Peptidase neurosin enhancer and methods of making and using same

Abstract

This invention discloses a peptidase neurolysin enhancer, its preparation method, and its applications. The general structural formula is: where R is one of hydrogen, alkyl, alkoxy, or fluoroalkyl; and Ar is one of phenyl, 4-hydroxyphenyl, or 3-indole. This invention provides a class of small-molecule peptidase neurolysin enhancers with novel structures that can enhance the degradation of α-synuclein, thereby reducing α-synuclein levels in cells, reversing mitochondrial damage and cytotoxicity, and effectively protecting cells from α-synuclein accumulation. The compounds provided by this invention alleviate mitochondrial damage and cell death caused by α-synuclein overexpression and can be used to treat various neurodegenerative diseases such as Parkinson's disease. The compounds provided by this invention can also alleviate peripheral inflammatory diseases such as ischemic stroke, traumatic brain injury, and autism by enhancing peptidase neurolysin function.

Description

Technical Field

[0001] This invention relates to the field of small molecule compound technology, specifically to a compound, its preparation method and application; it also relates to a pharmaceutical composition. Background Technology

[0002] Parkinson's disease (Parkinson's disease) is the second most common neurodegenerative disease worldwide after Alzheimer's disease. Its two main pathological features are the progressive degeneration of dopaminergic neurons in the substantia nigra due to the aggregation of α-synuclein to form Lewy bodies, and mitochondrial damage. The pathogenesis of Parkinson's disease is complex, and currently there are no sufficiently effective drugs for treatment. Current clinical practice typically uses levodopamine to increase dopamine levels and improve symptoms, but it does not slow the progression of the disease. Finding effective drugs to alleviate the symptoms of Parkinson's disease is a pressing technical problem that needs to be solved in this field.

[0003] Research has found that mitochondrial peptidase neurolysin (Nln) plays a major role in the degradation of α-synuclein by mitochondrial proteases after it enters the mitochondria. However, α-synuclein cannot be cleared promptly in cases of aging, protein homeostasis imbalance, or gene defects associated with neurodegenerative diseases. α-synuclein accumulates in mitochondria, affecting mitochondrial function and causing damage such as mitochondrial breakage, decreased mitochondrial membrane pressure, and reduced mitochondrial DNA, ultimately leading to nerve cell death.

[0004] Correspondingly, when cells are treated with Nln enhancers, the degradation of α-synuclein is enhanced, thereby reducing the level of α-synuclein in cells, reversing mitochondrial damage and cytotoxicity, and effectively protecting cells from α-synuclein accumulation. Nln enhancers hold promise as a novel drug for the treatment of Parkinson's disease, alleviating mitochondrial damage and cell death caused by α-synuclein overexpression. Summary of the Invention

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

[0006] According to a first aspect of the present invention, a compound is provided, the compound having the following general structural formula:

[0007]

[0008] Wherein, R is one of hydrogen, alkyl, alkoxy, or fluoroalkyl; and Ar is one of phenyl, 4-hydroxyphenyl, or 3-indole.

[0009] In one embodiment of the present invention, when R is a fluoroalkyl group, the general structural formula of the compound is:

[0010]

[0011] Where X is one of oxygen, nitrogen, sulfur, and carbon; n is any integer selected from the range of 1 to 4.

[0012] In one embodiment of the present invention, the compound has the structure shown in structural formula (I).

[0013]

[0014] In one embodiment of the invention, the compound has a structure shown in structural formula (II) or structural formula (III):

[0015]

[0016] According to a second aspect of the present invention, a method for preparing a compound is also provided, for preparing the compound provided in the first aspect of the present invention, the method comprising the following steps:

[0017] (1) Amino acid protection reaction: Take 18-25 parts of amino acid by molar amount, add 1,4-dioxane and water; add NaOH and 20-40 parts of Boc anhydride dropwise under ice bath conditions; raise to room temperature for reaction, after the reaction is completed, adjust the reaction solution to acidity, extract the organic phase and remove the solvent to obtain the first intermediate product.

[0018] (2) Amino acid condensation reaction: Dissolve 2-4 parts of the first intermediate product in molar amounts, and add 3-6 parts of EDCI, 2-4 parts of HOBt, and 7-12 parts of DIPEA to the solution and stir at room temperature; add 3-4 parts of NHR1R2 to the reaction solution, where R1 is hydrogen and R2 is selected from phenyl, fluorophenyl, and alkoxyphenyl; carry out the reaction at room temperature, and after the reaction is completed, extract and remove the solvent to separate and obtain the second intermediate product;

[0019] (3) Deprotection reaction: Dissolve 0.7-1.9 parts of the second intermediate product by molar amount, and then add dioxane hydrochloride or trifluoroacetic acid dropwise under ice bath conditions. Stir the reaction under room temperature conditions, and extract the compound after treatment.

[0020] In one embodiment of the present invention, when the compound is S-2-amino-N-(4-(2-fluoroethoxy)phenyl)-3-phenylpropionic acid amide as shown in structural formula (Ⅰ), the method includes:

[0021]

[0022] Among them, NHR1R2 is 4-(2-fluoroethoxy)aniline;

[0023] The amino acid in the amino acid acid protection reaction step is L-phenylalanine, and the first intermediate product is N-Boc-Phe-OH.

[0024] In one embodiment of the present invention, the extraction process after treatment in the deprotection reaction step includes: removing the organic solvent by rotary evaporation, diluting with water, adjusting the solution to alkaline with sodium hydroxide, extracting the organic phase and removing the solvent therein, and separating the S-2-amino-N-(4-(2-fluoroethoxy)phenyl)-3-phenylpropionic acid amide by silica gel column chromatography.

[0025] In one embodiment of the present invention, the method for synthesizing 4-(2-fluoroethoxy)aniline includes:

[0026] Dissolve 4-nitrophenol in DMF, add potassium carbonate, stir in an oil bath, and add 1-bromo-2-fluoroethane dropwise to react. After the reaction, extract the intermediate product. Dissolve ammonium chloride in water, add iron powder, and reflux at high temperature. Dissolve the intermediate product in methanol. After the ammonium chloride solution has been refluxed, add the intermediate product solution dropwise to the ammonium chloride solution and continue reflux to react. After the reaction is complete, remove methanol by rotary evaporation, dilute with water, and adjust the solution to neutral. Remove iron powder by suction filtration, and extract the organic phase. Dry to remove water from the organic phase, and rotary evaporate to obtain 4-(2-fluoroethoxy)aniline.

[0027] In one embodiment of the present invention, when the compound is of structural formula (II) as (S)-2-amino-3-(4-hydroxyphenyl)-N-phenylpropionamide, the method includes:

[0028]

[0029] Among them, NHR1R2 aniline;

[0030] The amino acid in the amino acid acid protection reaction step is L-tyrosine, and the first intermediate product is Boc-L-Tyr.

[0031] In one embodiment of the present invention, the extraction process after treatment in the deprotection reaction step includes: filtration to obtain (S)-2-amino-3-(4-hydroxyphenyl)-N-phenylpropionamide.

[0032] In one embodiment of the present invention, when the compound is (S)-2-amino-3-(1H-indol-3-yl)-N-phenylpropionamide as shown in structural formula (Ⅲ), the method includes:

[0033]

[0034] Among them, NHR1R2 aniline;

[0035] The amino acid in the amino acid acid protection reaction step is L-tryptophan, and the first intermediate product is N-Boc-L-Trp.

[0036] In one embodiment of the present invention, the extraction process after treatment in the deprotection reaction step includes: removing the organic solvent by rotary evaporation, diluting with water, adjusting the pH of the solution to alkaline using sodium hydroxide, and extracting the organic phase; removing the solvent from the extracted organic phase, and scraping a silica gel plate to obtain S-2-amino-N-(4-(2-fluoroethoxy)phenyl)-3-phenylpropionic acid amide.

[0037] In one embodiment of the present invention, the extraction process in the amino acid protection reaction includes: extraction with ethyl acetate; the solvent removal process includes: drying the organic phase with anhydrous sodium sulfate to remove water from the organic phase, and after drying, removing the organic solvent from the organic phase by vacuum distillation.

[0038] According to a third aspect of the invention, the use of the compound provided in the first aspect of the invention in the preparation of a peptidase neurolysin enhancer is also provided.

[0039] In one embodiment of the present invention, 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.

[0040] According to a fourth aspect of the invention, a pharmaceutical composition is also provided, comprising the compound provided in the first aspect of the invention and a pharmaceutically acceptable carrier.

[0041] One beneficial effect of this invention is that it provides a class of small-molecule peptidase neurolysin enhancers with novel structures that can enhance the degradation of α-synuclein, thereby reducing α-synuclein levels in cells, reversing mitochondrial damage and cytotoxicity, and effectively protecting cells from α-synuclein accumulation. The compounds provided by this invention alleviate mitochondrial damage and cell death caused by α-synuclein overexpression and can be used to treat various neurodegenerative diseases such as Parkinson's disease. Furthermore, the compounds provided by this invention can also alleviate peripheral inflammatory diseases such as ischemic stroke, traumatic brain injury, and autism by enhancing peptidase neurolysin function.

[0042] Other features and advantages of the invention will become clear from the following detailed description of exemplary embodiments of the invention with reference to the accompanying drawings. Attached Figure Description

[0043] The accompanying drawings, which are incorporated in and form part of this specification, illustrate embodiments of the invention and, together with their description, serve to explain the principles of the invention.

[0044] Figure 1 This is a statistical graph of quantitative analysis of α-synuclein-split GFP fluorescence signals;

[0045] Figure 2 This is a confocal microscope imaging result from Example 4;

[0046] Figure 3 This is a statistical graph showing the levels of α-synuclein in cells as detected by Western blotting.

[0047] Figure 4 This is a confocal microscope imaging result from Example 6. Detailed Implementation

[0048] To make the inventive objectives, technical solutions, and beneficial technical effects of this application clearer, the following detailed description is provided in conjunction with specific embodiments. It should be understood that the embodiments described in this specification are merely illustrative and not intended to limit the scope of this application.

[0049] For simplicity, this paper only explicitly discloses some numerical ranges. However, any lower limit can be combined with any upper limit to form an unspecified range; and any lower limit can be combined with other lower limits to form an unspecified range, just as any upper limit can be combined with any other upper limit to form an unspecified range. Furthermore, although not explicitly stated, every point or individual value between the endpoints of a range is included within that range. Therefore, each point or individual value can serve as its own lower or upper limit and be combined with any other point or individual value, or with other lower or upper limits, to form an unspecified range.

[0050] In the description of this article, it should be noted that, unless otherwise stated, "above" and "below" include the number itself, and "several" in "one or more" means two or more.

[0051] The foregoing description of this invention is not intended to describe every disclosed embodiment or implementation. Instead, the following description provides more specific examples of exemplary embodiments. Throughout the application, guidance is provided through a series of embodiments that can be used in various combinations. The examples listed are merely representative and should not be construed as exhaustive.

[0052] This invention provides a compound with the following general structural formula:

[0053]

[0054] Wherein, R is one of hydrogen, alkyl, alkoxy, or fluoroalkyl; and Ar is one of phenyl, 4-hydroxyphenyl, or 3-indole.

[0055] In one embodiment of the present invention, when R is a fluoroalkyl group, the general structural formula of the compound is:

[0056]

[0057] Where X is one of oxygen, nitrogen, sulfur, and carbon; n is any integer selected from the range of 1 to 4.

[0058] In one embodiment of the present invention, when R is a fluoroalkyl group, X is oxygen, n=1, and Ar is phenyl, the compound has the structure shown in structural formula (I).

[0059]

[0060] The compound shown in structural formula (Ⅰ) is S-2-amino-N-(4-(2-fluoroethoxy)phenyl)-3-phenylpropionic acid amide.

[0061] In one embodiment of the invention, the compound has the structure shown in structural formula (II) or structural formula (III):

[0062]

[0063] Specifically, when R is hydrogen and Ar is 4-hydroxyphenyl, the compound has the structure shown in formula (II), namely (S)-2-amino-3-(4-hydroxyphenyl)-N-phenylpropionamide. When R is hydrogen and Ar is 3-indole, the compound has the structure shown in formula (III), namely (S)-2-amino-3-(1H-indole-3-yl)-N-phenylpropionamide.

[0064] The present invention also provides a method for preparing the aforementioned compound, the method comprising the following steps:

[0065] (1) Amino acid protection reaction: Take 18-25 parts of amino acid by molar amount, add 1,4-dioxane and water; add NaOH and 20-40 parts of Boc anhydride dropwise under ice bath conditions; raise to room temperature for reaction, after the reaction is completed, adjust the reaction solution to acidity, extract the organic phase and remove the solvent to obtain the first intermediate product.

[0066] The reactants for the amino acid acid protection reaction are amino acids and Boc anhydride, which is (BOC)₂O (di-tert-butyl dicarbonate). After adding NaOH dropwise under ice bath conditions, the mixture needs to be kept in an ice bath for about 10 minutes before continuing to add Boc anhydride. After the addition is complete, the mixture also needs to be kept in an ice bath for about 10 minutes before the temperature is raised to room temperature for the reaction. After the reaction is complete, the reaction solution needs to be adjusted to acidity, for example, by using a saturated potassium bisulfate solution to adjust the pH to 4.

[0067] In one embodiment of the present invention, the extraction process in the amino acid protection reaction includes: extraction with ethyl acetate; the solvent removal process includes: drying the organic phase with anhydrous sodium sulfate to remove water from the organic phase, and after drying, removing the organic solvent from the organic phase by vacuum distillation.

[0068] The type of amino acid used in an amino acid protection reaction directly alters the first intermediate product. For example, when L-phenylalanine is used, the first intermediate product is N-Boc-Phe-OH; when L-tryptophan is used, the first intermediate product is N-Boc-L-Trp; and when L-tyrosine is used, the first intermediate product is Boc-L-Tyr.

[0069] (2) Amino acid condensation reaction: Dissolve 2-4 parts of the first intermediate product in molar amounts, and add 3-6 parts of EDCI, 2-4 parts of HOBt, and 7-12 parts of DIPEA to the solution and stir at room temperature; add 3-4 parts of NHR1R2 to the reaction solution, where R1 is hydrogen and R2 is selected from phenyl, fluorophenyl, and alkoxyphenyl; carry out the reaction at room temperature, and after the reaction is completed, extract and remove the solvent to separate and obtain the second intermediate product.

[0070] The extraction steps can be designed based on the different solvents used for the first intermediate, and extraction can be performed multiple times. For example, extraction can be performed first with ethyl acetate, followed by two extractions with water, then three extractions with ammonium chloride solution, and finally two extractions with saturated brine. The solvent removal step can involve drying with anhydrous sodium sulfate followed by vacuum distillation to remove the organic solvent. There are various methods for separating the second intermediate, such as silica gel column chromatography.

[0071] (3) Deprotection reaction: Dissolve 0.7-1.9 parts of the second intermediate by molar amount, then add dioxane hydrochloride or trifluoroacetic acid dropwise under ice bath conditions, stir the reaction at room temperature, and extract the compound after treatment. The final treatment and extraction method will be adjusted according to the different second intermediate products.

[0072] In one embodiment of the present invention, when the compound is S-2-amino-N-(4-(2-fluoroethoxy)phenyl)-3-phenylpropionic acid amide as shown in structural formula (Ⅰ), the method includes:

[0073]

[0074] Among them, NHR1R2 is 4-(2-fluoroethoxy)aniline;

[0075] The amino acid in the amino acid protection reaction step is L-phenylalanine, and the first intermediate product is N-Boc-Phe-OH.

[0076] In one embodiment of the present invention, the extraction process after treatment in the deprotection reaction step includes: removing the organic solvent by rotary evaporation, diluting with water, adjusting the solution to alkaline with sodium hydroxide, extracting the organic phase and removing the solvent therein, and separating the S-2-amino-N-(4-(2-fluoroethoxy)phenyl)-3-phenylpropionic acid amide by silica gel column chromatography.

[0077] In one embodiment of the present invention, the method for synthesizing 4-(2-fluoroethoxy)aniline includes:

[0078]

[0079] Dissolve 4-nitrophenol in DMF, add potassium carbonate, stir in an oil bath, and add 1-bromo-2-fluoroethane dropwise to react. After the reaction, extract the intermediate product. Dissolve ammonium chloride in water, add iron powder, and reflux at high temperature. Dissolve the intermediate product in methanol. After the ammonium chloride solution has been refluxed, add the intermediate product solution dropwise to the ammonium chloride solution and continue reflux to react. After the reaction is complete, remove methanol by rotary evaporation, dilute with water, and adjust the solution to neutral. Remove iron powder by suction filtration, and extract the organic phase. Dry to remove water from the organic phase, and rotary evaporate to obtain 4-(2-fluoroethoxy)aniline.

[0080] In one embodiment of the present invention, when the compound is of structural formula (II) as (S)-2-amino-3-(4-hydroxyphenyl)-N-phenylpropionamide, the method includes:

[0081]

[0082] Among them, NHR1R2 aniline;

[0083] The amino acid in the amino acid protection reaction step is L-tyrosine, and the first intermediate product is Boc-L-Tyr.

[0084] In one embodiment of the present invention, the extraction process after treatment in the deprotection reaction step includes: filtration to obtain (S)-2-amino-3-(4-hydroxyphenyl)-N-phenylpropionamide.

[0085] In one embodiment of the present invention, when the compound is (S)-2-amino-3-(1H-indol-3-yl)-N-phenylpropionamide as shown in structural formula (Ⅲ), the method includes:

[0086]

[0087] Among them, NHR1R2 aniline;

[0088] The amino acid in the amino acid protection reaction step is L-tryptophan, and the first intermediate product is N-Boc-L-Trp.

[0089] In one embodiment of the present invention, the extraction process after treatment in the deprotection reaction step includes: removing the organic solvent by rotary evaporation, diluting with water, adjusting the pH of the solution to alkaline using sodium hydroxide, and extracting the organic phase; removing the solvent from the extracted organic phase, and scraping a silica gel plate to obtain S-2-amino-N-(4-(2-fluoroethoxy)phenyl)-3-phenylpropionic acid amide.

[0090] The present invention also provides the application of the aforementioned compound in the preparation of peptidase neurolysin enhancers.

[0091] In one embodiment of the present invention, a 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. The peptidase neurolysin enhancer can enhance the degradation of α-synuclein, thereby reducing the level of α-synuclein in cells, reversing mitochondrial damage and cytotoxicity, and effectively protecting cells from α-synuclein accumulation. This can alleviate mitochondrial damage and cell death caused by α-synuclein overexpression, thereby treating various neurodegenerative diseases such as Parkinson's disease. Furthermore, the compounds provided by the present invention can also alleviate peripheral inflammatory diseases such as ischemic stroke, traumatic brain injury, and autism by enhancing peptidase neurolysin function.

[0092] According to the present invention, a pharmaceutical composition is also provided, comprising the aforementioned compounds and a pharmaceutically acceptable carrier. The carrier, which can be arbitrarily mixed, can be varied depending on the dosage form, administration method, etc. Examples of carriers include excipients, binders, disintegrants, lubricants, flavoring agents, fragrances, colorants, and sweeteners. The pharmaceutical composition can be in pharmaceutically conventional forms such as capsules, powders, tablets, granules, pills, injections, syrups, oral liquids, inhalers, ointments, suppositories, and patches.

[0093] Example 1

[0094] Synthesis of S-2-amino-N-(4-(2-fluoroethoxy)phenyl)-3-phenylpropionic acid amide

[0095] The structural formula of the target compound:

[0096] The general synthetic route for phenylalanine series compounds is as follows:

[0097]

[0098] 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.

[0099] Step 1: Amino acid protection reaction

[0100] Referring to the above synthetic route, compound 1 is L-phenylalanine and compound 2 is N-Boc-Phe-OH.

[0101] Take 54.00 g (24.23 mmol) of L-phenylalanine (compound 1), add 40.0 mL of 1,4-dioxane and 40.0 mL of water. Then, under ice bath conditions, add 40.0 mL of 1N NaOH dropwise, and continue the ice bath for 10 min. Next, under ice bath conditions, add 7.93 g (36.35 mmol) of Boc anhydride (i.e., di-tert-butyl dicarbonate, (BOC)₂O in the above synthetic route). After the addition is complete, continue the ice bath for 10 min, then slowly raise the temperature to room temperature and react at room temperature for 8 h.

[0102] After 8 hours of reaction, the reaction solution was analyzed using thin-layer chromatography (TLC). TLC can be used to track the reaction progress. The reaction solution is coated onto a glass plate, plastic, or aluminum substrate to form a uniform thin layer. After spotting and development, the developed plate is observed under ultraviolet light or by spraying a colorimetric reagent. Observing the spots of the raw materials and products can determine the reaction status. When the spots of the raw materials disappear, the reaction is considered complete.

[0103] The reaction solution was concentrated to approximately 5 mL, and then cooled to room temperature. 20 mL of water was added to the concentrate, and the pH was adjusted to 4 using saturated potassium hydrogen sulfate solution. Extraction was then performed with ethyl acetate (50 mL × 3), and the organic phases were combined. The organic phase was dried using anhydrous sodium sulfate to remove water. After drying, the organic solvent was removed from the organic phase by vacuum distillation, yielding a pale yellow liquid. Upon standing at room temperature, the pale yellow liquid crystallized to obtain the product N-Boc-Phe-OH (compound 2), with a yield of 66.36%. N-Boc-Phe-OH (compound 2) will be used in the third step of the reaction.

[0104] Step 2: Preparation of 4-(2-fluoroethoxy)aniline

[0105] Following the general synthetic route for phenylalanine compounds, NHR1R2 will be introduced as a reactant in the N-Boc-Phe-OH (compound 2) reaction to generate compound 3. In the synthesis of S-2-amino-N-(4-(2-fluoroethoxy)phenyl)-3-phenylpropionic acid amide, NHR1R2 is 4-(2-fluoroethoxy)aniline.

[0106] Specifically, the synthetic route for 4-(2-fluoroethoxy)aniline is as follows:

[0107]

[0108] Referring to the above synthetic route, compound 5 is 4-nitrophenol, compound 7 is the target product, namely 4-(2-fluoroethoxy)aniline, and compound 6 is an intermediate.

[0109] 4.64 g (33.35 mmol) of 4-nitrophenol (compound 5) was dissolved in a 100 mL round-bottom flask with an appropriate amount of N,N-dimethylformamide (DMF). Then, 6.91 g (50.02 mmol) of potassium carbonate (K₂CO₃) was added, and the mixture was stirred in an oil bath. 4.62 g (36.68 mmol) of 1-bromo-2-fluoroethane was added dropwise, and the reaction was carried out at 80 °C in an oil bath for 8 hours.

[0110] After 8 hours of reaction, the reaction solution was analyzed by thin-layer chromatography (TLC). Once the results indicated the reaction was complete, an equal volume of water was added to the reaction solution, followed by extraction with ethyl acetate (50 mL × 3). The organic phases were combined. The mixture was washed with saturated brine (50 mL × 2), and then dried with anhydrous sodium sulfate to remove water. After drying, the organic solvent was removed from the organic phase by vacuum distillation, yielding 6.16 g of intermediate (compound 6), with a yield of 119%.

[0111] 10.53 g (198.5 mmol) of ammonium chloride (NH4Cl) was dissolved in a 250 mL round-bottom flask with an appropriate amount of water. Then, 6.66 g (119.16 mmol) of iron powder was added, and the mixture was refluxed at 100 °C for 40 minutes. During reflux, 6.16 g (39.72 mmol) of the intermediate (compound 6) was dissolved in an appropriate amount of methanol for later use.

[0112] After reflux, the reaction solution temperature was lowered to 80°C, and then a methanol-soluble solution of compound 6 was added dropwise to the reaction solution. The mixture was then refluxed at 85°C for 8 hours. After 8 hours of reaction, the reaction solution was analyzed by thin-layer chromatography (TLC). Once the results indicated the reaction was complete, methanol was removed from the reaction solution using a rotary evaporator. The solution was then diluted with water, and the pH was adjusted to 7 using approximately 5 mL of sodium bicarbonate (NaHCO3).

[0113] Iron powder was removed by vacuum filtration, followed by extraction with ethyl acetate (50 mL × 3). After extraction, the organic phase was dried with anhydrous sodium sulfate and filtered. Finally, rotary evaporation yielded 4.36 g of the target product 4-(2-fluoroethoxy)aniline (compound 7), with a yield of 84.50%. 4-(2-fluoroethoxy)aniline (compound 7) will be used in subsequent reactions.

[0114] Step 3: Amino acid condensation reaction

[0115] Referring to the general synthetic route of phenylalanine series compounds, the amino condensation reaction is the process of reacting N-Boc-Phe-OH (compound 2) with 4-(2-fluoroethoxy)aniline (compound 7) to generate the intermediate (compound 3).

[0116] Take 1.00 g and 3.80 mmol of N-Boc-Phe-OH (compound 2) and place it in a 100 mL round-bottom flask. Add 30.0 mL of dichloromethane (CH2Cl2, DMC) to dissolve it. Then add 1.09 g and 5.70 mmol of EDCI (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride), 0.51 g and 3.80 mmol of HOBt (hydroxybenzotriazole), and 2.00 mL and 11.40 mmol of DIPEA (N,N-diisopropylethylamine) to the solution and stir at room temperature.

[0117] After 30 minutes, 0.65 g (4.18 mmol) of 4-(2-fluoroethoxy)aniline (compound 7) was added, and the reaction was allowed to proceed overnight at room temperature. After the overnight reaction, the reaction solution was analyzed by thin-layer chromatography (TLC). Once the results indicated the reaction was complete, an equal volume of dichloromethane (CH2Cl2, DMC) solution was added for dilution. The solution was then extracted twice with water (50 mL × 2), three times with ammonium chloride solution (NH4Cl, 50 mL × 3), and finally twice with saturated saline solution (50 mL × 2).

[0118] After extraction, the sample was dried with anhydrous sodium sulfate, and the organic solvent was removed by vacuum distillation. Subsequently, 575 mg of the intermediate (compound 3) was obtained by silica gel column chromatography (PE:EA = 15:1–7:1), with a yield of 37.58%. The intermediate (compound 3) will be used in the next reaction step.

[0119] Step 4: Deprotection reaction

[0120] Referring to the general synthetic route of phenylalanine series compounds, the deprotection reaction is the process of reacting intermediate (compound 3) to generate target product (compound 4). In this example, compound 4 is S-2-amino-N-(4-(2-fluoroethoxy)phenyl)-3-phenylpropionic acid amide.

[0121] Dissolve 0.62 g (1.74 mmol) of the intermediate (compound 3) in 15.0 mL of acetone. Then, slowly add 4 M, 4.50 mL of dioxane hydrochloride solution under ice bath conditions. Stir overnight at room temperature. After the reaction has proceeded overnight, the reaction solution is analyzed by thin-layer chromatography (TLC). Once the results indicate that the reaction is complete, remove the acetone from the reaction solution using a rotary evaporator, then dilute with twice the amount of water, and adjust the pH to 11 using sodium hydroxide (NaOH, approximately 6 mL).

[0122] The product was extracted three times (50 mL × 3) with dichloromethane (CH2Cl2). After extraction, the product was dried over anhydrous sodium sulfate, and the organic solvent was removed by vacuum distillation. Subsequently, 0.34 g of the target product (compound 4), namely S-2-amino-N-(4-(2-fluoroethoxy)phenyl)-3-phenylpropionic acid amide, was obtained by silica gel column chromatography (PE:EA = 1:1 to EA:CH3OH = 10:1), with a yield of 79.41% and mp values ​​of 207.2–208.2 °C. Its NMR data are shown below.

[0123] 1H NMR(400MHz,Chloroform-d)δ9.24(s,1H),7.49-7.42(m,2H),7.32-7.18(m,5H),6.89-6.82(m,2H),4.79-4.59(m ,2H),4.22-4.07(m,2H),3.68(dd,J=9.4,3.9Hz,1H),3.32(dd,J=13.8,3.9Hz,1H),2.74(dd,J=13.8,9.5Hz,1H). 13 C NMR(101MHz,Chloroform-d)δ172.22,155.16,137.88,131.68,129.43,128.95,127.07,1 21.23,115.16,82.08(d,J=170.6Hz),67.59(d,J=20.5Hz),56.86,40.88.HRMS(ESI):m / z calcd for C17H19N2O2,[M+Na]+325.1322, found 325.1322.

[0124] Example 2

[0125] Synthesis of (S)-2-amino-3-(1H-indol-3-yl)-N-phenylpropionamide

[0126] The structural formula of the target compound:

[0127] The general synthetic route for tryptophan series compounds is as follows:

[0128]

[0129] It should be noted that when the R1 and R2 groups in NHR1R2 are changed, the specific structures of compounds 10, 11 and 12 will also change accordingly.

[0130] Step 1: Amino acid protection reaction

[0131] Referring to the above synthetic route, compound 8 is L-tryptophan and compound 9 is N-Boc-L-Trp.

[0132] Take 4.00 g (19.60 mmol) of L-tryptophan (compound 8), add 40.0 mL of 1,4-dioxane and 40.0 mL of water. Then, add 40.0 mL of 2N NaOH dropwise while in an ice bath, and continue the ice bath for 10 min. Next, add 5.13 g (23.52 mmol) of Boc anhydride (i.e., di-tert-butyl dicarbonate, (BOC)₂O in the above synthetic route) dropwise while in an ice bath. After the addition is complete, continue the ice bath for 10 min, then slowly raise the temperature to room temperature and react at room temperature for 11 h.

[0133] After 11 hours of reaction, the reaction solution was analyzed by thin-layer chromatography (TLC). Once the results indicated the reaction was complete, the reaction solution was concentrated to approximately 5 mL, and then cooled to room temperature. 20 mL of water was added to the concentrate, and the pH was adjusted to 4 using saturated potassium hydrogen sulfate solution. The solution was then extracted with ethyl acetate (50 mL × 3), and the organic phases were combined. The organic phase was dried over anhydrous sodium sulfate to remove water. After drying, the organic solvent was removed by vacuum distillation, yielding 3.47 g of a creamy-white solid, which was identified as N-Boc-L-Trp (compound 9), with a yield of 58.2%. N-Boc-L-Trp (compound 9) will be used in subsequent reactions.

[0134] Step 2: Amino acid condensation reaction

[0135] Following the general synthetic route for tryptophan-based compounds, NHR1R2 will be introduced as a reactant in the reaction of N-Boc-L-Trp (compound 9) to generate compound 10. In the synthesis of (S)-2-amino-3-(1H-indol-3-yl)-N-phenylpropionamide, NHR1R2 is aniline; the preparation method of aniline is omitted. The amino acid condensation reaction is the process of reacting N-Boc-L-Trp (compound 9) with aniline to generate the intermediate (compound 10).

[0136] Take 1.00 g of N-Boc-L-Trp (compound 9) at 2.48 mmol and place it in a 100 mL round-bottom flask. Add an appropriate amount of N,N-dimethylformamide (DMF) to dissolve it. Then add 0.95 g of EDCI (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) at 3.72 mmol, 0.55 g of HOBt (hydroxybenzotriazole) at 2.48 mmol, and 1.72 mL of DIPEA (N,N-diisopropylethylamine) at 7.43 mmol to the solution and stir at room temperature.

[0137] After 30 minutes, 0.46 g (3.72 mmol) of aniline was added, and the reaction was allowed to proceed overnight at room temperature. After the overnight reaction, the reaction solution was analyzed by thin-layer chromatography (TLC). Once the result indicated the reaction was complete, an equal volume of water was added for dilution. The solution was then extracted with ethyl acetate (50 mL × 3), and the organic phases were combined. The mixture was then extracted twice more with water (50 mL × 2), three times with ammonium chloride solution (NH₄Cl, 50 mL × 3), and finally twice with saturated brine (50 mL × 2).

[0138] After extraction, the sample was dried with anhydrous sodium sulfate, and the organic solvent was removed by vacuum distillation. Subsequently, 514 mg of the intermediate (compound 10) was obtained by silica gel column chromatography (PE:EA = 20:1–5:1), with a yield of 41.12%. The intermediate (compound 10) will be used in the next reaction step.

[0139] Step 3: Deprotection reaction

[0140] Referring to the general synthetic route of tryptophan series compounds, the deprotection reaction is the process of reacting the intermediate (compound 10) to generate the target product (compound 11 or compound 12). In this embodiment, the configuration of the target product is consistent with compound 11, namely (S)-2-amino-3-(1H-indol-3-yl)-N-phenylpropionamide.

[0141] 0.514 g (1.36 mmol) of the intermediate (compound 10) was dissolved in 10.0 mL of dichloromethane (CH2Cl2, DMC) and incubated on ice for 10 min. Then, 6.0 mL of trifluoroacetic acid was slowly added dropwise while in an ice bath, and the mixture was stirred at room temperature for 4 h. After 4 h of reaction, the reaction solution was analyzed by thin-layer chromatography (TLC). Once the results indicated the reaction was complete, dichloromethane (CH2Cl2, DMC) was removed from the reaction solution using a rotary evaporator. The solution was then diluted with twice the amount of water, and the pH was adjusted to 11 using approximately 6 mL of sodium hydroxide (NaOH).

[0142] The mixture was extracted three times (50 mL × 3) with dichloromethane (CH2Cl2). After extraction, the product was dried over anhydrous sodium sulfate, and the organic solvent was removed by vacuum distillation. 180 mg of the product was scraped onto a silica gel plate to yield 125 mg of the target product (compound 11), namely (S)-2-amino-3-(1H-indol-3-yl)-N-phenylpropionamide, with a yield of 94.3% and mp values ​​of 177.3–177.8 °C. Its NMR data are shown below.

[0143] 1H NMR(400MHz, Methanol-d4)δ7.66(d,J=7.9Hz,1H),7.48(dd,J=8.6,1.1Hz,2H),7.37(d,J=8.2Hz,1H),7.32-7.25(m,2H), 7.19(s,1H),7.13-7.07(m,2H),7.02-6.97(m,1H),4.13(t,J=6.9Hz,1H),3.42(dd,J=14.6,6.4Hz,1H),3.30-3.25(m,1H). 13 C NMR (101MHz, Methanol-d4) δ170.74,139.01,138.20,129.81,128.48,125.67,125. 40,122.71,121.54,120.12,119.35,112.45,108.88,56.19,29.94.HRMS(ESI):m / z calcd forC 17 H 17 N3O, [M+H] + 280.1444, found 280.1444.

[0144] Example 3

[0145] Synthesis of (S)-2-amino-3-(4-hydroxyphenyl)-N-phenylpropionamide

[0146] The structural formula of the target compound:

[0147] The general synthetic route for tyrosine series compounds is as follows:

[0148]

[0149] It should be noted that when the R1 and R2 groups in NHR1R2 are changed, the specific structures of compounds 15 and 16 will also change accordingly.

[0150] Step 1: Amino acid protection reaction

[0151] Referring to the above synthetic route, compound 13 is L-tyrosine, and compound 14 is Boc-L-Tyr.

[0152] Take 18.1 g, 0.1 mol of L-tyrosine (compound 13) and 4.0 g, 0.1 mol of sodium hydroxide, and dissolve them in 80.0 mL of 1,4-dioxane and 80.0 mL of water. Add 26.2 g, 0.12 mol of Boc anhydride (i.e., di-tert-butyl dicarbonate, (BOC)₂O in the above synthetic route) dropwise at 0-5 °C. After the addition is complete, stir at room temperature for 16 hours to allow the reaction to proceed.

[0153] After 16 hours of reaction, the reaction solution was analyzed by thin-layer chromatography (TLC). Once the results indicated the reaction was complete, the reaction solution was concentrated under reduced pressure to remove most of the 1,4-dioxane. The residual solution was then adjusted to pH 4 using saturated potassium hydrogen sulfate solution. Extraction was then performed with ethyl acetate (200 mL × 3), and the organic phases were combined. The organic phase was dried using anhydrous sodium sulfate to remove water. After drying, the organic solvent was removed from the organic phase by vacuum distillation, yielding 23.8 g of product, namely Boc-L-Tyr (compound 14), with a yield of 85%. Boc-L-Tyr (compound 14) will be used in subsequent reactions.

[0154] Step 2: Amino acid condensation reaction:

[0155] Following the general synthetic route for tyrosine compounds, NHR1R2 will be introduced as a reactant in the reaction of Boc-L-Tyr (compound 14) to generate compound 15. In the synthesis of (S)-2-amino-3-(4-hydroxyphenyl)-N-phenylpropionamide, NHR1R2 is aniline; the preparation method of aniline is omitted. The amino condensation reaction is the process of reacting Boc-L-Tyr (compound 14) with aniline to generate the intermediate (compound 15).

[0156] Take 1.00 g of Boc-L-Tyr (compound 14) at 3.55 mmol and place it in a 100 mL round-bottom flask. Dissolve it in an appropriate amount of N,N-dimethylformamide (DMF). Then add 1.02 g of EDCI (1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride) at 5.33 mmol, 0.48 g of HOBt (hydroxybenzotriazole) at 3.55 mmol, and 1.86 mL of DIPEA (N,N-diisopropylethylamine) at 10.66 mmol to the solution and stir at room temperature.

[0157] After 30 minutes, 0.40 g (3.91 mmol) of aniline was added, and the reaction was allowed to proceed overnight at room temperature. After the overnight reaction, the reaction solution was analyzed by thin-layer chromatography (TLC). Once the results indicated the reaction was complete, an equal volume of water was added for dilution. The solution was then extracted with ethyl acetate (50 mL × 3), and the organic phases were combined. The mixture was then extracted twice with water (50 mL × 2), three times with ammonium chloride solution (NH₄Cl, 50 mL × 3), and finally twice with saturated saline solution (50 mL × 2).

[0158] After extraction, the sample was dried with anhydrous sodium sulfate, and the organic solvent was removed by vacuum distillation. Subsequently, 312 mg of the intermediate (compound 15) was obtained by silica gel column chromatography (PE:EA = 10:1–3:1), with a yield of 24.17%. The intermediate (compound 15) will be used in the next reaction step.

[0159] Step 3: Deprotection reaction:

[0160] Referring to the general synthetic route of tyrosine series compounds, the deprotection reaction is the process of reacting intermediate (compound 15) to generate target product (compound 16). In this example, compound 16 is (S)-2-amino-3-(4-hydroxyphenyl)-N-phenylpropionamide.

[0161] 0.312 g (0.88 mmol) of the intermediate (compound 15) was dissolved in 10.0 mL of acetone and stirred in an ice bath for 10 min. Then, 3.0 mL of 4 M dioxane hydrochloride solution was slowly added dropwise. The mixture was stirred overnight at room temperature. After the reaction, the reaction solution was analyzed by thin-layer chromatography (TLC). Once the results indicated the reaction was complete, 22.8 mg of the target product (compound 16), namely (S)-2-amino-3-(4-hydroxyphenyl)-N-phenylpropionamide, was obtained by filtration, with a yield of 10.18%. Its NMR data are shown below.

[0162] 1 H NMR(400MHz,Methanol-d4)δ7.53–7.48(m,2H),7.35–7.30(m,2H),7.16-7.10(m,3H),6.79– 6.75(m,2H),4.13–4.09(m,1H),3.20(dd,J=14.0,6.9Hz,1H),3.05(dd,J=14.0,7.7Hz,1H); 13C NMR(101MHz,Methanol-d4)δ169.77,138.97,135.79,130.55,130.02,129.55,128.83,128.69,128.40,44.28,38.94.HRMS(ESI):m / z calcd for C 15 H 16 N₂O₂, [M+H] + 257.1285, found 257.1285.

[0163] Example 4

[0164] Confocal microscopy imaging and quantitative statistics of α-synuclein-splitGFP signal

[0165] 1. Experimental materials

[0166] 1.1 Cells

[0167] Yeast cell BY4741(MATa his3Δ1leu2Δ0met15Δ0ura3Δ0)

[0168] Source: From Rong Li's laboratory at Johns Hopkins University, USA

[0169] 1.2. Peptidase neurolysin (Nln) enhancer

[0170]

[0171]

[0172] 2. Experimental Methods

[0173] The α-synuclein and mitochondrial proteins were labeled using the fluorescent probe splitGFP system, specifically the 11th β-strand of GFP. 11 ) is used to label the COOH terminus of α-synuclein, the first 10 β-strands of GFP (GFP 1-10 The COOH terminus of the mitochondrial matrix protein Grx5 was used to label Fis1 on the outer mitochondrial membrane. The red fluorescent protein mCherry was used to label Fis1.

[0174] When GFP 1-10 With GFP 11 There was no green fluorescence when the two parts were separated, but when α-synuclein-GFP was separated... 11 After entering the mitochondrial matrix, it interacts with Grx5-GFP in the matrix. 1-10The combination forms a complete GFP that emits green fluorescence, allowing for fluorescence tracking imaging and quantification of green α-synuclein-GFP in mitochondria. This enables the detection of whether Nln enhancer small molecule compounds can effectively degrade the aggregation of α-synuclein in mitochondria.

[0175] This embodiment utilizes the α-synuclein-splitGFP system to conduct cell experiments in yeast cells (BY4741) for verification.

[0176] Human α-synuclein-splitGFP system was continuously overexpressed in BY4741 cells using PCR homologous recombination technology, while the pleiotropic resistance transporter Prd5 was knocked out to prevent drug efflux. Specific gene information is as follows:

[0177] Δura3::pGAP-α-Syn-HA-GFP11-His3MX6; trp::pGAP-mCherry-Fis1TM-KanM X6; GRX5-GFP1-10-NatMX6; Δpdr5::HygMX6

[0178] Six experimental groups were formed by adding 225 μM peptidase neurolysin (Nln) enhancers #1 to #6 to SC medium of yeast cells (BY4741), with DMSO solvent as the control group. All seven cell groups were incubated overnight at 30°C with shaking for 19 hours. The next morning, 100 μl of the overnight cultured cells were added to 5 ml of fresh medium for resuscitation and incubation for 3 hours until the absorbance (OD600) reached 0.1-0.25. After a cumulative treatment of 22 hours, the cells were dropped onto clean 1.5 NA glass slides, covered with coverslips, and live-cell imaging was performed on each of the 12 cell groups.

[0179] The equipment used for live-cell imaging was a Yokagawa CSU-10 spinning disc confocal rotor combined with a Carl Zeiss 200M inverted microscope. GFP / mCherry fluorescence was excited by 488 / 561nm lasers, and imaging was performed using a Hamamatsu C9100-13 EMCCD camera. 3D imaging of yeast cells (BY4741) was performed using a 100×1.45NA objective lens, with each layer spaced 0.5μm, for a total Z-axis thickness of 5-6μm. Image acquisition was performed using MetaMorph (version 7.0; MDSAnalytical Technologies) software, and analysis was performed using NIH's ImageJ software. The experimental results are presented as Z-projection images.

[0180] Quantitative analysis of α-synuclein-split GFP fluorescence signal was performed using published Python code. The signal intensity of green splitGFP in mitochondria labeled with red fluorescent protein mcherry in each cell was statistically analyzed, and the average values ​​were calculated for comparison.

[0181] 3. Experimental Results

[0182] 3.1 Quantitative Analysis of α-synuclein-split GFP Fluorescence Signal

[0183] Table 1: Quantitative analysis data of α-synuclein-split GFP fluorescence signal

[0184]

[0185]

[0186]

[0187]

[0188]

[0189] The specific readings of the α-synuclein-split GFP fluorescence signal quantitative analysis are shown in Table 1. The data in Table 1 were processed using one-way ANOVA unpaired multiple comparisons (P<0.01). It should be noted that due to the difference in the number of imaging cells in each experimental group, the amount of data (number of rows) in each group (each column) varies.

[0190] The average signal intensity of green splitGFP in mitochondria labeled with red fluorescent protein mcherry in the 7 groups of cells and the decrease in signal intensity of the 6 experimental groups relative to the control group are as follows:

[0191] Group Average signal strength Signal strength drop Control (DMSO) 0.002282375 —— #1 0.00120664 0.001075735 #2 0.001209923 0.001072452 #3 0.001287255 0.00099512 #4 0.00131444 0.000967935 #5 0.001417045 0.00086533 #6 0.001501033 0.000781342

[0192] See Figure 1 , Figure 1 The values ​​shown are the mean and SEM values ​​of the splitGFP intensity in mitochondria in three biostatistical experiments, calculated using one-way ANOVA unpaired multiple comparisons (P<0.01).

[0193] Depend on Figure 1It can be seen that all six peptidase neurolysin (Nln) enhancers used in this experiment can reduce the signal intensity of splitGFP, which means that all six peptidase neurolysin (Nln) enhancers effectively degraded the aggregation of α-synuclein in mitochondria.

[0194] Among the six peptidase neurolysin (Nln) compounds, the two most effective were #1 and #2, namely: (S)-5-((indol-3-yl)methyl)-3-(4-(2-fluoroethoxyphenyl)-2,2-dimethyl-3-phenylimidazolin-4-one, and S-2-amino-N-(4-(2-fluoroethoxy)phenyl)-3-phenylpropionic acid amide. Therefore, the confocal microscopy imaging experiments below only present the imaging results of the two most effective groups.

[0195] 3.2 Confocal Microscopy Imaging

[0196] Yeast cell culture medium expressing the human α-synuclein-splitGFP system was supplemented with 225 μM of compounds #1 and #2. After overnight incubation and recovery treatment for 22 hours (DMSO was used as a control), confocal microscopy imaging results were performed as follows: Figure 2 As shown (scale bar size is 5μM).

[0197] Figure 2 The left column shows cell imaging images of the DMSO control group, the middle column shows cell imaging images of experimental group #1, and the right column shows cell imaging images of experimental group #2. Figure 2 In the top row of images, the white areas represent green fluorescence, which is α-synuclein labeled with splitGFP; in the bottom row of images, the white areas represent red fluorescence, which is mitochondria labeled with mCherry.

[0198] Depend on Figure 2 It was found that the control group (DMSO) cells overexpressed α-synuclein. Compound #1 (S)-5-((indol-3-yl)methyl)-3-(4-(2-fluoroethoxyphenyl)-2,2-dimethyl-3-phenylimidazolin-4-one) and compound #2 S-2-amino-N-(4-(2-fluoroethoxy)phenyl)-3-phenylpropionic acid amide significantly degraded the aggregation of α-synuclein in mitochondria.

[0199] Example 5

[0200] Western blot analysis of α-synuclein levels in cells

[0201] 1. Experimental materials

[0202] The cells and peptidase neurolysin (Nln) enhancer used in this embodiment are exactly the same as those in Example 4. In addition, the following reagents are also involved:

[0203]

[0204] 2. Experimental Methods

[0205] Six experimental groups were formed by adding 225 μM peptidase neurolysin (Nln) enhancers #1 to #6 to YPD medium for yeast cells (BY4741), with DMSO solvent serving as the control group. The yeast cells in all seven groups were cultured overnight at 30°C, and then revived in YPD medium for 3 hours the following day until the absorbance (OD600) reached 0.1–0.25.

[0206] Cells were collected by centrifugation at 21000g at 4°C to completely remove YPD medium. Then, 1 ml of ice water was added, and the mixture was centrifuged again to wash away the liquid; cell pelleting was visible at the bottom of the centrifuge tube. 100 μl of 1xLDS and 40 mM DTT were added, and the mixture was rapidly vortexed to lyse the cells. Then, 100 μl of glass beads was added, and the mixture was vortexed again for one minute. After vortexing, the cells were boiled at 100°C for 8-10 minutes, followed by centrifugation at full speed at room temperature for 2 minutes, retaining the supernatant.

[0207] Each time, take 10 μl of supernatant and run it on a 4-12% SDS-PAGE protein gel. Transfer it to a PVDF membrane using iBlot (Thermo Fishier Scientific) according to the instructions. Block with Odyssey blocking TBS buffer. Use HA-tag rabbit antibody as the primary antibody and HRP-conjugated rabbit antibody as the secondary antibody.

[0208] The LI-COR imaging system was used to image and analyze the strips formed by gel running (LI-CORBiosciences).

[0209] 3. Experimental Results

[0210] The statistical results of the gray values ​​of the α-synuclein antibody band in cells, divided by the gray value of the PGK1 antibody band (control) in the protein imprinting experiment detected by the above experimental method are shown in Table 2.

[0211] Table 2: Quantitative statistics on α-synuclein levels

[0212] DMSO #1 #2 #3 #4 #5 #6 first 1 0.238 0.496 0.673 0.73 0.658 0.53 The second 1 0.748 0.83 —— —— —— —— The third 1 0.576 0.878 —— —— —— —— Fourth 1 —— 0.824 —— —— —— ——

[0213] See Figure 3 , Figure 3The values ​​shown are the mean and SEM values ​​of intracellular α-synuclein levels obtained using Western blotting. (Example:) Figure 3 As shown, all six peptidase neurolysin (Nln) enhancers used in this experiment effectively degraded α-synuclein.

[0214] Example 6

[0215] Mitochondrial membrane potential detection

[0216] 1. Experimental materials

[0217] The cells used in this embodiment are exactly the same as those in Example 4.

[0218] The peptidase neurolysin (Nln) enhancer used in this embodiment is:

[0219]

[0220]

[0221] In addition, this embodiment also involves the following reagents:

[0222] Tetramethylrhodamine methyl ester (TMRM) dye

[0223] Source: Sigma-Aldrich, Product Number: Cat#T5428-25MG

[0224] 2. Experimental Methods

[0225] Tetramethylrhodamine methyl ester (TMRM) stain is a cell-permeable red dye that stains active mitochondria with high membrane potential. The staining disappears when the mitochondrial membrane potential decreases. Therefore, TMRM staining can be used to detect mitochondrial membrane potential and function, thereby detecting the mitochondrial membrane potential of yeast cells overexpressing α-synuclein-splitGFP, to determine the effect of Nln enhancer small molecules on mitochondrial membrane potential, and further to determine the effect of Nln enhancer small molecules on mitochondrial function.

[0226] The α-synuclein and mitochondrial proteins were labeled using the fluorescent probe splitGFP system, specifically the 11th β-strand of GFP. 11 ) is used to label the COOH terminus of α-synuclein, the first 10 β-strands of GFP (GFP 1-10 The COOH terminus of the mitochondrial matrix protein Grx5 was used to label Fis1 on the outer mitochondrial membrane. The red fluorescent protein mCherry was used to label Fis1.

[0227] When GFP1-10 With GFP 11 There was no green fluorescence when the two parts were separated, but when α-synuclein-GFP was separated... 11 After entering the mitochondrial matrix, it interacts with Grx5-GFP in the matrix. 1-10 The combination forms a complete GFP that emits green fluorescence, allowing for fluorescence tracking imaging and quantification of green α-synuclein-GFP in mitochondria. This enables the detection of whether Nln enhancer small molecule compounds can effectively degrade the aggregation of α-synuclein in mitochondria.

[0228] This embodiment utilizes the α-synuclein-splitGFP system to conduct cell experiments in yeast cells (BY4741) for verification.

[0229] Human α-synuclein-splitGFP system was continuously overexpressed in BY4741 cells using PCR homologous recombination technology, while the pleiotropic resistance transporter Prd5 was knocked out to prevent drug efflux. Specific gene information is as follows:

[0230] Δura3::pGAP-α-Syn-HA-GFP11-His3MX6; trp::pGAP-mCherry-Fis1TM-KanM X6; GRX5-GFP1-10-NatMX6; Δpdr5::HygMX6

[0231] Two experimental groups were formed by adding 225 μM peptidase neurolysin (Nln) enhancers #1 and #2 to SC medium of yeast cells (BY4741), with DMSO solvent as the control group. All three groups of cells were incubated overnight at 30°C with shaking. The next morning, 100 μl of the overnight cultured cells were added to 5 ml of fresh medium for resuscitation and incubation for 3 hours until the absorbance (OD600) reached 0.1-0.25.

[0232] After a cumulative treatment of 22 hours, 2.5 μM TMRM staining solution was added to the three groups of cell culture media, and yeast cells expressing α-synuclein-split GFP signaling were stained for ten minutes at 30°C. Ten minutes later, the three groups of cells were washed three times with PBS, and then the cells were dropped onto clean 1.5 NA glass slides, covered with coverslips, and imaged.

[0233] Cell imaging was performed using a Yokagawa CSU-10 spinning disc confocal microscope combined with a Carl Zeiss 200M inverted microscope. GFP / mCherry fluorescence was excited by 488 / 561 nm lasers, and imaging was performed using a Hamamatsu C9100-13 EMCCD camera. 3D imaging of yeast cells (BY4741) was performed using a 100×1.45NA objective lens, with 0.5 μm layer spacing, resulting in a total Z-axis thickness of 5–6 μm. Image acquisition was performed using MetaMorph (version 7.0; MDSAnalytical Technologies) software, and analysis was performed using NIH's ImageJ software. The experimental results are presented as Z-projection images.

[0234] 3. Experimental Results

[0235] Yeast cell culture medium expressing the human α-synuclein-splitGFP system was treated with 225 μM of compounds #1 and #2 for 22 hours (DMSO was used as a control). The cells were then stained with TMRM staining solution, and the results of confocal microscopy imaging are shown below. Figure 4 As shown (scale bar size is 5μM).

[0236] Figure 4 The left column shows cell imaging images of the DMSO control group, the middle column shows cell imaging images of experimental group #1, and the right column shows cell imaging images of experimental group #2. Figure 4 The white areas in the top row of images represent green fluorescence, i.e., splitGFP-labeled α-synuclein; the white areas in the bottom row represent red fluorescence, i.e., TMRM-stained mitochondria.

[0237] Depend on Figure 4 As shown in the top row of imaging images, the control group (DMSO) cells overexpressed α-synuclein, while compound #1 (S)-5-((indol-3-yl)methyl)-3-(4-(2-fluoroethoxyphenyl)-2,2-dimethyl-3-phenylimidazolin-4-one) and compound #2 S-2-amino-N-(4-(2-fluoroethoxy)phenyl)-3-phenylpropionic acid amide both significantly degraded α-synuclein in mitochondria.

[0238] At the same time Figure 4As shown in the image below, the control group (DMSO) TMRM-stained cells (yeast cells overexpressing α-synuclein-splitGFP) had a low mitochondrial membrane potential and impaired mitochondrial function; while the cells treated with peptidase neurolysin (Nln) enhancers #1 and #2 (yeast cells where α-synuclein in the mitochondria was degraded) had an increased mitochondrial membrane potential and restored mitochondrial function.

[0239] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, and are not limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or technical improvements to the embodiments in the market, or to enable others skilled in the art to understand the embodiments disclosed herein. The scope of the invention is defined by the appended claims.

Claims

1. A compound, characterized in that, The compound has the structure shown in structural formula (Ⅰ):

2. A method for preparing a compound, characterized in that, The method for preparing the compound as described in claim 1 comprises the following steps: (1) Amino acid protection reaction: Take 18-25 parts of amino acid by molar amount, add 1,4-dioxane and water; add NaOH and 20-40 parts of Boc anhydride dropwise under ice bath conditions; raise to room temperature for reaction, after the reaction is completed, adjust the reaction solution to acidity, extract the organic phase and remove the solvent to obtain the first intermediate product. (2) Amino acid condensation reaction: Dissolve 2-4 parts of the first intermediate product in molar amounts, and add 3-6 parts of EDCI, 2-4 parts of HOBt, and 7-12 parts of DIPEA to the solution and stir at room temperature; add 3-4 parts of NHR1R2 to the reaction solution, wherein NHR1R2 is 4-(2-fluoroethoxy)aniline; carry out the reaction at room temperature, and after the reaction is completed, extract and remove the solvent to separate and obtain the second intermediate product; (3) Deprotection reaction: Dissolve 0.7-1.9 parts of the second intermediate product by molar amount, and then add dioxane hydrochloride or trifluoroacetic acid dropwise under ice bath conditions. Stir the reaction under room temperature conditions, and extract the compound after treatment.

3. The preparation method according to claim 2, characterized in that, When the compound is S-2-amino-N-(4-(2-fluoroethoxy)phenyl)-3-phenylpropionic acid amide as shown in structural formula (Ⅰ), the method includes: The amino acid in the amino acid acid protection reaction step is L-phenylalanine, and the first intermediate product is N-Boc-Phe-OH.

4. The preparation method according to claim 3, characterized in that, The deprotection reaction step includes the following extraction process: rotary evaporation to remove organic solvent, followed by dilution with water, adjusting the solution to alkaline using sodium hydroxide, extracting the organic phase and removing the solvent, and then separating the S-2-amino-N-(4-(2-fluoroethoxy)phenyl)-3-phenylpropionic acid amide by silica gel column chromatography.

5. The preparation method according to claim 3, characterized in that, The methods for synthesizing 4-(2-fluoroethoxy)aniline include: Dissolve 4-nitrophenol in DMF, add potassium carbonate, stir in an oil bath, and add 1-bromo-2-fluoroethane dropwise to react. After the reaction, extract the intermediate product. Dissolve ammonium chloride in water, add iron powder, and reflux at high temperature. Dissolve the intermediate product in methanol. After the ammonium chloride solution has been refluxed, add the intermediate product solution dropwise to the ammonium chloride solution and continue reflux to react. After the reaction is complete, remove methanol by rotary evaporation, dilute with water, and adjust the solution to neutral. Remove iron powder by suction filtration, and extract the organic phase. Dry to remove water from the organic phase, and rotary evaporate to obtain 4-(2-fluoroethoxy)aniline.

6. The preparation method according to claim 2, characterized in that, The extraction process in the amino acid acid protection reaction includes: extraction with ethyl acetate; the solvent removal process includes: drying the organic phase with anhydrous sodium sulfate to remove moisture from the organic phase, and after drying, removing the organic solvent from the organic phase by vacuum distillation.

7. The use of the compound as described in claim 1 in the preparation of a peptidase neurolysin enhancer.

8. The application according to claim 7, 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.

9. A pharmaceutical composition, characterized in that, It comprises the compound as described in claim 1 and a pharmaceutically acceptable carrier.

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