An indolepiperazine compound and its application in drugs for neurodegenerative diseases

Abstract

This invention belongs to the field of biomedical technology and provides an indolepiperazine compound and its application in drugs for neurodegenerative diseases. The structural formula of the indolepiperazine compound is: [structural formula would be inserted here]. This compound can be used to prepare drugs for neurodegenerative diseases and pharmaceutical intermediates. The above compound can be... Aspergillus tanneir The strain was obtained by fermentation of UJNMF0417. The yield can be further increased by optimizing the fermentation conditions and modifying the strain, which has good commercial prospects.

Description

Technical Field

[0001] This invention belongs to the field of biomedical technology, specifically relating to an indolepiperazine compound and its pharmaceutical applications. Background Technology

[0002] The information disclosed in this background section is intended to enhance understanding of the overall background of the invention and is not necessarily to be construed as an admission or in any way implying that such information constitutes prior art known to those skilled in the art.

[0003] Neurodegeneration (ND) is a phenomenon characterized by the slow and progressive loss of function and structure of neurons in specific brain regions. It is a major pathophysiological feature of various neurodegenerative diseases (NDDs), including Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), and Huntington's disease (HD). Neurodegeneration is a complex process influenced by genetic, environmental, and age-related endogenous factors. However, the exact pathological effects and underlying molecular mechanisms remain not fully understood. Neurodegenerative diseases primarily affect the elderly, and their treatment and prevention represent a significant challenge that my country will face and need to address for a considerable period. Therefore, in-depth research into the etiology and mechanisms of neurodegenerative diseases, exploring effective prevention and treatment methods, and finding effective preventative and therapeutic drugs are crucial for improving the quality of life of the elderly, reducing the social burden, and even promoting national development. Summary of the Invention

[0004] To address the limitation of existing drugs for treating neurodegenerative diseases, this invention provides an indolepiperazine compound with neuroprotective activity.

[0005] Another object of the present invention is to provide a method for preparing the above-mentioned indolepiperazine compounds, which can be obtained by microbial fermentation.

[0006] To achieve the above objectives, the present invention adopts the following technical solution.

[0007] An indolepiperazine compound has the structure shown in formula (I):

[0008]

[0009] Formula (I).

[0010] The preparation method of the above-mentioned indolepiperazine compounds includes the following steps:

[0011] (1) Aspergillus spp. Aspergillus tanneir UJNMF0417, obtained fermentation product;

[0012] (2) The fermentation product obtained in step (1) was successively extracted with ethanol and ethyl acetate to obtain a crude extract;

[0013] (3) The crude extract obtained in step (2) was separated by D101 macroporous resin column chromatography and eluted with 30%, 50%, 75% and 95% ethanol to obtain four fractions Fr.1-Fr.4;

[0014] Fr.2 was subjected to normal-phase silica gel column chromatography with a mobile phase of dichloromethane / methanol with a volume ratio of 1:0-0:1 to obtain eight fractions, Fr.2.1-Fr.2.8.

[0015] Fr.2.3 was subjected to gradient elution silica gel column chromatography with dichloromethane / methanol at a volume ratio of 1:0 to 0:1 to obtain six fractions: Fr.2.3.1-Fr.2.3.6.

[0016] Fr.2.3.6 was separated by Sephadex LH-20 chromatography to obtain five fractions: Fr.2.3.6.1-Fr.2.3.6.3.

[0017] Fr.2.3.6.3 was obtained by semi-preparative high performance liquid chromatography with acetonitrile / water at a volume ratio of 52:48 as the mobile phase, a flow rate of 3.0 mL / min, and collection of the eluent with a retention time of 36.5 min. The mobile phase was then removed.

[0018] The Aspergillus tanneir The accession number of UJNMF0417 is CGMCC No.41647.

[0019] Preferably, the ethanol in step (2) is 90%-95% ethanol.

[0020] The present invention also provides a fungus of the genus Aspergillus. Aspergillus tanneir Uses of UJNMF0417 and the above-mentioned indolepiperazine compounds in the preparation of drugs for neurodegenerative diseases and pharmaceutical intermediates.

[0021] A medicament for neurodegenerative diseases containing the aforementioned indolepiperazine compounds and their pharmaceutically acceptable salts. The medicament also includes other active ingredients and / or pharmaceutically acceptable excipients. The salts are hydrochlorides, sulfates, or sodium and potassium salts formed from the -NH- group of the indolepiperazine compounds.

[0022] The present invention has the following advantages:

[0023] The indolepiperazine compounds of this invention possess neuroprotective activity and have potential applications in the preparation of drugs for neurodegenerative diseases and pharmaceutical intermediates; the above compounds can be... Aspergillus tanneirThe strain was obtained by fermentation of UJNMF0417. The yield can be further increased by optimizing the fermentation conditions and modifying the strain, which has good commercial prospects.

[0024] Biological Preservation Information

[0025] Aspergillus tanneir UJNMF0417 was deposited on December 2, 2024, at the China General Microbiological Culture Collection Center (CGMCC), located at Institute of Microbiology, Chinese Academy of Sciences, No. 3, No. 1 Beichen West Road, Chaoyang District, Beijing, China, with accession number CGMCC No. 41647. Attached Figure Description

[0026] Figure 1 The main target compound 1 H- 1 Information related to H COSY and HMBC.

[0027] Figure 2 The main NOESY-related information for the target compound.

[0028] Figure 3 The effect of indolepiperazine compounds on 6-OHDA-induced PC-12 cell proliferation.

[0029] Figure 4 The effect of indolepiperazine compounds on 6-OHDA-induced apoptosis in PC-12 cells.

[0030] Figure 5 The effect of indolepiperazine compounds on 6-OHDA-induced intracellular ROS levels in PC-12 cells.

[0031] Figure 6 The effects of indolepiperazine compounds on the levels of MAD, SOD, and GSH induced by 6-OHDA in PC-12 cells. Detailed Implementation

[0032] The present invention will be further described below with reference to embodiments and accompanying drawings, but the present invention is not limited to the following embodiments. Unless otherwise specified, the experimental methods in the embodiments employ conventional techniques in the art, and the experimental reagents are all commercially available.

[0033] Example 1: Preparation of indolepiperazine compounds

[0034] 1. Preparation of fermented products

[0035] Preparation of seed culture medium: 200 mL potato extract, 20 g glucose, 30 g sea salt, and water to a final volume of 1 L. Pour the culture medium into 20 500 mL Erlenmeyer flasks, approximately 150 mL per flask, and autoclave at 121°C for 25 minutes.

[0036] Rice culture medium preparation method: 80g rice, 0.4g yeast extract, 0.4g glucose, 120mL water, 3% sea salt. Place in 1L Erlenmeyer flasks, for a total of 150 flasks. Autoclave at 121℃ for 25 minutes.

[0037] Use a sterile bamboo skewer to pick up an appropriate amount Aspergillus tanneir The UJNMF0417 strain was inoculated into the above culture medium and cultured on a shaker (200 rpm) at 28°C for 3 days to obtain the seed liquid. Then, 10 mL of the seed liquid was inoculated into a 1 L Erlenmeyer flask containing rice culture medium and cultured statically at 28°C for 30 days before the fermentation product was collected.

[0038] After fermenting for 30 days, the rice culture medium was transferred out and crushed. It was then soaked in 95% ethanol four times (each soaking lasted 6-7 days). The 95% ethanol was collected after each soaking and concentrated into an aqueous solution using a large rotary evaporator. Then, it was extracted with an equal volume of ethyl acetate four times. After further concentration using a rotary evaporator, a crude extract was obtained.

[0039] 2. Isolation of the target compound

[0040] The crude extract obtained above (90 g) was subjected to D101 macroporous resin column chromatography and eluted with 30%, 50%, 75%, and 95% ethanol to obtain four fractions (Fr.1-Fr.4).

[0041] Fr.2 was eluted by silica gel column chromatography with a gradient of dichloromethane:methanol (1:0-0:1). During elution, thin-layer chromatography silica gel plates were used for detection and similar fractions of compounds were combined, resulting in a total of 8 fractions (Fr.2.1-Fr.2.8).

[0042] Fr.2.3 Gradient elution silica gel column chromatography with dichloromethane:methanol (1:0-0:1) was performed, and detection was performed using a silica gel plate for thin-layer chromatography. The fractions were combined by rotary evaporation to obtain 6 fractions (Fr.2.3.1-Fr.2.3.6).

[0043] Fr.2.3.6 was analyzed by gel column chromatography using Sephadex LH-20 combined with thin-layer chromatography using silica gel plates, and the fractions were combined by rotary evaporation to obtain 5 fractions (Fr.2.3.6.1-Fr.2.3.6.3).

[0044] Fr.2.3.6.3 The target compound (t) was collected by semi-preparative high-performance liquid chromatography (acetonitrile / water, v / v 52:48, 3.0 mL / min). R = 36.5 min, 3.0 mg), is a colorless gelatinous substance.

[0045] High-resolution mass spectrometry (HR-ESIMS) was used to analyze the compound. 1 H NMR, 13 C NMR, 2D 1 H- 1 H COSY and HMBC analyses determined the planar structure, and the relative configuration of the compound was determined by combining the NOESY correlation signal. 1 H and 13 The C NMR data are shown in Table 1, mainly... 1 H- 1 For information regarding H COSY and HMBC, please see [link / reference]. Figure 1 See NOESY spectrum Figure 2 .

[0046] Table 1. Target compounds in CDCl3 1 H (600 MHz) and 13 C (150 MHz) NMR data

[0047]

[0048] Structural identification: High-resolution mass spectrometry (HRESIMS) revealed a quasi-molecular ion peak at m / z 388.1635 ([M + Na)). + The calculated value is 388.1632. Combined with NMR spectral data, the molecular formula of the compound is inferred to be C. 21 H 23 N3O3. 1 H-spectral data show that it contains 2 active hydrogen atoms ( δ H 7.84, 4.34), 4 aromatic protons ( δ H 7.42, 7.10, 7.16, 7.33), 1 olefin proton ( δ H 5.72) 2 methyl groups ( δ H 1.80, 2.04) and 3 methylene signals ( δ H 4.17, 6.62, 5.72). 13 C10 NMR and DEPT135 spectra show that this compound contains 21 carbons, including 2 methyl carbons (C10 NMR and DEPT135 NMR). δc 26.2, 18.3), 4 methylene carbons ( δ c 33.6, 30.3, 22.4, 46.2), 7 methylene carbons ( δ c 118.0, 120.0, 122.2, 111.1, 60.1, 49.8, 123.5) and 8 seasonal carbon signals ( δ c 132.5, 103.8, 127.5, 136.2, 83.4, 165.4, 166.5,138.1).

[0049] By comparing with literature data, combined with 1 H- 1 The ¹H COSY and HMBC spectra show that this compound is similar to 13-dehydroxycyclotryprostatin C, but the chemical shifts of the C-8~C-12 and C-17~C-19 segments differ significantly in 1D NMR. Further analysis of the 2D NMR data reveals that this compound has the same planar structure as 13-dehydroxycyclotryprostatin C, therefore the two compounds have different stereoconfigurations.

[0050] Its relative configuration was determined by the NOESY spectrum and the chemical shifts of key hydrogen atoms: In the NOESY spectrum, 9-OH ( δ H 4.34) and H-8a ( δ H 3.25) and H-19 ( δ H 5.72), and H-8b ( δ H 3.08) and H-12 ( δ H 4.17) Related Explanation: H-18 and H-12 are on the same side of the diketopiperazine ring, while H-19 and 9-OH are on the other side. The influence of different configurations on the chemical shifts of H-18 and H-19 in the literature further determined their relative configurations: when 9-OH and H-19 are on the same side, the chemical shifts of H-18 and H-19 are 6.65 and 5.60, respectively; when 9-OH and H-19 are on opposite sides, the chemical shifts of H-18 and H-19 are 5.91 and 4.80, respectively. The chemical shift values ​​of the target compound and 13-dehydroxycyclotryprostatin C, H-18 (δ... H 6.62, 6.06), H-19 (δ) H(5.72, 4.92), suggesting that 9-OH and H-19 are on the same side in the target compound. Biogenic analysis indicates that the C-11~C-15 fragment originates from L-proline, therefore the compound is presumed to have a 9-OH configuration. S ,12 S 18 S :

[0051] .

[0052] Example 2 Evaluation of neuroprotective activity

[0053] Using 6-OHDA-induced damage to rat adrenal pheochromocytoma (PC-12) cells as a model, the neuroprotective activity and antioxidant capacity of the compounds obtained in Example 1 were evaluated.

[0054] 1. Neuroprotective effect

[0055] Cell viability was determined using the MTT assay to evaluate the neuroprotective activity of the analyte. Rat adrenal pheochromocytoma cells (PC-12) were cultured at a concentration of 1.0 × 10⁻⁶ cells / cells. 5 Cells were seeded at a density of 100 μL / well in 96-well plates and cultured for 24 hours at 37°C in a 5% CO2 incubator. Control and experimental groups were set up. The experimental groups were incubated for 6 hours with 6.25 μM, 12.5 μM, 25 μM, 50 μM, and 100 μM DMSO solutions of indopiperazine compounds obtained in Example 1 (the control group received an equal volume of DMSO solution). Then, 6-OHDA was added to the culture medium to a concentration of 75 μM to damage the cells for 12 hours. Subsequently, 10 μL of MTT solution was added to each well, and the cells were incubated at 37°C for 4 hours. After removing the culture medium, 150 μL of DMSO was added, and the optical density (OD) was measured at 490 nm using a microplate reader to calculate cell viability.

[0056] Experimental results are as follows Figure 3 As shown, concentration gradient experiments on the target compound revealed that at concentrations of 6.26–100 μM, it could reduce 6-OHDA-induced PC-12 cell death, with PC-12 cell survival rates of 57% (6.25 μM), 64% (12.5 μM), 74% (25 μM), 64% (50 μM), and 49% (100 μM), which were superior to the 48% survival rate in the damage group (6-OHDA).

[0057] The effect of the compound shown in formula (I) on 6-OHDA-induced apoptosis in PC-12 cells was observed using Annexin V-PI double staining. PC-12 cells in logarithmic growth phase were collected and cultured at a cell density of 5 × 10⁶ cells / year. 4Cells were seeded at 1 / mL in 6-well plates and cultured at 37°C with 5% CO2 for 24 hours. The experimental groups were incubated with medium containing different concentrations of indolepiperazine compounds (6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM) for 6 hours, followed by the addition of 6-OHDA. The damage group received only 6-OHDA and was incubated for 12 hours. Cells were then centrifuged at 1200 rpm for 3 minutes, the supernatant was discarded, and the cells were resuspended in PBS at a concentration adjusted to 1×10⁻⁶. 6 Take 100 μL of the suspension and add 5 μL of FITC Annexin V dye to incubate in the dark for 15 min, followed by 5 μL of PI dye to incubate in the dark for 5 min. After incubation, add 400 μL of 1× Binding Buffer and mix well, then analyze using flow cytometry.

[0058] Depend on Figure 4 It can be seen that the compound has a good ameliorative effect on the damage of PC-12 cells treated with 6-OHDA, and the effect is best at 25 μM.

[0059] 2. Antioxidant effect

[0060] 6-OHDA can induce reactive oxygen species (ROS) production, leading to cell damage and apoptosis. Therefore, DCFH-DA and flow cytometry were used to detect the effects of compounds on ROS induced by 6-OHDA in PC-12 cells. PC-12 cells in logarithmic growth phase were harvested and cultured at a cell density of 5 × 10⁶ cells / year. 4 Cells were seeded at 1 mL / mL in 6-well plates and cultured at 37°C with 5% CO2 for 24 hours. The experimental groups were incubated for 6 hours with culture medium containing different concentrations of indolepiperazine compounds (6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM), followed by the addition of 6-OHDA. The damaged group received only 6-OHDA and was incubated for 12 hours. The DCFH-DA probe was then mixed with the culture medium at a 1:1000 ratio, and 1 mL was added to each well. After incubation for 30 minutes, the cells were centrifuged at 1200 rpm for 3 minutes, the supernatant was removed, and the cells were resuspended in culture medium for flow cytometry analysis.

[0061] The results are as follows Figure 5 As shown, compared with the 6-OHDA group, pretreatment of PC-12 cells induced by 6-OHDA with compounds can significantly reduce the generation of intracellular ROS.

[0062] PC-12 cells in the logarithmic growth phase were harvested at a cell density of 5 × 10⁻⁶ cells / year. 4Cells were seeded at / mL in 6-well plates and cultured at 37℃ with 5% CO2 for 24 hours. The experimental groups were incubated for 6 hours with medium containing different concentrations of indolepiperazine compounds (6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM), followed by the addition of 6-OHDA. The damaged group was incubated with only 6-OHDA for 12 hours. The levels of SOD, GSH, and MDA in PC-12 cells were measured according to the kit instructions (Wuhan Yilairuit Biotechnology Co., Ltd.). The results are as follows: Figure 6 As shown, compared with the damaged group (6-OHDA), the levels of SOD and GSH in PC-12 cells treated with the compound at 6.25-25 μM increased, while the level of MDA decreased. This indicates that the compound can restore the function of the intracellular antioxidant system and maintain the dynamic balance between intracellular oxidation and antioxidation.

[0063] The above description is merely an embodiment of this application and is not intended to limit the scope of this application. Various modifications and variations can be made to this application by those skilled in the art. Any modifications, equivalent substitutions, improvements, etc., made within the spirit and principles of this application should be included within the scope of the claims of this application.

Claims

1. An indolepiperazine compound, with the following structural formula: 。 2. A method for preparing an indolepiperazine compound as described in claim 1, characterized in that, Includes the following steps: (1) Fermentation Aspergillus tanneir UJNMF0417, obtained fermentation product; (2) The fermentation product obtained in step (1) was successively extracted with ethanol and ethyl acetate to obtain a crude extract; (3) The crude extract obtained in step (2) was separated by D101 macroporous resin column chromatography and eluted with 30%, 50%, 75% and 95% ethanol to obtain four fractions Fr.1-Fr.4; Fr.2 was subjected to normal-phase silica gel column chromatography with a mobile phase of dichloromethane / methanol with a volume ratio of 1:0-0:1 to obtain eight fractions, Fr.2.1-Fr.2.

8. Fr.2.3 was subjected to gradient elution silica gel column chromatography with dichloromethane / methanol at a volume ratio of 1:0 to 0:1 to obtain six fractions: Fr.2.3.1-Fr.2.3.

6. Fr.2.3.6 was separated by Sephadex LH-20 chromatography column to obtain fractions Fr.2.3.6.1-Fr.2.3.6.3; Fr.2.3.6.3 was obtained by semi-preparative high performance liquid chromatography with acetonitrile / water at a volume ratio of 52:48 as the mobile phase, a flow rate of 3.0 mL / min, and collection of the eluent with a retention time of 36.5 min. The mobile phase was then removed. The Aspergillus tanneir The accession number of UJNMF0417 is CGMCC No.41647; In step (1), the fermentation step is as follows: 10 mL of seed liquid is inoculated into a 1 L Erlenmeyer flask containing rice culture medium, and after static culture at 28℃ for 30 days, the fermentation product is collected. Rice culture medium preparation method: 80g rice, 0.4g yeast extract, 0.4g glucose, 120mL water, 3% sea salt; In step (2), the extraction method is as follows: the rice culture medium after fermentation for 30 days is transferred out and crushed, and soaked in 95% ethanol for 4 times, with each soaking time being 6-7 days. The 95% ethanol after each soaking is collected and concentrated into an aqueous solution using a large rotary evaporator. Then, an equal amount of ethyl acetate is used for extraction, and the extraction is performed four times. After concentration in a rotary evaporator, a crude extract is obtained.

3. Use of an indolepiperazine compound as described in claim 1 in the preparation of a medicament for neurodegenerative diseases.

4. A medicament for neurodegenerative diseases containing an indolepiperazine compound as described in claim 1.

5. The drug according to claim 4, characterized in that, It also includes other active ingredients and / or medically acceptable excipients.

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