H NMR (600 MHz) and 13 C NMR (150 MHz) data (DMSO- d 6)

[0053] Table 5. Compound 6 1 H NMR (600 MHz) and 13 C NMR (150 MHz) data (DMSO- d 6)

[0054] The above results indicate that the structural formulas of compounds 1-6 are as follows ( Figure 1 ):

[0055] Example 2: Compound 1 6. Oxygen-glucose deprivation injury H9c2(2) 1) Protective effect on cardiac myoblasts This embodiment examines compound 1. 6. In oxygen-glucose deprivation injury H9c2(2) 1) Effects of this study on cardiomyocyte viability, mitochondrial ROS levels, and antioxidant gene mRNA expression in a rat cardiomyocyte model.

[0056] (1) Cell culture H9c2(2 1) Rat cardiac myoblasts were purchased from the American Type Culture Collection (ATCC, Manassas, Virginia, USA) and cultured in Dulbecco modified Eagle medium containing 10% fetal bovine serum (FBS) in a 37°C, humidified incubator containing 5% CO2. Cells were cultured at a rate of 5 × 10⁶ cells / year. 4 CCK cells were seeded at a density of cells / well in 6-well plates and incubated for 24 h to allow them to adhere before being used in subsequent experiments; 8. Detection experiment: H9c2 cells were stored at 5 × 10⁸ cells per well. 3 Cells were seeded at a density of 1,000 cells per well in 96-well plates and incubated overnight at 37°C until the cells adhered.

[0057] (2) Establishment of cell damage model and drug treatment A cardiomyocyte injury model was established using conventional OGD methods in this field. After cell adhesion, the cells were treated with appropriate compounds and then subjected to OGD injury treatment, followed by recovery culture. Cell viability, mitochondrial ROS, total RNA extraction, and qPCR detection were then performed.

[0058] (3) CCK 8. Detecting cell viability Using a cell counting kit 8 (CCK) 8) Assess cell viability. After the cells have been treated as specified, add 10 μL of CCK to each well containing 100 μL of culture medium. 8 solutions; incubate at 37℃ in the dark for 3 h. Before detection, centrifuge the culture plates at 28℃ and 3000 r / min for 5 min to eliminate air bubbles; measure the absorbance at 450 nm using an ELISA reader, and cell viability is expressed as a percentage of the absorbance of the control group.

[0059] (4) MitoSOX staining to detect mitochondrial ROS levels MitoSOX Red mitochondrial superoxide indicator was used to determine mitochondrial ROS levels. H9c2 cells were seeded in confocal culture dishes or 6-well plates. After drug administration and OGD treatment, the cells were gently washed three times with phosphate-buffered saline (PBS). 5 μM MitoSOX working solution was added, and the cells were incubated at 37°C in the dark for 30 min. The cells were washed three times with PBS to remove excess dye, and images were immediately acquired using a fluorescence microscope with an excitation wavelength of 510 nm and an emission wavelength of 580 nm. The mean fluorescence intensity was quantitatively analyzed using ImageJ software.

[0060] (5) RNA extraction, reverse transcription and real-time quantitative PCR Total RNA was extracted from H9c2 cells using AG RNAex Pro reagent according to the kit instructions. RNA concentration and purity were determined using a NanoDrop spectrophotometer, with an A260 / A280 ratio of 1.8 selected. 2.1 RNA samples with an A260 / A230 ratio greater than 2.0 were used for subsequent experiments.

[0061] 1 μg of total RNA was used to synthesize cDNA using the MIKX reverse transcription kit; then, real-time quantitative PCR was performed on a LightCycler 480 II system using MIKX premixed PCR buffer, with amplification conditions set according to the manufacturer's instructions. The relative expression levels of the target gene were determined using a 2-1... - Ct Calculation by method, β Actin was used as an internal control.

[0062] (6) Experimental Results like Figure 2 As shown in Figure A, under OGD-induced cardiomyocyte injury conditions, compounds 1, 2, and 6 can reduce the loss of cardiomyocyte viability and have a direct cardiomyocyte protective effect, among which compound 1 has the most significant cardiomyocyte protective effect.

[0063] like Figure 2 As shown in Figure B, all six tested compounds could alleviate OGD-induced excessive ROS production in cardiomyocyte mitochondria. The fluorescence intensity of MitoSOX was significantly lower than that of the model group, indicating that all of these compounds have the ability to scavenge mitochondrial ROS.

[0064] like Figure 2 C As shown in F, the test compound upregulated SOD1, SOD2, CAT, and HO in cardiomyocytes. The mRNA expression level of endogenous antioxidant genes suggests that these compounds exert their antioxidant cardioprotective effect by enhancing the myocardial endogenous antioxidant defense system.

[0065] Example 3: In vivo cardioprotective effect of compound 1 on a mouse model of myocardial infarction In this embodiment, a mouse model of myocardial infarction was established by ligation of the left anterior descending coronary artery to evaluate the effects of compound 1 on cardiac function, ventricular remodeling, myocardial fibrosis, and expression of antioxidant genes in myocardial tissue in mice with myocardial infarction.

[0066] (1) Laboratory animals Select 6 8 weeks old, weighing 20 25 g male C57BL / 6 mice. Mice were housed in an SPF-grade animal facility at an ambient temperature of 22±2℃ and a relative humidity of 55±5%, with a 12-hour light / dark cycle and free access to food and water; mice were acclimatized for at least one week before the experiment.

[0067] (2) Construction and administration of myocardial infarction model A myocardial infarction model was established by ligation of the left anterior descending coronary artery. Mice were anesthetized with 2% isoflurane inhalation, fixed in a supine position, and connected to a small animal ventilator via endotracheal intubation; after pericardial incision, 8... The left anterior descending coronary artery was permanently ligated with 0-silk sutures. Intraperitoneal administration of compound 1 was initiated on postoperative day 3. The compound 1 was dissolved in normal saline and administered at a dose of 10 mg / kg / day for 7 consecutive days. Two groups were set up: the MI group (myocardial infarction + normal saline) and the MI + compound 1 group (myocardial infarction + 10 mg / kg / day of compound 1), with 5 patients in each group. Six mice.

[0068] (3) Echocardiographic examination After the last administration, mice were anesthetized with 1.5% isoflurane and subjected to M-mode ultrasound using a Vevo 2100 small animal ultrasound imaging system. Five consecutive cardiac cycles were recorded to measure the left ventricular anterior wall thickness (LVAW; s, LVAW; d) at end-systole and end-diastole; the left ventricular diameter (Diameters; s, Diameters; d) at end-systole and end-diastole; the left ventricular ejection fraction (EF); and the left ventricular fractional shortening (FS).

[0069] (4) Sample collection, tissue staining and RT qPCR Mice were euthanized after ultrasound examination, and cardiac arrest was induced by 0.1 M potassium chloride. Hearts were harvested and weighed, and the heart weight / body weight ratio (HW / BW) was calculated. A portion of ventricular tissue was flash-frozen in liquid nitrogen. Store at 80℃, extract RNA according to the aforementioned in vitro cell experiment method, and perform RT. qPCR was used to detect the expression of antioxidant genes; the remaining myocardial tissue was fixed with 4% paraformaldehyde, embedded in paraffin, and 5 μm sections were prepared for Masson trichrome staining to observe myocardial fibrosis and infarct area.

[0070] (5) Experimental Results like Figure 3 China A As shown in B, compared to the model group, mice treated with compound 1 showed increased left ventricular ejection fraction and left ventricular fractional shortening, indicating improved cardiac pumping function. Figure 3 As shown in A, C, and D, compound 1 can increase the thickness of the left ventricular anterior wall at end-systole and diastole, alleviating the thinning of the ventricular wall in the infarct area; it can also reduce the left ventricular diameter at end-systole and diastole, inhibiting pathological dilation of the left ventricular cavity.

[0071] like Figure 3 China E As shown in F, the heart weight / body weight ratio decreased and the myocardial infarction area shrank in the compound 1 treatment group; Figure 3 China G Masson staining of H showed a reduction in fibrotic scarring and an increase in surviving cardiomyocytes in the infarct boundary area, suggesting that compound 1 can inhibit pathological ventricular remodeling after myocardial infarction.

[0072] like Figure 3 As shown in Figure I, compound 1 moderately upregulated the mRNA expression of SOD1, SOD2, CAT, and HO-1 antioxidant genes in myocardial tissue, consistent with the results of in vitro cell experiments, confirming that compound 1 exerts a cardioprotective effect in vivo by activating antioxidant pathways.

[0073] The above description is merely a preferred embodiment of the present invention and is not intended to limit the invention. Various modifications and variations can be made to the present invention by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of the present invention should be included within the scope of protection of the present invention.

Claims

1. A class of sponge-derived 2-pyrrolidone alkaloids, characterized in that, Selected from at least one of the following compounds: 。 2. The method for preparing sponge-derived 2-pyrrolidone alkaloids according to claim 1, characterized in that, Includes the following steps: sponge P. suberitoides After pulverization, it is extracted with methanol and concentrated to obtain a crude extract; The crude extract was dissolved in water, extracted with ethyl acetate, and concentrated under reduced pressure to obtain the aqueous primary fraction containing the target 2-pyrrolidone alkaloids. The primary aqueous fraction was subjected to gradient elution using a forward silica gel column to obtain the secondary aqueous fraction containing the target 2-pyrrolidone alkaloids; The aqueous secondary fraction was rapidly prepared by medium-pressure liquid chromatography elution to obtain the aqueous tertiary fraction containing the target 2-pyrrolidone alkaloids; The aqueous tertiary fraction was eluted by gel column chromatography and then by secondary medium-pressure liquid chromatography to obtain the aqueous quaternary fraction containing the target 2-pyrrolidone alkaloids. The target 2-pyrrolidone alkaloids were obtained by high performance liquid chromatography separation of the aqueous tertiary and quaternary fractions.

3. The method for preparing sponge-derived 2-pyrrolidone alkaloids according to claim 2, characterized in that, The aqueous phase tertiary fraction was purified by high performance liquid chromatography to obtain compound 3, compound 4, compound 5, or compound 6; Alternatively, the aqueous quaternary fraction can be purified by high performance liquid chromatography to obtain compound 1 or compound 2.

4. The method for preparing sponge-derived 2-pyrrolidone alkaloids according to claim 2, characterized in that, The process involves gradient elution via a forward silica column, with the eluent being a dichloromethane / methanol mixture, the ratio changing from 10:1 to 0:1, v / v, followed by elution with methanol / trifluoroacetic acid, the methanol / trifluoroacetic acid gradient ratio being 1000:1 to 1000:2, v / v.

5. The method for preparing sponge-derived 2-pyrrolidone alkaloids according to claim 2, characterized in that, The process involves rapid preparative chromatography using medium-pressure liquid chromatography and secondary rapid preparative chromatography elution. The eluent is a mixture of methanol / water containing 0.09%-0.11% trifluoroacetic acid. During elution, the gradient ranges from 5:95 to 100:0, v / v, and the flow rate is 16.0-20.0 mL / min.

6. The method for preparing sponge-derived 2-pyrrolidone alkaloids according to claim 2, characterized in that, The gel column chromatography was performed using pure methanol as the eluent and the gel was separated by Sephadex LH-20 gel column chromatography.

7. The method for preparing sponge-derived 2-pyrrolidone alkaloids according to claim 2, characterized in that, The high-performance liquid chromatography separation uses a mobile phase of methanol / water containing 0.09-0.11% trifluoroacetic acid, an XDB-C18 column, a flow rate of 1.9-2.1 mL / min, a detection wavelength of 254±2 nm, and a column temperature of 24.5-25.5℃.

8. The use of the sponge-derived 2-pyrrolidone alkaloids as described in claim 1 in the preparation of cardioprotective drugs.

9. The application according to claim 8, characterized in that, The cardiac protection includes at least one of anti-myocardial ischemia, improved cardiac function, and inhibition of pathological remodeling after myocardial infarction; The cardiac protection is achieved through an antioxidant mechanism, which includes scavenging mitochondrial reactive oxygen species and / or upregulating the expression of endogenous antioxidant enzymes; The endogenous antioxidant enzyme is selected from at least one of superoxide dismutase, catalase, and heme oxygenase-1. The pathological remodeling following myocardial infarction includes at least one of the following: fibrotic scar formation at the infarct boundary, ventricular dilation, and decreased cardiac function.

10. A cardioprotective pharmaceutical composition, characterized in that, It comprises a therapeutically effective amount of the sponge-derived 2-pyrrolidone alkaloid of claim 1 and a pharmaceutically acceptable carrier or excipient.

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