A pyrrole alkaloid compound with anti-fibrosis effect and its preparation method and application

Abstract

The present invention discloses a pyrrole alkaloid compound with anti-fibrosis properties, a preparation method and an application thereof, and belongs to the field of medical technology. The present invention extracts and separates a series of pyrrole alkaloid compounds from the fermentation product of the mangrove-derived fungus Penicillium sp.DM27. In the anti-renal fibrosis experiment, the compounds can inhibit α-SMA and Fibronectin in HK-2 cells (human proximal tubular epithelial cells) treated with TGF-β, and have the potential to be developed into anti-renal fibrosis drugs. The present invention provides a lead compound for the development of anti-renal fibrosis drugs, which is beneficial to the development and utilization of natural medicinal resources. The alkaloids of the present invention can be produced by liquid fermentation using microorganisms, with a simple operating process, a short cycle, low cost, and a guaranteed source; the present invention utilizes biological synthesis and is environmentally friendly.

Description

Technical Field

[0001] The present invention belongs to the field of medical technology, and specifically relates to pyrrole alkaloid compounds from the fermentation product of mangrove-derived fungus Penicillium sp. DM27, a preparation method thereof, and application thereof in drugs for treating chronic kidney disease. Background Art

[0002] Chronic renal failure is one of the major diseases that threaten human life, and its incidence has been increasing year by year in recent years. Chronic kidney disease (CKD) is the main cause of chronic renal failure. Epidemiology shows that the prevalence of CKD in my country is about 10.8%, and there are more than 100 million CKD patients. Since there is still no effective treatment for chronic renal failure, relying on replacement therapies such as hemodialysis and kidney transplantation not only increases the patient's pain and family economic burden, but also increases the social medical burden. Therefore, the prevention and treatment of CKD is one of the important public health issues faced by countries around the world. Renal interstitial fibrosis is the only way for chronic kidney disease to progress to end-stage renal disease, and its pathological manifestations are mainly glomerular sclerosis and tubulointerstitial fibrosis. The only way for CKD to develop into chronic renal failure is tubulointerstitial fibrosis (TIF). In the early stage of renal interstitial fibrosis, local inflammation of the kidney may occur. As the disease progresses, tubular epithelial-to-mesenchymal transition (EMT) and abnormal accumulation of extracellular matrix occur, which promote glomerular sclerosis and TIF, and eventually lead to a gradual decline in renal function until it is lost and develops into chronic renal failure. Therefore, clarifying the pathological mechanism of renal interstitial fibrosis and finding corresponding drugs to delay renal interstitial fibrosis are the key to reducing the incidence of chronic renal failure.

[0003] Natural products have always been an important source of drugs. Most of the drugs currently used in clinical practice are derived from the modification and optimization of the structure of natural products, and some drugs are directly derived from natural products. According to statistics, from 1981 to 2020, drugs directly or indirectly derived from natural products accounted for an average of 50-60% of the drugs launched each year.

[0004] Mangrove-derived fungi refer to fungi that grow in the soil of mangrove plants or in the root system. This type of fungus has a rich variety of secondary metabolites with novel structures and significant biological activity. More and more secondary metabolites with significant activity have been discovered, making mangrove fungi one of the important sources of novel active medicinal compounds. Therefore, searching for novel compounds in the secondary metabolites of mangrove fungi has become an important way to discover lead compounds for the treatment of chronic kidney disease and renal fibrosis.

[0005] So far, there is no report on the isolation of anti-renal fibrosis compounds from this fungus. Summary of the invention

[0006] In order to overcome the deficiencies of the prior art, the purpose of the present invention is to provide a pyrrole alkaloid compound with anti-fibrosis extracted from the fermentation product of the mangrove-derived fungus Penicillium sp.DM27, and its preparation method and application. The pyrrole alkaloid provided by the present invention can inhibit α-SMA and fibronectin in HK-2 cells (human proximal tubular epithelial cells) treated with TGF-β, and has the potential to be developed into an anti-renal (lung, heart) fibrosis drug. The present invention provides a lead compound for the development of anti-renal fibrosis drugs, which is conducive to the development and utilization of natural medicinal resources. The Penicillium sp.DM27 strain has been published in the following articles: He LM, Zhao ZF, Zhang MK, et al, Peniciisoquinoline A: A new tetrahydroisoquinoline from mangrove-derived Penicillium sp.DM27 fungus [J]. Journal of Holistic Integrative Pharmacy. 2022, 3 (3): 222-227; Dong Zhitong, Isolation and screening of two special habitat fungi and research on the metabolites of Aspergillus 094102 [D]. Wuhan University, 2017.

[0007] The purpose of the present invention is achieved through the following technical solutions:

[0008] The present invention extracts and separates 22 new pyrrole alkaloid compounds from the fermentation product of mangrove-derived fungus Penicillium sp.DM27, including: 3 pyrrolizidine alkaloids (compounds 1-3), 3 piperidone alkaloids (compounds 4-6), 13 pyrrolidine alkaloids (compounds 7-19), and 3 ring-opening derivatives of pyrrolidine alkaloids (compounds 20-22). The structural formulas of the compounds are as follows.

[0009] The structural formula of compounds 1 and 2:

[0010] The group combinations of compounds 1 and 2 are shown in the following table:

[0011] serial number <![CDATA[R1]]> <![CDATA[R2]]> <![CDATA[R3]]> 2′ configuration 8′,9′ double bond Compound 1 H H OH R have Compound 2 H H OH S have

[0012] The structural formula of compound 3:

[0013] The group combinations of compound 3 are shown in the following table:

[0014] serial number <![CDATA[R1]]> <![CDATA[R2]]> <![CDATA[R3]]> 8′,9′ double bond Compound 3 H H H have

[0015] The structural formula of compound 4-6:

[0016] The group combinations of compounds 4-6 are shown in the following table:

[0017]

[0018] The structural formula of compounds 7 and 8:

[0019] The group combinations of compounds 7 and 8 are shown in the following table:

[0020] serial number <![CDATA[R1]]> <![CDATA[R2]]> <![CDATA[R3]]> <![CDATA[R4]]> 3,4 double bond 8′,9′ double bond Compound (±)-7 H H O O none have Compound (±)-8 H H O O have have

[0021] The structural formula of compound 9:

[0022] The group combinations of compound 9 are shown in the following table:

[0023] serial number <![CDATA[R1]]> 2,3 double bond Compound (±)-9 OH have

[0024] The structural formula of compound 10-18:

[0025] The group combinations of compounds 10-18 are shown in the following table:

[0026]

[0027] The structural formula of compound 19:

[0028] The group combinations of compound 19 are shown in the following table:

[0029] serial number <![CDATA[R1]]> <![CDATA[R2]]> <![CDATA[R3]]> 2′,3′ double bond Compound 19 O H H have

[0030] The structural formula of compound 20-22:

[0031] The group combinations of compounds 20-22 are shown in the following table:

[0032] serial number <![CDATA[R1]]> <![CDATA[R2]]> <![CDATA[R3]]> 8′,9′ double bond Compound 20 H H H have Compound 21 H H <![CDATA[CH2CH2COOH]]> have Compound 22 H H <![CDATA[CH2CH2COCH2OH]]> have

[0033] The preparation method of the pyrrole alkaloid compound comprises the following steps: inoculating Penicillium sp. DM27 strain into a liquid culture medium for fermentation, separating the fermentation liquid from the mycelium, crushing the mycelium and extracting with acetone ultrasonically, recovering the acetone, combining the remaining liquid with the fermentation liquid, and adding a macroporous resin for adsorption. The macroporous resin adsorbing the metabolites is subjected to column chromatography and methanol / water gradient elution to obtain four components with methanol contents of 30%, 50%, 70%, and 100%, respectively, and the characteristic absorption peaks of the pyrrole alkaloids are detected by HPLC-DAD for the above four components, and the presence of nitrogen atoms is analyzed by LC-MS to determine the component where the pyrrole alkaloids are located. Then, silica gel column chromatography is used to separate the components to obtain smaller components, and then Sephadex LH-20 gel column chromatography is used for separation, and the part containing the characteristic peaks of the pyrrole alkaloid compounds is subjected to semi-preparative liquid phase purification to obtain the above pyrrole alkaloid compound.

[0034] The pyrrole alkaloid compound has an anti-fibrosis (fibrosis of the kidney, lung, heart and other internal organs), and the present invention also provides the use of the pyrrole alkaloid compound in the preparation of an anti-fibrosis drug. Further, the pyrrole alkaloid compound is used in the preparation of a drug for treating chronic kidney disease, diabetic nephropathy, cardiopulmonary fibrosis and the like.

[0035] An anti-fibrosis drug comprises the pyrrole alkaloid compound and a pharmaceutically acceptable carrier or excipient.

[0036] Compared with the prior art, the present invention has the following advantages:

[0037] Firstly, the culture conditions of the strain Penicillium sp. DM27 used in the present invention are easy to control and can be fermented repeatedly, and a sufficient amount of pyrrole alkaloid series compounds can be effectively enriched through fermentation, thereby solving the problem that such pyrrole alkaloids are difficult to synthesize artificially due to their complex structures.

[0038] Secondly, in the preparation method of the pyrrole alkaloid compound of the present invention, mycelium extraction, combined fermentation broth, macroporous resin HP20 adsorption, macroporous resin column chromatography, ODS column chromatography, Sephadex LH-20 gel column chromatography, and high performance liquid chromatography separation are successively used to successfully prepare a series of pyrrole alkaloids. The equipment requirements are simple and easy, and the purification steps are concise and efficient. Among them, the HP20 adsorption method uses less solvent than the traditional extraction method, is environmentally friendly, and is fast and efficient.

[0039] Thirdly, the pyrrole alkaloids prepared by the present invention have a significant inhibitory effect on the proliferation of α-SMA, Fibroncetin (fibronectin) and Col-I induced by TGF-β1 stimulation in HK-2 and NIH-3T3 cells, and have the potential to be developed into anti-fibrosis (chronic kidney disease fibrosis, pulmonary fibrosis, myocardial fibrosis) drugs.

[0040] Fourthly, the pyrrole alkaloid compound prepared by the present invention has a significant anti-fibrosis effect at the cellular level, and has potential economic benefits as there are almost no drugs available for treating fibrosis on the market. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] Figure 1 The EMT of renal tubules in HK-2 cells induced by TGF-β1 was observed in different treatments. (A) The mRNA content of α-SMA, FN and Col I in HK-2 cells was determined by fluorescence quantitative PCR. (B) The expression of α-SMA and FN in HK-2 cells was determined by Western blotting. The quantification of α-SMA and FN was compared with GAPDH (glyceraldehyde phosphate dehydrogenase). The calculated values ​​are: ±SD (n=4-6). *p<0.05; **p<0.01; ***p<0.001.

[0042] Figure 2 Blockade of TGF-β1-induced fibroblast activation in NIH-3T3 cells under different treatments. (A) mRNA expression of α-SMA, FN and Col I in NIH-3T3 cells determined by quantitative PCR (B) Quantification of α-SMA and FN in NIH-3T3 cells determined by Western blotting was performed by comparing with GAPDH (glyceraldehyde phosphate dehydrogenase), and the calculated values ​​were: ± SD (n = 4-8). *p < 0.05; **p < 0.01; ***p < 0.001. DETAILED DESCRIPTION

[0043] The following examples are used to further illustrate the present invention, but should not be construed as limiting the present invention. Unless otherwise specified, the technical means used in the examples are conventional means well known to those skilled in the art.

[0044] Example 1: Preparation of pyrrole alkaloid compounds and their derivatives

[0045] A. Preparation of strain fermentation broth:

[0046] The Penicillium sp.DM27 strain (the strain has been published in the following articles: He LM, Zhao ZF, Zhang MK, et al, Peniciisoquinoline A: A new tetrahydroisoquinoline from mangrove-derived Penicillium sp.DM27 fungus [J]. Journal of Holistic Integrative Pharmacy. 2022, 3 (3): 222-227; Dong Zhitong, Isolation and screening of two special habitat fungi and study of the metabolites of Aspergillus 094102 [D]. Wuhan University, 2017.) was inoculated on PDA medium for 7 days, cut into 0.5 cm × 0.5 cm pieces, and placed in PDB medium for large-scale liquid fermentation. The fermentation treatment temperature was 28 ° C, the fermentation time was 28 days, and the total fermentation volume was 30 L. PDA medium formula: 200g potato extract, 20g glucose, 20g agar, 5g peptone, 3g potassium dihydrogen phosphate, 1.5g magnesium sulfate, 1000mL water, pH 6.5-7.0; PDB medium formula: 200g potato extract, 20g glucose, 5g peptone, 3g potassium dihydrogen phosphate, 1.5g magnesium sulfate, 1000mL water, pH 6.5-7.0.

[0047] B. Preparation of total crude extract

[0048] The fermented mycelium and the fermentation liquid were separated by filter cloth, and the mycelium was crushed and extracted with acetone by ultrasonic method. Each ultrasonic time was 20 minutes, and the extraction was carried out three times. The acetone was recovered from the extract by rotary evaporation, and the remaining water layer was combined with the fermentation liquid.

[0049] C. Separation and purification

[0050] HP20 macroporous resin was added to the combined fermentation broth, stirred and adsorbed for 8 hours, and then the fermentation broth was removed, and the adsorbed HP20 resin was loaded into a glass column. The HP20 macroporous resin chromatography column was eluted with methanol / water (MeOH / H2O) at elution ratios of 30:70, 50:50, 70:30 and 100:0, thereby obtaining 4 corresponding components, namely Fr.A, Fr.B, Fr.C and Fr.D.

[0051] Component Fr.B (6.64 g) was passed through a silica gel column, loaded with 250 g of 200-300 mesh silica gel powder, and gradient eluted with CH2Cl2 / MeOH (40:1, 30:1, 20:1, 15:1, 10:1, 5:1, 0:1, v / v), each gradient elution was 1500 mL, and each small portion of 250 mL was collected. The small portions with the same color were tracked and detected by TLC (thin layer chromatography), and the small portions were combined to form components Fr.B.1 to Fr.B.21.

[0052] Component Fr.B.2 (26 mg) was separated by semi-preparative HPLC under the conditions of MeOH:H2O=70:90 and flow rate of 3.0 mL / min to obtain compound 18 (5 mg, t R =18.7min).

[0053] Component Fr.B.4 (127 mg) was eluted through a gel column Sephadex LH-20 (MeOH / CH2Cl2=1:1), with a total eluate of 750 mL. 15 mL of each aliquot was collected and followed by TLC detection. The aliquots with the same color were combined and divided into 6 components Fr.B.4.1 to Fr.B.4.6. Component Fr.B.4.5 (45 mg) was separated by semi-preparative high performance liquid separation under the conditions of MeOH-H2O=55:45 and a flow rate of 3.0 mL / min, and compound 10 (13 mg, t R =35.6min).

[0054] Component Fr.C (5.60 g) was passed through a silica gel column and eluted with PE / EA (from 4:1, 3:1, 2:1, 1:1, 0:1, v / v), 1500 mL for each gradient elution, 250 mL for each aliquot was collected, followed by TLC detection, and the aliquots with the same color were combined to be divided into 13 components Fr.C.1 to Fr.C.13. Component Fr.C.9 (45 mg) was passed through a silica gel column and eluted with CH2Cl2 / MeOH (40:1, 20:1, 0:1), 300 mL for each gradient elution, 15 mL for each aliquot was collected, followed by TLC detection, and the aliquots with the same color were combined to be divided into 4 components Fr.C.9.1 to Fr.C.9.4. Component Fr.C.9.2 (15 mg) was separated by semi-preparative HPLC under the conditions of MeOH:H2O (50:50, v / v, 30 min) to obtain compound 3 (2 mg, t R =20.4min). Component Fr.C.9.3 (15 mg) was separated by semi-preparative HPLC using MeOH:H2O (70:30 to 80:20, 20 min) to obtain compound 11 (4 mg, t R =15.6min).

[0055] Component Fr.D (5.80 g) was passed through a silica gel column and collected in 250 mL portions using CH2Cl2 / MeOH (100:1, 80:1, 50:1, 25:1, 10:1, 0:1, v / v). The mixture was followed and detected by TLC, and the portions with the same color were combined and eluted into 19 components Fr.D.1 to Fr.D.19.

[0056] Component Fr.D.2 (495 mg) was passed through a gel column Sephadex LH-20 (MeOH / CH2Cl2=1:1), and the total eluate was 750 mL. 15 mL of each aliquot was collected and detected by TLC tracking. The aliquots with the same color were combined and divided into 6 components Fr.D.2.1 to Fr.D.2.6. Component Fr.D.2.3 (71 mg) was passed through the liquid phase to obtain compound 15 (5 mg, t R = 20.6 min, 30% MeCN). Component Fr.D.2.4 (20 mg) was passed through the liquid phase to give compound 14 (5 mg, t R =23.7 min, 35% MeCN), compound 16 (8 mg, t R =28.7min, 30% MeCN). Component Fr.D.2.5 (65 mg) was separated by semi-preparative HPLC under the conditions of MeCN:H2O=30:70, 30 min, flow rate 3.0 mL / min to obtain compound 12 (6 mg, t R =20.0min) and compound 13 (2 mg, t R =24.5min).

[0057] Component Fr.D.3 (498 mg) was passed through a gel column Sephadex LH-20 (MeOH-CH2Cl2 / 1:1) with a total eluent of 750 mL. 15 mL of each aliquot was collected and followed by TLC detection. The aliquots with the same color were combined to obtain only one subcomponent, which was then separated by semi-preparative high performance liquid phase separation under the conditions of MeCN:H2O=30:70, 30 minutes, and a flow rate of 3.0 mL / min to obtain compound 9 (8 mg, t R =20.6min).

[0058] Component Fr.D.5 (153 mg) was passed through a gel column Sephadex LH-20 (MeOH-CH2Cl2 / 1:1) with a total eluent of 750 mL. 15 mL of each aliquot was collected and followed by TLC detection. The aliquots with the same color were combined to separate into 5 components Fr.D.5.1 to Fr.D.5.5. Component Fr.D.5.5 (21 mg) was separated by semi-preparative high performance liquid phase separation under the conditions of MeOH-H2O (70:30, v / v) to obtain compound 8 (2 mg, t R=18.7min); Compound 8 was then separated by OD-H chiral column under the conditions of 1 mL / min, n-hexane:isopropanol=95:5 to obtain enantiomer (+)-8 (0.5 mg, t R =12.0min) and (-)-8 (0.5mg, t R =14.5min).

[0059] Component Fr.D.6 (233 mg) was passed through a gel column Sephadex LH-20 (MeOH-CH2Cl2 / 1:1) with a total eluent of 750 mL. 15 mL of each aliquot was collected and detected by TLC tracking. The aliquots with the same color were combined to separate into 8 components Fr.D.6.1 to Fr.D.6.8. Component Fr.D.6.4 (50 mg) was passed through the liquid phase to obtain compound 17 (3 mg, t R =18.7min, 40% MeCN). Component Fr.D.6.7 (53 mg) was separated by semi-preparative HPLC using MeCN-H2O (65%-95%, v / v, 30 min) to obtain component 6.7.1 (5 mg) and compound 7 (1 mg, t R =32.3min). Compound 7 was then separated by OJ-H chiral column under the condition of n-hexane:isopropanol=97:3 to obtain the corresponding isomer (+)-7 (0.5mg, t R 21.5min) and (-)-7 (0.5mg, t R 22.7min). Component 6.7.1 was separated by semi-preparative HPLC using MeCN-H2O (36%:74%, v / v) to give compound 2 (2 mg, t R =30.6min) and compound 1 (2mg, t R =31.7min). Component Fr.D.6.8 (58mg) was separated by semi-preparative HPLC using MeCN-H2O MeCN-H2O (55%:15%. v / v). Compound 20 (1mg, t R =24.8min).

[0060] Component Fr.D.7 (100 mg) was passed through a gel column Sephadex LH-20 (MeOH-CH2Cl2 / 1:1) with a total eluent of 750 mL. 15 mL of each aliquot was collected and detected by TLC tracking. The aliquots with the same color were combined to separate into 8 components Fr.D.7.1 to Fr.D.7.8. Component Fr.D.7.3 (40 mg) was separated by semi-preparative high performance liquid phase separation under the conditions of MeOH:H2O (50% to 100%, v / v, 20 min) to obtain compound 19 (15 mg, t R=11.0min). Component Fr.D.7.6 was separated by semi-preparative HPLC (MeOH:H2O, 20%-60%, 40min) to give compound 21 (11 mg, t R =26.0min) and compound 18 (2 mg, t R = 25.2 min, 45% MeCN).

[0061] Component Fr.D.8 (458 mg) was passed through a gel column Sephadex LH-20 (MeOH-CH2Cl2 / 1:1) with a total eluent of 750 mL. 15 mL of each aliquot was collected and detected by TLC tracking. The aliquots with the same color were combined to separate into 5 components Fr.D.8.1 to Fr.D.8.5. Component Fr.D.8.5 was separated by semi-preparative high performance liquid phase separation under the conditions of MeCN:H2O (25% to 55%, 30 min) to obtain component 8.5.1 (5 mg) and compound 6 (1 mg, t R =34.1min). Compound 6 was then separated by an OJ-H chiral column (n-hexane:isopropanol=92.5:7.5, 20 minutes, 1 mL / min) to obtain the corresponding isomer (+)-6 (0.5 mg, t R =16.2min) and (-)-6 (0.5mg, t R =19.7min). Component Fr.D.8.5.1 was separated by semi-preparative HPLC using MeCN:H2O (30%, 30min) to give compound 4 (2 mg, t R =26.3min) and compound 5 (2mg, t R =26.9min). Compound 4 was then separated by an OJ-H chiral column (n-hexane:isopropanol=95%:5%, 20 minutes, 1 mL / min) to obtain the corresponding isomer (+)-4 (0.5 mg, t R 12.3min) and (-)-4 (0.5mg, t R =14.4min). Compound 5 was also separated by OD-H chiral column (n-hexane:isopropanol=92.5:7.5%, 30min, 1mL / min) to obtain the corresponding isomer (+)-5 (0.5mg, t R 14.4min) and (-)-5 (0.5mg, t R 17.3min).

[0062] Component Fr.D.9 (421 mg) was passed through a gel column Sephadex LH-20 (MeOH-CH2Cl2 / 1:1) with a total eluent of 750 mL. 15 mL of each aliquot was collected and followed by TLC detection. The aliquots with the same color were combined to elute into 7 components Fr.D.9.1 to Fr.D.9.7. Component Fr.D.9.7 was separated by semi-preparative HPLC under the conditions of MeCN:H2O=30:70%, 30 minutes, and a flow rate of 3 mL / min to obtain compound 22 (2 mg, t R =25.2min).

[0063] D. Structural Elucidation

[0064] Each compound was analyzed by NMR, mass spectrometry and other methods, and the results are shown below.

[0065] Compound 1: colorless oil; ECD{MeOH,λ[nm](Δε),c=0.71×10 -4 M}236(-3.32),220(+3.54),211(+1.01), 1 H and 13 C NMR data are shown in Table 1; HRESIMS m / z 280.1905 [M+H] + (cald 280.1913).

[0066] Compound 2: colorless oil; ECD{MeOH,λ[nm](Δε),c=0.71×10 -4 M}287(-7.58),252(-1.54),236(-4.05),215(+8.73), 1 H and 13 C NMR data are shown in Table 1; HRESIMS m / z 280.1906 [M+H] + (cald 280.1913).

[0067] Compound 3: colorless oil; ECD{MeOH,λ[nm](Δε),c=1.13×10 -4 M}223(+60.15),298(-22.36), 1 H and 13 C NMR data are shown in Table 1; HR-ESIMS 266.1749 [M+H] + (cald 266.1756).

[0068] Compound (+)-4: colorless oil; ECD{MeOH,λ[nm](Δε),c=0.02×10 -4 M}224(+1.19), 1 H and 13 C NMR data are shown in Table 1; HR-ESIMS 254.1749 [M+H] + (cald254.1756).

[0069] Compound (-)-4: colorless oil; ECD{MeOH,λ[nm](Δε),c=0.02×10 -4 M}225(-1.57), 1 H and 13 C NMR data are shown in Table 2; HR-ESIMS 254.1749 [M+H] + (cald254.1756).

[0070] Compound (+)-5: colorless oil; ECD{MeOH,λ[nm](Δε),c=0.02×10 -4 M}292(+0.42); 233(-1.04); 210(+1.34), 1 H and 13 C NMR data are shown in Table 2; HR-ESIMS 254.1748 [M+H] + (cald 254.1756).

[0071] Compound (-)-5: colorless oil; ECD{MeOH,λ[nm](Δε),c=0.02×10 -4 M}295(-0.53); 231(+1.02); (204 -1.48), 1 H and 13 C NMR data are shown in Table 2; HR-ESIMS 254.1748 [M+H] + (cald 254.1756).

[0072] Compound (+)-6: colorless oil; ECD{MeOH,λ[nm](Δε),c=0.02×10 -4 M}233(-1.11), 1 H and 13 C NMR data are shown in Table 2; HR-ESIMS 256.1905 [M+H] + (cald256.1913).

[0073] Compound (-)-6: colorless oil; ECD{MeOH,λ[nm](Δε),c=0.02×10 -4 M}230(+0.72), 1 H and 13 C NMR data are shown in Table 2; HR-ESIMS 256.1905 [M+H] + (cald256.1913).

[0074] Compound (+)-7: colorless oil; ECD{MeOH,λ[nm](Δε),c=0.02×10 -4 M}240(-1.76);218(+1.25), 1 H and 13 C NMR data are shown in Table 3; HR-ESIMS 266.1749 [M+H] + (cald 266.1756).

[0075] Compound (-)-7: colorless oil; ECD{MeOH,λ[nm](Δε),c=0.02×10 -4 M}239(+1.85);215(-1.66), 1 H and 13 C NMR data are shown in Table 3; HR-ESIMS 266.1749 [M+H] + (cald 266.1756).

[0076] Compound (+)-8: colorless oil; ECD{MeOH,λ[nm](Δε),c=0.02×10 -4 M}240(-2.97); 244(-1.42); 204(-3.81), 1 H and 13 C NMR data are shown in Table 3; HR-ESIMS 264.1587 [M+H] + (cald 264.1599).

[0077] Compound (-)-8: colorless oil; ECD{MeOH,λ[nm](Δε),c=0.02×10 -4 M}242(+3.06);226(+1.28);205(+3.81), 1 H and 13 C NMR data are shown in Table 3; HR-ESIMS 264.1587 [M+H]+ (cald 264.1599).

[0078] Compound 9: colorless oil; UV(MeOH)λ max (logε)287(1.83),226(1.61),203(2.60)nm; ECD{MeOH,λ[nm](Δε),c=0.10 10 -4 M}232.5(-2.00),215(+3.25),233(+60.40),201(-17.75),190(-34.85); IR(KBr)ν max 3419,3013,2913,2361,1626,1512,1384,1317,1274,1132,1011cm -1 ; 1 H and 13 C NMR data are shown in Table 3; HR-ESIMS 164.0687 [M+Na] + (calcd[2M+Na] + 305.1472).

[0079] Compound 10: colorless oil; 1 H and 13 C NMR data are shown in Table 3; HR-ESIMS 296.1853 [M+H] + (calcd296.1862).

[0080] Compound 11: colorless oil; 1 H and 13 C NMR data are shown in Table 3; HRESIMS 266.1749 [M+H] + (cald266.1756).

[0081] Compound 12: colorless oil; 1 H and 13 C NMR data are shown in Table 4; HRESIMS 284.1849 [M+H] + (cald284.1862).

[0082] Compound 13: colorless oil; 1 H and 13 C NMR data are shown in Table 4; HRESIMS 320.1823 [M+Na] + (cald320.1838).

[0083] Compound 14: colorless oil; UV(MeOH)λ max (logε)206(3.87),286(3.03)nm; ECD{MeOH,λ[nm](Δε),c=0.1×10 -4 M}216(-7.00),202.4(-7.86),196.4(-7.50),189.8(-17.73); 1 H and 13 C NMR data are shown in the table; HRESIMS m / z 252.1964 [M+H] + (calcd for C 15 H 25 NO2,252.1958).

[0084] Compound 15: colorless oil; UV(MeOH)λ max (logε)287(2.01),224(2.42),203(3.10)nm; ECD{MeOH,λ[nm](Δε),c=0.1×10 -4 M}241(-13.00),206.9(-7.25),191.8(-8.98); IR(KBr)ν max 3446,2926,1624,1515,1384,1318,1122,1053cm -1 ; 1 H and 13 C NMR data are shown in the table; HRESIMS m / z 290.1732 [M+H] + (calcd for C 15 H 25 NO3,290.1727).

[0085] Compound 16: colorless oil; UV(MeOH)λ max (logε)203(4.42),273(3.23)nm; ECD{MeOH,λ[nm](Δε),c=0.1×10 -4 M}211.6(+9.47),198.4(-17.30),189(+42.80); 1 H and 13 C NMR data are shown in the table; HRESIMS m / z 288.1576 [M+H] + (calcd for C 15 H 23 NO3,288.1570).

[0086] Compound 17: colorless oil; 1 H and 13 C NMR data are shown in Table 4; HRESIMS m / z 268.1904 [M+H] + (calcd for C 15 H 23 NO3,268.1913).

[0087] Compound 18: colorless oil; 1 H and 13 C NMR data are shown in Table 5; HRESIMS m / z 268.1904 [M+H] + (calcd for C 15 H 23 NO3,268.1913).HRESIMS m / z 298.2019[M+H] + (calcd for C 15 H 23 NO3,298.2018).

[0088] Compound 19: colorless oil; 1 H and 13 C NMR data are shown in Table 4; HRESIMS 288.1563 [M+Na] + (cald288.1576).

[0089] Compound 20: colorless oil; 1 H and 13 C NMR data are shown in Table 4; HRESIMS 198.1489 [M+H] + (calcd198.1494).

[0090] Compound 21: colorless oil; 1 H and 13 C NMR data are shown in Table 4; HRESIMS 270.1696 [M+H] + (calcdfor270.1705).

[0091] Compound 22: colorless oil; 1 H and 13 C NMR data are shown in Table 4; HRESIMS 284.1859 [M+H] + (calcdfor284.1862).

[0092] Table 1. Compounds 1-3 1 H and 13C NMR data, deuterated reagent is CDCl3 (δ in ppm, J in Hz)

[0093]

[0094]

[0095] Table 2. Compounds (±)-4, (±)-5 and (±)-6 1 H and 13 C NMR data, deuterated reagent is CDCl3 (δ in ppm, J in Hz)

[0096]

[0097] Table 3. Compounds (±) 7-9 1 H and 13 C NMR data, deuterated reagent is CDCl3 (δ in ppm, J in Hz)

[0098]

[0099] Table 4 Compounds 10-12 1 H and 13 C NMR data, deuterated reagent is CDCl3 (δ in ppm, J in Hz)

[0100]

[0101]

[0102] Table 5. Compounds 13-15 1 H and 13 C NMR data, deuterated reagent is CDCl3 (δ in ppm, J in Hz)

[0103]

[0104] Table 6 Compounds 16-18 1 H and 13 C NMR data, deuterated reagent is CDCl3 (δ in ppm, J in Hz)

[0105]

[0106]

[0107] Table 7 Compounds 19 to (±)-22 1 H and 13 C NMR data, deuterated reagent is CDCl3 (δ in ppm, J in Hz)

[0108]

[0109] Example 2: Application of pyrrole alkaloid compounds in anti-fibrosis

[0110] 1. Cell culture and treatment.

[0111] Human renal proximal tubule epithelial cells (HK-2) and mouse fibroblasts (NIH-3T3) were cultured in Dulbecco's modified Eagle's medium (DMEM) containing 10% fetal bovine serum at 37°C and 5% CO2. 10 ng / mL TGF-β1 (PeproTech, USA) was selectively added with or without the addition of 100 μM test compounds and dissolved in DMSO (0.1%) for 48 h.

[0112] 2. Quantitative real-time PCR (qPCR). Total RNA was isolated from cells using Trizol reagent (Vazyme, China), and then cDNA was synthesized using a reverse transcription system kit (Vazyme, China). The expression of the target gene was detected using SYBR qPCR Master Mix Kit (Vazyme, China). The expression of β-actin was normalized and 2- ΔΔ The relative expression of genes was calculated by the Ct method. The primer pair sequences are:

[0113] humanβ-actin forward:5′-CATGTACGTTGCTATCCAGGC-3′,

[0114] humanβ-actin reverse: 5′-CTCCTTAATGTCACGCACGAT-3′;

[0115] human a-SMA forward: 5′-AAAAGACAGCTACGTGGGTGA-3′,

[0116] human a-SMA reverse: 5′-GCCATGTTCTATCGGGTACTTC-3′;

[0117] human collagen I forward: 5′-GAGGGCCAAGACGAAGACATC-3′,

[0118] human collagen I reverse: 5′-CAGATCACGTCATCGCACAAC-3′;

[0119] human fibronectin forward:5′-CGGTGGCTGTCAGTCAAAG-3′,

[0120] human fibronectin reverse:5′-AAACCTCGGCTTCCTCCATAA-3′;

[0121] mouseβ-actin forward:5′-GGCTGTATTCCCCTCCATCG-3′,

[0122] mouseβ-actin reverse:5′-CCAGTTGGTAACAATGCCATGT-3′;

[0123] mouse a-SMA forward:5′-GTCCCAGACATCAGGGAGTAA-3′,

[0124] mouse a-SMA reverse:5′-TCGGATACTTCAGCGTCAGGA-3′;

[0125] mouse collagen I forward:5′-GCTCCTCTTAGGGGCCACT-3′,

[0126] mouse collagen I reverse:5′-CCACGTCTCACCATTGGGG-3′;

[0127] mouse fibronectin forward:5′-TTCAAGTGTGATCCCCATGAAG-3′,

[0128] mouse fibronectin reverse:5′-CAGGTCTACGGCAGTTGTCA-3′。

[0129] 3. Western blot analysis. Cells were lysed on ice with RIPA buffer (Beyotime, China) and protease inhibitor cocktail reagent, and then centrifuged at 12000 rpm for 10 minutes. Equal amounts of protein were separated by 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore, USA). Cell membranes were blocked with 5% skim dry milk for 1 hour at room temperature and incubated overnight at 4°C with the following primary antibodies: α-SMA (1:1000, Abcam, UK), Fibronectin (1:1000, Cell Signaling Technology, USA), GAPDH (1:1000, Servicebio, China). Subsequently, cell membranes were incubated with horseradish peroxidase (HRP)-conjugated secondary antibodies for 1 hour and observed using chemiluminescent reagents (Biosharp, China). ImageJ software (NIH, USA) was used to quantify and normalize the target bands relative to GAPDH.

[0130] 4. Experimental Results

[0131] Many studies have shown that EMT (epithelial to mesenchymal cell transition) plays an important role in the pathogenesis of chronic kidney disease. In this process, epithelial cells lose their specific marker E-cadherin, thereby acquiring the characteristics of mesenchymal cells or myofibroblasts, resulting in increased expression of α-SMA (α-smooth muscle actin), and the production of more fibronectin and collagens. If the expression of the above proteins is inhibited, it may indicate a potential therapeutic effect on chronic kidney disease.

[0132] The inhibitory effect of compound 14, whose structure is shown below, on fibroblast activation or epithelial to mesenchymal transition (EMT) was evaluated: After culturing HK-2 cells (human renal tubular epithelial cells) with transforming growth factor-β (TGF-β1) stimulation for 48 h, the expression of mRNA corresponding to α-SMA, fibronectin and type I collagen (Col I) was significantly increased, while the experimental group with the addition of 10 μM compound 14 inhibited this effect ( Figure 1 A). Western blotting to detect α-SMA and fibronectin in HK-2 cells ( Figure 1 B) is consistent with qPCR, further proving that compound 14 does inhibit the expression of the above proteins. This indicates that compound 14 has the potential to treat chronic kidney disease.

[0133]

[0134] In typical cases of renal fibrosis, fibroblasts differentiate into α-SMA-positive myofibroblasts, and changes in the expression of proteins such as α-SMA can reflect the effects of drugs on renal fibrosis.

[0135] The effect of compound 14 on fibroblast activation was evaluated using TGF-β1-induced NIH-3T3 cells (mouse embryonic fibroblasts). Real-time quantitative PCR results showed that the expression of α-SMA in TGF-β1-activated fibroblasts was significantly higher than that in the normal control group. In the experimental group treated with 10μM concentration of compound 14, the expression of α-SMA stimulated by TGF-β1 was downregulated. The experimental group treated with compound 14 also consistently counteracted the expression of FN and Col I in NIH-3T3 cells stimulated by TGF-β1 ( Figure 2 A). This is consistent with the Western blot results of α-SMA and FN observed in NIH-3T3 cells ( Figure 2 B). This indicates that compound 14 has the potential to treat renal fibrosis.

Claims

1. A pyrrole alkaloid compound, characterized in that: The compound has the following structure: 。 2. The use of the pyrrole alkaloid compound according to claim 1 in the preparation of an anti-fibrotic drug, characterized in that: The fibrosis is renal fibrosis.

3. The use of the pyrrole alkaloid compound according to claim 1 in the preparation of medicines, characterized in that: The medicine is a medicine for treating chronic kidney disease.

4. An anti-fibrotic drug, characterized in that: The method comprises the pyrrole alkaloid compound according to claim 1; wherein the fibrosis is renal fibrosis.

5. The anti-fibrotic drug according to claim 4, characterized in that: It also contains a pharmaceutically acceptable carrier or excipient.

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