Indole alkaloid compounds, methods of making, and use in the manufacture of medicaments for the treatment of chronic kidney disease

Abstract

The application discloses an indole alkaloid compound, a preparation method and application thereof in preparation of a medicine for treating chronic kidney disease, and the structure of the indole alkaloid compound is shown as formula I. The new indole alkaloid compound is extracted from fruits of a plant for the first time, the compound can significantly reverse the increase of alpha-smooth muscle actin, type I collagen and vimentin expression in HK-2 cells induced by TGF-beta, and the compound shown as formula I has a drug development potential for preventing or treating chronic kidney disease, in particular, anti-kidney fibrosis, at a concentration of 10 muM.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to indole alkaloid compounds, their preparation methods, and their application in the preparation of drugs for treating chronic kidney disease. Background Technology

[0002] Bousigonia mekongensis is a plant belonging to the genus Bousigonia in the family Apocynaceae. It is mainly distributed in southwestern my country. Plants of the genus Bousigonia are rich in monoterpenoid indole alkaloids with novel structures and significant activities. Some of these compounds have biological activities such as anti-chronic kidney disease and anti-tumor effects.

[0003] Chronic kidney disease (CKD) seriously threatens human health, with its prevalence increasing year by year over the past few decades, becoming a major global public health challenge. According to my country's largest epidemiological survey, the prevalence of CKD in the adult population is approximately 10.8%, causing a huge health and economic burden. There is an inseparable and causally linked relationship between the progression of CKD and renal fibrosis. Renal fibrosis is a crucial pathological process in the development of almost all CKD cases to their end-stage. This process involves damage to the renal parenchyma and interstitium, fibroblast activation, inflammatory cell infiltration, the production of various active substances and cytokines, and the deposition of extracellular matrix. Recent research on modern drugs targeting this area has progressed rapidly. While these drugs can alleviate inflammation and fibrosis, they often target single points with "precision strikes," which has limitations. Furthermore, the potent intervention in specific pathways leads to various side effects, and there is a lack of treatment for the root cause of the disease. Therefore, there is an urgent need to develop safer and more effective therapeutic drugs. Naturally derived lead compounds have always been a source of inspiration for the development of anti-renal fibrosis drugs. Therefore, finding and developing safe, effective, and low-adverse-effect anti-renal fibrosis drug lead compounds from natural medicinal plants is an important approach to the development of anti-CKD drugs. Summary of the Invention

[0004] To address the problems existing in the prior art, this invention provides an indole alkaloid compound, a preparation method, and its application in the preparation of drugs for treating chronic kidney disease. This compound can significantly reverse the TGF-β-induced increase in the expression of α-smooth muscle actin, type I collagen, and vimentin in HK-2 cells, and has the potential to develop drugs for the prevention or treatment of chronic kidney disease, especially for the treatment of renal fibrosis.

[0005] This invention is achieved through the following technical solution: In a first aspect, the present invention provides an indole alkaloid compound, the structure of which is shown in Formula I: Formula I.

[0006] In a second aspect, the present invention provides a method for preparing the indole alkaloid compound, wherein the indole alkaloid compound is extracted and isolated from the fruit of the plant *Lysimachia christinae*, comprising the following steps: (1) The dried milk vine fruit was extracted by percolation with organic solvent 1 1-4 times. The solvent was recovered until it was tasteless. After being suspended in water, it was extracted with organic solvent 2 under acidic conditions (pH 2-3). The aqueous layer was extracted with organic solvent 3 under alkaline conditions (pH 9-10). The total alkaloids were obtained by recovering organic solvent 3. (2) The total alkaloids in step (1) were separated by MPLC chromatography and the indole alkaloid compound (bousimekofruine B) of formula I was obtained by liquid chromatography.

[0007] Furthermore, in step (1), organic solvent 1, organic solvent 2, and organic solvent 3 are each independently selected from C 1-6 alcohols, C 3-6 Ketones, C 2-6 Ether, C 2-6 Ester and C 1-6 Halogenated hydrocarbons.

[0008] Furthermore, C 1-6 The alcohol is selected from methanol, ethanol, n-propanol, isopropanol, n-butanol, isobutanol, tert-butanol, n-pentanol, isopentanol, n-hexanol, and cyclohexanol; the C 3-6 The ketone is selected from acetone, methyl ethyl ketone, and methyl isobutyl ketone; the C 2-6 The ether is selected from dimethyl ether and diethyl ether; the C 2-6 The ester is selected from ethyl formate, ethyl acetate, and ethyl propionate; the C 1-6 The halogenated hydrocarbons are selected from dichloromethane, trichloromethane, and dichloroethane.

[0009] Furthermore, in step (1), organic solvent 1 is ethanol, and organic solvent 2 and organic solvent 3 are dichloromethane.

[0010] Furthermore, in step (2), the chromatographic column used in the MPLC chromatographic separation is a C18 column, and the eluent is an aqueous methanol solution or an aqueous acetonitrile solution.

[0011] Further, the eluent is an aqueous methanol solution, with a methanol-to-water volume ratio ranging from 15:85 to 100:0, yielding 7 fractions. Fractions 5 to 7 are combined and further separated by MOLC chromatography, with the methanol-to-water volume ratio ranging from 10:90 to 100:0, yielding 5 fractions. The fifth fraction is then eluted by preparative liquid chromatography with 72% aqueous methanol solution (10 mL / min) to obtain the indole alkaloid compound represented by Formula I. During the separation process, the selected flow segment can be determined by thin-layer chromatography (TLC) or other commonly used detection methods in the art, followed by further separation and purification.

[0012] In a third aspect, the present invention provides the use of the aforementioned indole alkaloid compound in the preparation of a medicament for treating chronic kidney disease.

[0013] Furthermore, the medication for treating chronic kidney disease is an anti-renal fibrosis drug.

[0014] Furthermore, the indole alkaloid compound also includes a pharmaceutically acceptable salt of the indole alkaloid compound shown in Formula I.

[0015] Furthermore, the pharmaceutically acceptable salt is an acid addition salt derived from inorganic and organic acids; the inorganic acid is selected from hydrochloric acid, sulfuric acid, phosphoric acid, hydrobromic acid, and hydroiodic acid, and the organic acid is selected from citric acid, tartaric acid, formic acid, oxalic acid, methanesulfonic acid, carbonic acid, succinic acid, benzoic acid, acetic acid, oxalic acid, p-toluenesulfonic acid, and p-bromobenzenesulfonic acid.

[0016] Compared with the prior art, the beneficial effects achieved by the present invention are as follows: This invention is the first to extract a novel indole alkaloid compound from the fruit of the plant *Lysimachia christinae*. This compound can significantly reverse the TGF-β-induced increase in the expression of α-smooth muscle actin, type I collagen, and vimentin in HK-2 cells. The compound described in Formula I has the potential to be developed into a drug for the prevention or treatment of chronic kidney disease, especially for the treatment of renal fibrosis, at a concentration of 10 μM. Attached Figure Description

[0017] Figure 1 The ultraviolet absorption spectrum of the indole alkaloid compound (bousimekofruine B); Figure 2 For bousimekofruine B 1 H NMR spectrum; Figure 3 For bousimekofruine B 13 C NMR spectrum; Figure 4For bousimekofruine B 1 H- 1 H COSY spectrum; Figure 5 The HSQC spectrum of bousimekofruine B; Figure 6 The HMBC spectrum of bousimekofruine B; Figure 7 The NOESY spectrum of bousimekofruine B; Figure 8 HRESIMS plot for bousimekofruine B; Figure 9 Experimental and calculation of ECD for bousimekofruine B; Figure 10 This is a graph showing the gene expression levels of renal fibrosis factors (α-SMA, ColⅠ, Vimentin). Detailed Implementation

[0018] The present invention is further illustrated below with reference to specific embodiments. It should be understood that these embodiments are for illustrative purposes only and are not intended to limit the scope of the invention. Experimental methods not specifically described in the following examples are generally performed under conventional conditions or as recommended by the manufacturer.

[0019] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of skill in the art. All reagents and materials used in this invention are readily available through conventional means, and unless otherwise specified, they shall be used in accordance with conventional methods in the art or as per the product instructions.

[0020] Example 1 Preparation (extraction) of indole alkaloid compound (bousimekofruine B): (1) Take 15 kg of dried milk vine fruit, extract with 95% ethanol by percolation, recover the solvent until there is no alcohol taste, add water to suspend, adjust the pH to 2.5 with 2% HCl, extract with dichloromethane 4 times, adjust the pH of the aqueous layer to 9-10 with 10% NaOH, extract with dichloromethane, and recover the dichloromethane to obtain total alkaloids (95 g); (2) The total alkaloids in step (1) were separated by MPLC (C18 column) chromatography with methanol / water solution as eluent (15:85, 35:65, 50:50, 80:20, 100:0). After HPLC analysis, Fr.5-7 were combined to obtain 35 g, which was further separated by MPLC (C18 column) chromatography with methanol / water solution as eluent (10:90, 20:80, 30:70, 50:50, 70:30, 80:20, 100:0). After HPLC analysis and combination, 5 fractions (Mi.1~Mi.5) were obtained. Among them, Mi.5 (206 mg) was eluted by preparative liquid chromatography with 72% methanol-water (10 mL / min) to obtain indole alkaloid compound (bousimekofruine B).

[0021] The UV absorption spectrum of the indole alkaloid compound (bousimekofruine B) is shown below. Figure 1 As shown, 1 H NMR image as follows Figure 2 As shown, 13 C NMR spectrum as shown Figure 3 As shown, 1 H- 1 H COSY spectrum as follows Figure 4 As shown, the HSQC spectrum is as follows Figure 5 As shown, the HMBC spectrum is as follows Figure 6 As shown, the NOESY spectrum is as follows Figure 7 As shown, the HRESIMS diagram is as follows: Figure 8 As shown, the experimental and calculated ECD plots are derived from... Figure 9 As shown; UV (CH3OH): λ max (logε): 264 (3.50), 242 (3.92), 215 (4.26) nm; CD (MeOH) λ max 200 (Δ -0.65), 201 (Δ -1.58), 204 (Δ -0.11), 216 (Δ -3.51), 231 (Δ 7.34), 245 (Δ -2.21), 255 (Δ 0.68) nm; mass spectrometry data: HR-ESI-MS (m / z): 593.3862 [M+H] + (Calcd. 593.3856 [M+H]) + ); 1 H NMR and 13 The C NMR data are shown in Table 1.

[0022] Table 1. Bousimekofruine B 1 H NMR and13 C NMR data (CDCl3, * indicates overlapping signals) .

[0023] The structural formula of the indole alkaloid compound was determined by ultraviolet spectroscopy, circular dichroism spectroscopy, mass spectrometry, and one-dimensional and two-dimensional nuclear magnetic resonance spectroscopy, as shown in Formula I. The indole alkaloid compound (bousimekofruine B) has a molecular weight of 592.3 and a molecular formula of C. 38 H 48 N4O2; pale yellow powder; readily soluble in chloroform, dichloromethane, and methanol.

[0024] Formula I.

[0025] Example 2 The indole alkaloid compound (bousimekofruine B) prepared in Example 1 was added to a 4% hydrochloric acid solution until pH = 4, filtered, and dried to prepare the hydrochloride salt of the indole alkaloid compound.

[0026] Example 3 The indole alkaloid compound (bousimekofruine B) prepared in Example 1 was added to a 4% sulfuric acid ethanol solution until pH = 4, filtered, and dried to prepare the sulfate of the indole alkaloid compound.

[0027] Example 4 The indole alkaloid compound (bousimekofruine B) prepared in Example 1 was added to a 4% ethanolic phosphate solution until pH = 4, filtered, and dried to prepare the phosphate of the indole alkaloid compound.

[0028] Example 5 The indole alkaloid compound (bousimekofruine B) prepared in Example 1 was added to a 4% citric acid solution until pH = 4, filtered, and dried to prepare the citrate of the indole alkaloid compound.

[0029] Example 6 The indole alkaloid compound (bousimekofruine B) prepared in Example 1 was added to a 4% tartaric acid solution until pH = 4, filtered, and dried to prepare the tartrate salt of the indole alkaloid compound.

[0030] Example 7 The indole alkaloid compound (bousimekofruine B) prepared in Example 1 was added to a 4% formic acid solution until pH = 4, filtered, and dried to prepare the formate salt of the indole alkaloid compound.

[0031] Example 8 The indole alkaloid compound (bousimekofruine B) prepared in Example 1 was added to a 4% oxalic acid solution until pH = 4, filtered, and dried to prepare the oxalate of the indole alkaloid compound.

[0032] Example 9 Anti-renal fibrosis activity test (1) Experimental materials Cell line: HK-2 cell line; Reagents and materials are shown in Table 2 below: Table 2. Reagents and Materials for Anti-renal Fibrosis Activity Test .

[0033] The experimental group consisted of the indole alkaloid compound (bousimekofruine B) prepared in Example 1, and the positive control was oridonin.

[0034] (2) Experimental methods HK-2 cells were cultured in complete MEM medium (89% MEM basal medium + 10% serum + 1% penicillin-streptomycin solution); an in vitro renal fibrosis model was constructed by inducing HK-2 cells at 10 ng / mL. Groups were set up: Ctrl, Model, BMF28, and a positive control group treated with oridonine A. Cells were cultured at a rate of 6 × 10⁻⁶. 5 Two mL of HK-2 cell suspension was seeded into each well of a 6-well plate. After 8 h of cell adhesion, the original culture medium was discarded, and the following drugs were administered: 2 mL of culture medium was added to the Ctrl group; 1 mL of 10 ng / mL TGF-β + 1 mL of culture medium was added to the Model group; 1 mL of 10 ng / mL TGF-β + 1 mL of 10 μM bousimekofruine B was added to the bousimekofruine B group; and 1 mL of 10 ng / mL TGF-β + 1 mL of 10 μM oridonin was added to the oridonin group. After co-culturing for 24 h, the cells were collected, centrifuged, and the supernatant was discarded (MEM culture medium was used for both TGF-β and drug dilutions). RNA was extracted, reverse transcribed into cDNA, and qPCR was performed to detect the gene expression levels of renal fibrosis factors (α-SMA, ColⅠ, Vimentin). The corresponding primer sequences are shown in Table 3. Table 3 qPCR primer sequences The gene expression levels of renal fibrosis factors (α-SMA, ColⅠ, Vimentin) are shown in the figure below. Figure 10 As shown, by Figure 10It was found that at a dosage of 10 μM, the compound bousimekofruine B could significantly reduce the mRNA expression levels of renal fibrosis factors α-SMA, ColⅠ, and Vimentin in HK-2 cells, showing good potential for anti-renal fibrosis applications.

[0035] The above description is merely a preferred embodiment of this application and is not intended to limit this application. Although this application has been described in detail with reference to the foregoing embodiments, those skilled in the art can still modify the technical solutions I described in the foregoing embodiments or make equivalent substitutions for some of the technical features. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the protection scope of this application.

Claims

1. An indole alkaloid compound, characterized in that, The structure of the indole alkaloid compound is shown in Formula I: Formula I.

2. The method for preparing the indole alkaloid compound according to claim 1, characterized in that, The indole alkaloid compound is extracted and isolated from the fruit of the plant *Lysimachia christinae*, and includes the following steps: (1) The dried milk vine fruit was extracted by percolation with organic solvent 1 1-4 times, the solvent was recovered until it was tasteless, water was added to suspend it, and then extracted with organic solvent 2 under acidic conditions. The aqueous layer was extracted with organic solvent 3 under alkaline conditions. The total alkaloids were obtained by recovering organic solvent 3. (2) The total alkaloids in step (1) were separated by MPLC chromatography and the indole alkaloid compound shown in Formula I was obtained by preparative liquid chromatography.

3. The method for preparing the indole alkaloid compound according to claim 2, characterized in that, In step (1), organic solvent 1, organic solvent 2, and organic solvent 3 are each independently selected from C 1-6 alcohols, C 3-6 Ketones, C 2-6 Ether, C 2-6 Ester and C 1-6 Halogenated hydrocarbons.

4. The method for preparing the indole alkaloid compound according to claim 3, characterized in that, In step (1), organic solvent 1 is ethanol, and organic solvent 2 and organic solvent 3 are dichloromethane.

5. The method for preparing the indole alkaloid compound according to claim 2, characterized in that, In step (2), the chromatographic column used in MPLC separation is a C18 column, and the eluent is an aqueous methanol solution or an aqueous acetonitrile solution.

6. The method for preparing the indole alkaloid compound according to claim 5, characterized in that, The eluent is an aqueous methanol solution, with the volume ratio of methanol to water varying from 15:85 to 100:0, yielding 7 fractions. Fractions 5 to 7 are combined and further separated by MOLC chromatography, with the volume ratio of methanol to water varying from 10:90 to 100:0, yielding 5 fractions. The fifth fraction is then eluted by preparative liquid chromatography with a 72% aqueous methanol solution to obtain the indole alkaloid compound represented by Formula I.

7. The use of the indole alkaloid compound of claim 1 or the indole alkaloid compound prepared by the preparation method of any one of claims 2 to 6 in the preparation of drugs for treating chronic kidney disease.

8. The application according to claim 7, characterized in that, The medication mentioned for treating chronic kidney disease is an anti-renal fibrosis drug.

9. The application according to claim 7, characterized in that, The indole alkaloid compounds also include pharmaceutically acceptable salts of the indole alkaloid compounds shown in Formula I.

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