Peptidase neurosin enhancer and methods of making and using same

Abstract

The application discloses a kind of peptidase neurosin enhancer and its preparation method and application, it has the structure shown in structural formula (I):The application provides a kind of small molecular peptidase neurosin enhancer, its structure is novel, can enhance the degradation of alpha-synuclein, and reduce the level of alpha-synuclein in cell reverse mitochondrial damage and cytotoxicity, effectively protect cell from alpha-synuclein aggregation.The compound provided in the application relieves mitochondrial damage and cell death caused by alpha-synuclein overexpression, and can be used to treat various neurodegenerative diseases such as Parkinson's disease.The compound provided in the application can also relieve peripheral inflammatory diseases such as ischemic stroke, traumatic brain injury and autism by enhancing the function of peptidase neurosin.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of small molecule compounds, in particular to a compound, a preparation method and application thereof, and a pharmaceutical composition. BACKGROUND

[0002] Parkinson's disease is the second most common neurodegenerative disease in the world after Alzheimer's disease, and its two major pathological features are that the accumulation of alpha-synuclein protein forms Lewy bodies, leading to the progressive degeneration of dopaminergic neurons in the substantia nigra and mitochondrial damage. Epidemiological studies show that the prevalence of Parkinson's disease in people over 65 years old in China is 1.7%, and with the aggravation of population aging, the number of patients in China will reach 5 million in 2030, which will bring a huge medical burden to society and patient families. The pathogenesis of Parkinson's disease is complex, and there is currently no effective treatment drug. The current clinical treatment usually uses levodopa drugs to increase dopamine levels and improve patient symptoms, but it cannot delay the progression of the disease. Seeking an effective drug to relieve Parkinson's disease is a technical problem to be solved in the art.

[0003] Studies have found that during the process of alpha-synuclein entering mitochondria and being degraded by mitochondrial proteases, mitochondrial peptidase neurolysin (Nln) plays a major role in degradation. However, when aging, protein homeostasis imbalance or neurodegenerative disease gene defects occur, alpha-synuclein cannot be immediately cleared. Alpha-synuclein will accumulate in mitochondria, affect mitochondrial function, cause mitochondrial fragmentation, mitochondrial membrane pressure reduction, mitochondrial DNA reduction and other damage, and ultimately lead to the death of nerve cells.

[0004] Correspondingly, when the cell is treated with an Nln enhancer, the degradation of alpha-synuclein can be enhanced, the level of alpha-synuclein in the cell is reduced, mitochondrial damage and cytotoxicity are reversed, and the cell is effectively protected from alpha-synuclein aggregation. The Nln enhancer is expected to become a new drug for the treatment of Parkinson's disease, and to alleviate mitochondrial damage and cell death caused by overexpression of alpha-synuclein. SUMMARY

[0005] In order to solve the problems in the prior art, the present application provides a peptidase neurolysin enhancer, a preparation method and application thereof.

[0006] According to a first aspect of the present application, a compound is provided, which has a structure shown in structural formula (I):

[0007]

[0008] According to a second aspect of the present application, there is further provided a method for preparing a compound according to the first aspect of the present application, the method comprising:

[0009]

[0010] (1) Amino acid protection reaction: 18-25 parts of L-Tyrosine are taken in mole fraction, and 1,4-dioxane and water are added; NaOH and 20-40 parts of Boc anhydride are added dropwise in sequence under ice bath condition; the reaction is carried out at room temperature to obtain Boc-L-Tyr;

[0011] (2) Amino acid condensation reaction: 2-5 parts of Boc-L-Tyr are dissolved in mole fraction, and 4-7 parts of EDCI, 2-5 parts of HOBt and 8-12 parts of DIPEA are added to the solution and stirred at room temperature; 4-7 parts of NHR1R2 are added to the reaction solution, wherein R1 is hydrogen and R2 is phenethyl, and NHR1R2 is phenethylamine; the reaction is carried out at room temperature to obtain an intermediate product;

[0012] (3) Deprotection reaction: 1-2 parts of the intermediate product are dissolved in mole fraction, and hydrochloric acid dioxane is added dropwise under ice bath condition, and the reaction is stirred at room temperature to obtain the compound.

[0013] In an embodiment of the present application, the amino acid protection reaction step further comprises the following steps after the reaction is completed:

[0014] S01, adjusting the reaction solution to be acidic;

[0015] S02, extracting the acidic reaction solution to obtain an organic phase;

[0016] S03, removing water from the organic phase;

[0017] S04, removing organic solvents from the organic phase to obtain Boc-L-Tyr.

[0018] In an embodiment of the present application, the step S01 comprises adjusting the solution to pH=4 by using saturated potassium bisulfate solution.

[0019] The step S02 comprises extracting the acidic reaction solution by using ethyl acetate;

[0020] The step S03 comprises drying the organic phase by using anhydrous sodium sulfate to remove water;

[0021] The step S04 comprises removing the organic solvents in the organic phase by distillation under reduced pressure.

[0022] In one embodiment of the present application, the amino acid condensation reaction step further comprises the following steps after the reaction is completed:

[0023] S11, extracting the reaction solution to obtain an organic phase;

[0024] S12, removing water from the organic phase;

[0025] S13, removing the organic solvent from the organic phase;

[0026] S14, separating the intermediate product by silica gel column chromatography.

[0027] In one embodiment of the present application, the step S11 comprises the following steps: first extracting the reaction solution with ethyl acetate, then extracting twice with water, extracting three times with ammonium chloride solution, and extracting twice with saturated brine.

[0028] The step S12 comprises the following step: drying the organic phase with anhydrous sodium sulfate to remove water.

[0029] The step S13 comprises the following step: removing the organic solvent from the organic phase by distillation under reduced pressure.

[0030] In one embodiment of the present application, the deprotection reaction step further comprises the following steps after the reaction is completed:

[0031] S21, drying the reaction solution with anhydrous sodium sulfate;

[0032] S22, removing the organic solvent by distillation under reduced pressure;

[0033] S23, separating the compound by silica gel column chromatography.

[0034] According to a third aspect of the present application, there is also provided a use of the compound provided by the first aspect of the present application in the preparation of a peptidase neurosin enhancer.

[0035] In one embodiment of the present application, the peptidase neurosin enhancer is used for treating neurodegenerative diseases and peripheral inflammatory diseases; the neurodegenerative diseases include Parkinson's disease, Alzheimer's disease, Lewy body dementia, multiple system atrophy, pure autonomic failure and rapid eye movement sleep behavior disorder; and the peripheral inflammatory diseases include ischemic stroke, traumatic brain injury and autism.

[0036] According to a fourth aspect of the present application, there is also provided a pharmaceutical composition comprising the compound provided by the first aspect of the present application and a pharmaceutically acceptable carrier.

[0037] One beneficial effect of the present application is that the present application provides a small molecule enhancer of peptidase neurosin, which has a novel structure and can enhance the degradation of alpha-synuclein, reduce the level of alpha-synuclein in cells, reverse mitochondrial damage and cytotoxicity, and effectively protect cells from alpha-synuclein aggregation. The compound provided by the present application can alleviate mitochondrial damage and cell death caused by overexpression of alpha-synuclein, and can be used to treat various neurodegenerative diseases such as Parkinson's disease. In addition, the compound provided by the present application can also alleviate peripheral inflammatory diseases such as ischemic stroke, traumatic brain injury and autism by enhancing the function of peptidase neurosin.

[0038] Other features of the present application, and its advantages, will become apparent in the course of the following detailed description of exemplary embodiments of the application. BRIEF DESCRIPTION OF DRAWINGS

[0039] The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the application and, together with the description, serve to explain the principles of the application.

[0040] Figure 1 is a statistical chart of alpha-synuclein-split GFP fluorescence signal quantitative analysis;

[0041] Figure 2 is a confocal microscope imaging result chart of Example 2;

[0042] Figure 3 is a statistical chart of alpha-synuclein level detected by Western blotting in cells;

[0043] Figure 4 is a confocal microscope imaging result chart of Example 4. DETAILED DESCRIPTION

[0044] In order to make the purposes, technical solutions and beneficial technical effects of the present application clearer, the present application will be described in detail below in combination with specific embodiments. It should be understood that the embodiments described in the specification are only for the purpose of explaining the present application, and are not intended to limit the present application.

[0045] For the sake of simplicity, only some numerical ranges are explicitly disclosed herein. However, any lower limit can be combined with any upper limit to form a range that is not explicitly recited; and any lower limit can be combined with other lower limits to form a range that is not explicitly recited, as can any upper limit with any other upper limit. In addition, even though not explicitly recited, every point or individual number within a range is included in that range. Hence, every midpoint or individual number can serve as its own lower limit or upper limit to combine with any other point or individual number or to combine with other lower or upper limits to form a range that is not explicitly recited.

[0046] In the description herein, it is to be understood that the terms "above" and "below" are inclusive of the number, and the term "several" means two or more, unless otherwise specified.

[0047] The above summary of the application is not intended to describe each disclosed embodiment or implementation of the present application. The following description more specifically illustrates example embodiments. Throughout this application, guidance is provided by a series of examples, which can be used in various combinations. In each instance, the recited list is representative only and should not be construed as exhaustive.

[0048] The present application provides a compound having a structure shown in structural formula (I):

[0049]

[0050] The compound shown in structural formula (I) is (S)-2-amino-3-(4-hydroxyphenyl)-N-phenethylpropanamide.

[0051] The present application also provides a method for preparing the aforementioned compound, comprising:

[0052]

[0053] (1) Amino acid protection reaction: take 18-25 parts of L-tyrosine in terms of mole fraction, add 1,4-dioxane and water; add NaOH and 20-40 parts of Boc anhydride dropwise under ice bath condition; raise the temperature to room temperature to carry out the reaction, and obtain Boc-L-Tyr.

[0054] In an embodiment of the present application, after the reaction in the amino acid protection reaction step, the method further comprises:

[0055] S01, adjusting the reaction solution to be acidic; specifically, step S01 comprises: using saturated potassium bisulfate solution to adjust the solution to pH=4.

[0056] S02, extracting the acidic reaction solution to obtain an organic phase; specifically, step S02 comprises: using ethyl acetate to extract the acidic reaction solution.

[0057] S03, removing water from the organic phase; specifically, using anhydrous sodium sulfate to dry the organic phase to remove water.

[0058] S04, removing the organic solvent in the organic phase to obtain Boc-L-Tyr; specifically, step S04 comprises: removing the organic solvent in the organic phase by distillation under reduced pressure.

[0059] (2) amino acid condensation reaction: 2-5 parts of Boc-L-Tyr is dissolved in mole fraction, and 4-7 parts of EDCI, 2-5 parts of HOBt and 8-12 parts of DIPEA are added to the solution and stirred at room temperature; 4-7 parts of NHR1R2 is added to the reaction solution, wherein R1 is hydrogen and R2 is phenethyl, NHR1R2 is phenethylamine; the reaction is carried out at room temperature, and the intermediate product is separated.

[0060] In an embodiment of the present application, the amino acid condensation reaction step further comprises the following steps after the reaction is completed:

[0061] S11, extracting the reaction solution to obtain an organic phase; specifically, step S11 comprises: first extracting the reaction solution with ethyl acetate, then extracting twice with water, then extracting three times with ammonium chloride solution, and then extracting twice with saturated brine.

[0062] S12, removing water from the organic phase; specifically, step S12 comprises: drying the organic phase with anhydrous sodium sulfate to remove water.

[0063] S13, removing organic solvents from the organic phase; specifically, step S13 comprises: removing the organic solvents from the organic phase by distillation under reduced pressure.

[0064] S14, separating the intermediate product by silica gel column chromatography.

[0065] (2) deprotection reaction: 1-2 parts of the intermediate product is dissolved in mole fraction, and then hydrochloric acid dioxane is added dropwise under ice bath condition, and the reaction is stirred at room temperature to obtain the compound.

[0066] In an embodiment of the present application, the deprotection reaction step further comprises the following steps after the reaction is completed:

[0067] S21, drying the reaction solution with anhydrous sodium sulfate;

[0068] S22, removing the organic solvents by distillation under reduced pressure;

[0069] S23, separating the compound by silica gel column chromatography.

[0070] The present application also provides a use of the aforementioned compound in the preparation of a peptidase neurosin enhancer.

[0071] In one embodiment of the present application, the peptidase neurosin enhancer is used to treat neurodegenerative diseases, including Parkinson's disease, Alzheimer's disease, Lewy body dementia, multiple system atrophy, pure autonomic failure and rapid eye movement sleep behavior disorder, and peripheral inflammatory diseases, including ischemic stroke, traumatic brain injury and autism. The peptidase neurosin enhancer can enhance the degradation of alpha-synuclein, reduce the level of alpha-synuclein in cells, reverse mitochondrial damage and cytotoxicity, and effectively protect cells from alpha-synuclein aggregation. This can alleviate mitochondrial damage and cell death caused by overexpression of alpha-synuclein, thereby treating various neurodegenerative diseases such as Parkinson's disease. In addition, the compound provided by the present application can also alleviate peripheral inflammatory diseases such as ischemic stroke, traumatic brain injury and autism by enhancing the function of peptidase neurosin.

[0072] According to the present application, a pharmaceutical composition is also provided, comprising the aforementioned compound and a pharmaceutically acceptable carrier. The carrier can be arbitrarily mixed and can vary depending on the dosage form, administration form, etc. Examples of the carrier include excipients, binders, disintegrants, lubricants, flavoring agents, odorants, colorants, and sweeteners, etc. The pharmaceutical composition can be in the form of a conventional pharmaceutical preparation such as capsules, powders, tablets, granules, pills, injections, syrups, oral solutions, inhalants, ointments, suppositories, and patches, etc.

[0073] Example 1

[0074] Synthesis of (S)-2-amino-3-(4-hydroxyphenyl)-N-phenethylpropanamide

[0075] Structural formula of the target synthesis:

[0076] The general synthetic route of the tyrosine series compounds is as follows:

[0077]

[0078] It should be noted that when the R1 and R2 groups in NHR1R2 are changed, the specific structures of compounds 3 and 4 will also change accordingly.

[0079] First step: amino acid acid protection reaction

[0080] Referring to the above synthesis route, compound 1 is L-tyrosine and compound 2 is Boc-L-Tyr.

[0081] Take 18.1 g, 0.1 mol of L-tyrosine (compound 1) and 4.0 g, 0.1 mol of sodium hydroxide, dissolved in 80.0 mL of 1,4-dioxane and 80.0 mL of water. At 0-5°C, add 26.2 g, 0.12 mol of Boc anhydride (i.e. di-tert-butyl dicarbonate, (BOC)2O in the above synthesis path). After the dropwise addition is complete, the reaction is stirred at room temperature for 16 hours.

[0082] After the reaction is carried out for 16 hours, the current reaction solution is detected by thin layer chromatography (TLC), and after the detection result shows that the reaction is complete, the reaction solution is concentrated under reduced pressure to remove most of the 1,4-dioxane. Then the residual solution is adjusted to pH = 4 with saturated potassium bisulfate solution. Then extract with ethyl acetate (200 mL x 3), and combine the organic phases. Dry the organic phase with anhydrous sodium sulfate to remove water from the organic phase. After drying is complete, remove the organic solvent from the organic phase by reduced pressure distillation to obtain 23.8 g of product, Boc-L-Tyr (compound 2), with a yield of 85%. Boc-L-Tyr (compound 2) will be used in subsequent reactions.

[0083] Second step: amino acid condensation reaction:

[0084] Referring to the general synthesis route of tyrosine series compounds, NHR1R2 will be introduced as a reactant in the reaction of Boc-L-Tyr (compound 2) to form compound 3. In the synthesis of (S)-2-amino-3-(4-hydroxyphenyl)-N-phenethylpropionamide, NHR1R2 is phenethylamine, and the preparation method of phenethylamine is described below. The amino condensation reaction is the process of reacting Boc-L-Tyr (compound 2) with phenethylamine to form the intermediate (compound 3).

[0085] Take 1.00 g, 3.55 mmol of Boc-L-Tyr (compound 2) and place it in a 100 mL round-bottom flask, and add an appropriate amount of N,N-dimethylformamide (DMF) to dissolve. Then add 1.02 g, 5.33 mmol of EDCI (1-ethyl-(3-dimethylaminopropyl) carbodiimide hydrochloride), 0.48 g, 3.55 mmol of HOBt (hydroxybenzotriazole), and 1.86 mL, 10.66 mmol of DIPEA (N,N-diisopropylethylamine) to the solution, and stir at room temperature.

[0086] After 30 minutes, 0.65 g, 5.33 mmol of phenethylamine was added and the reaction was allowed to proceed at room temperature overnight. After the overnight reaction, the reaction solution was detected by thin layer chromatography (TLC). TLC can be used to track the progress of the reaction. The reaction solution was applied to a glass plate, plastic or aluminum substrate to form a uniform thin layer. After spotting and developing, the developed plate was observed under a UV lamp or by spraying a color reagent. The reaction was considered complete when the spots of the starting materials disappeared.

[0087] An equal amount of water was added for dilution. Then, ethyl acetate (50 mL x 3) was used for extraction, and the organic phase was combined. The combined organic phase was extracted twice with water (50 mL x 2), three times with ammonium chloride solution (NH4Cl, 50 mL x 3), and twice with saturated brine (50 mL x 2).

[0088] After extraction, anhydrous sodium sulfate was used for drying, and the organic solvent was removed by reduced pressure distillation. Then, 510 mg of intermediate (compound 3) was obtained by silica gel column chromatography (PE:EA = 10:1-3:1), with a yield of 41.13%. The intermediate (compound 3) will be used in the next reaction.

[0089] Step 3: Deprotection reaction

[0090] Referring to the general synthesis route of tyrosine series compounds, the deprotection reaction is the process of generating the target product (compound 4) from the intermediate (compound 3). In this embodiment, compound 4 is (S)-2-amino-3-(4-hydroxyphenyl)-N-phenethylpropanamide.

[0091] 0.480 g, 1.38 mmol of intermediate (compound 3) was dissolved in 10.0 mL of acetone and stirred in an ice bath for 10 min. Then, 4M, 3.0 mL of hydrochloric acid dioxane (HCl dioxane) solution was slowly added dropwise. The reaction was stirred overnight at room temperature. After the overnight reaction, the reaction solution was detected by thin layer chromatography (TLC). After the detection results indicated that the reaction was complete, the acetone was removed by rotary evaporation. The solution was diluted with twice the amount of water and dried with anhydrous sodium sulfate. The organic solvent was removed by reduced pressure distillation. Then, 101 mg of target product (compound 4), (S)-2-amino-3-(4-hydroxyphenyl)-N-phenethylpropanamide, was obtained by silica gel column chromatography (PE:EA = 1:1-EA:CH3OH = 10:1), with a yield of 25.77%, m.p. 155.3-156.1°C, and the following nuclear magnetic resonance data:

[0092] 1H NMR (400 MHz, Methanol-d4) δ 7.29 - 7.13 (m, 5H), 7.07 - 7.01 (m, 2H), 6.77 - 6.73 (m, 2H), 3.73 (t, J = 7.0 Hz, 1H), 3.51 - 3.42 (m, 1H), 3.36 - 3.32 (m, 1H), 2.94 (dd, J = 13.8, 6.7 Hz, 1H), 2.81 (dd, J = 13.8, 7.5 Hz, 1H), 2.74 - 2.68 (m, 2H); 13 C NMR (101 MHz, Methanol-d4) δ 157.35, 140.41, 131.41, 129.77, 129.49, 129.40, 127.34, 116.30, 57.92, 41.80, 41.73, 36.50.

[0093] Example Two

[0094] Confocal microscope imaging and quantification of a-synuclein-splitGFP signal

[0095] 1. Experimental materials

[0096] 1.1. Cells

[0097] Yeast cell BY4741 (MATa his3A leu2A met15A ura3A)

[0098] Source: from Rong Li lab at Johns Hopkins University, USA

[0099] 1.2. Peptidase neurosin (Nln) enhancer

[0100]

[0101]

[0102] 2. Experimental methods

[0103] The splitGFP system was used to label a-synuclein and mitochondria. Specifically, the 11th beta-strand of GFP (GFP 11 ) was used to label the COOH terminus of a-synuclein, and the first 10 beta-strands of GFP (GFP 1-10 ) were used to label the COOH terminus of the mitochondrial matrix protein Grx5. The red fluorescent protein mCherry was used to label Fis1 on the outer membrane of mitochondria.

[0104] When GFP 1-10 and GFP 11Two parts do not emit green fluorescence when separated, while the alpha-synuclein-GFP 11 After entering the mitochondrial matrix, it binds with Grx5-GFP 1-10 in the matrix to form a complete GFP emitting green fluorescence, so that green alpha-synuclein-GFP in mitochondria can be imaged and quantified by fluorescence tracking, so as to detect whether the Nln enhancer small molecule compound can effectively degrade the aggregation of alpha-synuclein in mitochondria.

[0105] This embodiment uses the alpha-synuclein-splitGFP system to perform cell experiment verification in yeast cells (BY4741).

[0106] Through PCR homologous recombination technology, human alpha-synuclein-splitGFP system is continuously overexpressed in BY4741 cells, and multi-effect drug transporter Prd5 is knocked out to prevent drug efflux. The specific gene information is as follows:

[0107] Δura3::pGAP-α-Syn-HA-GFP11-His3MX6; trp::pGAP-mCherry-Fis1TM-KanMX6; GRX5-GFP1-10-NatMX6; Δpdr5::HygMX6

[0108] Take 225μM of peptidase neurolysin (Nln) enhancer #1 to #6, respectively, and add them to the SC culture medium of yeast cells (BY4741) to form 6 test groups, with DMSO solvent as the control group. Put the 7 groups of cells into a 30℃ incubator and shake culture overnight for 19 hours. The next morning, take out 100μl of overnight cultured cells and add them to 5ml of fresh culture medium for recovery culture for 3 hours until the optical density (OD600) reaches 0.1-0.25. After 22 hours of cumulative treatment, drop the cells on a clean 1.5NA glass slide and cover it with a cover glass. Perform live cell imaging on the 12 groups of cells, respectively.

[0109] Live cell imaging was performed using a Yokagawa CSU-10 spinning disc confocal combined with a Zeiss Carl Zeiss 200M inverted microscope. GFP / mCherry fluorescence was excited by 488 / 561 nm lasers, respectively, and imaged by a Hamamatsu C9100-13 EMCCD camera. 100x 1.45 NA objective was used for 3D imaging of yeast cells (BY4741) with 0.5 pm interval between each layer, and a total of 5-6 pm Z-axis thickness. Image acquisition was performed using MetaMorph (version 7.0; MDS Analytical Technologies) software, and analysis was performed using ImageJ software (NIH). The results were shown as Zprojection pictures.

[0110] Quantification of a-synuclein-split GFP fluorescence signal was performed using published python code. The signal intensity of green split GFP in mitochondria labeled by red fluorescent protein mcherry in each cell was counted, and the average value was calculated for comparison.

[0111] 3. Experimental results

[0112] 3.1. Quantification of a-synuclein-split GFP fluorescence signal

[0113] Table 1: Quantification of a-synuclein-split GFP fluorescence signal data

[0114]

[0115]

[0116]

[0117]

[0118] The detailed results of quantification of a-synuclein-split GFP fluorescence signal are shown in Table 1. The data in Table 1 were processed using one-way ANOVA unpaired multiple comparisons method (P < 0.01). It should be noted that due to the difference in the number of imaged cells in each experimental group, the data volume (number of rows) in each group (each column) is different.

[0119] The average value of the signal intensity of green split GFP in mitochondria labeled by red fluorescent protein mcherry in 7 groups of cells and the signal intensity decrease value of 6 experimental groups relative to the control group are as follows:

[0120]

[0121]

[0122] Referring to Figure 1 , Figure 1 The mean and SEM values of splitGFP intensity in mitochondria in three biological statistical experiments are shown, and one-way ANOVA unpaired multiple comparisons are calculated (P < 0.01).

[0123] It can be seen that the six peptidase neurosin (Nln) enhancers used in the experiment can reduce the signal intensity of splitGFP, that is, the six peptidase neurosin (Nln) enhancers can effectively degrade the aggregation of α-synuclein in mitochondria. Figure 1

[0124] Among the six peptidase neurosin (Nln) compounds, the two compounds with the best effect are #1 and #2, that is, (S)-5-((indol-3-yl)methyl)-3-(4-(2-fluoroethoxyphenyl)-2,2-dimethyl-3-phenyl imidazoline-4-ketone, and S-2-amino-N-(4-(2-fluoroethoxy) phenyl)-3-phenylpropionic acid amide. Therefore, the following confocal microscope imaging experiment only presents the imaging results of the two groups with the best effect.

[0125] 3.2, Confocal microscope imaging

[0126] The confocal microscope imaging results of the yeast cells expressing human α-synuclein-splitGFP system after adding 225 μM of compounds #1 and #2 in the culture medium, overnight culture and resuscitation for 22 hours (solvent DMSO as control) are shown in Figure 2 (scale bar size is 5 μM).

[0127] Figure 2 The left column is the cell imaging picture of the DMSO control group, the middle column is the cell imaging picture of the #1 experimental group, and the right column is the cell imaging picture of the #2 experimental group. Figure 2 The part shown as white in the upper row of pictures is green fluorescence, that is, splitGFP labeled α-synuclein; the part shown as white in the lower row of pictures is red fluorescence, that is, mCherry labeled mitochondria.

[0128] It can be seen that the six peptidase neurosin (Nln) enhancers used in the experiment can reduce the signal intensity of splitGFP, that is, the six peptidase neurosin (Nln) enhancers can effectively degrade the aggregation of α-synuclein in mitochondria. Figure 2 ​It can be seen that the control group (DMSO) cells overexpress α-synuclein, and both the #1 compound (S)-5-((indol-3-yl)methyl)-3-(4-(2-fluoroethoxyphenyl)-2,2-dimethyl-3- phenylimidazolin-4-one, and the #2 compound S-2-amino-N-(4-(2-fluoroethoxy)phenyl)-3- phenylpropanoic amide significantly degrade the aggregation of α-synuclein in mitochondria.

[0129] Example Three

[0130] Western blotting to detect α-synuclein levels in cells

[0131] 1. Experimental materials

[0132] The cells used in this example and the neprilysin (Nln) enhancer are exactly the same as in Example Two, in addition, the following reagents are also involved:

[0133]

[0134] 2. Experimental method

[0135] Take 225 μM of neprilysin (Nln) enhancer #1 to #6, respectively, add to the YPD medium of yeast cells (BY4741) to form 6 test groups, and take DMSO solvent as the control group. The 7 groups of yeast cells were cultured overnight in a 30°C incubator, and the next day they were resuscitated and cultured for 3 hours with YPD medium until the optical density (OD600) reached 0.1-0.25.

[0136] Centrifuge the cells at 21000g at 4°C, completely remove the YPD medium, then add 1 ml of ice water and centrifuge again to wash off the liquid, at this time the cell precipitate can be seen at the bottom of the centrifuge tube. Add 100 μl of 1xLDS and 40 mM DTT and shake quickly to break the cells, then add 100 ul of glass beads and shake again for 1 minute. After shaking, boil at 100°C for 8-10 minutes, then centrifuge at room temperature for 2 minutes, and retain the supernatant.

[0137] Take 10 μl of supernatant each time for 4-12% SDS-PAGE protein gel loading, transfer to PVDF membrane according to the instructions using iBlot (Thermo Fishier Scientific), block with Odyssey blocking TBS buffer, use HA-tag rabbit antibody as the first antibody, and use HRP-conjugated rabbit antibody as the second antibody.

[0138] Imaging and data analysis of the bands formed by the running of the gels was performed using a LI-COR imaging system (LI-COR Biosciences).

[0139] 3. Results of the experiments

[0140] The results of the statistical analysis of the values obtained by dividing the grey value of the band of the alpha-synuclein antibody by the grey value of the band of the PGK1 antibody (control) in the cells tested by Western blot are shown in Table 2.

[0141] Table 2: Statistical data of the quantification of the alpha-synuclein levels

[0142] DMSO #1 #2 #3 #4 #5 #6 First time 1 0.238 0.496 0.673 0.73 0.658 0.53 Second time 1 0.748 0.83 —— —— —— —— Third time 1 0.576 0.878 —— —— —— —— Fourth time 1 —— 0.824 —— —— —— ——

[0143] See Figure 3 , Figure 3 The mean and SEM values of the alpha-synuclein levels in the cells, shown as Western blot, are shown in Table 3. Figure 3 As shown in Table 3, all the six neurosin (Nln) enhancers used in this experiment were effective in degrading alpha-synuclein.

[0144] Example 4

[0145] Mitochondrial membrane potential assay

[0146] 1. Materials used in the experiment

[0147] The cells used in this example were identical to those used in Example 2.

[0148] The neurosin (Nln) enhancers used in this example were:

[0149]

[0150]

[0151] In addition, the following reagents were used in this example:

[0152] Tetramethylrhodamine methyl ester (TMRM) dye

[0153] Origin: Sigma-Aldrich Cat# T5428-25MG

[0154] 2. Method used in the experiment

[0155] Tetramethylrhodamine methyl ester (TMRM) dye is a cell-permeant red dye that stains active mitochondria with higher membrane potential, and the staining disappears when the mitochondrial membrane potential decreases. Therefore, TMRM staining can be used to detect mitochondrial membrane potential and function, so as to detect the mitochondrial membrane potential of the yeast cell overexpressing α-synuclein-splitGFP, so as to judge the influence of the Nln enhancer small molecule on the mitochondrial membrane potential, and further judge the influence of the Nln enhancer small molecule on the mitochondrial function.

[0156] The splitGFP system is used to label α-synuclein and mitochondria. Specifically, the 11th β-strand of GFP (GFP 11 ) is used to label the COOH end of α-synuclein, and the first 10 β-strands of GFP (GFP 1-10 ) are used to label the COOH end of the mitochondrial matrix protein Grx5. The red fluorescent protein mCherry is used to label Fis1 on the outer membrane of mitochondria.

[0157] When the two parts of GFP 1-10 and GFP 11 are separated, there is no green fluorescence, and when α-synuclein-GFP 11 enters the mitochondrial matrix, it combines with Grx5-GFP 1-10 in the matrix to form a complete GFP emitting green fluorescence, so that the green α-synuclein-GFP in the mitochondria can be imaged and quantified by fluorescence tracking, so as to detect whether the Nln enhancer small molecule compound can effectively degrade the aggregation of α-synuclein in the mitochondria.

[0158] This example uses the α-synuclein-splitGFP system to perform cell experiments in yeast cells (BY4741).

[0159] Through PCR homologous recombination technology, human α-synuclein-splitGFP system is continuously overexpressed in BY4741 cells, and multi-effect drug transporter Prd5 is knocked out to prevent drug efflux. The specific gene information is as follows:

[0160] Δura3::pGAP-α-Syn-HA-GFP11-His3MX6; trp::pGAP-mCherry-Fis1TM-KanMX6; GRX5-GFP1-10-NatMX6; Δpdr5::HygMX6

[0161] Take 225 μM of peptidase neurosin (Nln) enhancer #1 and #2, respectively, add to the SC medium of yeast cells (BY4741) to form two test groups, and take DMSO solvent as a control group. Put the three groups of cells into a 30°C incubator overnight and shake culture, and the next morning, take 100 μl of the overnight cultured cells and add to 5 ml of fresh culture medium for recovery culture for 3 hours until the optical density (OD600) reaches 0.1-0.25.

[0162] After cumulative processing for 22 hours, 2.5 μM of TMRM dye solution is added to the culture medium of the three groups of cells, and the yeast cells expressing α-synuclein-split GFP signal are dyed at 30°C for ten minutes. After ten minutes, the three groups of cells are washed with PBS three times, and then the cells are dropped on a clean 1.5NA glass slide and covered with a cover glass, and the three groups of cells are imaged.

[0163] The equipment used for cell imaging is: Yokagawa CSU-10 spinning disc confocal turntable combined with Zeiss Carl 200M inverted microscope. 488 / 561 nm laser excites GFP / mCherry fluorescence, respectively, and imaging is performed by Hamamatsu C9100-13 EMCCD camera. 100x1.45NA objective is used for 3D imaging of yeast cells (BY4741), with an interval of 0.5 μm between each layer, and a total imaging thickness of 5-6 μm Z axis. Image acquisition uses MetaMorph (version 7.0; MDS Analytical Technologies) software, analysis uses ImageJ software of NIH, and experimental results are shown as Zprojection pictures.

[0164] 3、Experimental results

[0165] After adding 225 μM of compounds #1 and #2 to the culture medium of yeast cells expressing human α-synuclein-split GFP system and processing for 22 hours (solvent DMSO as a control), TMRM dye is used for staining, and then confocal microscope imaging results are as shown in Figure 4 (scale bar size is 5 μM).

[0166] Figure 4 The left column is the cell imaging picture of the DMSO control group, the middle column is the cell imaging picture of the #1 test group, and the right column is the cell imaging picture of the #2 test group. Figure 4 The part shown as white in the upper row of pictures is green fluorescence, i.e., split GFP labeled α-synuclein; the part shown as white in the lower row of pictures is red fluorescence, i.e., TMRM stained mitochondria.

[0167] By Figure 4 As can be seen from the imaging pictures above, the control group (DMSO) cells overexpress α-synuclein, while both #1 compound (S)-5-((indol-3-yl)methyl)-3-(4-(2-fluoroethoxyphenyl)-2,2-dimethyl-3-phenylimidazolin-4-one, and #2 compound S-2-amino-N-(4-(2-fluoroethoxy)phenyl)-3-phenylpropanoic acid amide significantly degrade α-synuclein in mitochondria.

[0168] Meanwhile, as can be seen from the imaging pictures below, the control group (DMSO) TMRM-stained cells (yeast cells overexpressing α-synuclein-splitGFP) have low mitochondrial membrane potential and impaired mitochondrial function; while the cells treated with peptidase neurosin (Nln) enhancer #1 and #2 (yeast cells with degraded α-synuclein in mitochondria) have high mitochondrial membrane potential and restored mitochondrial function. Figure 4

[0169] The foregoing description of various embodiments of the application has been presented for the purposes of illustration and description. It is not intended to be exhaustive or to limit the application to the precise form disclosed, and various modifications and variations are possible in light of the above teachings. The concepts disclosed herein can be used in any number of embodiments and applications outside of the specific examples discussed. It is intended that the specification and examples be considered as exemplary only, with a genuine intellectual contribution being set forth. It is also possible in light of these teachings to produce yet further embodiments based on the following claims.​

Claims

1. The use of a compound in the preparation of a peptidase neurolysin enhancer, said compound having the structure shown in structural formula (I): 。 2. The application according to claim 1, characterized in that, The peptidase neurolysin enhancer is used to treat neurodegenerative diseases and peripheral inflammatory diseases; the neurodegenerative diseases include: Parkinson's disease, Alzheimer's disease, Lewy body dementia, multiple system atrophy, pure autonomic failure, and rapid eye movement sleep behavior disorder; the peripheral inflammatory diseases include: ischemic stroke, traumatic brain injury, and autism.

3. A pharmaceutical composition, characterized in that, The compound comprises a compound and a pharmaceutically acceptable carrier, the compound having the structure shown in formula (I): 。

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