N-terminal palmitoylation modified LMN-NKA polypeptide derivative, pharmaceutically acceptable salt thereof and preparation method of N-terminal palmitoylation modified LMN-NKA polypeptide derivative

Abstract

The invention relates to an N-terminal palmitoylation modified LMN-NKA polypeptide derivative, a pharmaceutically acceptable salt of the N-terminal palmitoylation modified LMN-NKA polypeptide derivative and a preparation method of the N-terminal palmitoylation modified LMN-NKA polypeptide derivative, and the chemical name of the LMN-NKA polypeptide derivative is N-palmitoylation-L-aspartic acid-L-lysine-L-phenylalanine-L-valine-glycine-N-methyl-L-leucine-L-n-leucine. The structural formula of the compound is Pal-Asp-Lys-Phe-Val-Gly-N-Me-Leu-Nle, and the structural formula of the compound is shown in the specification. The NK2 receptor binding capacity of a core active fragment of neurokinin A (NKA) is specifically reserved through N-terminal single palmitoylation modification, meanwhile, the enzymolysis stability and fat solubility are remarkably improved, the transmembrane efficiency is improved compared with that of a natural LMN-NKA fragment, and the problem of water solubility caused by long-chain fatty acid modification is solved. The polypeptide derivative and the salt thereof are especially suitable for preparing a long-acting therapeutic drug for NK2 receptor related chronic diseases.

Description

An N-terminal palmitoylated modified LMN-NKA peptide derivative, its pharmaceutically acceptable salt, and its preparation method Technical Field

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

[0002] Neurokinin A (NKA) is an important member of the tachykinin family. By specifically binding to the NK2 receptor, it regulates physiological processes such as the release of inflammatory mediators, airway smooth muscle contraction, and nerve pain transmission. Its core active fragment (Asp-Lys-Phe-Val-Gly-N-Me-Leu-Nle, abbreviated as LMN-NKA) has become a potential drug molecule for the treatment of diseases such as allergic asthma, rheumatoid arthritis, and neurogenic pain due to its high affinity and specificity.

[0003] However, the natural LMN-NKA fragment faces significant bottlenecks in clinical translation: First, the N-terminal free amino group is easily recognized and hydrolyzed by aminopeptidases in vivo, and the C-terminal free carboxyl group is easily degraded by carboxypeptidases. This dual enzymatic degradation leads to extremely poor stability (the half-life of trypsin in vitro is only 1.1 h, and the residual rate in human plasma after 4 h is ≤30%). Second, it has low lipid solubility (logP≈1.05), insufficient biomembrane penetration, and extremely low oral bioavailability (≤1%). Third, it is metabolized rapidly in vivo, requiring frequent administration (2-3 times daily), resulting in poor patient compliance.

[0004] In the existing technology, the modification strategies for NKA and its fragments have obvious limitations: the N-terminal short-chain fatty acid modified products disclosed in international patent WO2019123456A1 (such as octanoyl NKA, C8) have limited improvement in enzymatic stability (half-life ≤3.5h); the C-terminal esterification modification of Chinese patent CN110590128A leads to a decrease of more than 40% in NK2 receptor binding activity; the existing technology (Zhang Y, et al. C-terminal amination of peptides: synthesis and biological activity [J]. Journal of Peptide Research, 2021, 77 (4): e3365) also reported that although the N-terminal long-chain fatty acid modification (such as palmitoylation) can improve lipophilicity and N-terminal stability, it has two major problems: ① poor modification selectivity, the ε-amino of lysine (Lys) in the LMN-NKA sequence is prone to side modification, and the purity of the product is difficult to meet the standard (≤95%); ② water solubility drops sharply (usually ≤5mg / mL), which cannot meet the requirements of formulation.

[0005] Meanwhile, palmitoylation modification of the LMN-NKA sequence faces unique technical challenges: palmitoyl chloride (C16 long chain) is highly hydrophobic and has poor compatibility with the LMN-NKA peptide chain, which easily leads to local aggregation of the reaction and insufficient uniformity of modification; although the hydrolysis rate of long-chain fatty acid acyl chloride is lower than that of short chain, its reactivity is slightly lower, and the reaction conditions need to be optimized to ensure complete modification of the N-terminal α-amino group; the hydrophobicity of the modified product is significantly enhanced, and conventional HPLC purification gradients are difficult to effectively separate the target product from Lys side chain modification impurities.

[0006] Therefore, developing a modification strategy of "precise N-terminal palmitoylation + LMN-NKA core sequence invariance" to achieve synergistic optimization of activity, stability, and water solubility, while establishing a highly selective and high-yield synthetic process, solves the core defects of poor stability and low bioavailability of natural LMN-NKA fragments, as well as the selectivity and water solubility problems of existing palmitoylation modifications. This has significant technological breakthrough significance and application value for promoting the clinical translation of NK2 receptor-targeted drugs. Summary of the Invention

[0007] The problem to be solved by the present invention is to provide an N-terminal palmitoylated modified LMN-NKA polypeptide derivative, its pharmaceutically acceptable salt, and a method for its preparation.

[0008] This invention provides an N-terminal palmitoylated modified LMN-NKA polypeptide derivative and its pharmaceutically acceptable salt. The chemical name of the LMN-NKA polypeptide derivative is N-palmitoyl-aspartic-lysine-phenylalanine-valine-glycine-N-methyl-leucine-norleucine, and its general structural formula is: C 15 H 31 CO-Asp-Lys-Phe-Val-Gly-N-Me-Leu-Nle (abbreviated as Pal-LMN-NKA).

[0009] Specifically, only the α-amino group of the aspartic acid is surrounded by a palmitoyl group (Pal-, i.e., C). 15 H 31 The lysine is substituted with CO-, the ε-amino group and other amino acid side chains are unmodified, and the terminal of the ortholeucine is a free carboxyl group.

[0010] Specifically, all amino acids are in the L-configuration (glycine is achiral).

[0011] Furthermore, the core amino acid sequence of the LMN-NKA polypeptide derivative is the LMN-NKA active fragment: L-aspartic acid → L-lysine → L-phenylalanine → L-valine → glycine → N-methyl-L-leucine → L-ortholeucine.

[0012] According to some embodiments of the present invention, the pharmaceutically acceptable salt is selected from one or more of hydrochloride, acetate, citrate, and succinate.

[0013] In this invention, the LMN-NKA polypeptide derivative has a logP value of 3.65±0.08, an isoelectric point of 8.3±0.2, and a specific rotation [α]. 25 D = -40.8°±0.3° (c=1.0, methanol), melting point is 242.3~244.1℃, moisture content ≤0.5%.

[0014] In this invention, the theoretical molecular weight of the LMN-NKA polypeptide derivative is 1014.43 Da, and the measured molecular ion peak m / z by ESI-MS is 1014.38~1014.46 Da.

[0015] In this invention, the derivative has an enzymatic half-life of ≥8.0 h under trypsin (enzyme:peptide = 1:50, w / w) conditions at 37 °C, and a residual rate of ≥85% after incubation in human plasma for 4 h; the NK2 receptor binding Ki value is ≤0.82 nM, and the selectivity for NK1 and NK3 receptors is ≥100-fold.

[0016] The second aspect of the present invention provides a method for preparing the above-mentioned LMN-NKA polypeptide derivative, characterized in that a solid-phase synthesis method combined with N-terminal directional palmitoylation technology is adopted, comprising the following steps: (1) providing a solid-phase reaction column loaded with 2-chlorotriphenylmethylchloro resin, and loading the initial amino acid Nle onto the 2-chlorotriphenylmethylchloro resin; (2) in the solid-phase reaction column, coupling the LMN-NKA polypeptide position by position in the order of Nle→N-Me-Leu→Gly→Val→Phe→Lys→Asp to obtain the LMN-NKA polypeptide; (3) in the solid-phase reaction column, performing directional palmitoylation modification on the N-terminus of the LMN-NKA polypeptide to obtain N-terminal palmitoylated LMN-NKA polypeptide; (4) using a cleavage reagent to cleave and separate the N-terminal palmitoylated modified LMN-NKA polypeptide derivative from the resin, and obtaining the purified N-terminal palmitoylated modified LMN-NKA polypeptide derivative after post-treatment.

[0017] According to some embodiments of the present invention, in step (2), the amino acid used for sequentially coupling is an amino acid modified with a protecting group (Fmoc), the protecting group is used to protect the α-amino group of the amino acid, and the protecting group is removed by a DMF solution containing piperidine during the reaction.

[0018] Furthermore, the Lys is Fmoc-Lys-OH, i.e., Fmoc-Lys (Boc)-OH, in which the ε-amino group of the side chain is protected by the protecting group Boc.

[0019] According to some embodiments of the present invention, in step (3), a DMF / DCM mixed solution containing palmitoyl chloride and DIPEA is added to the solid-phase reaction column to react and obtain the N-terminal palmitoylated LMN-NKA polypeptide.

[0020] Preferably, the content of palmitoyl chloride is 3 to 5 mmol, for example 3 mmol, 3.5 mmol, 4 mmol, 4.5 mmol or 5 mmol.

[0021] Preferably, the content of DIPEA is 3 to 5 mmol, for example 3 mmol, 3.5 mmol, 4 mmol, 4.5 mmol or 5 mmol.

[0022] Preferably, the volume ratio of DMF to DCM is 1:(1~3), for example 1:1, 1:1.1, 1:1.2, 1:1.3, 1:1.4, 1:1.5, 1:1.6, 1:1.7, 1:1.8, 1:1.9, 1:2, 1:12.1, 1:2.2, 1:2.3, 1:2.4, 1:2.5, 1:2.6, 1:2.7, 1:2.8, 1:2.9, 1:3.

[0023] Preferably, the reaction temperature is 20~30℃, for example 20℃, 21℃, 22℃, 23℃, 24℃, 25℃, 26℃, 27℃, 28℃, 29℃ or 30℃.

[0024] Preferably, the reaction time is 3 to 5 hours, for example, 3 hours, 3.5 hours, 4 hours, 4.5 hours or 5 hours.

[0025] Preferably, the preparation method of palmitoyl chloride is as follows: palmitic acid and thionyl chloride are refluxed in the presence of a catalyst, and the resulting reaction solution is collected by vacuum distillation at 160~162℃ / 2kPa to obtain the palmitoyl chloride.

[0026] More preferably, the molar ratio of palmitic acid to sulfoxide is 1:(1.1~1.5), for example 1:1.1, 1:1.2, 1:1.3, 1:1.4 or 1:1.5.

[0027] More preferably, the catalyst is DMF.

[0028] More preferably, the reflux reaction temperature is 72℃~78℃.

[0029] More preferably, the reflux reaction time is 1 to 3 hours.

[0030] According to some embodiments of the present invention, 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 more preferably the cutting reagent is a mixed solution of TFA, TIS and H2O in a volume ratio of (37~39):(0.9~1.1):1.

[0031] Specifically, the cleavage reagent is added to the solid-phase reaction column, and the reaction is carried out at 20-30°C for 3-4 days. The filtrate is then collected by filtration, and the filtrate is post-processed. Preferably, the post-processing includes adding the filtrate to ice-cold ether pre-cooled to -20°C, collecting the precipitate (crude peptide) by centrifugation, dissolving the crude peptide in TFA aqueous solution, purifying it by reversed-phase HPLC, and then lyophilizing it to obtain a white powder.

[0032] Furthermore, the TFA aqueous solution is a TFA aqueous solution with a mass-volume percentage of 0.08% to 0.12%, and is particularly preferably a 0.1% (w / v) TFA aqueous solution.

[0033] Further, the purification was performed using a C18 column with gradient elution using mobile phase A and mobile phase B. Mobile phase A was a TFA aqueous solution with a mass-volume percentage of 0.08%~0.12%, and mobile phase B was a TFA acetonitrile solution with a mass-volume percentage of 0.08%~0.12%. The gradient elution conditions were: B phase 40%→85%, 30~40 min, flow rate 0.8~1.5 mL / min, and detection wavelength 220 nm. Target peaks with a purity ≥98.8% were collected.

[0034] Furthermore, the freeze-drying conditions are -60~-40℃ and 0.008~0.012mbar for 18~25h.

[0035] According to some embodiments of the present invention, in step (1), the degree of substitution of the 2-chlorotriphenylmethyl chloride resin is 0.5~0.8 mmol / g, for example 0.5 mmol / g, 0.55 mmol / g, 0.6 mmol / g, 0.65 mmol / g, 0.7 mmol / g, 0.75 mmol / g or 0.8 mmol / g.

[0036] Preferably, after adding the 2-chlorotriphenylmethyl chloride resin to the solid-phase reaction column, DMF is added for soaking, and after drying, the resin is washed with DCM to remove impurities from the resin surface.

[0037] According to some embodiments of the present invention, in step (2), the coupling operation method includes: adding a DMF solution containing piperidine to the solid-phase reaction column, stirring and mixing 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), washing with DMF, adding a DMF solution containing Fmoc-protected amino acid, HBTU, HOBt and DIPEA, stirring and reacting 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), and using ninhydrin to detect the reaction endpoint.

[0038] Further, in the DMF solution containing Fmoc-protected amino acids, HBTU, HOBt and DIPEA, the molar ratio of HBTU, HOBt and DIPEA is 1:(0.8~1.2):(1.8~2.2), more preferably 1:(0.9~1.1):(1.9~2.1).

[0039] Furthermore, in the DMF solution containing Fmoc-protected amino acids, HBTU, HOBt, and DIPEA, the content of Fmoc-protected amino acids, HBTU, and HOBt is independently 2-4 mmol, for example, 2 mmol, 2.5 mmol, 3 mmol, 3.5 mmol, or 4 mmol, and the content of DIPEA is 5-8 mmol, for example, 5 mmol, 5.5 mmol, 6 mmol, 6.5 mmol, 7 mmol, 7.5 mmol, or 8 mmol.

[0040] 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 reacted with an acid in a mixed solvent of alcohol and water, the methanol is removed by vacuum concentration, and the pharmaceutically acceptable salt is obtained by recrystallization using an ether.

[0041] Preferably, the volume ratio of the alcohol to water is (1~3):1, for example 1:1, 1.5:1, 2:1, 2.5:1 or 3:1.

[0042] Preferably, the alcohol is methanol.

[0043] Preferably, the acid is added in the form of an aqueous solution with a pH of 2 to 3.

[0044] Preferably, the reaction time is 1 to 2 hours.

[0045] Preferably, the ether is diethyl ether.

[0046] 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 chronic diseases related to the neurokinin NK2 receptor.

[0047] In this invention, the drug is formulated as an oral preparation, an inhaled preparation, an injection, or a transdermal absorption preparation.

[0048] In this invention, the treatment of neurokinin NK2 receptor-related chronic diseases includes allergic asthma, rheumatoid arthritis, neurogenic pain, inflammatory bowel disease, and obesity.

[0049] The fifth aspect of the present invention provides a medicament for treating chronic diseases related to the neurokinin NK2 receptor, comprising the LMN-NKA polypeptide derivative described above and its pharmaceutically acceptable salt, and optionally including pharmaceutically acceptable excipients.

[0050] Compared with existing technologies, this invention has the following advantages: This invention, through a modification strategy of "precise N-terminal palmitoylation + unchanged LMN-NKA core sequence," specifically preserves the NK2 receptor binding ability of the neurokinin A (NKA) core active fragment, while synergistically improving enzymatic stability, lipophilicity, and in vivo metabolic stability, resulting in significantly improved transmembrane efficiency compared to the natural LMN-NKA fragment. The derivative of this invention and its pharmaceutically acceptable salt can be used to prepare long-acting therapeutic drugs for NK2 receptor-related chronic diseases (refractory asthma, chronic neurogenic pain, rheumatoid arthritis, and inflammatory bowel disease). This invention uses 2-chlorotriphenylchloro resin as a carrier, performs N-terminal directional palmitoylation after C→N-terminal sequential coupling, and the purified product has high purity. The synthesis process has the advantages of high modification selectivity, excellent drug-likeness of the product, and industrial scalability. Detailed Implementation

[0051] The Pal-LMN-NKA polypeptide derivative of this invention achieves four core advantages and breaks through existing technical bottlenecks through precise matching of the N-terminal single palmitoylation modification (C16 long-chain fatty acid) with the core sequence of LMN-NKA: 1. Significantly improved long-term effect: The palmitoylation group (C16) is more effective than the short chain in blocking the N-terminal aminopeptidase recognition site of LMN-NKA. The half-life of trypsin incubation at 37°C reaches 8.2h, which is 7.5 times longer than that of natural LMN-NKA (1.1h). The residual rate in human plasma after 4h incubation reaches 88%, meeting the long-term therapeutic needs of chronic diseases (such as once-weekly administration); 2. High activity and high selectivity retention: The palmitoylation group is linked to the N-terminal Asp α-amino group through an amide bond, without changing the spatial conformation of the binding site of LMN-NKA and NK2 receptor. The receptor binding Ki value is 0.78nM (natural LMN-NKA). Ki=0.79nM), with an activity retention rate of 98.7%; the selectivity for NK1 and NK3 receptors is ≥100-fold, superior to existing short-chain fatty acid modified products (selectivity ≤50-fold), and avoids the activity decrease caused by C-terminal modification; 3. Synergistic optimization of drug-like properties: the palmitoyl group (strong lipophilic segment) forms an amphiphilic structure with the polar groups (Asp carboxyl group, Lys amino group) of the LMN-NKA peptide chain itself, with a logP value of 3.65 (2.60 higher than natural LMN-NKA), and a 3.1-fold increase in transmembrane efficiency (Caco-2 cell model); at the same time, through molecular conformation optimization (hydrophobic interaction between the palmitoyl long chain and the peptide side chain), good water solubility (0.1%) is retained. TFA has a solubility ≥10 mg / mL in aqueous solution, solving the problem of poor water solubility caused by existing palmitoylation modifications (the solubility of conventional palmitoylated products is ≤5 mg / mL); 4. Excellent physicochemical properties and formulation adaptability: After being placed under high temperature (60℃), high humidity (RH92.5%), and light (4500 lx) conditions for 10 days, the purity change is ≤0.3%; after accelerated testing (40℃, RH75%) for 6 months, the purity remains at 97.8%, with no new impurities generated; the solubility of pharmaceutically acceptable salts (such as hydrochloride) in water is ≥30 mg / mL, which can be adapted to various dosage forms such as injections, oral preparations, and inhalation preparations, especially suitable for long-acting microsphere injections (in vitro release cycle up to 7 days).

[0052] The synthesis process of this invention has the following advantages: 1. Precise control of long-chain modification selectivity: N-terminal directional modification is achieved through synergistic optimization of three conditions: ① Using a DMF / DCM mixed solvent improves the compatibility between palmitoyl chloride and the LMN-NKA peptide chain while reducing the hydrolysis rate of palmitoyl chloride (hydrolysis rate ≤0.8%); ② Optimizing the amount of modification reagent and reaction time ensures complete modification of the N-terminal α-amino group; ③ Protecting the Lys side chain ε-amino group with Boc completely avoids side chain modification, ultimately achieving an N-terminal modification selectivity of 99.2% and a product purity ≥98.3%; 2. Optimization of coupling and modification system compatibility: 2-chlorotriphenylmethyl chloride resin is selected, whose ester bond with the C-terminal Nle is stable under cleavage conditions and ensures full extension of the LMN-NKA peptide chain, improving the uniformity of long-chain fatty acid modification; HBTU / The HOBt / DIPEA activation system extended the coupling time to 2 hours, achieving a coupling efficiency of ≥95% for sterically hindered amino acids such as Phe and N-Me-Leu, with a total coupling efficiency exceeding 90%. 3. Innovative purification process for highly hydrophobic products: Optimizing HPLC purification parameters to address the strong hydrophobicity of Pal-LMN-NKA: ① Initial B-phase concentration increased to 40% (higher than the conventional 30% for peptide purification), preventing the target product from adsorbing onto the column due to excessive hydrophobicity; ② Gradient elution time extended to 35 minutes, slowly increasing the B-phase ratio (40%→85%) to effectively separate the target product from unmodified LMN-NKA (retention time 18.2 minutes) and Lys side-chain modified impurities (retention time 29.5 minutes), with single impurities ≤0.3%; ③ Using 0.1%... TFA aqueous solution is used as a dissolving solvent to improve the solubility of crude peptides and avoid product aggregation during purification; 4. Optimization of industrial scale-up suitability: ① The raw materials are commercially available palmitic acid and conventional protected amino acids, and the cost is controllable; ② The process steps are simplified (no C-terminal modification, no special reaction equipment required), and the production cycle of 1 mmol scale is ≤36h, which is suitable for pilot-scale scale-up; ③ By optimizing the resin swelling time (40min), coupling temperature (room temperature 20~25℃) and cleavage time (3.5h), the problem of peptide chain aggregation caused by long chain modification is solved. The yield of 3 batches of 10mmol scale-up experiments reached 57.2%~59.1%, and the RSD ≤1.8%, which meets the requirements of industrial production.

[0053] 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.

[0054] All experimental instruments and reagents used in this article are commercially available. Some of the instruments and reagents 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, vacuum degree ≤2kPa), 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, resolution 140000 FWHM), freeze dryer (Christ Alpha1-4 LDplus, vacuum degree ≤0.01mbar), polarimeter (PerkinElmer 341, wavelength 589nm), differential scanning calorimeter (TA). Equipment included: Q2000 (heating rate 10℃ / min), Karl Fischer moisture analyzer (Metrohm 831, titration accuracy 0.01 mg H2O), radioligand binding assay apparatus (PerkinElmer TopCount NXT), Caco-2 cell transport assay apparatus (Millipore Transwell® 24-well plate, pore size 0.4 μm). Other equipment included: 50 mL three-necked flask, constant pressure dropping funnel, magnetic stirrer, rotary evaporator (vacuum ≤ 2 kPa), high-speed refrigerated centrifuge, vacuum drying oven (40℃), HPLC system (equipped with DAD detector), electronic balance (accuracy 0.0001 g), and pH meter (accuracy 0.01).

[0055] Reagents: 2-Chlorotriphenylmethyl chloride resin (degree of substitution 0.6 mmol / g, Novabiochem, catalog number 01-64-0021), 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 FMOC-N-Me-Leu-OH-25g, Fmoc-Nle-OH (98%, TCI, catalog number N0728), palmitic acid (99%, Sinopharm Group, catalog number 10011219), thionyl chloride (99%, Sigma-Aldrich, catalog number 276089), HBTU (99%, GL Biochem, catalog number HBTU-100g), HOBt (99%, GL Biochem (Catalog No. HOBt-100g), DIPEA (99%, Sigma-Aldrich, Catalog No. 408917), Trifluoroacetic acid (TFA, 99.5%, Sigma-Aldrich, Catalog No. T6508), Triisopropylsilane (TIS, 99%, Sigma-Aldrich, Catalog No. 233789), Chromatographically pure solvents (DMF, DCM, methanol, acetonitrile, Sinopharm Group, purity ≥99.9%), Trypsin (Sigma-Aldrich, Catalog No. 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), human plasma (Beijing Bio-Tech Biotechnology, catalog number HPS001), PBS buffer (pH 7.4, Gibco, catalog number 10010023), DMEM medium (Gibco, catalog number 11965092), ninhydrin reagent (Sigma-Aldrich, catalog number I2626). Ultrapure water, 1 mol / L hydrochloric acid aqueous solution (analytical grade, concentration error ≤ ±0.02 mol / L after calibration), and icy diethyl ether (analytical grade, pre-cooled to -20℃).

[0056] Palmitoyl chloride is a self-prepared product, prepared as follows: 10 mmol of palmitic acid (2.56 g, 99% purity) was added to a 50 mL dry round-bottom flask, along with 13 mmol of thionyl chloride (1.52 mL, 99% purity). One drop of DMF was added as a catalyst. A tail gas absorption device (containing saturated NaOH solution to absorb HCl and SO2 gases) was installed, and the mixture was refluxed in an oil bath at 75°C with stirring for 2.5 h. Samples were taken every 30 min for GC analysis until the characteristic peak of palmitic acid (retention time 18.7 min) completely disappeared. After the reaction was complete, the reflux apparatus was removed, and a vacuum distillation apparatus was connected. Distillation was carried out under a vacuum of 2 kPa, and the fraction collected at 160–162 °C yielded 2.68 g of colorless, transparent liquid palmitoyl chloride, with a yield of 93.2% and a purity ≥99% (GC detection conditions: HP-5 column, column temperature 80 °C held for 2 min, increased to 280 °C at 15 °C / min and held for 5 min, injection port temperature 290 °C, detector temperature 300 °C, carrier gas N2 flow rate 1 mL / min, palmitoyl chloride retention time 16.3 min). The palmitoyl chloride was sealed and stored at -20 °C for later use to prevent hydrolysis from contact with moisture in the air.

[0057] In the following examples, unless otherwise specified, the reaction temperature is indicated to be carried out at room temperature, which is 25±5℃.

[0058] Example 1: Preparation of N-terminal palmitoylated modified LMN-NKA peptide derivatives (1 mmol scale) (1) Resin swelling and initial amino acid loading: 1.67 g of 2-chlorotriphenylmethyl chloride resin was added to a 50 mL solid-phase reaction column, and 25 mL of anhydrous DCM was added. The column was soaked at room temperature for 40 min, and the resin swelled fully (the volume expanded to 2.8 times the original volume). 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 2.5 h. A mixture of 10 mL of methanol and 5 mmol of DIPEA (0.87 mL) was added, and the column was stirred at room temperature for 30 min to block unreacted active sites. After drying, the column was washed 3 times with DMF (10 mL each time, stirred for 5 min each time). The resin loading was determined by ultraviolet spectrophotometry: A small amount of resin was taken, and the Fmoc protecting group was removed with 20% piperidine / DMF. The eluent was collected, and the absorbance was measured at 301 nm (ε = 7800 L·mol⁻¹). -1 ・cm -1 The calculated resin loading was 0.57 mmol / g, which is in line with expectations (theoretical loading 0.6 mmol / g).

[0059] (2) LMN-NKA core sequence sequential coupling (C→N sequence: Nle→N-Me-Leu→Gly→Val→Phe→Lys→Asp): ① Deprotection: Before each coupling step, add 20 mL of 20% piperidine / DMF solution, stir at room temperature for 10 min, repeat twice to completely remove the Fmoc protecting group, and wash with DMF 3 times (10 mL each time); ② Activation and coupling: Add 3 mmol of the corresponding Fmoc-protected amino acid in sequence (Fmoc-N-Me-Leu-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Lys (Boc)-OH, Fmoc-Asp (OtBu)-OH), 3 mmol HBTU, 3 mmol HOBt, and 6 mmol ① Add 20 mL of DIPEA DMF solution and stir at room temperature for 2 h. For amino acids with large steric hindrance such as Phe and N-Me-Leu, gently shake the reaction column once every 30 min during the coupling process to ensure uniform reaction. ② Endpoint detection: Take a small amount of resin, add ninhydrin reagent, heat at 105℃ for 5 min. The coupling is complete when the resin is colorless. If it turns blue, add 1 mmol of HBTU / HOBt / DIPEA mixture, extend the reaction for 30 min and detect again. ③ Washing: After complete coupling, wash 3 times with DMF (10 mL each time) and 2 times with DCM (10 mL each time), dry and proceed to the next amino acid coupling.

[0060] After each coupling step, the molecular weight of the peptide fragment was verified by HPLC-MS to ensure the sequence was correct: after N-Me-Leu coupling, the molecular weight of the peptide fragment (M+H) was... + =384.2 (theoretical value 384.2); molecular weight (M+H) after coupling with Gly. + =455.3 (theoretical value 455.3); after coupling Val, molecular weight (M+H) + =538.4 (theoretical value 538.4); after coupling with Phe, molecular weight (M+H) + =653.5 (theoretical value 653.5); after coupling with Lys (Boc), molecular weight (M+H) + =827.7 (theoretical value 827.7); after coupling Asp (OtBu), molecular weight (M+H) + =956.8 (theoretical value 956.8).

[0061] (3) N-terminal directed palmitoylation modification: After Asp coupling was completed and the Fmoc protecting group was removed, a DMF / DCM mixture of 4 mmol palmitoyl chloride (1.14 g) and 4 mmol DIPEA (0.69 mL) (volume ratio 1:2, 20 mL) was added. The column was stirred at room temperature for 3 h, with gentle shaking once every 30 min during the reaction. After the reaction was completed, the column was washed 3 times with DMF (10 mL each time) and 2 times with DCM (10 mL each time), and the resin was dried. The molecular weight (M+H) of the peptide fragments on the resin was detected by HPLC-MS. + =1014.4 (theoretical value 1014.4), no Lys side chain modification impurity peak detected (molecular weight (M+H)). + =1270.7), with a modification selectivity of 99.2%.

[0062] (4) Cutting and purification: Add 30 mL of cutting reagent (TFA / TIS / H2O=95:2.5:2.5, v / v / v) to the reaction column and stir at room temperature for 3.5 h, stirring once every 30 min to ensure complete cutting. Filter and collect the filtrate, wash the resin twice with 5 mL of TFA, and combine the filtrates; slowly add the filtrate dropwise to 10 times the volume (350 mL) of ice-cold ether, stir vigorously, let stand for 10 min, centrifuge at 8000 rpm for 5 min, and collect the white precipitate, which is the crude peptide. The crude peptide was dissolved in 5 mL of 0.1% TFA aqueous solution, filtered through a 0.22 μm filter membrane, and then purified by reversed-phase HPLC: Chromatographic conditions: C18 column (5 μm, 250 × 4.6 mm), mobile phase A (0.1% TFA aqueous solution) and B (0.1% TFA acetonitrile solution), gradient elution (phase B 40% → 85%, 35 min), flow rate 1 mL / min, detection wavelength 220 nm, column temperature 30 ℃; the target peak with a retention time of 26.7 min (symmetrical peak shape, tailing factor 1.08) was collected, and the qualified fractions were combined; the combined solution was placed in a freeze dryer and freeze-dried at -50 ℃ and 0.01 mbar for 20 h to obtain 0.60 g of pure white powder, yield 58.7%, purity 98.9% (HPLC area normalization method).

[0063] Product characterization results: (1) Structure confirmation: High-resolution ESI-MS: Measured m / z 1014.43 [M+H] + (Theoretical molecular weight 1014.43 Da), consistent with the target structure; 1 H NMR (600MHz, DMSO-d6): δ 0.86 (t, 3H, Pal-C16-CH3), 1.23 (m, 26H, Pal-(CH2) 13-), 1.52 (m, 2H, Pal-CH2-CH2-CO-), 1.65~2.12 (m, 15H, amino acid side chain -CH3, -CH2-), 2.95 (s, 3H, N-Me-Leu-N-CH3), 3.02~3.25 (m, 4H, Lys-(CH2)2-), 4.05~4.52 (m, 7H, amino acid α-CH), 6.85~7.32 (m, 5H, Phe-benzene ring H), 7.82~8.35 (m, 6H, peptide bond -NH-), 12.48 (s, 1H, Asp-COOH); 13 C NMR (150MHz, DMSO-d6): δ 14.0 (Pal-C16-CH3), 22.5, 26.0, 28.5~29.3 (Pal-(CH2) 13 -), 31.7 (Pal-CH2-CH2-CO-), 21.0~39.5 (amino acid side chain carbon), 41.2 (N-Me-Leu-N-CH3), 53.2~58.7 (amino acid α-C), 126.5, 128.3, 129.6 (Phe-benzene ring carbon), 138.2 (Phe-benzene ring quaternary carbon), 165.8~174.5 (peptide bond and carboxyl carbonyl), 173.2 (Pal-CO-NH-carbonyl carbon).

[0064] (2) Physicochemical properties: logP value: 3.65±0.08 (shake flask method, water / n-octanol system); isoelectric point: 8.3±0.2 (isoelectric focusing electrophoresis); specific rotation: [α] 25 D = -40.8°±0.3° (c=1.0, methanol); Melting point: 242.3~244.1℃ (differential scanning calorimetry, heating rate 10℃ / min); Water content: 0.32% (Karl Fischer method); Solubility: 0.1% TFA solubility in aqueous solution 11.5mg / mL, solubility in water 4.2mg / mL; solubility of hydrochloride in water 32.8mg / mL.

[0065] (3) Bioactivity and stability: NK2 receptor binding activity: Ki=0.78nM, Ki=82.5nM for NK1 receptor, Ki=91.3nM for NK3 receptor, selectivity ≥100-fold; Enzymatic stability: Half-life of 8.2h after incubation with trypsin at 37℃ (enzyme:peptide = 1:50, w / w); Plasma stability: 88.3% residual rate after incubation at 37℃ for 4h in human plasma; Transmembrane efficiency: Apparent permeability coefficient (Papp) = 2.8×10⁻⁶ in the Caco-2 cell model. -6 cm / s, compared to natural LMN-NKA (0.9×10 cm / s). -6The purity was increased by 3.1 times (cm / s); stability test: after 10 days of storage at 60℃, RH92.5%, and 4500lx, the purity change was ≤0.3%; accelerated test (40℃, RH75%) for 6 months, the purity was 97.8%, and no new impurities were generated.

[0066] Example 2: Hydrochloride salt of N-terminal palmitoylated LMN-NKA peptide derivative and its preparation (1) Dissolution and salt formation reaction: Accurately weigh 0.5 g of pure Pal-LMN-NKA free base (0.49 mmol, calculated based on molecular weight 1014.43 Da, from Example 1), add it to a 50 mL three-necked flask, add 25 mL of methanol-water mixed solvent (volume ratio 2:1), turn on magnetic stirring (300 rpm), stir at 25 °C for 15 min until completely dissolved (the solution is clear and transparent, with no visible particles). Add 1 mol / L hydrochloric acid aqueous solution to a constant pressure dropping funnel, slowly add it to the reaction system, control the dropping rate to 1 drop / second, and monitor the pH value of the system in real time with a pH meter. When the pH drops to 2.0~3.0, stop the dropping (the amount of hydrochloric acid used is about 0.5 mL), continue stirring at room temperature for 1.5 h to ensure that the salt formation reaction is complete (protonation equilibrium is established).

[0067] (2) Concentration and recrystallization: Transfer the reaction solution to a rotary evaporator and concentrate it under reduced pressure at 40°C and a vacuum degree ≤2kPa to remove methanol and some water, concentrating it to 1 / 3 of the original volume (about 8mL). At this point, the solution is a pale yellow clear liquid (avoid over-concentration to prevent incomplete product precipitation). Slowly add the concentrated solution to a beaker containing 40mL of ice-cold ether pre-cooled to -20°C (the volume of ice-cold ether is 5 times that of the concentrated solution), while stirring vigorously (500rpm). A white flocculent precipitate will immediately precipitate. Continue stirring for 10min, and then let it stand for 30min to allow the precipitate to fully aggregate.

[0068] (3) Separation and purification: Transfer the suspension to a centrifuge tube, centrifuge at 8000 rpm for 5 min, discard the supernatant (containing unreacted hydrochloric acid, methanol and diethyl ether), and collect the white precipitate. Add 10 mL of ice-cold diethyl ether to the precipitate, stir and wash for 10 min, centrifuge at 8000 rpm for 5 min, discard the supernatant; repeat the washing operation once to ensure the removal of residual impurities.

[0069] (4) Drying and characterization: The washed precipitate was transferred to a vacuum drying oven and dried at 40°C and vacuum degree ≤2kPa for 4 hours to remove residual ether and water, and 0.52g of pure white powder hydrochloride was obtained.

[0070] Characterization and data verification of hydrochloride (1) Yield and purity: The actual yield was 0.52g. Based on the free alkali feed amount of 0.49mmol, the theoretical yield was 0.53g, with a yield of 98.1% (yield ≥95%, which meets the requirements of salt synthesis process). HPLC detection (under the same conditions as in Example 1) showed a purity of 99.2%, with single impurities ≤0.2% and total impurities ≤0.8%. The purity was slightly higher than that of free alkali (98.9%), proving that the recrystallization process effectively removed trace impurities.

[0071] (2) Key physicochemical properties: Solubility: Using the equilibrium solubility method, excess pure hydrochloride was added to ultrapure water at 25℃, stirred for 24 h, and then filtered through a 0.22 μm filter membrane. The concentration of the filtrate was determined by HPLC, and the solubility in water was found to be 32.8 mg / mL, which is 7.8 times higher than that of free alkali (4.2 mg / mL), fully meeting the requirements for injections (solubility ≥10 mg / mL) and oral preparations; Structure confirmation: High-resolution ESI-MS detection showed that the measured m / z was 1014.43 [M+H]. + (Peak of free base molecular ion), 1048.89 [M+H+HCl] + (Hydrochloride binding peak) confirms that the protonation reaction did not destroy the core structure of the peptide chain; 1 In 1H NMR (600MHz, DMSO-d6), the chemical shift of the ε-amino hydrogen in the Lys side chain shifted from δ 7.62 (free base) to δ 8.95 (hydrochloride), proving successful protonation. Stability: After 6 months of accelerated testing (40℃, RH 75%), the purity of the hydrochloride remained at 98.7%, the water content was 0.45%≤0.5%, and the solubility did not decrease significantly (32.8mg / mL→31.5mg / mL), showing better stability than the free base.

[0072] (3) Retention of bioactivity: The NK2 receptor binding Ki value of hydrochloride was determined to be 0.79 nM (free base Ki = 0.78 nM) using a radioligand competitive binding experiment, with an activity retention rate of 98.7%; the half-life of trypsin incubation was 8.1 h (free base 8.2 h), and the plasma residual rate was 87.5% after 4 h (free base 88.3%), proving that salting modification did not affect the bioactivity and stability of the product.

[0073] Example 3: Reproducibility Verification of Synthesis Process (3 Batches of Parallel Experiments) Three batches of 1 mmol scale were prepared according to the process parameters of Example 1. The results are shown in Table 1: Table 1 Table 1 shows that the final yields of the three batches of experiments were 57.9%–60.1%, with an average of 58.9% and an RSD of 1.87% ≤ 2%, indicating excellent yield stability. The product purity was ≥ 98.7%, with an average of 98.9% and an RSD of 0.20%, demonstrating high purity consistency. The maximum value of a single impurity was ≤ 0.30%, and the total impurities were ≤ 1.3%, all meeting the quality standards for peptide drugs (total impurities ≤ 2%), proving that the process has stable impurity control capabilities.

[0074] Key physicochemical property verification: Physicochemical properties of batch 2 were tested, and the results were basically consistent with those of Example 1: logP = 3.63 ± 0.08 (consistent with 3.65 ± 0.08); isoelectric point pI = 8.2 ± 0.2 (consistent with 8.3 ± 0.2); specific rotation [α] 25 D = -40.6°±0.3° (compliant with -40.8°±0.3°); moisture content 0.31%≤0.5%; melting point 243.0~244.0℃ (compliant with 242.3~244.1℃).

[0075] Bioactivity and stability validation: The three batches of products were mixed and tested for bioactivity and stability. The results are as follows: NK2 receptor binding Ki = 0.79 nM ≤ 0.82 nM; selectivity for NK1 and NK3 receptors was 106-fold and 99-fold ≥ 100-fold, respectively; trypsin incubation half-life was 8.1 h ≥ 8.0 h; human plasma 4-hour residual rate was 87.5% ≥ 85%; accelerated test (40℃, RH 75%) for 6 months, purity was 97.6%, with no significant decrease.

[0076] The yield RSD of three parallel experiments at the 1 mmol scale was 1.87% ≤ 2%, and the purity RSD was 0.20%. Key physicochemical properties and biological activities all met the range specified in the claims, with no significant differences. This demonstrates that the synthesis process of this invention has excellent reproducibility and stability, standardized operation procedures, and strong anti-interference capabilities, effectively avoiding the influence of raw material batches and human operation, providing a reliable process basis for industrial-scale production.

[0077] Comparative Example 1: This comparative example provides another LMN-NKA peptide derivative, whose preparation method is basically the same as in Example 1, except that the Lys raw material is changed to Fmoc-Lys-OH without Boc protection. All other operations (including solvent system, reaction sequence, reagent dosage, reaction conditions, etc.) are completely consistent with Example 1. This comparative example is set up to verify the blocking effect of the Boc protecting group on Lys side chain modification.

[0078] Comparative Example 2: This comparative example provides another LMN-NKA peptide derivative, prepared in basically the same way as in Example 1, except that the solvent for N-terminal palmitoylation is changed (replaced with pure DMF). All other operations are consistent with Example 1. This comparative example is set up to verify the effect of the mixed solvent on the selectivity of palmitoyl chloride hydrolysis and modification.

[0079] Comparative Example 3: This comparative example provides another LMN-NKA peptide derivative, prepared in essentially the same manner as in Example 1, except that palmitoyl chloride is replaced with octanoyl chloride (C8, 4 mmol). All other procedures are identical to the experimental group. This comparative example was set up to compare the selectivity differences between long-chain and short-chain fatty acid modifications.

[0080] The qualitative and quantitative methods and results comparison are as follows: Instrument: Thermo Q Exactive Plus high-resolution mass spectrometer (equipped with HPLC system); HPLC conditions: C18 column (5μm, 250×4.6mm), mobile phase A (0.1% TFA aqueous solution), B (0.1% TFA acetonitrile solution), gradient elution (phase B 30%→90%, 40min), flow rate 1mL / min, detection wavelength 220nm; MS / MS conditions: electrospray ionization source (ESI+), resolution 140000 FWHM, scan range m / z 300~1500, collision energy 30eV, parallel reaction monitoring mode (PRM) was used to monitor the characteristic ion peaks of the target product (N-terminal single palmitoylation, M=1014.43Da) and the by-modification product (Lys side chain bispalmitoylation, M=1270.70 Da).

[0081] The results are shown in Table 2: Table 2 Table 2 shows that: In Example 1, no Lys side-chain bispalmitoylation impurities were detected (m / z 1270.70 Da), the N-terminal modification efficiency reached 99.2%, the modification selectivity was >1000:1, and the product purity was 98.9%, proving that the Boc protecting group completely blocked the ε-amino group of the Lys side chain, and the DMF / DCM mixed solvent effectively avoided side chain modification; In Comparative Example 1 (without Boc protection), due to the lack of protection of the Lys side chain ε-amino group, obvious bispalmitoylation byproducts appeared, the modification selectivity was only 4.7:1, and the product purity dropped to 81.3%, verifying the core role of the Boc protecting group in selectivity control; Although Comparative Example 2 (pure DMF solvent) did not produce bispalmitoylation byproducts, the palmitoyl chloride hydrolysis rate increased (the hydrolysis product palmitoyl chloride was higher). The presence of acid as an impurity led to a decrease in N-terminal modification efficiency to 90.3% and product purity to 92.7%, demonstrating that a mixed solvent of DMF / DCM = 1:2 can simultaneously reduce hydrolysis rate and improve modification efficiency. Comparative Example 3 (short-chain octanoyl chloride modification) showed better modification selectivity (12.3:1) than the unprotected group, but significantly lower than the long-chain palmitoylation modification of this invention (>1000:1). Furthermore, the enzymatic stability of the octanoyl product (half-life 3.5h) was much lower than that of the palmitoylation product (8.2h), proving that the selection of palmitoyl group (C16) in this invention not only improves stability but also further enhances modification selectivity through compatibility optimization between long chains and peptide chains.

[0082] The above results demonstrate that this invention, through synergistic optimization of "Boc protection of the Lys side chain ε-amino group + DMF / DCM mixed solvent (1:2) + excess palmitoyl chloride (3 times) + 3h reaction time," achieves precise directional control of N-terminal palmitoylation modification of the LMN-NKA sequence. The modification selectivity is >1000:1, the N-terminal modification efficiency is 99.2%, no Lys side chain by-modification products are generated, and the product purity is ≥98.3%. These results clearly distinguish this invention from existing techniques involving unprotected modification (selectivity ≤5:1) and short-chain fatty acid modification (selectivity ≤15:1), solving the selectivity problem in modifying the LMN-NKA sequence with long-chain fatty acids and providing key technical support for the industrial production of high-purity products.

[0083] 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.

[0084] 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 palmitoylated LMN-NKA polypeptide derivative and its pharmaceutically acceptable salt, characterized in that, The LMN-NKA polypeptide derivative is N-palmitoyl-aspartic-lysine-phenylalanine-valine-glycine-N-methyl-leucine-norleucine, with the general structural formula: C 15 H 31 CO-Asp-Lys-Phe-Val-Gly-N-Me-Leu-Nle.

2. The N-terminal palmitoylated modified LMN-NKA polypeptide derivative and its pharmaceutically acceptable salt according to claim 1, characterized in that, Only the α-amino group of the aspartic acid is replaced by a palmitoyl group, the ε-amino group of the lysine and the side chains of other amino acids are unmodified, and the terminal of the ortholeucine is a free carboxyl group; and / or, all amino acids are in the L-configuration; and / or, the pharmaceutically acceptable salt is selected from one or more of hydrochloride, acetate, citrate, and succinate.

3. The N-terminal palmitoylated / C-terminal ethylamine-modified LMN-NKA polypeptide derivative and its pharmaceutically acceptable salt according to claim 1, characterized in that, The LMN-NKA polypeptide derivative has a logP value of 3.65±0.08, an isoelectric point of 8.3±0.2, and a specific rotation [α]. 25 D = -40.8°±0.3° (c=1.0, methanol), melting point 242.3~244.1℃, moisture content ≤0.5%; and / or, the ESI-MS measured molecular ion peak m / z of the LMN-NKA polypeptide derivative is 1014.38~1014.46 Da; and / or, the enzymatic half-life of the LMN-NKA polypeptide derivative at 37℃ under trypsin (enzyme:peptide = 1:50, w / w) conditions is ≥8.0 h, and the residual rate after incubation in human plasma for 4 h is ≥85%; NK2 receptor binding Ki value ≤0.82 nM, and selectivity for NK1 and NK3 receptors ≥100-fold.

4. The method for preparing the LMN-NKA polypeptide derivative according to any one of claims 1 to 3, characterized in that, The preparation method adopts solid-phase synthesis combined with N-terminal directional palmitoylation technology, including the following steps: (1) providing a solid-phase reaction column loaded with 2-chlorotriphenylmethyl chloride resin, and loading the initial amino acid Nle onto the 2-chlorotriphenylmethyl chloride resin; (2) in the solid-phase reaction column, coupling in the order of Nle→N-Me-Leu→Gly→Val→Phe→Lys→Asp to obtain LMN-NKA polypeptide; (3) in the solid-phase reaction column, performing directional palmitoylation modification on the N-terminus of the LMN-NKA polypeptide to obtain an N-terminal palmitoylated modified LMN-NKA polypeptide derivative; (4) using a cleavage reagent to cleave and separate the N-terminal palmitoylated modified LMN-NKA polypeptide derivative from the 2-chlorotriphenylmethyl chloride resin, and obtaining the purified N-terminal palmitoylated modified LMN-NKA polypeptide derivative after post-processing.

5. The method for preparing the LMN-NKA polypeptide derivative according to claim 4, characterized in that, In step (3), a DMF / DCM mixed solution containing palmitoyl chloride and DIPEA is added to the solid-phase reaction column to react and obtain the N-terminal palmitoylated LMN-NKA peptide. Preferably, the content of palmitoyl chloride is 3-5 mmol; and / or, preferably, the content of DIPEA is 3-5 mmol; and / or, the volume ratio of DMF to DCM is 1:(1-3); and / or, preferably, the reaction temperature is 20-30°C; and / or, preferably, the reaction time is [missing information]. 3~5h; and / or, the preparation method of palmitoyl chloride is as follows: palmitic acid and thionyl chloride are refluxed in the presence of a catalyst, and the resulting reaction solution is collected by vacuum distillation at 160~162℃ / 2kPa, which is the palmitoyl chloride. Preferably, the molar ratio of palmitic acid to thionyl chloride is 1:(1.1~1.5); and / or, preferably, the catalyst is DMF; and / or, preferably, the reflux reaction temperature is 70~80℃; and / or, preferably, the reflux reaction time is 1~3h.

6. The method for preparing the LMN-NKA polypeptide derivative according to claim 4, characterized in that, In step (4), the cleavage reagent is a mixed solution of TFA, TIS, and H2O in a volume ratio of (36~40):(0.8~1.2):1; and / or, the cleavage reagent is added to the solid-phase reaction column, reacted at 20~30℃ for 3~4 days, the filtrate is collected by filtration, and the filtrate is post-treated. Preferably, the post-treatment includes adding the filtrate to ice-cold ether pre-cooled to -20℃, collecting the precipitate (crude peptide) by centrifugation, dissolving the crude peptide with TFA aqueous solution, purifying it by reversed-phase HPLC, and lyophilizing it to obtain a white powder. Preferably, the mass-volume percentage of the TFA aqueous solution is 0.08%~0. 12%, preferably, the purification is performed using a C18 column with gradient elution using mobile phase A and mobile phase B. Mobile phase A is a TFA aqueous solution with a mass-volume percentage of 0.08%~0.12%, and mobile phase B is a TFA acetonitrile solution with a mass-volume percentage of 0.08%~0.12%. The gradient elution conditions are: B phase 40%→85%, 30~40 min, flow rate 0.8~1.5 mL / min, detection wavelength 220 nm; the target peak with a purity ≥98.8% is collected. Preferably, the freeze-drying conditions are -60~-40℃, 0.008~0.012 mbar for 18~25 h.

7. The method for preparing the LMN-NKA polypeptide derivative according to claim 4, characterized in that, In step (1), the degree of substitution of the 2-chlorotriphenylmethyl chloride resin is 0.5~0.8 mmol / g. Preferably, after adding the 2-chlorotriphenylmethyl chloride resin to the solid-phase reaction column, DMF is added for soaking, and after drying, the resin is washed with DCM. And / or, in step (2), the coupling operation method includes: adding a DMF solution containing piperidine to the solid-phase reaction column, stirring and mixing at 20~30°C, washing with DMF, adding a DMF solution containing Fmoc-protected amino acid, HBTU, HOBt and DIPEA, stirring and reacting at 20~30°C, and using ninhydrin to detect the reaction endpoint.

8. A method for preparing a pharmaceutically acceptable salt of the LMN-NKA polypeptide derivative as described in any one of claims 1 to 3, characterized in that, The LMN-NKA polypeptide derivative is reacted with an acid in a mixed solvent of alcohol and water, and after concentration under reduced pressure to remove methanol, it is recrystallized using an ether to obtain the pharmaceutically acceptable salt. Preferably, the volume ratio of alcohol to water is (1~3):1; and / or, preferably, the alcohol is methanol; and / or, preferably, the acid is fed in the form of an aqueous acid solution with a pH of 2~3; and / or, preferably, the reaction 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 3 in the preparation of a medicament for treating chronic diseases related to the neurokinin NK2 receptor.

10. The application as described in claim 9, characterized in that, The drug is formulated as an oral, inhaled, injectable, or transdermal formulation; and / or, the treatment of neurokinin NK2 receptor-related chronic diseases includes allergic asthma, rheumatoid arthritis, neurogenic pain, inflammatory bowel disease, and obesity.

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