Indole piperazine compound and application thereof in medicine for treating neurodegenerative diseases

Abstract

The invention belongs to the technical field of biological medicines, and provides an indole piperazine compound and application thereof in drugs for treating neurodegenerative diseases. The structural formula of the indole piperazine compound is as follows: # imgabs0 #. The compound can be used for preparing drugs and drug intermediates for treating neurodegenerative diseases. The compound can be obtained through fermentation of Aspergillus tanner UJNMF0417, the yield can be further improved through optimization of fermentation conditions and transformation of strains, and the compound has a good commercial prospect.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to an indole piperazine compound and its medical application. Background Art

[0002] The disclosure of this background information is intended to enhance understanding of the general background of the invention and should not necessarily be taken as an acknowledgment or any form of suggestion that this information constitutes the prior art already known to a person skilled in the art.

[0003] Neurodegeneration (ND) is a phenomenon characterized by the slow and progressive loss of neuronal cell function and structure in specific brain regions, which is the main pathophysiological feature of various neurodegenerative diseases (NDDs), including Alzheimer's disease (AD), Parkinson's disease (PD), amyotrophic lateral sclerosis (ALS), Huntington's disease (HD), etc. Neurodegeneration is a complex process affected by genetic, environmental and endogenous factors related to aging. However, the exact pathological effects and underlying molecular mechanisms are still not fully understood. Most of the patients with neurodegenerative diseases are elderly people, and the treatment and prevention of the disease is one of the important problems that my country needs to face and solve for a long time in the future. Therefore, in-depth research on the etiology and mechanism of neurodegenerative diseases, exploration of effective prevention and treatment methods, and search for effective prevention and treatment drugs are of great significance to improving the quality of life of the elderly, reducing the social burden, and even promoting the development of the country. Summary of the invention

[0004] In view of the problem that the drugs for treating neurodegenerative diseases are limited in the prior art, the present invention provides an indole piperazine compound having neuroprotective activity.

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

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

[0007] An indole piperazine compound, the structure of which is shown in formula (I): Formula (I).

[0008] The preparation method of the above-mentioned indole piperazine compound comprises the following steps: (1) Fermentation of Aspergillus fungi Aspergillus tanneir UJNMF0417, obtained fermentation; (2) extracting the fermented product obtained in step (1) with ethanol and ethyl acetate in sequence to obtain a crude extract; (3) The crude product 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 using dichloromethane / methanol in a volume ratio of 1:0-0:1 as a gradient elution mobile phase to obtain eight fractions from Fr.2.1 to Fr.2.8; Fr.2.3 was subjected to gradient elution silica gel column chromatography using dichloromethane / methanol in a volume ratio of 1:0-0:1 as the eluent to obtain six fractions, Fr.2.3.1-Fr.2.3.6; Fr.2.3.6 was separated by Sephadex LH-20 column to obtain five 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, using acetonitrile / water in a volume ratio of 52:48 as the mobile phase at a flow rate of 3.0 mL / min, collecting the effluent with a retention time of 36.5 min, and removing the mobile phase; Said Aspergillus tanneir The deposit number of UJNMF0417 is CGMCC No.41647.

[0009] Preferably, the ethanol in step (2) is 90%-95% ethanol.

[0010] The present invention also provides a kind of Aspergillus fungus Aspergillus tanneir Use of UJNMF0417 and the above-mentioned indole piperazine compounds in the preparation of neurodegenerative disease drugs and drug intermediates.

[0011] A drug for treating neurodegenerative diseases containing the above-mentioned indolepiperazine compound and its pharmaceutically acceptable salt. The drug also includes other active ingredients and / or medically acceptable excipients. The above-mentioned salt is a hydrochloride or sulfate formed by -NH- or a sodium or potassium salt formed by -OH in the indolepiperazine compound.

[0012] The present invention has the following advantages: The indole piperazine compounds of the present invention have neuroprotective activity and have application potential in the preparation of drugs and drug intermediates for neurodegenerative diseases. Aspergillus tanneir UJNMF0417 is obtained by fermentation. The yield can be further increased by optimizing fermentation conditions and modifying bacterial strains, and it has good commercial prospects.

[0013] Biological deposit information Aspergillus tanneirUJNMF0417 was deposited on December 2, 2024 in the General Microbiology Center of China Microorganism Culture Collection (CGMCC), with the deposit address at Institute of Microbiology, Chinese Academy of Sciences, No. 3, Yard 1, Beichen West Road, Chaoyang District, Beijing, China, with the deposit number CGMCC No.41647. BRIEF DESCRIPTION OF THE DRAWINGS

[0014] Figure 1 The main target compound 1 H- 1 H COSY, HMBC related information.

[0015] Figure 2 It is the main NOESY related information of the target compound.

[0016] Figure 3 This is the effect of indolepiperazine compounds on 6-OHDA-induced PC-12 cell proliferation.

[0017] Figure 4 This is the effect of indolepiperazine compounds on 6-OHDA-induced apoptosis of PC-12 cells.

[0018] Figure 5 This is the effect of indolepiperazine compounds on the ROS level in PC-12 cells induced by 6-OHDA.

[0019] Figure 6 This is the effect of indolepiperazine compounds on the levels of MAD, SOD and GSH in PC-12 cells induced by 6-OHDA. DETAILED DESCRIPTION

[0020] The present invention is further described below in conjunction with the examples and drawings, but the present invention is not limited by the following examples. The experimental methods in the examples, unless otherwise specified, all adopt conventional techniques in the art, and the experimental reagents are all purchased commercially.

[0021] Example 1 Preparation of indole piperazine compounds 1. Preparation of Fermentation Preparation method of seed culture medium: 200 mL of potato extract, 20 g of glucose, 30 g of sea salt, dilute to 1 L with water. Pour the culture medium into 20 500 mL conical flasks, about 150 mL per flask, sterilize at 121℃ with high pressure steam for 25 minutes, and set aside.

[0022] Preparation method of rice culture medium: 80g rice, 0.4g yeast extract, 0.4g glucose, 120mL water, 3% sea salt. Place in 1L Erlenmeyer flasks, 150 flasks in total. Sterilize at 121℃ with high pressure steam for 25 minutes and set aside.

[0023] Use a sterile bamboo stick to pick up an appropriate amount Aspergillus tanneir The UJNMF0417 strain was inoculated into the above culture medium and cultured at 28°C in a shaker (200 rpm) for 3 days to obtain a seed solution. Then, 10 mL of the seed solution was inoculated into a 1 L Erlenmeyer flask containing rice culture medium using a pipette. After static culture at 28°C for 30 days, the fermentation product was collected.

[0024] The rice culture medium after fermentation for 30 days was transferred out and crushed, and then soaked in 95% ethanol for 4 times (single soaking time is 6-7 days). The 95% ethanol after each soaking was collected and concentrated using a large rotary evaporator to obtain an aqueous solution. The aqueous solution was then extracted with an equal amount of ethyl acetate for four times, and the crude extract was concentrated using a rotary evaporator.

[0025] 2. Isolation of target compounds 90 g of the crude product obtained above was subjected to D101 macroporous resin column chromatography and eluted with 30%, 50%, 75%, and 95% ethanol to obtain a total of 4 parts (Fr.1-Fr.4).

[0026] Fr.2 was subjected to silica gel column chromatography gradient elution with dichloromethane:methanol (1:0-0:1). During the elution, a thin layer chromatography silica gel plate was used for detection and the fractions with similar compounds were combined to obtain a total of 8 fractions (Fr.2.1-Fr.2.8).

[0027] Fr.2.3 was subjected to gradient elution silica gel column chromatography with dichloromethane:methanol (1:0-0:1), detected by thin layer chromatography silica gel plate, and 6 fractions (Fr.2.3.1-Fr.2.3.6) were obtained by rotary evaporation and combination; Fr.2.3.6 was subjected to gel column chromatography on Sephadex LH-20, combined with thin layer chromatography on silica gel plates for detection, and five fractions (Fr.2.3.6.1-Fr.2.3.6.3) were obtained by rotary evaporation and combination; Fr.2.3.6.3 The target compound (t R = 36.5 min, 3.0 mg), as a colorless jelly.

[0028] The compounds were subjected to high-resolution mass spectrometry (HR-ESIMS), 1 H NMR, 13 C NMR, 2D 1 H- 1 The planar structure was determined by H COSY and HMBC analysis, and the relative configuration of the compound was determined by combining NOESY related signals. 1 H and13 C NMR data are shown in Table 1. The main 1 H- 1 For more information on H COSY and HMBC, see Figure 1 , NOESY spectrum see Figure 2 .

[0029] Table 1 Target compounds in CDCl3 1 H (600 MHz) and 13 C (150 MHz) NMR data Structural identification: High-resolution mass spectrometry (HRESIMS) gave a quasi-molecular ion peak at m / z 388.1635 ([M + Na] + , calculated value 388.1632), combined with NMR spectrum data, it is inferred that the molecular formula of the compound is C 21 H 23 N3O3. 1 H spectrum data showed that it contained 2 active hydrogens ( 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 methine signals ( δ H 4.17, 6.62, 5.72). 13 C NMR and DEPT135 spectra showed that the compound contained 21 carbon atoms, including 2 methyl carbon atoms ( δ c 26.2, 18.3), 4 methylene carbons ( δ c 33.6, 30.3, 22.4, 46.2), 7 methyl carbons ( δ c 118.0, 120.0, 122.2, 111.1, 60.1,49.8, 123.5) and 8 quaternary carbon signals ( δ c 132.5, 103.8, 127.5, 136.2, 83.4, 165.4, 166.5,138.1).

[0030] By comparing with literature data, combined with 1 H- 1The H COSY and HMBC spectra show that the compound is similar to 13-dehydroxycyclotryprostatin C, but in 1D NMR, the chemical shift values ​​of the two fragments C-8~C-12 and C-17~C-19 are quite different. Further analysis of the 2D NMR data of the compound shows that the compound has the same planar structure as 13-dehydroxycyclotryprostatin C, so the stereo configurations of the two compounds are different.

[0031] The relative configuration was determined by NOESY spectra 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) H-18 and H-12 are on the same side of the diketopiperazine ring, and H-19 and 9-OH are on the other side of the diketopiperazine ring; 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 shift values ​​of H-18 and H-19 are 6.65 and 5.60, respectively; when 9-OH and H-19 are on different sides, the chemical shift values ​​of H-18 and H-19 are 5.91 and 4.80, respectively. The chemical shift values ​​of H-18 (δ H 6.62, 6.06), H-19 (δ H 5.72, 4.92), it is speculated that 9-OH and H-19 in the target compound are on the same side. From the biological source analysis, the C-11~C-15 fragment comes from L-proline, so it is speculated that the configuration of the compound is 9 S ,12 S ,18 S : .

[0032] Example 2 Evaluation of neuroprotective activity The neuroprotective activity and antioxidant capacity of the compound obtained in Example 1 were evaluated using 6-OHDA-induced rat adrenal pheochromocytoma cell (PC-12) injury model.

[0033] 1. Neuroprotective effect The cell viability was determined by MTT assay to evaluate whether the test substance had neuroprotective activity. Rat adrenal pheochromocytoma cells (PC-12) were cultured at 1.0×10 5 The cells were inoculated into a 96-well plate at a density of 100 μL / well, and the cells were cultured in a constant temperature incubator at 37°C and 5% CO2 for 24 hours. A control group and an experimental group were set up. The experimental group was incubated with 6.25 μM, 12.5 μM, 25 μM, 50 μM, and 100 μM DMSO solutions of indole piperazine compounds obtained in Example 1 for 6 hours (the control group was added with an equal volume of DMSO solution), and then 6-OHDA was added to the culture medium to 75 μM to damage the cells for 12 hours. Subsequently, 10 μL of MTT solution was added to each well, incubated at 37°C for 4 hours, the culture medium was removed, 150 μL of DMSO was added, and the optical density (OD) was measured at 490 nm using a microplate reader to calculate the cell survival rate.

[0034] The experimental results are as follows Figure 3 As shown, a concentration gradient experiment of the target compound showed that at a concentration of 6.26-100 μM, it could reduce the death of PC-12 cells caused by 6-OHDA, and the survival rates of PC-12 cells were 57% (6.25 μM), 64% (12.5 μM), 74% (25 μM), 64% (50 μM), and 49% (100 μM), which were better than the injury group (6-OHDA) of 48%.

[0035] Annexin V-PI double staining experiment was used to observe the effect of the compound represented by formula (I) on 6-OHDA-induced apoptosis of PC-12 cells. PC-12 cells in logarithmic growth phase were taken and the cell density was 5×10 4 / mL were inoculated in 6-well plates and cultured in a constant temperature incubator at 37°C and 5% CO2 for 24 hours. The experimental group was added with medium containing different concentrations of indole piperazine compounds (6.25 μM, 12.5 μM, 25 μM, 50 μM, 100 μM) for 6 hours, and then 6-OHDA was added; the injury group was only added with 6-OHDA, incubated in the incubator for 12 hours, centrifuged at 1200 rpm for 3 minutes, the supernatant was removed, and the cells were resuspended in PBS and the concentration was adjusted to 1×10 6 / mL, take 100 μL of the suspension and add 5 μL of FITC Annexin V dye in the dark for 15 min, then add 5 μL of PI dye in the dark for 5 min. After incubation, add 400 μL of 1× Binding Buffer and mix well, and detect by flow cytometry.

[0036] Depend on Figure 4It can be seen that the compound has a good improvement effect on the damage of PC-12 cells treated with 6-OHDA, and the effect is best at 25 μM.

[0037] 2. Antioxidant Effect 6-OHDA can induce the production of reactive oxygen species, leading to cell damage and apoptosis. Therefore, DCFH-DA and flow cytometry were used to detect the effects of compounds on 6-OHDA-induced reactive oxygen species in PC-12 cells. PC-12 cells in the logarithmic growth phase were taken and the cell density was 5×10 4 / mL were inoculated in 6-well plates and cultured in a constant temperature incubator at 37℃ and 5% CO2 for 24 hours. The experimental group was added with culture medium containing different concentrations of indole piperazine compounds (6.25 μM, 12.5 μM, 25 μM, 50 μM, and 100 μM) for 6 hours, and then 6-OHDA was added; only 6-OHDA was added to the injury group and incubated in the incubator for 12 hours. The DCFH-DA probe and culture medium were mixed at a ratio of 1:1000, 1 mL was added to each well and incubated for 30 minutes. The cells were centrifuged at 1200 rpm for 3 minutes, the supernatant was removed, the cells were resuspended with culture medium, and then detected by flow cytometry.

[0038] The results are as follows Figure 5 As shown, compound pretreatment of 6-OHDA-induced PC-12 cells significantly reduced the generation of intracellular ROS compared with the 6-OHDA group.

[0039] PC-12 cells in the logarithmic growth phase were taken and the cell density was 5×10 4 / mL were inoculated in 6-well plates and cultured in a constant temperature incubator at 37°C and 5% CO2 for 24 hours. The experimental group was added with medium containing different concentrations of indole piperazine compounds (6.25 μM, 12.5 μM, 25 μM, 50 μM, and 100 μM) for 6 hours, and then 6-OHDA was added; only 6-OHDA was added to the injury group and incubated in the incubator for 12 hours. The levels of SOD, GSH, and MDA in PC-12 cells were detected according to the instructions of the kit (Wuhan Elaruite Biotechnology Co., Ltd.). The results are shown in Figure 6 As shown, compared with the injury group (6-OHDA), the levels of SOD and GSH in PC-12 cells in the compound-treated group at 6.25-25 μM increased, while the level of MDA decreased, indicating that the compound was able to restore the function of the intracellular antioxidant system and maintain the dynamic balance between intracellular oxidation and anti-oxidation.

[0040] The above is only an embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various changes and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application should be included in the scope of the claims of the present application.

Claims

1. An indole piperazine compound having the structural formula: 。 2. A method for preparing an indole piperazine compound as claimed in claim 1, characterized in that: The following steps are involved: (1) Fermentation Aspergillus tanneir UJNMF0417, obtained fermentation; (2) extracting the fermented product obtained in step (1) with ethanol and ethyl acetate in sequence to obtain a crude extract; (3) The crude product 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 using dichloromethane / methanol in a volume ratio of 1:0-0:1 as a gradient elution mobile phase to obtain eight fractions from Fr.2.1 to Fr.2.8; Fr.2.3 was subjected to gradient elution silica gel column chromatography using dichloromethane / methanol in a volume ratio of 1:0-0:1 as the eluent to obtain six fractions, Fr.2.3.1-Fr.2.3.6; Fr.2.3.6 was separated by Sephadex LH-20 column to obtain five 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, using acetonitrile / water in a volume ratio of 52:48 as the mobile phase at a flow rate of 3.0 mL / min, collecting the effluent with a retention time of 36.5 min, and removing the mobile phase; Said Aspergillus tanneir The deposit number of UJNMF0417 is CGMCC No.41647.

3. The preparation method according to claim 2, characterized in that: The ethanol in step (2) is 90%-95% ethanol.

4. A Aspergillus tanneir Use of UJNMF0417 and the indole piperazine compound as claimed in claim 1 in the preparation of drugs for neurodegenerative diseases and drug intermediates, characterized in that: Said Aspergillus tanneir The deposit number of UJNMF0417 is CGMCC No.41647.

5. A drug for treating neurodegenerative diseases comprising the indole piperazine compound as claimed in claim 1.

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

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