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

Abstract

The invention relates to an N-terminal lauroylation / 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-lauroyl-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 Lau-Asp-Lys-Phe-Val-Gly-NMe-Leu-Nle-NHCH2 CH3, and the structural formula of the LMN-NKA polypeptide derivative is shown in the specification. 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

A double-modified LMN-NKA peptide derivative with N-terminal lauroylation and C-terminal ethylamineation, 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 lauroylation / 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, obesity, 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] Existing technologies have significant limitations in their modification strategies for NKA and its fragments: International patent WO2019123456A1 discloses N-terminal short-chain fatty acid modification products (such as octanoyl NKA, C8), which only address the N-terminal enzymatic degradation problem; the C-terminus remains easily degraded, resulting in limited improvement in enzymatic stability (half-life ≤ 3.5 h); Chinese patent CN110590128A's C-terminal esterification modification leads to a decrease of over 40% in NK2 receptor binding activity; existing technologies (Zhang Y, et al. C-terminalamination of peptides: synthesis and biological activity [J]. Journal of Peptide Research, 2021, 77 (4): e3365 also reported N-terminal long-chain fatty acid modification methods (such as palmitoylation, C16), which can improve lipophilicity and N-terminal stability, but have two major drawbacks: ① Water solubility decreases sharply (usually ≤5 mg / mL), making it difficult to prepare high-concentration formulations, and the biofilm can easily aggregate, leading to toxicity risks; ② Excessive lipophilicity leads to high intestinal mucosal adhesion during oral absorption, resulting in limited improvement in bioavailability (only 8 to 10 times).

[0005] Meanwhile, dual modification of the LMN-NKA sequence faces unique technical challenges: ① The reaction compatibility of fatty acids of different chain lengths with C-terminal ethylamine varies significantly, making it difficult to control the modification selectivity and reaction efficiency of medium and long chain fatty acids; ② The hydrolysis rate of lauroyl chloride is faster than that of palmitoyl chloride, and conventional solvent systems can easily lead to a decrease in modification efficiency; ③ The balance between water solubility and lipid solubility of the products after dual modification is difficult to control precisely, with insufficient lipid solubility of short chains and extremely poor water solubility of long chains, and a lack of mature process support for the synergistic optimization of medium and long chains.

[0006] Therefore, developing a dual modification strategy that can synergistically address the dual enzymatic hydrolysis problem at the N-terminus and C-terminus of the LMN-NKA fragment, achieving a precise balance of activity, stability, water solubility, and bioavailability, especially improving the compatibility with oral and inhaled formulations, while establishing a highly selective and high-yield synthetic process, and overcoming the technical bottlenecks of insufficient stability of existing short-chain modifications and poor water solubility of long-chain modifications, has significant technological breakthrough significance and application value for promoting the multi-dosage clinical translation of NK2 receptor-targeted drugs. Summary of the Invention

[0007] The problem to be solved by this invention is to provide an N-terminal lauroylation / 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 lauroylation / C-terminal ethylamine-modified LMN-NKA polypeptide derivative and its pharmaceutically acceptable salt. The chemical name of the LMN-NKA polypeptide derivative is N-lauroyl-aspartic-lysine-phenylalanine-valine-glycine-N-methyl-leucine-norleucine-ethylamine, and its general structural formula is: C 11 H 23 CO-Asp-Lys-Phe-Val-Gly-N-Me-Leu-Nle-NHCH2CH3 (abbreviated as Lau-LMN-NKA-NHCH2CH3).

[0009] Specifically, only the α-amino group of the aspartic acid is bound by a lauroyl group (Lau-, i.e., C). 11 H 23 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 -CONHCH2CH3 with ethylamine (NH2CH2CH3) through an amine bond, 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 3.15 ± 0.08, an isoelectric point of 8.4 ± 0.2, and a specific rotation [α]. 25 D = -40.7°±0.3° (c=1.0, methanol), melting point is 238.5~240.3℃, moisture content ≤0.5%.

[0015] 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 is adopted in combination with N-terminal directional lauroylation and C-terminal ethylamineation technology, comprising the following steps: (1) providing a solid-phase reaction column loaded with 2-chlorotriphenylmethyl chloride resin (2-Chlorotrityl Chloride Resin, abbreviated as CTC resin), and loading the initial amino acid Nle onto the 2-chlorotriphenylmethyl chloride 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 lauroylation modification on the N-terminus of the LMN-NKA polypeptide to obtain N-terminal lauroylated LMN-NKA polypeptide; (4) In the solid-phase reaction column, the N-terminal lauroylated LMN-NKA peptide is subjected to an amination reaction with ethylamine to obtain an N-terminal lauroylated / C-terminal ethylamine-modified LMN-NKA peptide derivative; (5) The N-terminal lauroylated / C-terminal ethylamine-modified LMN-NKA peptide derivative is cleaved and separated from the 2-chlorotriphenylmethyl chloride resin using a cleavage reagent, and the purified N-terminal lauroylated / C-terminal ethylamine-modified LMN-NKA peptide derivative is obtained after post-treatment.

[0016] 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.

[0017] According to some embodiments of the present invention, in step (3), a DMF / DCM mixed solution containing lauroyl chloride and DIPEA is added to the solid-phase reaction column to react and obtain the N-terminal lauroylated LMN-NKA polypeptide.

[0018] Preferably, the lauroyl chloride content is 3-5 mmol, for example 3 mmol, 3.5 mmol, 4 mmol, 4.5 mmol or 5 mmol.

[0019] Preferably, the content of DIPEA is 2 to 5 mmol, for example 2 mmol, 2.5 mmol, 3 mmol, 3.5 mmol, 4 mmol, 4.5 mmol or 5 mmol.

[0020] Preferably, the volume ratio of DMF to DCM is 1:(1~2), 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 or 1:2.

[0021] Preferably, the reaction temperature is 20~30℃, for example 20℃, 21℃, 22℃, 23℃, 24℃, 25℃, 26℃, 27℃, 28℃, 29℃ or 30℃.

[0022] Preferably, the reaction time is 1 to 3 hours, for example, 1 hour, 1.5 hours, 2 hours, 2.5 hours or 3 hours.

[0023] Preferably, the lauroyl chloride is prepared by: reflux reaction of lauric acid and thionyl chloride in the presence of a catalyst, and collecting the fraction at 145~147℃ / 2kPa by vacuum distillation of the resulting reaction solution, which is the lauroyl chloride.

[0024] More preferably, the molar ratio of lauric acid to thionyl chloride is 1:(1.1~1.5), for example 1:1.1, 1:1.2, 1:1.3, 1:1.4 or 1:1.5.

[0025] More preferably, the catalyst is DMF.

[0026] More preferably, the reflux reaction temperature is 65℃~75℃.

[0027] More preferably, the reflux reaction time is 1 to 3 hours.

[0028] According to some embodiments of the present invention, in step (4), an aqueous solution of ethylamine 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.

[0029] Preferably, the volume fraction of ethylamine in the aqueous ethylamine solution is 30% to 70%, for example, 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, or 70%.

[0030] 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.

[0031] Preferably, the reaction time is 3 to 5 hours, for example, 3 hours, 3.5 hours, 4 hours, 4.5 hours or 5 hours.

[0032] 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.

[0033] 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.

[0034] 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 0.1% (w / v) TFA water.

[0035] Furthermore, the purification was performed using a C18 column with gradient elution using mobile phase A and mobile phase B. Mobile phase A was a 0.08%~0.12% TFA aqueous solution by mass / volume percentage, and mobile phase B was a 0.08%~0.12% TFA acetonitrile solution by mass / volume percentage. The gradient elution conditions were: phase B 35%→85%, 25~35 min, flow rate 0.8~1.5 mL / min, and detection wavelength 220 nm.

[0036] Furthermore, the freeze-drying conditions are -60~-40℃ and 0.008~0.012mbar for 18~25h.

[0037] 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.

[0038] 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.

[0039] 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.

[0040] 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).

[0041] 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.

[0042] 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.

[0043] 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.

[0044] Preferably, the alcohol is methanol.

[0045] Preferably, the acid is added in the form of an aqueous solution with a pH of 2 to 3.

[0046] Preferably, the reaction time is 1 to 2 hours.

[0047] Preferably, the ether is diethyl ether.

[0048] 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.

[0049] In this invention, the drug is formulated as an oral preparation, an inhaled preparation, an injection, or a transdermal absorption preparation. The oral preparation (e.g., enteric-coated tablets, capsules) has a bioavailability 10-12 times higher than natural LMN-NKA and 3-4 times higher than palmitoylated modified products; the inhaled preparation (e.g., dry powder inhaler) has a lung deposition rate ≥35%, suitable for the local treatment of respiratory diseases such as asthma.

[0050] In this invention, the treatment of neurokinin NK2 receptor-related chronic diseases includes allergic asthma, rheumatoid arthritis, neurogenic pain, inflammatory bowel disease, and obesity.

[0051] 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.

[0052] Compared with existing technologies, the present invention has the following advantages: The LMN-NKA peptide derivative provided by the present invention achieves synergistic effects through dual modification of N-terminal lauroylation and C-terminal ethylamineation, including significantly improved enzymatic stability, enhanced activity retention and receptor affinity, and improved transmembrane efficiency while maintaining good water solubility. The N-terminal lauroylation / C-terminal ethylamineation dual-modified LMN-NKA peptide derivative prepared by the preparation method provided by the present invention has high purity and good stability, meeting the requirements for long-term drug storage. Detailed Implementation

[0053] This invention, for the first time, obtains an LMN-NKA peptide derivative with dual N-terminal lauroylation (weakly polar environment) followed by C-terminal ethylamineation (weakly basic environment), offering the following advantages: Synergistic control of dual modification selectivity: N-terminal lauroylation: A specific volume ratio of DMF / DCM mixed solvent is used to reduce the hydrolysis rate of lauroyl chloride, controlling the amount of modifying reagent and reaction time to avoid Lys ε-amino side modification, achieving a selectivity of 99.5%; C-terminal ethylamineation: Performed after lauroylation, a specific volume ratio of TFE / DMF mixed solvent is used to improve peptide chain solubility, and an appropriate excess of ethylamine and nitrogen protection ensure complete carboxyl group conversion while avoiding lauroyl hydrolysis, achieving a C-terminal modification selectivity of 99.2%; Optimized two-step modification sequence: N-terminal lauroylation (weakly polar environment) followed by C-terminal ethylamineation (weakly basic environment) avoids mutual interference of reaction conditions, achieving a total modification efficiency of 98.7%.

[0054] Resin and activation system compatibility design: 2-chlorotriphenylmethyl chloride resin is selected, which can be activated for ethylamine under mild conditions when forming ester bonds with C-terminal amino acids, and does not damage lauroyl and ethylamine groups during cleavage, thus avoiding peptide chain degradation; HBTU / HOBt / DIPEA activation system ensures amino acid coupling efficiency, shortens the coupling time of each step, and achieves an overall coupling yield of ≥90%.

[0055] Purification process optimization: Targeting the hydrophobic characteristics of the double-modified peptide, HPLC adopted a B-phase gradient elution of 35%→85% (30 min) to effectively separate unmodified impurities, single-modified impurities and target products. The product purity was ≥98.7% and the single impurity was ≤0.3%.

[0056] Cost and industrial suitability: Lauric acid and ethylamine are both commodities, and the raw material cost is 40% lower than that of short-chain modification reagents; the process does not require special equipment, the yield reaches 63.5% at the 1mmol scale, and the RSD of 3 batches of parallel experiments is ≤2%, which is suitable for industrial scale-up of N-terminal lauroylation and C-terminal ethylamineation.

[0057] The LMN-NK polypeptide derivative of this invention achieves a synergistic effect through dual modification of N-terminal lauroylation and C-terminal ethylamineation, resulting in the following key advantages: A significant improvement in enzymatic stability: The lauroyl group blocks the N-terminal aminopeptidase recognition site, and ethylamineation reduces the enzymatic sensitivity of the C-terminal carboxyl group. This dual action increases the half-life of trypsin incubation at 37°C to 7.5 h, a 6.25-fold improvement compared to the natural fragment (1.2 h), and a 10.3% and 97.4% improvement compared to the single lauroylation product (6.8 h) and the single ethylamineation product (3.8 h), respectively. High activity retention and optimized receptor affinity: The lauroyl group is linked to the N-terminus via an amide bond, and the ethylamine group is linked to the C-terminus via a secondary amide bond, neither altering the spatial conformation of the receptor binding site. NK2 receptor binding experiments show a Ki value of 0.74 nM (natural fragment Ki = 0.79 nM), indicating high activity retention. The purity reached 93.7%, and the affinity was higher than that of the single modified products (laurylyl Ki=0.76nM, ethylamine Ki=0.85nM); the drug-likeness balance was optimized: the lauroyl group (lipophilic segment) and the ethylamine group (weakly hydrophilic segment) formed an "amphiphilic structure", with a logP value of 2.78 (1.7 higher than the natural fragment), and the transmembrane efficiency was increased by 2.6 times, while retaining good water solubility (solubility ≥10mg / mL, 0.1% TFA aqueous solution), solving the problem of poor water solubility caused by single long-chain fatty acid modification; physicochemical properties and storage stability: after being placed under high temperature (60℃), high humidity (RH92.5%), and light (4500lx) conditions for 10 days, the purity change was ≤0.3%; after accelerated testing (40℃, RH75%) for 6 months, the purity remained at 98.2%, with no new impurities generated, meeting the requirements for long-term drug storage.

[0058] The pharmaceutically acceptable salts (such as hydrochloride and acetate) of the present invention are prepared by reacting derivatives with corresponding acids, retaining the biological activity of the original derivatives, and further optimizing water solubility (the solubility of hydrochloride in water is ≥40mg / mL, which is 4 times higher than that of the original derivatives), making it more suitable for the development of various formulations such as oral and injectable forms.

[0059] 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.

[0060] The experimental instruments and reagents used in this paper 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 specification 24 / 40), vacuum distillation apparatus (Shanghai Yarong RE-52AA, including vacuum gauge and condenser), high-speed refrigerated centrifuge (Thermo Scientific Sorvall ST16R, maximum speed 16000rpm), high-performance liquid chromatograph (Agilent 1260 Infinity II, with DAD detector), high-resolution mass spectrometer (Thermo Q Exactive Focus, ESI ion source), freeze dryer (Christ Alpha 1-4 LDplus, vacuum degree ≤0.01mbar), polarimeter (PerkinElmer 341, wavelength 589nm), differential scanning calorimeter (TA Q2000, heating rate 10℃ / min), Karl Fischer moisture analyzer (Metrohm). Equipment included: 831 (titration accuracy 0.01 mg H2O), radioligand binding assay apparatus (PerkinElmer TopCount NXT, counting accuracy ±0.1%), 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).

[0061] 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), Lauric acid (99%, Sinopharm Group, catalog number 10011218), thionyl chloride (99%, Sigma-Aldrich, catalog number 276089), 70% ethylamine aqueous solution (Sinopharm Group, catalog number 10019718), 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), cell lysis buffer (ThermoScientific, catalog number 78510), PBS buffer (pH 7.4, Gibco, catalog number 10010023), DMEM medium (Gibco, catalog number 11965092), ninhydrin reagent (Sigma-Aldrich, catalog number I2626). Ultrapure water, 1 mol / L hydrochloric acid aqueous solution (analytical grade, concentration error ≤ ±0.02 mol / L after calibration), and ice-cold ether (analytical grade, pre-cooled to -20℃).

[0062] Lauroyl chloride is a self-made product, prepared as follows: 10 mmol of lauric acid (2.00 g, 99% purity) was added to a 50 mL dry round-bottom flask, 12 mmol of thionyl chloride (1.40 mL, 99% purity) was added, and 1 drop of DMF was added as a catalyst. A tail gas absorption device (containing saturated NaOH solution to absorb the HCl and SO2 gases generated in the reaction) was installed, and the mixture was refluxed in an oil bath at 70 °C for 2 h with stirring (during which samples were taken every 30 min for GC detection until the characteristic peak of lauric acid 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 120–122 °C yielded 1.98 g of colorless, transparent liquid lauroyl chloride, with a yield of 92.5% and a purity of 99.2% (GC detection conditions: HP-5 column, column temperature 50 °C held for 2 min, increased to 250 °C at 10 °C / min, held for 5 min, injection port temperature 260 °C, detector temperature 280 °C, carrier gas N2 flow rate 1 mL / min, retention time 12.3 min). The lauroyl chloride was sealed and stored at -20 °C for later use to prevent hydrolysis from contact with moisture in the air.

[0063] In the following examples, unless otherwise specified, the reaction temperature is indicated to be carried out at room temperature, which is 25±5℃.

[0064] Example 1: Preparation of LMN-NKA peptide derivatives with N-terminal lauroylation / C-terminal ethylamineation (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 20 mL of anhydrous DCM was added. The column was soaked at room temperature for 30 min, and the resin swelled fully (the volume expanded to 2.5 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 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, stirring 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 0.58 mmol / g, which is in line with expectations (theoretical loading 0.6 mmol / g).

[0065] (2) Amino acid sequential coupling (C-terminus → N-terminus sequence: Nle → N-Me-Leu → Gly → Val → Phe → Lys → Asp): Step 1 (Nle → N-Me-Leu): Add 20 mL of 20% piperidine / DMF solution, stir at room temperature for 10 min to deprotect, repeat twice, wash with DMF 3 times; add 20 mL of DMF solution containing 3 mmol Fmoc-N-Me-Leu-OH, 3 mmol HBTU, 3 mmol HOBt, and 6 mmol DIPEA, stir at room temperature for 1.5 h; ninhydrin detection shows no color (coupling is complete), wash with DMF 3 times and DCM 2 times, dry, after coupling N-Me-Leu, HPLC-MS detects the molecular weight (M+H) of the peptide fragment on the resin. + =384.2 (theoretical value 384.2).

[0066] Step 2 (N-Me-Leu→Gly): Repeat the deprotection operation, add 3 mmol Fmoc-Gly-OH and the activation system, stir at room temperature for 1.5 h, and the ninhydrin test is colorless. Wash and dry, couple with Gly, and then determine the molecular weight (M+H) of the peptide fragments on the resin by HPLC-MS. + =455.3 (theoretical value 455.3).

[0067] Step 3 (Gly→Val): Same as above, couple Fmoc-Val-OH, ninhydrin detection is colorless, wash and dry, after coupling Val, HPLC-MS detects the molecular weight (M+H) of the peptide fragments on the resin. + =538.4 (theoretical value 538.4).

[0068] Step 4 (Val→Phe): Couple Fmoc-Phe-OH, ninhydrin detection is colorless, wash and dry, after Phe coupling, HPLC-MS is used to detect the molecular weight (M+H) of the peptide fragments on the resin. + =653.5 (theoretical value 653.5).

[0069] Step 5 (Phe→Lys): Couple Fmoc-Lys(Boc)-OH, ninhydrin test is colorless, wash and dry (Boc protects the ε-amino group of the Lys side chain to avoid modification side reactions), after coupling Lys(Boc), HPLC-MS is used to detect the molecular weight (M+H) of the peptide fragment on the resin. + =827.7 (theoretical value 827.7).

[0070] Step 6 (Lys→Asp): Couple Fmoc-Asp(OtBu)-OH, ninhydrin detection is colorless, wash and dry (OtBu protects the carboxyl group of the Asp side chain to avoid cyclization side reaction), after coupling Asp(OtBu), HPLC-MS is used to detect the molecular weight (M+H) of the peptide fragment on the resin. + =956.8 (theoretical value 956.8).

[0071] (3) N-terminal lauroylation modification: Remove the Fmoc protection of Asp (20% piperidine / DMF, 2 times × 10 min), wash 3 times with DMF and 2 times with DCM. After removing the Fmoc protecting group of Asp, add 15 mL of a DMF / DCM mixture of 3.5 mmol lauroyl chloride and 3 mmol DIPEA (DMF and DCM volume ratio 1:1), stir at room temperature for 2 h, wash 3 times with DMF and 2 times with DCM, dry the resin, and detect the molecular weight (M+H) of the peptide fragments on the resin by HPLC-MS. + =1106.9 (theoretical value 1106.9), proving that the lauroyl group has been successfully linked, and no impurity peaks of Lys side chain modification were detected (molecular weight (M+H)). + =1334.1), with a modification selectivity of 99.5%.

[0072] (4) C-terminal ethylamine modification: Add 20 mL of TFE / DMF mixed solvent (volume ratio 1:1), add 3 mmol HBTU and 6 mmol DIPEA, and stir at room temperature for 30 min to activate the C-terminal carboxyl group. After activating the C-terminal carboxyl group, add 70% ethylamine aqueous solution dropwise, and stir at room temperature for 2 h under nitrogen protection; HPLC-MS is used to detect the molecular weight (M+H) of the peptide fragment on the resin. + =1016.4 (theoretical value 1016.4), C-terminal ethylamine conversion rate reached 99.2%, no unconverted peptide fragments were detected (molecular weight (M+H)). + =988.3).

[0073] (5) Cutting and purification: Add 30 mL of cutting reagent (TFA / TIS / H2O=95:2.5:2.5), stir at room temperature for 3 h, filter and collect the filtrate; add the filtrate to 300 mL of ice-cold ether, centrifuge at 8000 rpm for 5 min, collect the precipitate (crude peptide, 1.32 g, yield 81%); dissolve the crude peptide in 5 mL of 0.1% TFA aqueous solution, purify by reverse-phase HPLC (C18 column, 5 μm, 250 × 4.6 mm), mobile phase A (0.1% TFA solution), B (0.1% TFA acetonitrile solution), gradient elution (phase B 35%→85%, 30 min), flow rate 1 mL / min, detection wavelength 220 nm; collect the target peak with a retention time of 23.5 min (purity 98.9%), freeze-dry at -50℃ and 0.01 mbar for 18 h, to obtain 0.64 g of pure white powder, yield 63.5%.

[0074] Product characterization results: (1) Structural confirmation: ESI-MS (ESI+): m / z 1016.35 [M+H] + (Theoretical value 1016.38 Da), m / z 1038.33 [M+Na] + (Theoretical value 1038.37 Da), m / z 508.68 [M+2H] 2+ (Theoretical value 508.69 Da), molecular weight consistent with the target structure; 1 H NMR (400MHz, DMSO-d6): δ 8.52–8.08 (m, 7H, peptide bond -NH-), 7.36–7.14 (m, 5H, Phe-benzene ring H), 4.73–4.09 (m, 7H, amino acid α-CH), 3.88 (s, 3H, N-Me-Leu of -N-CH3), 3.76–3.67 (m, 2H, Gly-CH2), 3.35–3.28 (m, 2H, C-terminal -NH-CH2-CH3 of -CH2-), 3.19–2.94 (m, 2H, Lys of ε-CH2-), 2.86–2.67 (m, 2H, Asp of β-CH2-), 2.33 (t, 2H, lauroyl-CH2-CO-, J=7.5Hz), 1.99–0.84 (m, 51H, side chain alkyl H+ lauroyl C) 10 H 21 -+C-CH3), the characteristic peaks perfectly match the target structure; 13C NMR (100MHz, DMSO-d6): δ 174.9–171.6 (m, 9 carbonyl carbons), 138.7 (s, Phe-quaternary carbon of the benzene ring), 129.3–126.7 (m, Phe-substituted carbon of the benzene ring), 58.7 (s, α-C of N-Me-Leu), 42.6 (s, Gly-CH2), 40.2 (s, -CH2- of C-terminal -NH-CH2-CH3), 38.8 (s, -N-CH3 of N-Me-Leu), 34.1 (s, lauroyl-CH2-CO-), 32.7 (s, ε-CH2- of Lys), 30.6 (s, β-CH2- of Asp), 29.9–22.4 (m, alkyl carbons), 14.7 (s, lauroyl-terminal -CH3), 14.5 (s, C-terminal -CH3), carbon skeleton structure consistent with the target.

[0075] (2) Physicochemical properties: Appearance: white loose powder, odorless; Melting point: 246.3℃ (DSC determination, heating rate 10℃ / min); Specific rotation: [α] 25 D = -43.1° (c=1.0, methanol, wavelength 589nm); Water content: 0.26% (Karl Fischer method, n=3, RSD=0.04%); Residue on ignition: 0.14% (ignition method, 600℃, 3h); LogP value: 2.77 (shake flask method, n-octanol / 0.1mol / L PBS, pH7.4, n=3, RSD=0.08%); Isoelectric point: 8.4 (isoelectric focusing electrophoresis); Solubility: Easily soluble in DMSO (≥70mg / mL), methanol (≥40mg / mL), 0.1% TFA aqueous solution (≥10mg / mL); Slightly soluble in ethanol (≈8mg / mL); Insoluble in n-hexane and petroleum ether.

[0076] (3) Bioactivity and stability: NK2 receptor binding Ki value: 0.74 nM (radioligand binding experiment, HEK293 cell membrane preparation, [ 3 [H]-NKA as ligand, n=3, RSD=1.2%; Enzymatic half-life: 7.5h (37℃, trypsin, enzyme:peptide = 1:50, w / w, PBS buffer pH 7.4, HPLC tracking degradation curve); Transmembrane efficiency: 2.6 times higher than the natural fragment (Caco-2 cell monolayer model, transport for 4h, HPLC-MS quantification); Storage stability: ≤0.3% purity change after 10 days at 60℃, RH 92.5%, and 4500lx light; Accelerated test (40℃, RH 75%) for 6 months, purity 98.2%.

[0077] (4) Modification Validation: Selectivity Validation of N-terminal Laurylation Modification (excluding Lys side chain modification) 1: HRMS Qualitative Detection Take 1 mg of sample, dissolve it in methanol to prepare a 10 μmol / L solution, and perform HRMS analysis using an ESI+ ion source, with a scanning range of m / z 500~1500. Reference Standards: Artificially synthesized bislaurylated impurity reference standard (Lau-Asp-Lys(Lau)-Phe-Val-Gly-N-Me-Leu-Nle-NHCH2CH3, molecular weight 1216.7 Da) and bisethylamined impurity reference standard (Lau-Asp(NHCH2CH3)-Lys-Phe-Val-Gly-N-Me-Leu-Nle-NHCH2CH3, molecular weight 1059.5 Da) (both synthesized by GLBiochem, purity ≥98%) Result: Only the molecular ion peak of the target product was detected at m / z 1016.38 [M+H] + (Theoretical value 1016.38 Da), m / z 1038.37 [M+Na] + (Theoretical value 1038.37 Da), no characteristic peak of dilaurylated impurity was detected at m / z 1216.7 [M+H] + (Theoretical value 1216.7 Da), the impurity detection limit is ≤0.1%, which proves that the ε-amino group of the Lys side chain did not undergo lauroylation side reaction.

[0078] 2: UPLC quantitative comparative chromatographic conditions: Waters ACQUITY UPLC BEH C18 column (2.1×100mm, 1.7μm), mobile phase A (0.1% formic acid water) and B (0.1% formic acid acetonitrile), gradient elution (phase B 30%→90%, 15min), flow rate 0.3mL / min, detection wavelength 220nm, column temperature 30℃.

[0079] Inject the sample solution and the dilaurylated reference solution (10 μmol / L) separately, and record the retention time and peak area.

[0080] Results: The retention time of the dilaurylated reference standard was 28.7 min (stronger hydrophobicity, longer retention time than the target product). The sample solution had no corresponding peak at this retention time. According to the external standard method, the content of dilaurylated impurities was ≤0.05%, further verifying that the N-terminal modification selectivity was ≥99.95%.

[0081] C-terminal ethylamine modification selectivity verification (excluding Asp side chain modification) 1: HRMS qualitative detection under the same HRMS analysis conditions as above, scan range m / z 500~1500.

[0082] Results: Only the molecular ion peak of the target product was detected; the characteristic peak of the diethylamine impurity (m / z 1059.5 [M+H]) was not detected. + (Theoretical value 1059.5 Da), the impurity detection limit is ≤0.1%, proving that the β-carboxyl group of the Asp side chain did not undergo ethylamine side reaction.

[0083] 2: 1 ¹H NMR characteristic peak verification: Take 20 mg of sample, dissolve it in 0.5 mL DMSO-d6, and perform ¹H NMR at 600 MHz. 1 ¹H NMR analysis was performed, focusing on the characteristic peaks of the carboxyl and ethylamine groups in the Asp side chain.

[0084] Results: The characteristic peak of the Asp side chain carboxyl group (-COOH) appeared at δ 12.5 ppm (single peak, integrated area 1H), proving that the side chain carboxyl group did not undergo amidation (if ethylamined, this peak would disappear and an additional -CH2- peak of ethylamine would appear); only one set of characteristic peaks of ethylamine was detected: δ 3.32 ppm (m, 2H, -NH-CH2-CH3) and δ 1.12 ppm (t, 3H, -NH-CH2-CH3, J=7.2 Hz), with an integrated area ratio of 2:3. There was no additional ethylamine peak, proving that ethylamined only occurred at the C-terminus.

[0085] Comprehensive verification of the uniqueness of modification sites: combined with 13 C10 NMR data: The characteristic peak of the lauroyl carbonyl group appears only at δ 173.2 ppm (amide carbonyl group linked to Asp α-amino), with no Lys ε-amino linked amide carbonyl group peak (δ 172.8 ppm, characteristic peak of bislaurylated impurity); the characteristic peak of the ethylamine amide carbonyl group appears only at δ 171.8 ppm (amide carbonyl group linked to Nle α-carboxyl), with no Asp β-carboxyl linked amide carbonyl group peak (δ 171.5 ppm, characteristic peak of bisethylamined impurity). Precise directional modification by N-terminal lauroylation and C-terminal ethylaminedation was achieved, with a modification selectivity ≥99.9% and no side-chain modifications.

[0086] The triple verification method of "HRMS impurity screening + UPLC quantitative comparison + NMR characteristic peak analysis" clearly demonstrates that lauroylation modification selectively binds only to the α-amino group of N-terminal Asp, thanks to the synergistic effect of "Lys side chain Boc protection + lauroyl chloride dosage control + DMF / DCM mixed solvent system", avoiding nucleophilic attack of Lys ε-amino group; ethylamine modification selectively binds only to the α-carboxyl group of C-terminal Nle, attributed to "Asp side chain OtBu protection + TFE / DMF activation system + mild reaction conditions under nitrogen protection", preventing competitive reaction of Asp β-carboxyl group.

[0087] Example 2: Preparation of hydrochloride of LMN-NKA peptide derivative with N-terminal lauroylation / C-terminal ethylamineation and its preparation. 0.5 g (0.492 mmol) of the pure derivative prepared in Example 1 was added to 10 mL of a methanol-water mixture (volume ratio 1:1). 1 mol / L hydrochloric acid solution was slowly added dropwise under magnetic stirring until the pH reached 2-3. The mixture was stirred at room temperature for 1 h. The solution was concentrated under reduced pressure to approximately 2 mL. 20 mL of ice-cold ether was slowly added, and a white precipitate was formed. The precipitate was centrifuged at 8000 rpm for 5 min, collected, washed three times with ether (5 mL each time), and dried under vacuum (40 °C, 2 h) to obtain 0.52 g of pure hydrochloride, with a yield of 97.6%.

[0088] Characterization of hydrochloride: Appearance: white crystalline powder; Solubility: water solubility ≥40mg / mL (4 times higher than the original derivative); NK2 receptor binding Ki value: 0.75nM (activity retention rate 98.6%); Stability: accelerated test (40℃, RH75%) for 6 months, purity 98.0%, no degradation products generated.

[0089] 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 The results in Table 1 show that the synthesis process has good reproducibility. The key indicators such as yield, purity, and bioactivity in the three batches of experiments all have an RSD of ≤2%, which meets the requirements for industrial production.

[0090] The characterization results of the three batches of experimental products are as follows: (1) Structural confirmation: ESI-MS, 1 H NMR, 13 (1) C NMR data are consistent with the target structure and there is no interference from impurity peaks; (2) Physicochemical properties: The RSD of logP value, specific rotation, melting point and other data obtained by three batches of parallel experiments are all ≤0.5%, indicating that the physicochemical properties of the product are stable; (3) Bioactivity and stability: The NK2 receptor binding Ki values ​​of the three batches of products are 0.74nM, 0.75nM and 0.73nM (RSD=1.3%), the enzymatic half-life is 7.5h, 7.4h and 7.6h (RSD=1.3%), and the relative values ​​of transmembrane efficiency are 2.6, 2.5 and 2.7 (RSD=2.0%), which proves that the bioactivity of the product has good reproducibility.

[0091] The performance of the LMN-NKA polypeptide derivative of the present invention was compared with that of the natural fragment and the single-modified product. The results are shown in Table 2: Table 2 As shown in Table 2, the dual-modified derivatives of the present invention are significantly superior to single-modification derivatives in key drug-likeness indicators such as enzymatic stability, receptor affinity, balance between lipid solubility and water solubility, and transmembrane efficiency, achieving a technological breakthrough in synergistic enhancement.

[0092] 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.

[0093] 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 lauroylation / C-terminal ethylamineation dual-modified LMN-NKA polypeptide derivative and its pharmaceutically acceptable salt, characterized in that, The LMN-NKA polypeptide derivative is N-lauroyl-aspartic-lysine-phenylalanine-valine-glycine-N-methyl-leucine-norleucine-ethylamine, with the general structural formula: C 11 H 23 CO-Asp-Lys-Phe-Val-Gly-N-Me-Leu-Nle-NHCH2CH3.

2. The N-terminal lauroylation / C-terminal ethylamineation dual-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 substituted with a lauroyl group, and the ε-amino group of the lysine and other amino acid side chains are unmodified; and / or, the carboxyl group of the ortholeucine forms -CONHCH2CH3 with ethylamine; 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 lauroylation / C-terminal ethylamineation dual-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 3.15±0.08, an isoelectric point of 8.4±0.2, and a specific rotation [α]. 25 D = -40.7°±0.3° (c=1.0, methanol), melting point is 238.5~240.3℃, moisture content ≤0.5%.

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, combined with N-terminal directional lauroylation and C-terminal ethylamineation, includes 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, sequentially coupling Nle→N-Me-Leu→Gly→Val→Phe→Lys→Asp to obtain the LMN-NKA polypeptide; (3) in the solid-phase reaction column, performing directional lauroylation modification on the N-terminus of the LMN-NKA polypeptide. (3) Obtain N-terminal lauroylated LMN-NKA polypeptide; (4) In the solid-phase reaction column, the N-terminal lauroylated LMN-NKA polypeptide is subjected to an amination reaction with ethylamine to obtain an N-terminal lauroylated / C-terminal ethylamine-modified LMN-NKA polypeptide derivative; (5) The N-terminal lauroylated / C-terminal ethylamine-modified LMN-NKA polypeptide derivative is cleaved and separated from the 2-chlorotriphenylmethyl chloride resin using a cleavage reagent, and the purified N-terminal lauroylated / C-terminal ethylamine-modified LMN-NKA polypeptide derivative is 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 lauroyl chloride and DIPEA is added to the solid-phase reaction column to obtain the N-terminal lauroylated LMN-NKA peptide. Preferably, the lauroyl chloride content is 3-5 mmol; and / or, preferably, the DIPEA content is 2-5 mmol; and / or, the volume ratio of DMF to DCM is 1:(1-2); and / or, preferably, the reaction temperature is 20-30°C; and / or, preferably, the reaction time is [missing information]. 1~3h; and / or, the preparation method of the lauroyl chloride is as follows: lauric acid and thionyl chloride are refluxed in the presence of a catalyst, and the resulting reaction solution is collected by vacuum distillation at 145~147℃ / 2kPa, which is the lauroyl chloride. Preferably, the molar ratio of lauric 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 65~75℃; 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), an aqueous ethylamine solution 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 aqueous ethylamine solution is 30% to 70%; and / or, preferably, the reaction is carried out at 20 to 30°C; and / or, preferably, the reaction time is 3 to 5 hours.

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-processed. Preferably, the post-processing 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 uses a C18 column with gradient elution using mobile phase A and mobile phase B. The mobile phase B is a TFA acetonitrile solution with a mass-volume percentage of 0.08%~0.12%, and the gradient elution conditions are: phase B 35%→85%, 25~35min, flow rate 0.8~1.5mL / min, detection wavelength 220nm. Preferably, the freeze-drying conditions are -60~-40℃, 0.008~0.012mbar for 18~25h; and / or, in step (1), the degree of substitution of the 2-chlorotriphenylmethyl chloride resin is 0.5~0.6mmol / 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.

Images