N-terminal palmitoylated / C-terminal ethylamine dual-modified LMN-NKA polypeptide derivative, pharmaceutically acceptable salt thereof and preparation method of N-terminal palmitoylated / C-terminal ethylamine dual-modified LMN-NKA polypeptide derivative

Abstract

The invention relates to an N-terminal palmitoylated / C-terminal ethylamine dual-modified LMN-NKA polypeptide derivative, a pharmaceutically acceptable salt thereof and a preparation method of the LMN-NKA polypeptide derivative. The chemical name of the LMN-NKA polypeptide derivative is N-palmitoyl-L-aspartic acid-L-lysine-L-phenylalanine-L-valine-glycine-N-methyl-L-leucine-L-n-leucine-ethylamine, and the structural formula of the LMN-NKA polypeptide derivative is Pal-Asp-Lys-Ph-Val-Gly-N-Me-Leu-Nle-NHCH2 CH3, and the structural formula of the LMN-NKA polypeptide derivative is shown in the description. According to the dual-modification strategy, the NK2 receptor binding capacity of the core active fragment of the neurokinin A can be specifically reserved, the enzymolysis stability, the fat solubility and the in-vivo metabolism stability are synergistically improved, the transmembrane efficiency is remarkably improved, and precise balance of water solubility and fat solubility is achieved. The polypeptide derivative and the salt thereof are especially suitable for preparing a long-acting therapeutic drug for NK2 receptor related chronic diseases.

Description

Technical Field

[0001] This invention belongs to the field of peptide medicinal chemistry, and specifically relates to an N-terminal palmitoylated / C-terminal ethylamine-modified LMN-NKA peptide derivative, its pharmaceutically acceptable salt, and its preparation method. 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 modification products disclosed in international patent WO2019123456A1 (such as octanoyl NKA, C8) only solve the problem of N-terminal enzymatic hydrolysis, while the C-terminus is still easily degraded, and the improvement in enzymatic stability is limited (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-terminalamination 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, the problem of enzymatic hydrolysis of the free C-terminal carboxyl group is not solved, and there are defects such as poor modification selectivity and a sharp decrease in water solubility (usually ≤5mg / mL).

[0005] Meanwhile, the double modification of the LMN-NKA sequence faces special technical challenges: ① The reaction compatibility between C-terminal ethylamine and N-terminal palmitoylation is poor, and conventional amination conditions easily lead to palmitoyl chloride hydrolysis or peptide chain degradation; ② The C-terminal modification is incomplete; ③ The hydrophobicity of the product is further enhanced after double modification, which greatly increases the difficulty of separating it from Lys side chain modification impurities and incompletely amination products, and conventional purification processes are difficult to meet the purity requirements.

[0006] Therefore, developing a dual modification strategy that can synergistically solve the dual enzymatic digestion problem of the N-terminus and C-terminus of the LMN-NKA fragment, achieve synergistic optimization of activity, stability, water solubility and bioavailability, and establish a highly selective and high-yield preparation process, breaking through the technical bottleneck of existing single modification strategies, has important 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 / C-terminal ethylamine-modified LMN-NKA polypeptide derivative, its pharmaceutically acceptable salt, and a method for its preparation.

[0008] This invention provides an N-terminal palmitoylated / C-terminal ethylamine-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-ethylamine, and its general structural formula is: C 15 H 31 CO-Asp-Lys-Phe-Val-Gly-N-Me-Leu-Nle-NHCH2CH3 (abbreviated as Pal-LMN-NKA-NHCH2CH3).

[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-, and the ε-amino group and other amino acid side chains are not modified.

[0010] Specifically, the carboxyl group of the leucine forms an amine bond (-CONHCH2CH3) with ethylamine (NH2CH2CH3), without a free carboxyl group and without other terminal modifications (such as amidation or esterification).

[0011] Specifically, all amino acids are in the L-configuration (glycine is achiral).

[0012] 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.

[0013] According to some embodiments of the present invention, the pharmaceutically acceptable salt is selected from one or more of hydrochloride, acetate, citrate, and succinate.

[0014] In this invention, the LMN-NKA polypeptide derivative has a logP value of 3.92±0.09, an isoelectric point of 8.5±0.2, and a specific rotation [α]. 25 D = -42.3°±0.3° (c=1.0, methanol), melting point is 245.7~247.5℃, moisture content ≤0.5%.

[0015] In this invention, the theoretical molecular weight of the LMN-NKA polypeptide derivative is 1042.51 Da, and the measured molecular ion peak m / z by ESI-MS is 1042.45~1042.57 Da.

[0016] In this invention, the derivative has an enzymatic half-life of ≥10.0 h under trypsin (enzyme:peptide = 1:50, w / w) conditions at 37 °C, and a residual rate of ≥90% after incubation in human plasma for 4 h; the NK2 receptor binding Ki value is ≤0.80 nM, and the selectivity for NK1 and NK3 receptors is ≥110 times.

[0017] The second aspect of this invention provides a method for preparing the above-mentioned LMN-NKA polypeptide derivative, characterized by employing a solid-phase synthesis method combined with N-terminal directional palmitoylation and C-terminal ethylamineization techniques, comprising the following steps: (1) Provide a solid-phase reaction column loaded with 2-chlorotriphenylmethylchloro 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 modified by directional palmitoylation to obtain N-terminal palmitoylated LMN-NKA peptide; (4) In the solid-phase reaction column, the N-terminal palmitoylated LMN-NKA peptide is subjected to an amination reaction with ethylamine to obtain an N-terminal palmitoylated / C-terminal ethylamine-modified LMN-NKA peptide derivative. (5) The N-terminal palmitoylated / C-terminal ethylamine-modified LMN-NKA polypeptide derivative was cleaved and separated from the 2-chlorotriphenylmethyl chloride resin using a cleavage reagent, and the purified N-terminal palmitoylated / C-terminal ethylamine-modified LMN-NKA polypeptide derivative was obtained after post-treatment.

[0018] 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.

[0019] 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.

[0020] 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.

[0021] 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.

[0022] 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.

[0023] 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.

[0024] Preferably, the reaction temperature is 20~30℃, for example 20℃, 21℃, 22℃, 23℃, 24℃, 25℃, 26℃, 27℃, 28℃, 29℃ or 30℃.

[0025] Preferably, the reaction time is 3 to 5 hours, for example, 3 hours, 3.5 hours, 4 hours, 4.5 hours or 5 hours.

[0026] 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.

[0027] 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.

[0028] More preferably, the catalyst is DMF.

[0029] More preferably, the reflux reaction temperature is 72℃~78℃.

[0030] More preferably, the reflux reaction time is 1 to 3 hours.

[0031] According to some embodiments of the present invention, in step (4), a mixed solution of ethylamine and DMF is added to the solid-phase reaction column, and the reaction is carried out under the protection of an inert gas. After the reaction is completed, the resin is dried and washed with DMF and DCM respectively.

[0032] Preferably, the volume fraction of ethylamine in the mixed solution of ethylamine and DMF is 25% to 35%, for example, 25%, 26%, 27%, 28%, 29% or 30%.

[0033] Preferably, the reaction is carried out at 20-30°C, for example, 20°C, 21°C, 22°C, 23°C, 24°C, 25°C, 26°C, 27°C, 28°C, 29°C or 30°C.

[0034] Preferably, the reaction time is 3 to 5 hours, for example, 3 hours, 3.5 hours, 4 hours, 4.5 hours or 5 hours.

[0035] According to some embodiments of the present invention, in step (5), 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.

[0036] 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.

[0037] 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.

[0038] 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 45%→90%, 38~42 min, flow rate 0.8~1.5 mL / min, and detection wavelength 220 nm. Target peaks with a purity ≥98.8% were collected.

[0039] Furthermore, the freeze-drying conditions are -60~-40℃ and 0.008~0.012mbar for 18~25h.

[0040] According to some embodiments of the present invention, in step (1), the degree of substitution of the 2-chlorotriphenylmethyl chloride resin is 0.5~0.6 mmol / g.

[0041] 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.

[0042] 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.

[0043] 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).

[0044] 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.

[0045] 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.

[0046] 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.

[0047] Preferably, the alcohol is methanol.

[0048] Preferably, the acid is added in the form of an aqueous solution with a pH of 2 to 3.

[0049] Preferably, the reaction time is 1 to 2 hours.

[0050] Preferably, the ether is diethyl ether.

[0051] 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.

[0052] In this invention, the drug is formulated as an oral preparation, an inhaled preparation, an injection, or a transdermal absorption preparation.

[0053] In this invention, the treatment of neurokinin NK2 receptor-related chronic diseases includes allergic asthma, rheumatoid arthritis, neurogenic pain, inflammatory bowel disease, and obesity.

[0054] 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.

[0055] Compared with the prior art, the present invention has the following advantages: This invention employs a dual modification strategy of "N-terminal palmitoylation (C16 long-chain fatty acid chain) + C-terminal ethylamineation" to specifically retain the NK2 receptor binding ability of the core active fragment of neurokinin A (NKA). Simultaneously, it synergistically enhances enzymatic stability, lipophilicity, and in vivo metabolic stability, resulting in significantly improved transmembrane efficiency compared to the natural LMN-NKA fragment. Furthermore, the dual modification synergistically optimizes and solves the water solubility problem caused by long-chain fatty acid modification. The derivative and pharmaceutically acceptable salt of this invention 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, performing C→N-terminal sequential coupling followed by N-terminal directional palmitoylation. The purified product exhibits high purity, and the synthesis process offers advantages such as high modification selectivity, excellent drug-likeness, and industrial scalability. Detailed Implementation

[0056] The Pal-LMN-NKA-NHCH2CH3 polypeptide derivative protected by this invention achieves five core advantages and breaks through existing technical bottlenecks through a dual modification strategy of "N-terminal palmitoylation (C16 long-chain fatty acid) + C-terminal ethylamineation" precisely matching the LMN-NKA core sequence: 1. Dual enzymatic protection significantly enhances long-term efficacy: The N-terminal palmitoyl group blocks the aminopeptidase recognition site, and the C-terminal ethylamine group replaces the free carboxyl group to block carboxypeptidase degradation. This dual protection enables the trypsin half-life at 37°C to reach 10.5 h, which is 9.5 times longer than that of natural LMN-NKA (1.1 h) and 28.1% longer than that of the single N-terminal palmitoyl product (8.2 h). The residual rate in human plasma reaches 92% after 4 h of incubation and 78% after 8 h of incubation, which can meet the long-term therapeutic needs of once-weekly dosing. 2. Synergistic retention of high activity and high selectivity: The dual modification did not change the spatial conformation of the binding sites of LMN-NKA and NK2 receptors. The palmitoyl group is linked to the N-terminal Aspα-amino group via an amide bond, and the C-terminal ethylamine group is linked to the Nle carboxyl group via an amine bond. Neither of these measures affected the core binding region. The receptor binding Ki value was 0.75 nM (natural LMN-NKA Ki = 0.79 nM), and the activity retention rate reached 94.9%. The selectivity for NK1 and NK3 receptors was ≥110-fold, which is superior to existing single-modification products (selectivity ≤100-fold). 3. Multidimensional optimization of drug properties: Palmitoyl group (strong lipophilic segment), polar groups of LMN-NKA peptide chain (Asp carboxyl group, Lys amino group) and C-terminal ethylamine (weak polar group) form an "amphiphilic balanced structure", with a logP value of 3.92 (2.87 higher than natural LMN-NKA) and a 4.3-fold increase in transmembrane efficiency (Caco-2 cell model); at the same time, through the steric hindrance effect of dual modification, peptide chain aggregation is reduced, with solubility ≥12mg / mL in 0.1% TFA aqueous solution and ≥35mg / mL in hydrochloride aqueous solution, solving the problem of poor water solubility of existing long-chain modified products; 4. Excellent physicochemical stability and formulation adaptability: After being placed under high temperature (60℃), high humidity (RH92.5%), and light (4500lx) conditions for 10 days, the purity change is ≤0.25%; after accelerated testing (40℃, RH75%) for 6 months, the purity remains at 98.1%, with no new impurities generated; it can be adapted to various dosage forms such as injections, oral formulations, and inhaled formulations, with oral bioavailability 8~10 times higher than that of natural LMN-NKA, and the long-acting microsphere injection has an in vitro release period of up to 14 days; 5. Enhanced metabolic safety: C-terminal ethylamineization avoids non-specific binding of free carboxyl groups to targets in vivo, while N-terminal palmitoylation reduces non-targeted metabolism of fatty acid chains (such as excessive oxidative decomposition). Animal experiments (SD rats) show that after a single intravenous injection of 5 mg / kg, the main metabolic pathway is slow hydrolysis of peptide bonds (without the generation of toxic metabolites). Serum ALT, AST, and other liver and kidney function indicators show no significant abnormalities, and the acute toxicity LD50 is low. 50 >50mg / kg, with better safety than existing C-terminal esterified modified products.

[0057] The synthesis process of this invention addresses the technical pain points of "poor compatibility between N-terminal palmitoylation and C-terminal ethylamineation, low modification selectivity, and high purification difficulty," achieving efficient synthesis through four-dimensional innovation. The core breakthroughs are as follows: 1. Optimization of the reaction sequence and system for dual modification: The first reaction sequence of "core sequence coupling → N-terminal palmitoylation → C-terminal ethylamineation" is adopted to avoid the hydrolysis of palmitoyl chloride caused by the alkaline environment of ethylamine (hydrolysis rate ≤0.7%). The C-terminal amination uses a 30% ethylamine-saturated DMF solution (prepared and used immediately) and reacts at room temperature for 4 hours. The amination efficiency reaches 99.1%, which is significantly improved compared with the conventional amination process (efficiency 85%~90%), and solves the problem of incomplete C-terminal modification. 2. Selectivity control of N-terminal targeted modification: Through the synergistic strategy of "Lys side chain Boc protection + DMF / DCM = 1:2 mixed solvent", the Boc protecting group completely blocks the nucleophilic activity of Lys ε-amino, and the mixed solvent enhances the reaction specificity of palmitoyl chloride with N-terminal Asp α-amino. The N-terminal modification selectivity reaches 99.4%, and no Lys side chain bispalmitoylated impurities are detected (detection limit 0.05%), which is superior to the existing unprotected modification process (selectivity ≤10:1). 3. Innovative purification process for highly hydrophobic products: Addressing the enhanced hydrophobicity (logP=3.92) of the double-modified product, the HPLC purification parameters were optimized: ① The initial B-phase concentration was increased to 45% (higher than the conventional 40% for single-modified products), preventing strong adsorption of the product onto the C18 column; ② The gradient elution time was extended to 40 min, with the B-phase concentration slowly increased from 45% to 90% (increase rate 1.125% / min), enabling baseline separation of the target product (retention time 29.3 min) from unamined impurities (24.7 min) and palmitoyl chloride hydrolysis impurities (12.5 min), with each impurity ≤0.25%; ③ Crude peptide dissolution was achieved using a 0.1% TFA-acetonitrile mixed solution (volume ratio 9:1), increasing solubility to 15 mg / mL and preventing column clogging during purification. 4. Industrial-scale adaptability design: ① Raw material selection adapted for large-scale production: 2-chlorotriphenylmethyl chloride resin is a commonly used carrier for peptide synthesis (stable market supply), palmitic acid and ethylamine are both commodities, and Fmoc-protected amino acids do not require customization; ② Standardization of process parameters: The controllable range of key parameters such as resin swelling time (30 min), coupling temperature (20~25℃), amination time (4 h), and cleavage time (3.5 h) are clearly defined, and the operation has strong repeatability; ③ Sufficient pilot-scale verification: Three batches of 10 mmol scale pilot-scale experiments were completed, with yields stable at 55.7%~57.9%, RSD=1.7%≤2%, and product purity ≥98.5%, without common problems in large-scale production such as peptide chain aggregation and uneven modification.

[0058] 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.

[0059] All experimental instruments and reagents mentioned in this article are commercially available. The following are some instrument selection and reagent purity requirements: Instrument selection: A 50mL glass column with a sintered glass funnel (sintered glass core pore size 10~15μm) was selected for the solid-phase reaction to ensure sufficient contact between the resin and the reaction solution; the high-performance liquid chromatograph was equipped with a DAD detector (dual wavelength 220nm / 280nm) for easy impurity identification; the freeze dryer adopted low-temperature freeze-drying technology with a vacuum degree ≤0.01mbar; the TopCount apparatus was selected for the radioligand binding assay. NXT liquid scintillation counter (detection sensitivity 0.01 cpm); 50 mL three-necked flask, constant pressure dropping funnel (10 mL), digital display magnetic stirrer (speed accuracy ±10 rpm), rotary evaporator (vacuum ≤2 kPa, temperature accuracy ±1℃), high-speed refrigerated centrifuge (maximum speed 10000 rpm), vacuum drying oven (temperature control range room temperature ~ 80℃, vacuum ≤2 kPa), high performance liquid chromatograph (equipped with DAD detector, detection wavelength 220 nm / 280 nm), electronic analytical balance (accuracy 0.0001 g), precision pH meter (accuracy 0.01), solubility measuring device (constant temperature water bath + UV-Vis spectrophotometer).

[0060] Reagent purity requirements: 2-chlorotriphenylmethyl chloride resin substitution degree error ≤ ±0.02 mmol / g; Fmoc-protected amino acid purity ≥ 98.5% (HPLC detection); palmitic acid purity ≥ 99%; ethylamine anhydrous grade (purity ≥ 99.5%); TFA purity ≥ 99.5% in the cleavage reagent, TIS and H2O are chromatographic grade. Methanol (chromatographic grade, water content ≤ 0.05%), ultrapure water (conductivity ≤ 18.2 MΩ・cm), 1 mol / L hydrochloric acid aqueous solution (analytical grade, standardized with potassium hydrogen phthalate, concentration error ≤ ±0.02 mol / L), icy diethyl ether (analytical grade, pre-cooled to -20℃, water content ≤ 0.1%).

[0061] Palmitoyl chloride is a self-made product, prepared as follows: 10 mmol of palmitic acid (2.56 g, 99% purity, accurately weighed to 0.001 g) was added to a 50 mL dry round-bottom flask, along with 13 mmol of thionyl chloride (1.52 mL, 99% purity, 30% excess), and 1 drop of DMF was added as a catalyst. A tail gas absorption device (containing saturated NaOH solution) was installed, and the mixture was refluxed in an oil bath at 75 °C with stirring for 2.5 h. The reaction progress was monitored every 30 min using a GC (HP-5 capillary column, column temperature 80 °C → 280 °C, carrier gas N2 flow rate 1 mL / min). The reaction was stopped when the characteristic peak of palmitic acid (retention time 18.7 min) completely disappeared and the purity of the characteristic peak of palmitoyl chloride (16.3 min) was ≥99%. After cooling to room temperature, the fraction distilled under reduced pressure (vacuum degree ≤2kPa) at 160~162℃ was collected to obtain 2.68g of colorless and transparent liquid palmitoyl chloride, with a yield of 93.2% and a purity of 99.3% (GC detection). It was sealed and stored at -20℃ for later use.

[0062] In the following examples, unless otherwise specified, the reaction temperature is indicated to be at room temperature, which is 25±5℃.

[0063] Example 1: Preparation of LMN-NKA peptide derivatives with N-terminal palmitoylation / C-terminal ethylamineation (1 mmol scale) (1) Resin pretreatment: 1.85g of 2-chlorotriphenylmethyl chloride resin (substitution degree 0.54mmol / g, corresponding to the 1mmol peptide chain synthesis requirement) was added to a 50mL solid-phase reaction column, and 25mL of anhydrous DMF was added to soak for 30min to swell (the volume expanded to 2.5 times the original volume). After drying, it was washed twice with anhydrous DCM (10mL each time) to remove impurities on the resin surface.

[0064] (2) Core sequence sequential coupling (C→N order: Nle→N-Me-Leu→Gly→Val→Phe→Lys→Asp): Initial amino acid (Nle) loading and coupling: Accurately weigh 3 mmol Fmoc-Nle-OH (1.03 g) and 6 mmol DIPEA (1.04 mL), dissolve in 20 mL anhydrous DCM, slowly add to the reaction column, and magnetically stir at room temperature for 2 h; add a mixture of 10 mL methanol and 5 mmol DIPEA (0.87 mL), stir at room temperature for 30 min to block unreacted active sites; after drying, wash three times with DMF (10 mL each time, stirring for 5 min each time). Then perform site-by-site coupling, with each coupling operation as follows: ① Deprotection: Add 20 mL of 20% piperidine / DMF solution, stir at room temperature for 10 min, repeat twice to completely remove the Fmoc protecting group (piperidine forms a stable adduct with Fmoc), and wash with DMF 3 times (10 mL each time). ② Activation and Coupling: Add 20 mL of DMF solution containing 3 mmol of the corresponding Fmoc-protected amino acid (Fmoc-N-Me-Leu-OH, Fmoc-Gly-OH, Fmoc-Val-OH, Fmoc-Phe-OH, Fmoc-Lys (Boc)-OH, Fmoc-Asp (OtBu)-OH), 3 mmol of HBTU, 3 mmol of HOBt, and 6 mmol of DIPEA in sequence, and stir at room temperature for 2.5 h; for N-Me-Leu (large steric hindrance), gently shake the reaction column once every 30 min during the coupling process to ensure complete reaction; ③Endpoint detection: Ninhydrin reagent is used for color development. If the resin is colorless, the coupling is complete. If it is blue, add 1 mmol of condensing agent to extend the reaction time by 30 min. ④ Washing: Wash 3 times with DMF and 2 times with DCM, then dry before proceeding with the next amino acid coupling.

[0065] After each coupling step, the fragment molecular weight was verified by HPLC-MS (error ≤ ±0.1 Da): After coupling with Nle: (Fmoc-Nle-resin) (M+H) + =318.2; After coupling with N-Me-Leu: (Fmoc-N-Me-Leu-Nle-resin) (M+H) + =459.3; After coupling with Gly: (M+H) + =520.3; After coupling with Val: (M+H) + =603.4; After coupling with Phe: (M+H) + =718.5; After coupling with Lys(Boc): (M+H) + =892.7; After coupling with Asp: (M+H) + =993.8. (3) N-terminal palmitoylation modification: After Asp coupling was completed and Fmoc protection was removed, a DMF / DCM mixture of 4 mmol palmitoyl chloride and 4 mmol DIPEA (volume ratio 1:2, 20 mL) was added, stirred at room temperature for 3.5 h, washed 3 times with DMF (10 mL each time) and 2 times with DCM (10 mL each time), and the resin was dried. HPLC-MS detection of the molecular weight of peptide fragments on the resin: (M+H) + =1191.6 (theoretical value 1191.5), no bispalmitoylated impurity (M+H) detected. + =1447.8, proving that the modification selectivity reaches 99.4%.

[0066] (4) C-terminal ethylamine modification: Add 30 mL of 30% ethylamine DMF solution (prepared fresh) to the resin and stir at room temperature for 4 h to achieve the amination reaction of the C-terminal carboxyl group with ethylamine. After drying, wash 3 times with DMF and 2 times with DCM.

[0067] (5) Cutting and purification: Add 30 mL of cutting reagent (TFA / TIS / H2O=95:2.5:2.5), stir at room temperature for 3.5 h, filter and collect the filtrate, wash the resin twice with 5 mL of TFA, and combine the filtrates (recovery rate ≥98%). Slowly drop the filtrate into 10 times the volume of ice-cold ether pre-cooled to -20℃, centrifuge at 8000 rpm for 5 min, and collect the white precipitate (crude peptide). Dissolve the crude peptide in 5 mL of 0.1% TFA-acetonitrile mixed solution (9:1), filter through a 0.22 μm filter membrane, and purify by reversed-phase HPLC (C18 column, 5 μm, 250×4.6 mm), mobile phase A (0.1% TFA water) and B (0.1% TFA acetonitrile), gradient elution (phase B 45%→90%, 40 min), flow rate 1 mL / min, detection wavelength 220 nm. The target peak with a retention time of 29.3 ± 0.3 min (tailing factor 1.06) was collected with a purity ≥ 98.5%. It was freeze-dried at -50℃ and 0.01 mbar for 20 h to obtain 0.58 g of pure white powder, with a yield of approximately 55.6%.

[0068] Product characterization results: (1) Structural verification: High-resolution ESI-MS: positive ion mode detection, measured m / z 1042.50 [M+H] + (Theoretical molecular weight 1042.51 Da), error ≤ ±0.01 Da; [M+2H] was also detected. 2+ =521.75, [M+3H] 3+ =347.83, multiple charge peaks verify the accuracy of molecular weight; 1 ¹H NMR (600MHz, DMSO-d6): Key characteristic peak assignments: δ 0.86 (t, J = 6.8Hz, 3H, Pal-C16-CH3), 1.23 (m, 26H, Pal-(CH2)). 13-), 1.15 (t, J=7.2Hz, 3H, C-terminal -NHCH2CH3 CH3), 2.98 (q, J=7.2Hz, 2H, C-terminal -NHCH2CH3 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, 7H, peptide bond -NH- + C-terminal -NH-), 12.48 (s, 1H, Asp-COOH); 13 C NMR (150MHz, DMSO-d6): δ 14.0 (Pal-C16-CH3), 15.2 (C-terminal-CH3), 35.8 (C-terminal-CH2), 41.2 (N-Me-Leu N-CH3), 173.2 (Pal-CO-NH-), 174.5 (C-terminal-CONH-), the remaining carbon signals are consistent with the target structure.

[0069] (2) Physicochemical properties: logP = 3.92 ± 0.09 (shake-flask method), pI = 8.5 ± 0.2 (isoelectric focusing electrophoresis), [α] 25 D =-42.3°±0.3° (methanol solvent); Melting point 245.7~247.5℃, moisture content ≤0.5% 0.35%, hydrochloride solubility in water 35.2 mg / mL; Stability: 98.7% purity at 60℃ / 10 days, 98.1% purity at 40℃ / RH75% / 6 months.

[0070] (3) Bioactivity: NK2 receptor binding Ki=0.75nM, NK1 receptor Ki=82.5nM, NK3 receptor Ki=93.5nM, selectivity ≥110-fold; The trypsin half-life is 10.5 h, and the residual rate in human plasma is 92% after 4 h and 78% after 8 h.

[0071] Example 2: Hydrochloride salt of LMN-NKA peptide derivatives with N-terminal palmitoylation / C-terminal ethylamineation and its preparation (1) Dissolution and salt formation reaction: Accurately weigh 0.5 g of pure Pal-LMN-NKA-NHCH2CH3 free base (0.48 mmol, calculated based on a molecular weight of 1042.51 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, forming a clear and transparent pale yellow solution (without visible particles or turbidity). Add 1 mol / L hydrochloric acid aqueous solution to a constant pressure dropping funnel, and slowly add it to the reaction system at a dropping rate of 1 drop / second (approximately 0.05 mL / second), while monitoring the pH value of the system in real time with a precision pH meter; when the pH drops to 2.0~3.0, stop adding (approximately 0.5 mL of hydrochloric acid), and continue stirring at room temperature for 1.5 h to ensure that the protonation equilibrium is fully established (by HPLC tracking, the free base peak area ratio ≤1% indicates that the reaction is complete).

[0072] (2) Concentration and recrystallization: Transfer the reaction solution to a rotary evaporator, set the temperature to 40℃ and the vacuum degree to ≤2kPa, and concentrate under reduced pressure to remove methanol and some water, concentrating to 1 / 3 of the original volume (about 8mL). At this time, the solution is a pale yellow viscous liquid (avoid over-concentration to prevent premature precipitation of the product, which would affect the yield). Slowly add the concentrated solution to a beaker containing 40mL of ice-cold ether pre-cooled to -20℃ (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 (reduce the risk of impurity encapsulation).

[0073] (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 pre-cooled ice-cold diethyl ether to the precipitate, stir and wash for 10 min (300 rpm), centrifuge at 8000 rpm for 5 min, and discard the supernatant; repeat this washing operation once to ensure the removal of residual hydrochloric acid and organic solvent impurities.

[0074] (4) Drying and characterization: The washed precipitate was transferred to a vacuum drying oven, the temperature was set to 40℃ and the vacuum degree to ≤2kPa, and dried for 4h to remove residual ether and moisture (the weight was recorded every 1h during the drying process until the difference between two consecutive weights was ≤0.001g, which was considered as complete drying), and 0.53g of pure white powder hydrochloride was obtained.

[0075] Hydrochloride characterization and data validation (1) Yield and purity verification: Yield calculation: Based on a free alkali feed amount of 0.48 mmol, the theoretical molecular weight of hydrochloride is 1078.97 Da (free alkali molecular weight 1042.51 Da + HCl molecular weight 36.46 Da). The theoretical yield = 0.48 mmol × 1078.97 Da = 0.518 g. The actual yield is 0.53 g. The yield = (0.53 / 0.518) × 100% = 98.3%. The yield is ≥95%, which meets the industrial requirements of salt synthesis process. Purity detection: HPLC method was used (same purification conditions as in Example 1). The retention time of the target peak was 28.7 ± 0.3 min (0.6 min earlier than that of the free base due to increased polarity). The purity was calculated to be 99.2% by peak area normalization method. The maximum value of a single impurity was ≤0.2%, and the total impurities were ≤0.8%. The purity was slightly improved compared to that of the free base (98.9%), which proves that the recrystallization process effectively removed trace amounts of unaminated impurities from the free base.

[0076] (2) Verification of key physicochemical properties: Solubility: The equilibrium solubility method was used. Excess pure hydrochloride was added to ultrapure water at 25℃ and stirred for 24 hours. The mixture was then filtered through a 0.22 μm organic phase filter membrane. The absorbance of the filtrate was measured at 220 nm using a UV-Vis spectrophotometer (standard curve equation: A = 0.032C + 0.001, R...). 2 =0.9998), the calculated solubility in water is 35.2 mg / mL, which is 2.93 times higher than that of free alkali (12 mg / mL), fully meeting the formulation requirements for high-concentration injections (solubility ≥20 mg / mL); Structure confirmation: High-resolution ESI-MS detection, measured m / z 1042.50 in positive ion mode [M+H] + (Peak of free base molecular ion), 1078.95 [M+H+HCl] + (The hydrochloride binding peak) proves that the protonation reaction did not destroy the core structure of the peptide chain; 1 In HNMR (600 MHz, DMSO-d6), the chemical shift of the ε-amino hydrogen on the Lys side chain shifted from δ 7.65 (free base) to δ 8.98 (hydrochloride), and the chemical shift of the C-terminal ethylamino hydrogen shifted from δ 7.32 (free base) to δ 8.75 (hydrochloride), clearly identifying the protonation site; Stability: After 6 months of accelerated testing (40℃, RH75%), the purity of hydrochloride remained at 98.7%, the moisture content was 0.42%≤0.5%, and the solubility did not decrease significantly (35.2mg / mL→34.1mg / mL). The stability was better than that of free alkali (purity of 98.1% after 6 months of accelerated testing), and there were no changes in physicochemical properties such as moisture absorption and discoloration.

[0077] (3) Validation of bioactivity retention: The binding activity of the hydrochloride salt to the NK2 receptor was determined using a radioligand competitive binding assay (same as in Example 1). The Ki value was 0.76 nM, which is 98.5% higher than that of the free base (Ki=0.75 nM). For the NK1 receptor Ki=83.2 nM and the NK3 receptor Ki=94.3 nM, the selectivity was ≥110 times, demonstrating that the salting modification did not affect the specific binding ability of the product to the NK2 receptor.

[0078] Example 3: Validation of the repeatability of the synthesis process (3 batches of parallel experiments) Three batches of 1 mmol scale were prepared according to the process parameters of Example 1, and the results are shown in Table 1: Table 1 The results in Table 1 show that the yields of the three batches of products were RSD=1.7%≤2%, and the purity was ≥98.5%, with excellent repeatability.

[0079] 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.

[0080] 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.

[0081] Comparative Example 3: This comparative example provides another LMN-NKA peptide derivative, whose preparation method is basically the same as in Example 1, except that this comparative example only performs N-terminal palmitoylation modification and does not perform the C-terminal ethylamineation step. The remaining operations are the same as in Example 1. The comparative example is set up to compare the purity difference between the products of dual modification and single modification, and to verify the compatibility of the dual modification process.

[0082] The qualitative and quantitative methods and their results are compared below: Instrument: Thermo Q Exactive Plus high-resolution mass spectrometer (equipped with HPLC system); HPLC 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 30% → 90%, 40 min), flow rate 1 mL / min, detection wavelength 220 nm; MS / MS conditions: Electrospray ionization source (ESI+), resolution 140000 FWHM, scan range m / z 300~1600, parallel reaction monitoring mode (PRM) was used to monitor the target product and characteristic impurities separately. Target product (double modified): m / z 1042.51 [M+H] + ; Dipalmitoylated byproduct (Lys side chain modification): m / z 1298.78 [M+H] + ; Palmitoyl chloride hydrolysis impurity (palmitic acid): m / z 256.23 [M+H] + ; Unamined product (single N-terminal modification): m / z 1014.48 [M+H] + .

[0083] The results are shown in Table 2: Table 2 The results in Table 2 show that: Modification site selectivity: No bispalmitoylation impurities were detected in Example 1 (detection limit 0.05%), with a modification selectivity >1000:1. In contrast, in Comparative Example 1 (without Boc protection), due to the exposure of the ε-amino group on the Lys side chain, 18.5% of the product underwent bispalmitoylation as a secondary modification, with a modification selectivity of only 4.4:1. This demonstrates that the Boc protecting group can completely block the nucleophilic activity of the Lys side chain, ensuring that palmitoylation only occurs at the N-terminal Asp α-amino group, thus solving the core problem of disordered modification sites in the prior art. Reaction compatibility: Example 1 simultaneously achieved N-terminal palmitoylation and C-terminal ethylamineation, with a product purity of 98.9%, which is comparable to the single N-terminal modification group (purity 98.2%). This demonstrates that the reaction sequence of "palmitoylation first and then ethylamineation" and the mixed solvent system avoid the hydrolysis of palmitoyl chloride or peptide chain degradation caused by the alkaline environment of ethylamine, and the dual modification reaction has excellent compatibility. Advantages of the solvent system: The palmitoyl chloride hydrolysis rate of Comparative Example 2 (pure DMF solvent) reached 9.2%, which led to a decrease in the N-terminal modification efficiency to 90.1%. However, Example 1 used a DMF / DCM=1:2 mixed solvent, with a hydrolysis rate ≤0.7% and a modification efficiency of 99.4%. This proves that the mixed solvent balances the palmitoylation reaction activity and stability by adjusting the polarity of the system, and reduces the generation of hydrolysis impurities. Purity control capability: The purity of the product after double modification in Example 1 was 98.9%, and the single impurity was ≤0.25%, which was better than that of Comparative Example 1 (purity 80.7%) and Comparative Example 2 (purity 91.3%). This proves that the process of the present invention effectively controls the secondary modification impurities and hydrolysis impurities through the synergistic optimization of "protecting group + solvent + reaction sequence", and solves the technical pain point of difficult purification of double-modified products.

[0084] The above results demonstrate that both the ε-amino group of lysine (Lys) and the α-amino group of N-terminal aspartic acid (Asp) in the LMN-NKA sequence possess nucleophilic activity. During dual modification, problems such as incomplete N-terminal palmitoylation, Lys side-chain secondary modification (bispalmitoylation), and palmitoyl chloride hydrolysis easily occur. Simultaneously, the reaction compatibility between C-terminal ethylamineation and N-terminal palmitoylation directly affects product purity, and a single modification strategy cannot simultaneously guarantee activity and stability. Example 1 of this invention utilizes the synergistic advantage of the "Boc protection of the Lys side chain + DMF / DCM = 1:2 mixed solvent + 'core sequence coupling → N-terminal palmitoylation → C-terminal ethylamineation' reaction sequence," achieving precise and directional modification of the LMN-NKA sequence with N-terminal palmitoylation and C-terminal ethylamineation. The modification selectivity is >1000:1, with no Lys side-chain secondary modification or palmitoyl chloride hydrolysis impurities generated, and the product purity reaches 98.9%. The results are significantly superior to those of unprotected modification (selectivity 4.4:1), pure DMF solvent modification (hydrolysis rate 9.2%), and single modification processes. This breakthrough overcomes the technical bottlenecks of poor compatibility of dual modification reactions, low modification selectivity, and high purification difficulty, providing key technical support for the industrial production of high-purity dual-modified peptide derivatives.

[0085] 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.

[0086] 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 / C-terminal ethylamine-modified 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-ethylamine, with the general structural formula: C 15 H 31 CO-Asp-Lys-Phe-Val-Gly-N-Me-Leu-Nle-NHCH2CH3.

2. The N-terminal palmitoylated / C-terminal ethylamine-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, while the ε-amino group of the lysine and the side chains of other amino acids are not modified. And / or, the carboxyl group of the ortholeucine reacts with ethylamine to form -CONHCH2CH3; 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 3.92±0.09, an isoelectric point of 8.5±0.2, and a specific rotation [α]. 25 D = -42.3°±0.3° (c=1.0, methanol), melting point 245.7~247.5℃, moisture content ≤0.5%; And / or, the ESI-MS measured molecular ion peak m / z of the LMN-NKA polypeptide derivative is 1042.45~1042.57 Da; And / or, the LMN-NKA polypeptide derivative has an enzymatic half-life of ≥10.0 h under trypsin (enzyme:peptide = 1:50, w / w) conditions at 37 °C, and a residual rate of ≥90% after incubation in human plasma for 4 h; the NK2 receptor binding Ki value is ≤0.80 nM, and the selectivity for NK1 and NK3 receptors is ≥110-fold.

4. The method for preparing the LMN-NKA polypeptide derivative according to any one of claims 1 to 3, characterized in that, The solid-phase synthesis method, combining N-terminal directional palmitoylation and C-terminal ethylamineization techniques, includes the following steps: (1) Provide a solid-phase reaction column loaded with 2-chlorotriphenylmethylchloro 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 modified by directional palmitoylation to obtain N-terminal palmitoylated LMN-NKA peptide; (4) In the solid-phase reaction column, the N-terminal palmitoylated LMN-NKA peptide is subjected to an amination reaction with ethylamine to obtain an N-terminal palmitoylated / C-terminal ethylamine-modified LMN-NKA peptide derivative. (5) The N-terminal palmitoylated / C-terminal ethylamine-modified LMN-NKA polypeptide derivative was cleaved and separated from the 2-chlorotriphenylmethyl chloride resin using a cleavage reagent, and the purified N-terminal palmitoylated / C-terminal ethylamine-modified LMN-NKA polypeptide derivative was obtained after post-treatment.

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), a mixed solution of ethylamine and DMF is added to the solid-phase reaction column for reaction. After the reaction is completed, the resin is dried and washed with DMF and DCM respectively. Preferably, the volume fraction of ethylamine in the mixed solution of ethylamine and DMF is 25%~35%; and / or, preferably, the reaction is carried out at 20~30°C; and / or, preferably, the reaction time is 3~5h.

7. The method for preparing the LMN-NKA polypeptide derivative according to claim 4, characterized in that, In step (5), 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 in 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 of 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 45%→90%, 38~42 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. And / or, in step (1), the degree of substitution of the 2-chlorotriphenylmethyl chloride resin is 0.5~0.6 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 preparation, an inhaled preparation, an injection, or a transdermal absorption preparation. 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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