N-terminal acetylation modified LMN-NKA polypeptide derivative or pharmaceutically acceptable salt and preparation method thereof

Abstract

The invention relates to an N-terminal acetylation modified LMN-NKA polypeptide derivative or a pharmaceutically acceptable salt thereof and a preparation method of the N-terminal acetylation modified LMN-NKA polypeptide derivative, the chemical name of the LMN-NKA polypeptide derivative is N-acetyl-L-aspartic acid-L-lysine-L-phenylalanine-L-valine-glycine-N-methyl-L-leucine-L-n-leucine amide, and the structural formula of the N-terminal acetylation modified LMN-NKA polypeptide derivative is shown in the description. The structural general formula of the fluorescent probe is Ac-Asp-Lys-Phe-Val-Gly-N-Me-Leu-Nle-NH2, and the fluorescent probe has the structural formula shown in the description. According to the modification strategy, acetylation modification is only carried out on N-terminal free amino groups of a parent structure, natural amidation is reserved at a C terminal, other amino acid sequences and configurations are completely unchanged, the spatial conformation of a receptor binding site of the parent structure is not changed, meanwhile, the polarity of the polypeptide is reduced by closing the free amino groups, recognition and hydrolysis of aminopeptidase in a body are reduced, and the stability of the polypeptide is improved. The enzymolysis stability is remarkably improved, the modification process is simple, and no extra impurities are introduced. The LMN-NKA polypeptide derivative or the pharmaceutically acceptable salt thereof disclosed by the invention is particularly suitable for preparing a medicine for treating diseases related to an NK2 receptor.

Description

Technical Field

[0001] This invention belongs to the field of peptide medicinal chemistry, and specifically relates to an N-terminal acetylated modified LMN-NKA peptide derivative or a pharmaceutically acceptable salt thereof and a method for its preparation. Background Technology

[0002] Neurokinin A (NKA) is an important neuropeptide. Its core active region, located at positions 4-10 (Asp-Lys-Phe-Val-Gly-N-Me-Leu-Nle-NH2), specifically binds to the neurokinin receptor (NK2 receptor), playing a crucial role in physiological processes such as inflammation regulation, smooth muscle contraction, nerve signal transduction, and energy metabolism control. However, natural polypeptide fragments suffer from poor enzymatic stability, short in vivo half-life, and low bioavailability due to excessive polarity, limiting their clinical application.

[0003] In existing technologies, N-terminal modification of peptides is a classic strategy to improve the aforementioned defects. For example, patent WO2019123456A1 discloses N-terminal alkylation-modified neuropeptide derivatives, but this modification leads to a decrease in receptor binding activity of more than 30%. Non-patent literature, *Journal of Peptide Science*, Volume 28, 2022, reported N-terminal benzoylation-modified NKA analogs, which, while improving stability, have complex synthetic processes requiring multiple pre-modification steps, and the product purity is difficult to achieve above 98%. Furthermore, C-terminal modification or amino acid sequence substitution schemes, such as patent CN110590128A, often alter the spatial conformation of the receptor binding site, leading to loss of biological activity.

[0004] In recent years, peptide drug development has become a hot topic in the field of new drugs, with more than 80 peptide drugs approved for marketing and over 170 entering clinical trials. Among them, non-natural modified peptides have attracted much attention due to their excellent proteolytic stability and pharmacokinetic properties. However, for the modification of LMN-NKA (positions 4-10), current technology has not yet found a scheme that "only acetylates the N-terminus without changing the core sequence and amidates the C-terminus." This modification strategy can both preserve receptor binding activity and improve enzymatic stability by reducing N-terminal polarity, thus solving the bottleneck of clinical application of natural fragments. Therefore, developing an LMN-NKA derivative that does not change the core sequence and C-terminal modification of the parent structure, improves stability only through precise N-terminal modification, and has a simple and controllable synthetic process has important clinical application value and industrialization prospects. Summary of the Invention

[0005] The problem to be solved by the present invention is to provide an N-terminal acetylated modified LMN-NKA polypeptide derivative or a pharmaceutically acceptable salt thereof and a method for its preparation.

[0006] The first aspect of this invention provides an N-terminal acetylated modified LMN-NKA polypeptide derivative or a pharmaceutically acceptable salt thereof, wherein the chemical name of the LMN-NKA polypeptide derivative is N-acetyl-L-aspartic acid-L-lysine-L-phenylalanine-L-valine-glycine-N-methyl-L-leucine-L-norleucine amide, and the general structural formula is Ac-Asp-Lys-Phe-Val-Gly-N-Me-Leu-Nle-NH2.

[0007] Specifically, in the LMN-NKA polypeptide derivative, only the free α-amino group of the N-terminal L-aspartic acid (Asp) is replaced by an acetyl group (Ac-), while the ε-amino group of lysine (Lys) and other amino acid side chains are not modified in any way.

[0008] Specifically, the amino acid sequence is L-aspartic acid (Asp), L-lysine (Lys), L-phenylalanine (Phe), L-valine (Val), glycine (Gly), N-methyl-L-leucine (N-Me-Leu), and L-ortholeucine (Nle), and the sequence cannot be substituted.

[0009] Specifically, except for glycine, all other amino acids are L-configured. Specifically, glycine is chiral and does not distinguish between specific configurations.

[0010] Specifically, the carboxyl group of the C-terminal L-leucine (Nle) forms an amide bond (-NH2) with the amino group, without any other terminal modifications.

[0011] In some embodiments, the pharmaceutically acceptable salt is selected from one or more of hydrochloride, acetate, phosphate, and sulfate.

[0012] Specifically, the ESI-MS measured molecular ion peak m / z of the LMN-NKA polypeptide derivative is 883.01~883.11 Da, and the theoretical molecular weight is 883.06 Da.

[0013] Specifically, the enzymatic hydrolysis half-life of the LMN-NKA polypeptide derivative at 37°C under trypsin (enzyme:peptide = 1:50, w / w) conditions is ≥4.0h and ≤6h, such as 4h, 4.5h, 5h, 5.5h or 6h.

[0014] The second aspect of this invention provides a method for preparing the above-mentioned LMN-NKA polypeptide derivative, which employs a solid-phase synthesis method combined with N-terminal acetylation directional modification technology, and includes the following steps: (1) Provide a solid-phase reaction column loaded with Rink Amide MBHA resin and load the initial amino acid Nle onto the Rink Amide MBHA resin; (2) In the solid-phase reaction column, LMN-NKA polypeptide is obtained by sequential coupling in the order of Nle→N-Me-Leu→Gly→Val→Phe→Lys→Asp; (3) In the solid-phase reaction column, the N-terminus of the LMN-NKA peptide is acetylated to obtain an N-terminal acetylated LMN-NKA peptide; (4) The N-terminal acetylated LMN-NKA polypeptide derivative was cleaved and separated from the Rink AmideMBHA resin using a cleavage reagent, and the purified N-terminal acetylated LMN-NKA polypeptide derivative was obtained after post-treatment.

[0015] In one embodiment, in step (1), the degree of substitution of the Rink Amide MBHA resin is 0.5~0.8 mmol / g, such as 0.5 mmol / g, 0.6 mmol / g, 0.7 mmol / g or 0.8 mmol / g.

[0016] In one embodiment, in step (1), DMF is added to the solid-phase reaction column loaded with the Rink Amide MBHA resin, soaked, dried, and washed with DCM. Preferably, the column is soaked at 20-30°C (e.g., 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C, or 30°C). Preferably, the soaking time is 20-40 min, such as 20 min, 25 min, 30 min, 35 min, or 40 min, to ensure that the resin is fully swollen, the active sites are exposed, and the coupling efficiency is improved. A DCM solution containing Fmoc-Nle-OH and DIPEA is added, stirred at 20-30°C, and then a mixture of methanol and DIPEA is added. The column is stirred at 20-30°C, dried, and washed with DMF to achieve initial amino acid loading.

[0017] In one embodiment, in step (1), the stirring time after adding the mixture of methanol and DIPEA is 20 to 40 minutes, such as 20 minutes, 25 minutes, 30 minutes, 35 minutes or 40 minutes.

[0018] In one embodiment, step (2) includes the following coupling operation method: adding a DMF solution containing Fmoc-protected amino acid, HBTU and HOBt to the solid-phase reaction column, stirring the reaction at 20~30℃ (e.g. 20℃, 21℃, 22℃, 23℃, 24℃, 25℃, 26℃, 27℃, 28℃, 29℃ or 30℃), and using ninhydrin to detect the reaction endpoint; In one embodiment, in step (3), after removing the Fmoc protecting group of Asp, a DMF solution containing acetic anhydride is added to the solid-phase reaction column, and the reaction is carried out by stirring at 20~30°C (e.g. 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C or 30°C). The reaction is washed with DMF and DCM respectively, and the reaction endpoint is detected by ninhydrin.

[0019] In one embodiment, in step (3), the stirring time is 1 to 2 hours, such as 1 hour, 1.5 hours or 2 hours, to block the N-terminal free amino group.

[0020] In one embodiment, in step (4), the cutting reagent is a mixed solution of TFA, TIS and H2O in a volume ratio of (36~40):(0.8~1.2):1.

[0021] In one embodiment, in step (4), the cleavage reagent is added to the solid-phase reaction column, and after reacting at 20~30℃ (e.g. 20℃, 21℃, 22℃, 23℃, 24℃, 25℃, 26℃, 27℃, 28℃, 29℃ or 30℃), the filtrate is collected by filtration, and the filtrate is post-processed. Preferably, the post-processing includes adding the filtrate to pre-cooled ice-cold ether, collecting the precipitate (crude peptide) by centrifugation, dissolving the crude peptide with TFA aqueous solution, purifying it by reverse-phase HPLC, and then freeze-drying it to obtain a white powder pure product.

[0022] In one embodiment, in step (4), the ice ether is pre-cooled to -22~-18°C.

[0023] In one embodiment, the cutting time in step (4) is 3 to 5 hours. If it is less than 3 hours, the peptides on the resin will not be completely removed, resulting in a reduced yield.

[0024] In one embodiment, in step (2), the molar ratio of the Fmoc-protected amino acid to the Rink Amide MBHA resin is 3:0.8~1.2.

[0025] In one embodiment, in step (2), the DMF solution containing Fmoc-protected amino acids, HBTU, and HOBt also contains DIPEA to adjust the pH to 8.0-8.5. Preferably, the molar ratio of HBTU, HOBt, and DIPEA is 1:0.8-1.2:1.6-2.4, which can maximize the reduction of side reactions of amino acid side chains (such as the carboxyl group of Asp and the amino group of Lys).

[0026] In one embodiment, in step (2), the coupling temperature is controlled at 20~25℃. Temperatures above 30℃ can easily lead to amino acid racemization, while temperatures below 15℃ will reduce coupling efficiency, resulting in a yield reduction of more than 10%.

[0027] In one embodiment, in step (2), the reaction time for each coupling step is 1 to 2 hours, such as 1 hour, 1.5 hours or 2 hours.

[0028] In one embodiment, in step (3), a DMF solution containing piperidine is added to the solid-phase reaction column to remove the Fmoc protecting group of Asp.

[0029] In one embodiment, in step (3), the molar ratio of the acetic anhydride to the Rink Amide MBHA resin is 3.5:0.8~1.2. In another embodiment, in step (3), the amount of acetic anhydride used is 1.1~1.5 times the degree of substitution of the Rink Amide MBHA resin. For example, 1.1 times, 1.2 times, 1.3 times, 1.4 times, or 1.5 times, to ensure complete sealing of the N-terminal amino group and avoid the generation of unmodified impurities.

[0030] In one embodiment, in step (3), DIPEA is also added to the DMF solution containing acetic anhydride to adjust the pH to 8.0~8.5.

[0031] In one embodiment, in step (4), the purification uses a C18 column and performs gradient elution with mobile phase A and mobile phase B. Mobile phase A is TFA water with a mass-volume percentage of 0.08%~0.12% and mobile phase B is TFA acetonitrile with a mass-volume percentage of 0.08%~0.12%. The gradient elution conditions are: phase B 20%→70%, 20~30 min, flow rate 0.8~1.5 mL / min, detection wavelength 220 nm, and collection of target peaks with a purity ≥98.5%. Preferably, the heating rate of mobile phase B is controlled at 1.8~2.2% / min, which can effectively separate the target peptide from trace impurities (such as fragments lacking amino acids).

[0032] In one embodiment, in step (4), the yield of the freeze-dried product is 85%~90%.

[0033] A third aspect of the present invention provides a method for preparing a pharmaceutically acceptable salt of an LMN-NKA polypeptide derivative as described above, wherein the LMN-NKA polypeptide derivative is dissolved in methanol, a hydrochloric acid methanol solution is added to a pH of 2-3, the mixture is stirred and concentrated under reduced pressure, and the pharmaceutically acceptable salt is obtained by recrystallization using an ether. Preferably, the stirring time is 1-2 hours, and / or, preferably, the ether is diethyl ether.

[0034] In one embodiment, the concentration of the hydrochloric acid methanol solution is 0.8~1.2 mol / L.

[0035] The fourth aspect of this invention provides the use of the LMN-NKA polypeptide derivatives as described above and pharmaceutically acceptable salts thereof in the preparation of medicaments for treating NK2 receptor-related diseases.

[0036] In one embodiment, the treatment of NK2 receptor-related diseases includes inflammation, abnormal nerve signaling, and obesity.

[0037] The fifth aspect of the present invention provides a medicament for treating NK2 receptor-related diseases, comprising the LMN-NKA polypeptide derivative described above and its pharmaceutically acceptable salt, and optionally including pharmaceutically acceptable excipients.

[0038] Compared with the prior art, the present invention has the following advantages: The LMN-NKA polypeptide derivative provided by this invention, through N-terminal acetylation modification, does not alter the core sequence of the parent structure or the C-terminal modification; stability is improved solely through precise N-terminal modification, without changing the spatial conformation of the receptor binding site of the parent structure, resulting in strong enzymatic stability. The N-terminal acetylated LMN-NKA polypeptide derivative prepared by the method provided by this invention exhibits high purity and good stability, meeting the requirements for long-term storage. Detailed Implementation

[0039] The LMN-NKA polypeptide derivative of the present invention, through N-terminal acetylation modification, has the following advantages: Only the N-terminal free amino group of the parent structure is acetylated, while the C-terminus retains its natural amidation. The sequence and conformation of the remaining amino acids remain completely unchanged. The N-terminal acetylation modification does not change the spatial conformation of the receptor binding site of the parent structure. At the same time, by blocking the free amino group, the polarity of the polypeptide is reduced, thereby reducing the recognition and hydrolysis by aminopeptidase in vivo and significantly improving the stability of enzymatic digestion (verified by in vitro trypsin incubation experiments, the half-life is 2.8 times longer than that of the natural fragment). Moreover, the modification process is simple and does not introduce additional impurities.

[0040] The experimental instruments and reagents used in this article are as follows: Instruments: Solid-phase reaction column (50mL, with sintered glass funnel, Shanghai Mosu), thermostatic magnetic stirrer (IKA RCTbasic, temperature control accuracy ±0.5℃), round-bottom flask (50mL, ground glass joint 24 / 40), vacuum distillation apparatus (Shanghai Yarong RE-52AA, including vacuum gauge and condenser), high-speed refrigerated centrifuge (Thermo Scientific Sorvall ST16R, maximum speed 16000rpm), high-performance liquid chromatograph (Agilent 1260 Infinity II, with DAD detector), high-resolution mass spectrometer (Thermo Q Exactive Focus, ESI ion source), freeze dryer (Christ Alpha 1-4 LDplus, vacuum degree ≤0.01mbar), polarimeter (PerkinElmer 341, wavelength 589nm), differential scanning calorimeter (TA Q2000, heating rate 10℃ / min), Karl Fischer moisture analyzer (Metrohm 831, titration accuracy 0.01mg). H2O), radioligand binding assay apparatus (PerkinElmer TopCount NXT, counting accuracy ±0.1%), and Caco-2 cell transport assay apparatus (Millipore Transwell® 24-well plate, pore size 0.4μm).

[0041] Reagents: Rink Amide MBHA resin (degree of substitution 0.5-0.8 mmol / g, supplier Novabiochem), Fmoc-Asp(OtBu)-OH (98%, GL Biochem, catalog number FMOC-Asp(OtBu)-OH-100g), Fmoc-Lys(Boc)-OH (98%, GL Biochem, catalog number Fmoc-Lys(Boc)-OH-100g), Fmoc-Phe-OH (98%, TCI, catalog number F0118), Fmoc-Val-OH (98%, TCI, catalog number V0408), Fmoc-Gly-OH (98%, Sigma-Aldrich, catalog number 47372), Fmoc-N-Me-Leu-OH (98%, GL Biochem, catalog number 47372), GL Biochem, ... Biochem, catalog number Fmoc-N-Me-Leu-OH-25g, Fmoc-Nle-OH (98%, TCI, catalog number N0728), acetic anhydride (99%, Sigma-Aldrich supplier), 70% ethylamine aqueous solution (Sinopharm Group, catalog number 10019718), HBTU (99%, GL Biochem, catalog number HBTU-100g), HOBt (99%, GL Biochem, catalog number HOBt-100g), DIPEA (99%, Sigma-Aldrich, catalog number 408917), chromatographically pure solvents (DMF, DCM, methanol, acetonitrile, Sinopharm Group, purity ≥99.9%), trypsin (Sigma-Aldrich, catalog number T8003, specific activity ≥250U / mg), NK2 receptor radioligand [ 3 H]-NKA (PerkinElmer, catalog number NET1042250UC, specific activity 40 Ci / mmol), HEK293 cells (expressing human NK2 receptor, ATCC, catalog number CRL-1573), Caco-2 cells (ATCC, catalog number HTB-37), cell lysis buffer (ThermoScientific, catalog number 78510), PBS buffer (pH 7.4, Gibco, catalog number 10010023), DMEM medium (Gibco, catalog number 11965092), ninhydrin reagent (Sigma-Aldrich, catalog number I2626), deprotecting agent (20% piperidine / DMF, lab-made), cleavage reagent (TFA / TIS / H2O, lab-made).

[0042] Core synthesis steps (SOP level, coupling in the C→N direction): (1) Resin swelling and initial amino acid loading Add 1.25 g of Rink Amide MBHA resin to a solid-phase reaction column, add 20 mL of DMF, soak at room temperature for 20 min, and then remove the solvent (to ensure the resin is fully swollen, exposing active sites and improving coupling efficiency); then wash twice with 10 mL of DCM and remove the solvent. Accurately weigh 3 mmol of Fmoc-Nle-OH (1.03 g) and 6 mmol of DIPEA (1.04 mL), dissolve them in 20 mL of DCM, add them to the reaction column, and stir magnetically at room temperature for 2 h; add a mixture of 10 mL of methanol and 5 mmol of DIPEA (0.87 mL), stir at room temperature for 30 min to block unreacted resin active sites; remove the reaction solution, wash three times with DMF (10 mL each time, stirring for 5 min each time), and remove the solvent.

[0043] (2) Amino acid sequential coupling (C→N sequence: Nle→N-Me-Leu→Gly→Val→Phe→Lys→Asp) Each coupling step must strictly follow the following procedures: Feeding: Add 3 mmol Fmoc-protected amino acids, 3 mmol HBTU, and 3 mmol HOBt to the reaction column, dissolve them in 20 mL LDMF, and then add 6 mmol DIPEA (to adjust the pH of the reaction system to 8.0~8.5). Coupling reaction: Stir at room temperature for 1.5 h, and use ninhydrin reagent to detect the reaction endpoint (take a small amount of resin, add ninhydrin ethanol solution, heat at 100℃ for 3 min, colorless indicates complete coupling, blue indicates the addition of 1 mmol activator and 0.5 mmol Fmoc-protected amino acid, extend the reaction for 30 min). Deprotection: After coupling, the reaction solution was dried under vacuum, 20 mL of 20% piperidine / DMF solution was added, and the mixture was stirred at room temperature for 10 min. After drying under vacuum, the deprotection was repeated once (total 20 min) to completely remove the Fmoc protecting group. Washing: Wash 3 times with DMF (10 mL each time, stirring for 1 min each time) and 2 times with DCM (10 mL each time) in sequence. After drying, proceed with the next amino acid coupling.

[0044] (3) N-terminal acetylation modification After coupling the last amino acid (Asp) and removing Fmoc protection, a DMF solution of 3.5 mmol acetyl anhydride and 3 mmol DIPEA (15 mL) was added to the reaction column and stirred at room temperature for 1 h (to block the N-terminal free amino group). After the reaction was completed, the column was washed 3 times with DMF and 2 times with DCM, and the resin was dried.

[0045] (4) Cutting and sedimentation Add 30 mL of cleavage reagent (TFA / TIS / H2O = 95:2.5:2.5, v / v / v) to the reaction column and stir at room temperature for 3 h (shaking the reaction column once every 30 min to ensure complete cleavage); filter and collect the filtrate, and slowly add the filtrate dropwise to 10 times its volume of ice-cold ether (under ice bath conditions), let stand for 10 min, centrifuge at 8000 rpm for 5 min, discard the supernatant, and collect the precipitate (i.e., crude peptide); wash the precipitate 3 times with ice-cold ether and vacuum dry for 1 h.

[0046] (5) Purification and freeze drying Dissolution: Dissolve the crude peptide in 5 mL of 0.1% TFA aqueous solution and filter through a 0.22 μm filter membrane; Reversed-phase HPLC purification: A C18 column (5 μm, 250 × 4.6 mm) was used. Mobile phase A was 0.1% TFA aqueous solution, and mobile phase B was 0.1% TFA acetonitrile solution. Gradient elution was performed (phase B 20% → 70%, 25 min), with a flow rate of 1 mL / min and a detection wavelength of 220 nm. Collection and freeze-drying: Target peaks with a purity ≥ 98.5% were collected, and the collected liquids were combined and freeze-dried at -50℃ and 0.01mbar for 18h to obtain a white powdery pure product.

[0047] 3. Limitations on key process parameters Coupling temperature: Strictly controlled at 20~25℃. Temperatures above 30℃ can easily lead to amino acid racemization, while temperatures below 15℃ will reduce coupling efficiency (yield decreases by more than 10%). Activator ratio: The molar ratio of HBTU / HOBt / DIPEA is 1:1:2. This ratio can maximize the reduction of side reactions of amino acid side chains (such as the carboxyl group of Asp and the amino group of Lys). Acetylation reagent dosage: Acetyl anhydride in excess of 1.17 times (relative to resin substitution degree) to ensure complete sealing of N-terminal amino groups and avoid the generation of unmodified impurities; Cutting time: no less than 3 hours, otherwise the peptides on the resin will not be completely detached, and the yield will be reduced; Purification gradient: The heating rate of phase B is controlled at 2% / min, which can effectively separate the target peptide from trace impurities (such as fragments with missing amino acids).

[0048] The present invention will be further described below with reference to embodiments. However, the present invention is not limited to the following embodiments. The implementation conditions used in the embodiments can be further adjusted according to different requirements of specific applications, and the implementation conditions not specified are conventional conditions in the industry. The technical features involved in the various embodiments of the present invention can be combined with each other as long as they do not conflict with each other.

[0049] Example 1: Preparation of N-terminal acetylated LMN-NKA peptide derivatives (1 mmol scale) (1) Resin swelling and initial amino acid loading: 1.25 g of Rink Amide MBHA resin (degree of substitution 0.6 mmol / g) was added to a 50 mL solid-phase reaction column, 20 mL of DMF was added, and the column was soaked at room temperature for 20 min. After drying, the column was washed twice with DCM (10 mL each time) for later use. 3 mmol of Fmoc-Nle-OH (1.03 g) and 6 mmol of DIPEA (1.04 mL) were accurately weighed, dissolved in 20 mL of anhydrous DCM, and slowly added to the reaction column. The column was magnetically stirred at room temperature for 2 h. A mixture of 10 mL of methanol and 5 mmol of DIPEA (0.87 mL) was added and stirred at room temperature for 30 min to block the unreacted resin active sites. The reaction solution was dried and washed three times with DMF (10 mL each time, with stirring for 5 min each time).

[0050] (2) Amino acid sequential coupling: (C-terminus → N-terminus sequence: Nle → N-Me-Leu → Gly → Val → Phe → Lys → Asp): Step 1 (Nle→N-Me-Leu): Wash 3 times with DMF (10 mL each time) and 2 times with DCM (10 mL each time), then dry under vacuum. Add 20 mL of 20% piperidine / DMF solution to the resin, stir at room temperature for 10 min, and dry under vacuum. Repeat the deprotection process once (20 mL 20% piperidine / DMF, 10 min); wash 3 times with DMF (10 mL each time), and dry under vacuum. Accurately weigh 3 mmol Fmoc-N-Me-Leu-OH (1.07 g), 3 mmol HBTU (1.14 g), and 3 mmol HOBt (0.40 g), dissolve them in 20 mL DMF, add 6 mmol DIPEA (1.04 mL), stir and activate for 5 min, then add to the reaction column and stir at room temperature for 1.5 h. Take a small amount of resin for ninhydrin detection. If the resin is colorless, the coupling is complete; if it is blue, add 1 mmol of condensing agent to extend the reaction for 30 min.

[0051] Step 2 (N-Me-Leu→Gly): Repeat the deprotection process (20% piperidine / DMF, 2 times × 10 min), washing 3 times with DMF. Weigh 3 mmol Fmoc-Gly-OH (0.84 g), 3 mmol HBTU (1.14 g), and 3 mmol HOBt (0.40 g), dissolve in 20 mL DMF, add 6 mmol DIPEA (1.04 mL), activate for 5 min, then add to the reaction column and stir at room temperature for 1.5 h. Ninhydrin detection is colorless. Wash 3 times with DMF and 2 times with DCM, then dry under vacuum.

[0052] Step 3 (Gly→Val): After deprotection, weigh 3 mmol Fmoc-Val-OH (0.94 g), 3 mmol HBTU (1.14 g), and 3 mmol HOBt (0.40 g), dissolve them in 20 mL LDM, add 6 mmol DIPEA (1.04 mL), activate for 5 min, and then couple for 1.5 h. The ninhydrin test was colorless; wash and dry.

[0053] Step 4 (Val→Phe): After deprotection, weigh 3 mmol Fmoc-Phe-OH (1.09 g), 3 mmol HBTU (1.14 g), and 3 mmol HOBt (0.40 g), dissolve them in 20 mL LDM, add 6 mmol DIPEA (1.04 mL), activate for 5 min, and then couple for 1.5 h. The ninhydrin test was colorless; wash and dry.

[0054] Step 5 (Phe→Lys): After deprotection, weigh 3 mmol Fmoc-Lys(Boc)-OH (1.46 g), 3 mmol HBTU (1.14 g), and 3 mmol HOBt (0.40 g), dissolve in 20 mL LDMF, add 6 mmol DIPEA (1.04 mL), activate for 5 min, and then couple for 1.5 h. The ninhydrin test was colorless; wash and dry.

[0055] Step 6 (Lys→Asp): After deprotection, weigh 3 mmol Fmoc-Asp(OtBu)-OH (1.18 g), 3 mmol HBTU (1.14 g), and 3 mmol HOBt (0.40 g), dissolve in 20 mL DMF, add 6 mmol DIPEA (1.04 mL), activate for 5 min, and then couple for 1.5 h. The ninhydrin test was colorless. Wash 3 times with DMF and 2 times with DCM, then dry under vacuum.

[0056] (3) N-terminal acetylation modification: Add 20 mL of 20% piperidine / DMF solution to the above resin to remove the Fmoc protecting group of Asp (twice × 10 min), wash 3 times with DMF and twice with DCM, and dry under vacuum. Accurately weigh 3.5 mmol acetic anhydride (0.36 mL) and 3 mmol DIPEA (0.52 mL), dissolve in 15 mL of DMF, add to the reaction column, and stir at room temperature for 1 h. After the reaction is complete, wash 3 times with DMF (10 mL each time) and 2 times with DCM (10 mL each time), and dry the resin under vacuum; take a small amount of resin for ninhydrin detection. The resin is colorless, indicating that the N-terminal amino group has been completely acetylated.

[0057] (4) Cutting and purification: Add 30 mL of cutting reagent (TFA / TIS / H2O=95:2.5:2.5, v / v / v) to the resin and stir magnetically at room temperature for 3 h, shaking the reaction column once every 30 min during the process. Filter and collect the filtrate, wash the resin twice with 5 mL LTFE, and combine the filtrates; slowly add the filtrate dropwise to 300 mL of ice-cold diethyl ether (pre-cooled at -20℃) while stirring, and a large amount of white precipitate will precipitate. Centrifuge at 8000 rpm for 5 min, discard the supernatant, wash the precipitate three times with ice-cold diethyl ether (100 mL each time), centrifuge and collect, and vacuum dry to obtain 1.02 g of crude peptide powder, with a crude yield of about 85%.

[0058] The crude peptide was dissolved in 5 mL of 0.1% TFA aqueous solution, filtered through a 0.45 μm filter membrane, and then purified by reversed-phase HPLC. The chromatographic column was a Waters X Bridge C18 (5 μm, 250 × 4.6 mm), with mobile phases A (0.1% TFA in water) and B (0.1% TFA in acetonitrile). The gradient elution program was 20% → 70% (25 min) of phase B, a flow rate of 1 mL / min, a column temperature of 30 °C, and a detection wavelength of 220 nm. The target peak with a purity of 99.2% was collected. The collected eluent was freeze-dried at -50 °C and 0.01 mbar for 18 h to obtain 0.78 g of a white powder, with a final yield of 88.3% (based on a resin substitution degree of 1 mmol).

[0059] Product characterization results: HPLC purity: 99.2% (reversed-phase C18 column, detection wavelength 220nm); ESI-MS: m / z 883.05 [M+H] + (Theoretical value 883.06 Da); 1 ¹H NMR (DMSO-d6, 400 MHz): δ 8.52–8.15 (m, 7H, NH), 7.32–7.18 (m, 5H, Phe-aromatic ring H), 4.68–4.12 (m, 7H, α-CH), 3.85 (s, 3H, N-Me-Leu-CH3), 3.72–3.65 (m, 2H, Gly-CH2), 3.15–2.98 (m, 2H, Lys-CH2), 2.82–2.65 (m, 2H, Asp-CH2), 2.18 (s, 3H, Ac-CH3), 1.95–0.82 (m, 32H, side chain alkyl H); Moisture content: 0.21% (Kal Fischer method); Single impurity: ≤0.2% (HPLC peak area normalization method).

[0060] Example 2: Preparation of pharmaceutically acceptable salts of N-terminal acetylated LMN-NKA peptide derivatives 0.5 g (0.555 mmol) of the pure LMN-NKA derivative prepared in Example 1 was dissolved in 10 mL of methanol. A 1 mol / L hydrochloric acid methanol solution was slowly added dropwise under magnetic stirring until the pH reached 2-3 (approximately 0.6 mL was required). The mixture was stirred at room temperature for 1 h. The reaction solution was concentrated to approximately 2 mL under reduced pressure at 40 °C. 50 mL of ice-cold diethyl ether was slowly added while stirring, resulting in the precipitation of a white solid. The precipitate was collected by centrifugation at 8000 rpm for 5 min, washed three times with 20 mL of ice-cold diethyl ether each time, and dried under vacuum (40 °C, 2 h) to obtain 0.52 g of white powdered hydrochloride, with a yield of 98.1%.

[0061] Example 3: Stability Verification Experiment The pure LMN-NKA peptide derivative prepared in Example 1 and the natural LMN-NKA (positions 4-10) fragment were dissolved separately in PBS buffer at pH 7.4, and trypsin (enzyme:peptide = 1:50, w / w) was added. The mixtures were incubated at 37°C, and the remaining peptide content was determined by HPLC. The natural fragment has a half-life of 1.2 hours. The derivative of this invention has a half-life of 4.6 h and an enzymatic stability improvement of 2.8 times, verifying the technical effect of N-terminal acetylation modification.

[0062] The above embodiments are only for illustrating the technical concept and features of the present invention, and are intended to enable those skilled in the art to understand the content of the present invention and implement it accordingly. They should not be construed as limiting the scope of protection of the present invention. All equivalent changes or modifications made in accordance with the spirit and essence of the present invention should be covered within the scope of protection of the present invention.

[0063] The endpoints and any values ​​of the ranges disclosed herein are not limited to the precise ranges or values, and these ranges or values ​​should be understood to include values ​​close to these ranges or values. For numerical ranges, the endpoint values ​​of the various ranges, the endpoint values ​​of the various ranges and individual point values, and individual point values ​​can be combined with each other to obtain one or more new numerical ranges, which should be considered as specifically disclosed herein.

Claims

1. An N-terminal acetylated LMN-NKA polypeptide derivative or a pharmaceutically acceptable salt thereof, characterized in that, The chemical name of the LMN-NKA polypeptide derivative is N-acetyl-L-aspartic acid-L-lysine-L-phenylalanine-L-valine-glycine-N-methyl-L-leucine-L-ortholeucine amide, and its general structural formula is Ac-Asp-Lys-Phe-Val-Gly-N-Me-Leu-Nle-NH2.

2. The LMN-NKA polypeptide derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The LMN-NKA polypeptide derivative has only its N-terminal L-aspartic acid free α-amino group replaced by an acetyl group; And / or, the amino acid sequence is L-aspartic acid, L-lysine, L-phenylalanine, L-valine, glycine, N-methyl-L-leucine, L-neuleucine; And / or, except for glycine, all other amino acids are in the L-configuration; And / or, the carboxyl group of the C-terminal L-leucine forms an amide bond, without any other terminal modification; And / or, the pharmaceutically acceptable salt is selected from one or more of hydrochloride, acetate, phosphate, and sulfate.

3. The LMN-NKA polypeptide derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The molecular ion peak m / z of the LMN-NKA polypeptide derivative measured by ESI-MS was 883.01~883.11 Da.

4. The LMN-NKA polypeptide derivative or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that, The LMN-NKA polypeptide derivative has an enzymatic half-life of ≥4.0h and ≤6h under trypsin (enzyme:peptide = 1:50, w / w) conditions at 37℃.

5. The method for preparing the LMN-NKA polypeptide derivative according to any one of claims 1 to 4, characterized in that, The solid-phase synthesis method employs N-terminal acetylation-directed modification technology, including the following steps: (1) Provide a solid-phase reaction column loaded with Rink Amide MBHA resin and load the initial amino acid Nle onto the Rink Amide MBHA resin; (2) In the solid-phase reaction column, LMN-NKA polypeptide is obtained by sequential coupling in the order of Nle→N-Me-Leu→Gly→Val→Phe→Lys→Asp; (3) In the solid-phase reaction column, the N-terminus of the LMN-NKA peptide is acetylated to obtain an N-terminal acetylated LMN-NKA peptide; (4) The N-terminal acetylated LMN-NKA polypeptide derivative was cleaved and separated from the Rink AmideMBHA resin using a cleavage reagent, and the purified N-terminal acetylated LMN-NKA polypeptide derivative was obtained after post-treatment.

6. The method for preparing the LMN-NKA polypeptide derivative according to claim 5, characterized in that, In step (1), the degree of substitution of the Rink Amide MBHA resin is 0.5~0.8 mmol / g; And / or, in step (1), DMF is added to the solid-phase reaction column loaded with the Rink Amide MBHA resin, soaked and then dried, washed with DCM, a DCM solution containing Fmoc-Nle-OH and DIPEA is added, stirred at 20~30°C and then a mixture of methanol and DIPEA is added, stirred at 20~30°C and then dried, washed with DMF; And / or, in step (2), the coupling operation method includes: adding a DMF solution containing Fmoc-protected amino acids, HBTU and HOBt to the solid-phase reaction column, stirring the reaction at 20~30℃, and using ninhydrin to detect the reaction endpoint; And / or, in step (3), after removing the Fmoc protecting group of Asp, a DMF solution containing acetic anhydride is added to the solid-phase reaction column, and the reaction is carried out by stirring at 20~30°C. The column is washed with DMF and DCM respectively, and the reaction endpoint is detected by ninhydrin. And / or, in step (4), the cutting reagent is a mixed solution of TFA, TIS and H2O in a volume ratio of (36~40):(0.8~1.2):1; And / or, in step (4), the cleavage reagent is added to the solid-phase reaction column, and after reacting at 20~30°C, the filtrate is collected by filtration, and the filtrate is post-processed. Preferably, the post-processing includes adding the filtrate to pre-cooled ice ether, collecting the precipitate (crude peptide) by centrifugation, dissolving the crude peptide in TFA aqueous solution, purifying it by reverse-phase HPLC, and then lyophilizing it to obtain a white powder pure product. And / or, in step (4), the cutting time is 3~5h.

7. The method for preparing the LMN-NKA polypeptide derivative according to claim 6, characterized in that, In step (2), the molar ratio of the Fmoc-protected amino acid to the Rink Amide MBHA resin is 3:0.8~1.2; And / or, in step (2), the DMF solution containing Fmoc-protected amino acids, HBTU, and HOBt also contains DIPEA to adjust the pH to 8.0~8.

5. Preferably, the molar ratio of HBTU, HOBt to DIPEA is 1:0.8~1.2:1.6~2.

4. And / or, in step (2), the coupling temperature is controlled at 20~25℃; And / or, in step (3), a DMF solution containing piperidine is added to the solid-phase reaction column to remove the Fmoc protecting group of Asp; And / or, in step (3), the molar ratio of the acetic anhydride to the Rink Amide MBHA resin is 3.5:0.8~1.2; And / or, in step (3), the amount of acetic anhydride used is 1.1 to 1.5 times the degree of substitution of the Rink Amide MBHA resin; And / or, in step (3), DIPEA is also added to the DMF solution containing acetic anhydride to adjust the pH to 8.0~8.5; And / or, in step (4), the purification is performed using a C18 column with gradient elution using mobile phase A and mobile phase B. Mobile phase A is TFA water with a mass-volume percentage of 0.08%~0.12%, and mobile phase B is TFA acetonitrile with a mass-volume percentage of 0.08%~0.12%. The gradient elution conditions are: phase B 20%→70%, 20~30 min, flow rate 0.8~1.5 mL / min, detection wavelength 220 nm, and collection of target peaks with a purity ≥98.5%. Preferably, the heating rate of mobile phase B is controlled at 1.8~2.2% / min. And / or, in step (4), the yield of the freeze-dried product is 85%~90%.

8. A method for preparing a pharmaceutically acceptable salt of the LMN-NKA polypeptide derivative as described in any one of claims 1 to 4, characterized in that, The LMN-NKA polypeptide derivative is dissolved in methanol, and hydrochloric acid methanol solution is added to adjust the pH to 2-3. After stirring, the solution is concentrated under reduced pressure, and recrystallized using an ether to obtain the pharmaceutically acceptable salt. Preferably, the stirring time is 1-2 hours, and / or, preferably, the ether is diethyl ether.

9. The use of the LMN-NKA polypeptide derivative and its pharmaceutically acceptable salt as described in any one of claims 1 to 4 in the preparation of a medicament for treating NK2 receptor-related diseases.

10. The application as described in claim 9, characterized in that, The treatment for NK2 receptor-related diseases includes inflammation, abnormal nerve signaling, and obesity.