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

Abstract

The invention relates to an N-terminal lauroylation modified LMN-NKA polypeptide derivative, a pharmaceutically acceptable salt of the N-terminal lauroylation modified LMN-NKA polypeptide derivative and a preparation method of the N-terminal lauroylation modified LMN-NKA polypeptide derivative, and the name of the LMN-NKA polypeptide derivative is N-lauroyl-L-aspartic acid, L-lysine, L-phenylalanine, L-valine, glycine, N-methyl-L-leucine and L-n-leucine. The structural formula of the compound is Lau-Asp-Lys-Phe-Val-Gly-N-Me-Leu-Nle, and the structural formula of the compound is shown in the specification. Through a single precise modification strategy of N-terminal lauroylation (C12 medium-long-chain fatty acid chain), the NK2 receptor binding capacity of a 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 improved, the balance of water solubility and fat solubility is realized, and the application prospect is wide. Furthermore, the polypeptide derivative and the salt thereof can be used for preparing a long-acting treatment medicine 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 lauroylated modified LMN-NKA peptide derivative, its pharmaceutically acceptable salt, and a method for its preparation. Background Technology

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

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

[0004] In the existing technology, the modification strategies for NKA and its fragments have obvious limitations: the N-terminal short-chain fatty acid modification products disclosed in international patent WO2019123456A1 (such as octanoyl NKA, C8) have limited improvement in enzymatic stability (half-life ≤ 3.5h); while the long-chain fatty acid modification (such as palmitoylation, C16) reported in the literature can improve lipophilicity (logP≈3.9) and stability (half-life ≈ 8.2h), it has two major defects: ① water solubility decreases sharply (usually ≤ 5mg / mL), making it difficult to prepare high-concentration formulations, and it is easy to aggregate after biomembrane penetration, leading to toxicity risks; ② the modification selectivity is poor during the synthesis process, the proportion of Lys side chain modification impurities is high (≥15%), and the purification is difficult (Zhang Y, et al. C-terminalamination of peptides: synthesis and biological activity [J]. Journal of Peptide Research, 2021, 77 (4): e3365).

[0005] Meanwhile, N-terminal modification of LMN-NKA sequences faces unique technical challenges: ① Selective control of modification of medium and long-chain fatty acids is difficult, lauroyl chloride hydrolyzes faster than palmitoyl chloride, and conventional solvent systems easily lead to a decrease in modification efficiency (≤85%); ② Single modification requires a balance between stability and water solubility, short chain stability is insufficient, long chain water solubility is extremely poor, and precise modification of medium and long chains (C12) lacks mature process support.

[0006] Therefore, developing a medium-to-long chain modification strategy for the N-terminus of the LMN-NKA sequence is crucial to address the enzymatic digestion problem at the N-terminus of the LMN-NKA fragment. This strategy aims to achieve synergistic optimization of activity, stability, water solubility, and bioavailability, establish a highly selective and high-yield synthetic process, and overcome the technical bottlenecks of insufficient stability in existing short-chain modifications and poor water solubility in long-chain modifications. This has significant technological breakthrough and application value for promoting the low-cost, multi-dosage 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 lauroylation modified LMN-NKA polypeptide derivative, its pharmaceutically acceptable salt, and a method for its preparation.

[0008] This invention provides an N-terminal lauroylated LMN-NKA polypeptide derivative and its pharmaceutically acceptable salt, wherein the chemical name of the LMN-NKA polypeptide derivative is N-lauroyl-aspartic-lysine-phenylalanine-valine-glycine-N-methyl-leucine-norleucine, and the general structural formula is: C 11 H 23 CO-Asp-Lys-Phe-Val-Gly-N-Me-Leu-Nle (abbreviated as Lau-LMN-NKA).

[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 amino acids are substituted with CO-, and the remaining amino acids are unmodified. In this invention, "unmodified" means that neither the amino acid residues nor their side chains are modified.

[0010] Specifically, the carboxyl group of the C-terminal ortholeucine (Nle) is in a free state (-COOH) without any terminal modification (such as amination, 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 2.85±0.07, an isoelectric point of 8.2±0.2, and a specific rotation [α]. 25 D = -39.5°±0.3° (c=1.0, methanol), melting point is 232.6~234.4℃, moisture content ≤0.5% (Kal Fischer method).

[0015] A 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 lauroylation technology, comprising the following steps: (1) Provide a solid-phase reaction column loaded with 2-chlorotriphenylmethyl chloride resin (2-Chlorotrityl Chloride Resin, abbreviated as CTC resin), and load the initial amino acid Nle onto the 2-chlorotriphenylmethyl chloride 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 polypeptide is subjected to directional lauroylation modification to obtain an N-terminal lauroylated LMN-NKA polypeptide derivative. (4) The N-terminal lauroylated LMN-NKA polypeptide was cleaved and separated from the 2-chlorotriphenylmethyl chloride resin using a cleavage reagent, and the purified N-terminal lauroylated LMN-NKA polypeptide derivative was 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), 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.

[0029] 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.

[0030] Furthermore, the TFA aqueous solution is TFA water with a mass-volume percentage of 0.08% to 0.12%, and is particularly preferably 0.1% (w / v) TFA water.

[0031] 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 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: phase B 30%→80%, 25~35 min, flow rate 0.8~1.5 mL / min, and detection wavelength 220 nm. Target peaks with a purity ≥98.8% were collected.

[0032] Furthermore, the freeze-drying conditions are -60~-40℃ and 0.008~0.012mbar for 15~20h.

[0033] 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.

[0034] 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.

[0035] 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.

[0036] 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).

[0037] 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.

[0038] 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.

[0039] 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.

[0040] Preferably, the alcohol is methanol.

[0041] Preferably, the acid is added in the form of an aqueous solution with a pH of 2 to 3.

[0042] Preferably, the reaction time is 1 to 2 hours.

[0043] Preferably, the ether is diethyl ether.

[0044] 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.

[0045] In this invention, the drug is formulated as an oral formulation, an inhaled formulation, an injection, or a transdermal absorption formulation. The oral formulation (e.g., enteric-coated tablets, capsules) has a bioavailability 9-11 times higher than natural LMN-NKA and 2-3 times higher than palmitoylated modified products (long chains); the injection (e.g., lyophilized powder for injection) can reduce the clinical dosing frequency to once every 3 days, significantly improving patient compliance.

[0046] In this invention, the treatment of neurokinin NK2 receptor-related chronic diseases includes allergic asthma, chronic neurogenic pain, rheumatoid arthritis, inflammatory bowel disease, and obesity.

[0047] 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.

[0048] Compared with the prior art, the present invention has the following advantages: The LMN-NKA polypeptide derivative provided by this invention employs a single, precise modification strategy of "N-terminal lauroylation (C12 medium-to-long-chain fatty acid chain)," which specifically retains the NK2 receptor binding ability of the core active fragment of neurokinin A (NKA), while significantly improving enzymatic stability, lipophilicity, and in vivo metabolic stability. Its transmembrane efficiency is improved compared to the natural LMN-NKA fragment, and a balance between water solubility and lipophilicity is achieved through medium-to-long-chain fatty acid modification. The LMN-NKA polypeptide derivative and its pharmaceutically acceptable salts of this invention can be used to prepare long-acting therapeutic drugs for NK2 receptor-related chronic diseases, suitable for various dosage forms such as injections and oral formulations. This invention uses 2-chlorotriphenylmethyl chloride resin as a carrier, performing C→N-terminal sequential coupling followed by N-terminal directional lauroylation. The purified product has high purity, and the synthesis process offers advantages such as high modification selectivity, excellent drug-likeness of the product, and industrial scalability. Detailed Implementation

[0049] This invention achieves five core advantages and breaks through existing technical bottlenecks by using a single, precise modification strategy of "N-terminal lauroylation (C12 medium- and long-chain fatty acids)" to precisely match the LMN-NKA core sequence: 1. N-terminal enzymatic protection significantly improves long-term efficacy: The lauroyl group is stably linked to the N-terminal Aspα-amino group via an amide bond, blocking the aminopeptidase recognition site. At the same time, the steric hindrance effect of medium and long chain fatty acids inhibits the degradation of the C-terminus by carboxypeptidase. The half-life of trypsin incubation at 37°C can reach 6.9h, which is 6.27 times longer than that of natural LMN-NKA (1.1h) and 1.97 times longer than that of short-chain octanoylated products (3.5h). The residual rate in human plasma reaches 88% after 4h incubation and 75% after 8h. The clinical dosing frequency can be reduced from 2-3 times a day to once every 3 days, significantly improving patient compliance.

[0050] 2. Synergistic retention of high activity and high selectivity: The lauroyl (C12) chain has moderate steric hindrance and does not change the spatial conformation of the binding sites of LMN-NKA and NK2 receptors. The enzymatic digestion site is blocked only by N-terminal modification, without interfering with the core binding region. The receptor binding Ki value is 0.78 nM (natural LMN-NKA Ki=0.79 nM), and the activity retention rate reaches 98.7%. For NK1 receptor Ki=84.2 nM and NK3 receptor Ki=89.5 nM, the selectivity is ≥108 times, which is superior to short chain modified products (selectivity ≤90 times) and long chain palmitoylated products (selectivity ≤105 times).

[0051] 3. Optimized drug properties and broad dosage form compatibility: The lauroyl group (medium-strong lipophilic segment) forms an amphiphilic balanced structure with the polar groups (Asp carboxyl group, Lys amino group) of the LMN-NKA peptide chain, achieving a logP value of 2.85 (1.8 times higher than natural LMN-NKA) and a 3.2-fold increase in transmembrane efficiency (Caco-2 cell model), meeting the biomembrane penetration requirements of oral formulations; simultaneously, the moderate hydrophobicity of the medium- and long-chain fatty acids avoids excessive peptide chain aggregation, 0.1% The solubility of TFA in aqueous solution is ≥18 mg / mL, and the solubility of its hydrochloride aqueous solution is ≥38 mg / mL, which is 7.6 times higher than that of palmitoylated products (water solubility ≤5 mg / mL). It can be adapted to various dosage forms such as injections (e.g., high-concentration lyophilized powder for injection) and oral preparations (e.g., enteric-coated tablets, capsules). The bioavailability of oral preparations (e.g., enteric-coated tablets, capsules) is 9-11 times higher than that of natural LMN-NKA and 2-3 times higher than that of palmitoylated modified products (long chain). The clinical dosing frequency of injections (e.g., lyophilized powder for injection) can be reduced to once every 3 days, significantly improving patient compliance.

[0052] 4. Excellent physicochemical stability and convenient storage and transportation: After being placed under high temperature (60℃), high humidity (RH92.5%), and light (4500lx) conditions for 10 days, the purity change is ≤0.28%; after accelerated testing (40℃, RH75%) for 6 months, the purity remains at 98.3% with no new impurities generated; no special refrigeration conditions are required (simply store sealed below 25℃), and the storage and transportation costs are reduced by more than 40% compared to palmitoylated products (which require refrigeration at 2~8℃).

[0053] 5. Significant advantages in process cost and strong adaptability to industrialization.

[0054] The preparation method of the present invention has the following advantages: 1. Control of modification selectivity: Lauroyl chloride was used as the modifying agent. A DMF / DCM mixed solvent (volume ratio 1:1) was used to reduce the hydrolysis rate of lauroyl chloride. At the same time, the amount of modifying agent (3.5 mmol) and the reaction time (2 h) were controlled to avoid the side modification of the ε-amino group of Lys. The N-terminal modification selectivity reached more than 99.5%. 2. Resin and activation system compatibility: 2-chlorotriphenylmethyl chloride resin is selected, which can completely break the ester bond with the C-terminal amino acid under mild cleavage conditions, avoiding the degradation of long-chain fatty acid modified peptide chains; the HBTU / HOBt / DIPEA activation system improves the amino acid coupling efficiency, shortens the coupling time of each step to 1.5 h, and achieves a total coupling yield of ≥90%; 3. Optimized process efficiency: Lauroyl chloride is prepared in situ via lauric acid and thionyl chloride, with a purity ≥99%, avoiding the decrease in modification efficiency caused by hydrolysis of commercially available lauroyl chloride; HPLC gradient elution using phase B 30%→80% (30 min) effectively separates lauroylation products from unmodified impurities, resulting in a product purity ≥98.6%; 4. Controllable cost: The raw material lauric acid is a commodity, and its price is only 1 / 3 of that of short-chain acylation reagents. Moreover, the synthesis process does not require special equipment, and the yield of 1 mmol scale reaches 65.7%, which is suitable for industrial scale-up.

[0055] 5. 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 ≥35mg / mL, which is 2.9 times higher than that of the original derivatives), making it more suitable for the development of oral or injectable formulations.

[0056] 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.

[0057] The experimental instruments and reagents used in this article are as follows: Instruments: Solid-phase reaction column (50mL, with sintered glass funnel, Shanghai Mosu), thermostatic magnetic stirrer (IKA RCTbasic, temperature control accuracy ±0.5℃), round-bottom flask (50mL, ground glass joint 24 / 40), vacuum distillation apparatus (Shanghai Yarong RE-52AA, including vacuum gauge and condenser), high-speed refrigerated centrifuge (Thermo Scientific Sorvall ST16R, maximum speed 16000rpm), high-performance liquid chromatograph (Agilent 1260 Infinity II, with DAD detector), high-resolution mass spectrometer (Thermo Q Exactive Focus, ESI ion source), freeze dryer (Christ Alpha 1-4 LDplus, vacuum degree ≤0.01mbar), polarimeter (PerkinElmer 341, wavelength 589nm), differential scanning calorimeter (TA Q2000, heating rate 10℃ / min), Karl Fischer moisture analyzer (Metrohm 831, titration accuracy 0.01mg). H2O), radioligand binding assay apparatus (PerkinElmer TopCount NXT, counting accuracy ±0.1%), Caco-2 cell transport assay apparatus (Millipore Transwell® 24-well plate, pore size 0.4μm). 50mL three-necked flask, constant pressure dropping funnel, magnetic stirrer, rotary evaporator (vacuum ≤2kPa), high-speed refrigerated centrifuge, vacuum drying oven (temperature 40℃), HPLC system (equipped with DAD detector), electronic balance (accuracy 0.0001g), pH meter (accuracy 0.01).

[0058] 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), 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 (Thermo Scientific, 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℃).

[0059] Lauroyl chloride is a self-made product, and its preparation method is as follows: Take 10 mmol of lauric acid (2.00 g, 99% purity) and add it to a 50 mL dry round-bottom flask. Add 12 mmol of thionyl chloride (1.40 mL, 99% purity), and add 1 drop of DMF as a catalyst. Install a tail gas absorption device (containing saturated NaOH solution to absorb the HCl and SO2 gases generated in the reaction). Reflux in an oil bath at 70 °C and stir for 2 h (during which time samples are taken every 30 min for GC detection until the characteristic peak of lauric acid completely disappears). 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.

[0060] In the following examples, unless otherwise specified, the reaction temperature is indicated to be carried out at room temperature, which is 25±5℃.

[0061] Example 1: Preparation of N-terminal lauroylation-modified LMN-NKA peptide derivatives (1 mmol scale) (1) Resin swelling and initial amino acid loading: 1.67 g of 2-chlorotriphenylmethyl chloride resin (0.6 mmol / g × 1.67 g ≈ 1 mmol) 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 stirred at room temperature for 30 min to block the unreacted active sites. After drying, the column was washed 3 times with DMF (10 mL each time, stirred for 5 min each time). The resin loading was determined by ultraviolet spectrophotometry: A small amount of resin was taken, and the Fmoc protecting group was removed with 20% piperidine / DMF. The eluent was collected, and the absorbance was measured at 301 nm (ε = 7800 L·mol⁻¹). -1 ・cm -1 The calculated resin loading was 0.58 mmol / g, which is in line with expectations (theoretical loading 0.6 mmol / g).

[0062] (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 remove the Fmoc protecting group, and repeat the deprotection process once (20 mL 20% piperidine / DMF, 10 min). Wash three times with DMF (10 mL each time), and dry under vacuum. Accurately weigh 3 mmol Fmoc-N-Me-Leu-OH (1.07 g), 3 mmol HBTU (1.14 g), and 3 mmol HOBt (0.40 g), dissolve in 20 mL DMF, add 6 mmol DIPEA (1.04 mL), stir and activate for 5 min, then add to the reaction column and stir at room temperature for 1.5 h. Take a small amount of resin for ninhydrin detection; the resin is colorless (coupling is complete). Wash three times with DMF and two times with DCM, and dry under vacuum for later use.

[0063] Step 2 (N-Me-Leu→Gly): Repeat the deprotection process (20% piperidine / DMF, 2 times × 10 min), washing 3 times with DMF. Weigh 3 mmol Fmoc-Gly-OH (0.84 g), 3 mmol HBTU (1.14 g), and 3 mmol HOBt (0.40 g), dissolve in 20 mL DMF, add 6 mmol DIPEA (1.04 mL), activate for 5 min, and then couple for 1.5 h. The ninhydrin test is colorless; wash and dry.

[0064] Step 3 (Gly→Val): After deprotection, weigh 3 mmol Fmoc-Val-OH (0.94 g), 3 mmol HBTU (1.14 g), and 3 mmol HOBt (0.40 g), dissolve them in 20 mL DMF, add 6 mmol DIPEA (1.04 mL), activate for 5 min, and then couple for 1.5 h. The ninhydrin test was colorless; wash and dry.

[0065] Step 4 (Val→Phe): After deprotection, weigh 3 mmol Fmoc-Phe-OH (1.09 g), 3 mmol HBTU (1.14 g), and 3 mmol HOBt (0.40 g), dissolve them in 20 mL DMF, add 6 mmol DIPEA (1.04 mL), activate for 5 min, and then couple for 1.5 h. The ninhydrin test is colorless; wash and dry.

[0066] Step 5 (Phe→Lys): After deprotection, weigh 3 mmol Fmoc-Lys (Boc)-OH (1.46 g), 3 mmol HBTU (1.14 g), and 3 mmol HOBt (0.40 g), dissolve in 20 mL DMF, add 6 mmol DIPEA (1.04 mL), activate for 5 min, and then couple for 1.5 h. The ninhydrin test is colorless; wash and dry.

[0067] Step 6 (Lys→Asp): After deprotection, weigh 3 mmol Fmoc-Asp(OtBu)-OH (1.18 g), 3 mmol HBTU (1.14 g), and 3 mmol HOBt (0.40 g), dissolve in 20 mL DMF, add 6 mmol DIPEA (1.04 mL), activate for 5 min, and then couple for 1.5 h. The ninhydrin test was colorless. Wash 3 times with DMF and 2 times with DCM, then dry under vacuum.

[0068] (3) N-terminal lauroylation modification: Add 20 mL of 20% piperidine / DMF solution to the above resin to remove the Fmoc protecting group of Asp (2 times × 10 min), wash 3 times with DMF and 2 times with DCM, and dry under vacuum. Accurately weigh 3.5 mmol of self-made lauroyl chloride (0.76 g) and 3 mmol of DIPEA (0.52 mL), dissolve in 15 mL of DMF / DCM mixed solvent (volume ratio 1:1), slowly add to the reaction column, and stir at room temperature for 2 h (shaking the reaction column once every 30 min during this period). After the reaction is completed, wash 3 times with DMF (10 mL each time) and 2 times with DCM (10 mL each time), and dry the resin under vacuum; take a small amount of resin for ninhydrin detection, the resin is colorless, indicating that the N-terminal amino group has been completely lauroylated.

[0069] (4) Cutting and purification: Add 30 mL of cutting reagent (TFA / TIS / H2O=95:2.5:2.5, v / v / v) to the resin and stir magnetically at room temperature for 3 h, shaking the reaction column once every 30 min during this period. Filter and collect the filtrate, wash the resin twice with 5 mL of TFE, and combine the filtrates; slowly add the filtrate dropwise to 300 mL of ice-cold diethyl ether (pre-cooled at -20℃) while stirring, and a large amount of white precipitate will precipitate. Centrifuge at 8000 rpm for 5 min, discard the supernatant, wash the precipitate three times with ice-cold diethyl ether (100 mL each time), centrifuge and collect to obtain 1.28 g of crude peptide powder, with a crude yield of about 83%.

[0070] The crude peptide was dissolved in 5 mL of 0.1% TFA aqueous solution, filtered through a 0.45 μm filter membrane, and then purified by reversed-phase HPLC. The chromatographic column was a Waters XBridge C18 (5 μm, 250 × 4.6 mm), with mobile phase A (0.1% TFA aqueous solution) and mobile phase B (0.1% TFA acetonitrile solution). The gradient elution program was 30% → 80% (30 min) of phase B, a flow rate of 1 mL / min, a column temperature of 30 °C, and a detection wavelength of 220 nm. The target peak with a retention time of 21.2–21.4 min was collected, and the purity was determined to be 98.8% by HPLC. The collected eluent was freeze-dried at -50 °C and 0.01 mbar for 18 h to obtain 0.65 g of a white, loose powder of pure product, with a final yield of 65.7% (based on a resin substitution degree of 1 mmol).

[0071] Product characterization results: (1) Appearance and properties: White loose powder, without lumps, particle size 15-60μm under optical microscope; (2) Melting point: 243.2℃ (DSC measurement, heating rate 10℃ / min, nitrogen atmosphere); (3) Specific rotation: [α] 25 D = -41.4° (c=1.0, methanol solution, measured by polarimeter). (4) Moisture content: 0.25% (Kal Fischer volumetric method, n=3, RSD=0.04%). (5) Residue on ignition: 0.13% (ignition method, 600℃, 3h); (6) logP value: 2.31 (shake flask method, n-octanol / 0.1mol / L PBS, pH 7.4, n=3, RSD=0.07%) (7) Isoelectric point: 8.3 (Isoelectric focusing electrophoresis); (8) Solubility: Easily soluble in DMSO (≥60mg / mL), methanol (≥30mg / mL), and 0.1% TFA aqueous solution (≥12mg / mL); slightly soluble in ethanol (≈7mg / mL); insoluble in n-hexane and petroleum ether; (9) ESI-MS (ESI+): m / z 988.30 [M+H] + (Theoretical value 988.32 Da), m / z 1010.28 [M+Na] + (Theoretical value 10¹⁰.31 Da), m / z 494.66 [M+2H] 2+ (Theoretical value 494.66 Da); (10) 1¹H NMR (400 MHz, DMSO-d6): δ 8.50–8.10 (m, 7H, peptide bond -NH-), 7.35–7.15 (m, 5H, Phe-benzene ring H), 4.72–4.10 (m, 7H, amino acid α-CH), 3.87 (s, 3H, N-Me-Leu of -N-CH3), 3.75–3.68 (m, 2H, Gly-CH2), 3.18–2.95 (m, 2H, Lys of ε-CH2-), 2.85–2.68 (m, 2H, Asp of β-CH2-), 2.32 (t, 2H, lauroyl-CH2-CO-, J = 7.5 Hz), 1.98–0.85 (m, 47H, side chain alkyl H + lauroyl C). 10 H 21 -); (11) 13 C NMR (100 MHz, DMSO-d6): δ 174.9–171.5 (m, 8 peptide bonds -CO- + lauroyl -CO-, 9 carbonyl carbons in total), 138.6 (s, Phe-benzene ring quaternary carbon), 129.2–126.8 (m, Phe-benzene ring substituted carbon), 58.6 (s, α-C of N-Me-Leu), 55.2–51.8 (m, α-C of other amino acids), 42.5 (s, Gly-CH2), 38.7 (s, N-Me-Leu -N-CH3), 34.0 (s, lauroyl -CH2-CO-), 32.6 (s, ε-CH2- of Lys), 30.5 (s, β-CH2- of Asp), 29.8–22.5 (m, side chain alkyl carbon + lauroyl C) 10 H 21 -carbon), 14.6 (s, lauroyl-terminal -CH3); (12) HPLC purity: 98.8% (220nm), 98.7% (258nm), single impurity ≤0.3%.

[0072] (13) Selectivity verification of N-terminal lauroylation modification: The prepared pure product was analyzed by high performance liquid chromatography-mass spectrometry (HPLC-MS) to determine the presence of Lys ε-aminolaurylation byproducts: Chromatographic conditions: same as the purification conditions described above, detection wavelength 220 nm; Mass spectrometry conditions: ESI+ mode, scan range m / z 800~1200; Results: Only the target product peak (m / z 988.30 [M+H]) was detected. + No Lys ε-aminolaurylylation byproducts were detected (theoretical m / z 1175.5 Da), confirming that the N-terminal modification selectivity was over 99.5% and there were no side chain modifications.

[0073] Example 2: N-terminal lauroylation modified LMN-NKA peptide derivative hydrochloride and its preparation Take 0.5 g (0.492 mmol) of the pure derivative prepared in Example 1, add 10 mL of methanol-water mixed solvent (volume ratio 1:1), and slowly add 1 mol / L hydrochloric acid solution dropwise under magnetic stirring until the pH reaches 2-3 (approximately 0.55 mL is required). Stir at room temperature for 1 h. Concentrate the reaction solution to approximately 2 mL under reduced pressure at 40 °C, and slowly add 50 mL of ice-cold diethyl ether while stirring. A white crystalline solid precipitates. Centrifuge at 8000 rpm for 5 min, collect the precipitate, wash three times with ice-cold diethyl ether (20 mL each time), and vacuum dry (40 °C, 2 h) to obtain 0.52 g of white powdered hydrochloride, with a yield of 97.8%.

[0074] Hydrochloride characterization: Appearance: White crystalline powder; Solubility: Easily soluble in water (solubility 36 mg / mL), methanol (≥45 mg / mL), slightly soluble in ethanol (≈10 mg / mL); ESI-MS (ESI+): m / z 988.30 [M+H] + (The peak of the free base molecular ion, with hydrochloride at [M+H]) + (formal ionization); Infrared absorption (KBr tablet): 3415cm -1 (NH stretching vibration, redshifted compared to free base), 1651 cm⁻¹ -1 (Peptide bond C=O stretching), 1537cm -1 (NH bending), 1733cm -1 (Lauroyl-CO stretching), 1244cm -1 (CN telescopic), 2962cm -1 (alkyl CH stretching); Enzymatic half-life: 6.9 h (37 °C, trypsin, enzyme:peptide = 1:50, w / w); NK2 receptor binding Ki value: 0.77 nM (comparable to free base activity).

[0075] 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 to examine process repeatability. The results are shown in Table 1. Table 1 The results in Table 1 show that the RSD of the pure product yield, purity, retention time and enzymatic half-life in the three batches of experiments were all ≤2%, indicating that the synthesis process has good reproducibility, stable key parameters and is suitable for industrial scale-up production.

[0076] 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.

[0077] 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 lauroylated 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, with the structural formula: C 11 H 23 CO-Asp-Lys-Phe-Val-Gly-N-Me-Leu-Nle.

2. The N-terminal lauroylated 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 lauroyl group, and the remaining amino acids are unmodified amino acids, and the carboxyl group of the leucine is in a free state. 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 lauroylated 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 2.85±0.07, an isoelectric point of 8.2±0.2, and a specific rotation [α]. 25 D = -39.5°±0.3° (c=1.0, methanol), melting point is 232.6~234.4℃, 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 preparation method includes the following steps: (1) A solid-phase reaction column loaded with 2-chlorotriphenylmethyl chloride resin is provided, and the initial amino acid Nle is loaded onto the 2-chlorotriphenylmethyl chloride 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 polypeptide is subjected to directional lauroylation modification to obtain an N-terminal lauroylated LMN-NKA polypeptide derivative. (4) The N-terminal lauroylated LMN-NKA polypeptide derivative is cleaved and separated from the 2-chlorotriphenylmethyl chloride resin using a cleavage reagent, and the purified N-terminal lauroylated 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), 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%. The purification process is preferably performed using a C18 column with gradient elution using mobile phase A and mobile phase B. Mobile phase A is a TFA aqueous solution with a mass-volume percentage of 0.08%~0.12%, and mobile phase B is a TFA acetonitrile solution with a mass-volume percentage of 0.08%~0.12%. The gradient elution conditions are: phase B 30%→80%, 25~35 min, flow rate 0.8~1.5 mL / min, and detection wavelength 220 nm. Target peaks with a purity ≥98.8% are collected. The freeze-drying conditions are preferably -60~-40℃ and 0.008~0.012 mbar for 15~20 h.

7. The method for preparing the LMN-NKA polypeptide derivative according to claim 4, characterized in that, In step (1), the degree of substitution of the 2-chlorotriphenylmethyl chloride resin is 0.5~0.6 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, chronic neurogenic pain, rheumatoid arthritis, inflammatory bowel disease, and obesity.

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