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CN-122011101-A - N-terminal lauroyl modified LMN-NKA polypeptide derivative, pharmaceutically acceptable salt thereof and preparation method

CN122011101ACN 122011101 ACN122011101 ACN 122011101ACN-122011101-A

Abstract

The invention relates to an N-end lauroyl modified LMN-NKA polypeptide derivative, a pharmaceutically acceptable salt thereof and a preparation method thereof, wherein 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-L-norleucine, and the structural formula is Lau-Asp-Lys-Phe-Val-Gly-N-Me-Leu-Nle. According to the invention, through a single precise modification strategy of N-end lauroyl (long chain fatty acid chain in C12), NK 2 receptor binding capacity of a neurokinin A core active fragment can be specifically reserved, enzymolysis stability, fat solubility and in-vivo metabolic stability are synergistically improved, transmembrane efficiency is improved, water solubility and fat solubility balance is realized, and further the polypeptide derivative and salt thereof can be used for preparing long-acting therapeutic drugs of NK 2 receptor-related chronic diseases.

Inventors

  • ZHANG NAN
  • NIE JINGJIE
  • ZHONG RONG
  • TAN GUOLIANG

Assignees

  • 橡果美健实业投资股份有限公司
  • 张南
  • 聂静洁

Dates

Publication Date
20260512
Application Date
20260130
Priority Date
20251215

Claims (10)

  1. 1. An N-terminal lauroylation modified LMN-NKA polypeptide derivative and pharmaceutically acceptable salt thereof are characterized in that the LMN-NKA polypeptide derivative is N-lauroyl-aspartic acid-lysine-phenylalanine-valine-glycine-N-methyl-leucine-norleucine, and the structural formula is C 11 H 23 CO-Asp-Lys-Phe-Val-Gly-N-Me-Leu-Nle.
  2. 2. The N-terminal lauroylated modified LMN-NKA polypeptide derivative and pharmaceutically acceptable salts thereof according to claim 1, wherein only the α -amino group of the aspartic acid is replaced by lauroyl, the remaining amino acids are all unmodified amino acids, and the carboxy group of the norleucine 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. 3. The N-terminal lauroylated modified LMN-NKA polypeptide derivative according to claim 1, wherein the LMN-NKA polypeptide derivative has a log p value of 2.85±0.07, an isoelectric point of 8.2±0.2, a specific optical rotation [ α ] 25 D = -39.5 ° ±0.3° (c=1.0, methanol), a melting point of 232.6 to 234.4 ℃ and a moisture content of 0.5% or less.
  4. 4. The method of producing an LMN-NKA polypeptide derivative according to any one of claims 1 to 3, comprising the steps of: (1) Providing a solid phase reaction column loaded with 2-chlorotrityl chloride resin, and loading initial amino acid Nle onto the 2-chlorotrityl chloride resin; (2) In the solid phase reaction column, the LMN-NKA polypeptide is obtained by coupling in sequence of Nle, N-Me-Leu, gly, val, phe, lys and Asp; (3) In the solid-phase reaction column, performing directional lauroyl modification on the N end of the LMN-NKA polypeptide to obtain an LMN-NKA polypeptide derivative with N end lauroyl modification; (4) And cutting and separating the N-end lauroyl-modified LMN-NKA polypeptide derivative from the 2-chlorotrityl chloride resin by adopting a cutting reagent, and obtaining the purified N-end lauroyl-modified LMN-NKA polypeptide derivative after post treatment.
  5. 5. The method for preparing the LMN-NKA polypeptide derivative according to claim 4, wherein in the step (3), a DMF/DCM mixed solution containing lauroyl chloride and DIPEA is added to the solid phase reaction column, and the N-end lauroyl LMN-NKA polypeptide is obtained by reaction, wherein the lauroyl chloride content is preferably 3-5 mmol, and/or the DIPEA content is preferably 2-5 mmol, and/or the volume ratio of DMF to DCM is 1 (1-2), and/or the reaction temperature is preferably 20-30 ℃, and/or the reaction time is preferably 1-3 h, and/or the lauroyl chloride preparation method comprises reflux reaction of lauric acid and thionyl chloride in the presence of a catalyst, wherein the obtained reaction liquid is obtained by distillation and collection of 145-147 ℃/2kPa fraction under reduced pressure, namely lauroyl chloride, the molar ratio of lauric acid to thionyl chloride is preferably 1 (1.1-1.5), and/or the reaction temperature is preferably 20-30 ℃, and/or the reflux reaction time is preferably 1-3 h, and/or the reflux reaction time is preferably 65-75 ℃.
  6. 6. The preparation method of the LMN-NKA polypeptide derivative according to claim 4, wherein in the step (4), the cutting reagent is a mixed solution of TFA, TIS and H 2 O (0.8-1.2) in a volume ratio of (36-40): 1, and/or the cutting reagent is added into the solid phase reaction column to react at 20-30 ℃ for 3-4, and then the filtrate is collected by filtration, preferably the filtrate is subjected to post-treatment, the post-treatment comprises adding the filtrate into diethyl ether precooled to-20 ℃, centrifuging to collect a precipitate, namely crude peptide, dissolving the crude peptide by using a TFA aqueous solution, purifying by reverse phase HPLC, and then freeze-drying to obtain a pure white powder, preferably the TFA aqueous solution is subjected to gradient elution by using a C18 column, the mobile phase A is 0.08-0.12% aqueous solution, the mobile phase B is 0.08-20% aqueous solution, the gradient of TFA is 0.98.98-20 nm, the gradient of freeze-drying is 20nm, and the target freeze-drying is carried out at a gradient of 20 nm-220 nm, and the target freeze-drying is carried out at a flow rate of 20-220 nm, preferably the gradient of 0.98-220 nm.
  7. 7. The method for producing LMN-NKA polypeptide derivatives according to claim 4, wherein in step (1), the substitution degree of the 2-chlorotrityl chloride resin is 0.5-0.6 mmol/g, preferably, the 2-chlorotrityl chloride resin is added to a solid phase reaction column, then added to DMF for soaking, and after pumping, DCM is used for washing the resin; And/or in the step (2), the coupling operation method comprises the steps of adding a DMF solution containing piperidine into the solid phase reaction column, stirring and mixing uniformly at 20-30 ℃, washing by using DMF, adding a DMF solution containing Fmoc-protected amino acid, HBTU, HOBt and DIPEA, stirring and reacting at 20-30 ℃, and detecting the reaction end point by using ninhydrin.
  8. 8. The method for producing a pharmaceutically acceptable salt of an LMN-NKA polypeptide derivative according to any one of claims 1 to 3, wherein the LMN-NKA polypeptide derivative is reacted with an acid in a mixed solvent of an alcohol and water, and then the reaction is carried out by concentrating under reduced pressure to remove methanol, and then the pharmaceutically acceptable salt is obtained by recrystallization using an ether, preferably the volume ratio of the alcohol to water is (1 to 3): 1, and/or the alcohol is preferably methanol, and/or the acid is preferably fed in the form of an aqueous acid solution having a pH of 2 to 3, and/or the reaction time is preferably 1 to 2 hours, and/or the ether is preferably diethyl ether.
  9. 9. Use of the LMN-NKA polypeptide derivative as claimed in any one of claims 1 to 3 and pharmaceutically acceptable salts thereof for the manufacture of a medicament for the treatment of chronic diseases associated with the neurokinin NK 2 receptor.
  10. 10. The use according to claim 9, wherein the medicament is in the form of an oral formulation, an inhalant formulation, an injection formulation or a transdermal absorption formulation; And/or, the treatment of neurokinin NK 2 receptor-related chronic diseases includes allergic asthma, chronic neuropathic pain, rheumatoid arthritis, inflammatory bowel disease, obesity.

Description

N-terminal lauroyl modified LMN-NKA polypeptide derivative, pharmaceutically acceptable salt thereof and preparation method Technical Field The invention belongs to the field of polypeptide pharmaceutical chemistry, and in particular relates to an N-end lauroyl modified LMN-NKA polypeptide derivative, a pharmaceutically acceptable salt thereof and a preparation method thereof. Background Neurokinin A (NKA) is an important member of the tachykinin family, and through specific binding to NK 2 receptor, physiological processes such as inflammatory mediator release, airway smooth muscle contraction, neuropathic pain conduction and the like are regulated, and a core active fragment (Asp-Lys-Phe-Val-Gly-N-Me-Leu-Nle, abbreviated as LMN-NKA) is a potential drug molecule for treating diseases such as allergic asthma, rheumatoid arthritis, neuropathic pain and the like due to high affinity and specificity. However, the natural LMN-NKA fragment has obvious clinical conversion bottlenecks, namely that the N-terminal free amino is easy to be identified and hydrolyzed by aminopeptidase in vivo, the C-terminal free carboxyl is easy to be degraded by carboxypeptidase, the stability is extremely poor due to double enzymolysis (the half-life period of trypsin in vitro is only 1.1h, the residual rate of trypsin in human blood plasma is less than or equal to 30 percent), the lipid solubility is extremely low (log P is about 1.05), the biological membrane penetrating capacity is insufficient, the oral bioavailability is extremely low (less than or equal to 1 percent), the in vivo metabolism speed is high, the administration is required to be performed at high frequency (2-3 times per day), and the compliance of patients is poor. In the prior art, the modification strategy for NKA and fragments thereof has obvious limitations that N-terminal short-chain fatty acid modification products (such as octanoylated NKA and C8) disclosed in International patent WO2019123456A1 have limited enzymolysis stability improvement (half-life is less than or equal to 3.5 h), long-chain fatty acid modification (such as palmitoylation and C16) reported in the literature can improve fat solubility (log P is about 3.9) and stability (half-life is about 8.2 h), but has two major core defects that ① has drastically reduced water solubility (usually less than or equal to 5 mg/mL), high concentration preparation is difficult to prepare, toxicity risk is easily caused by aggregation after biological membrane penetration, modification selectivity in the ② synthesis process is poor, and the ratio of Lys side chain auxiliary modification impurities is high (more than or equal to 15 percent), and purification difficulty is high （Zhang Y, et al. C-terminal amination of peptides: synthesis and biological activity [J]. Journal of Peptide Research, 2021, 77 (4): e3365）. Meanwhile, N-terminal modification of LMN-NKA sequences faces special technical problems that the modification selectivity of long-chain fatty acid in ① is difficult to regulate and control, the hydrolysis rate of lauroyl chloride is faster than that of palmitoyl chloride, the modification efficiency is easy to be reduced (less than or equal to 85 percent) by a conventional solvent system, the single modification of ② needs to be balanced with stability and water solubility, short-chain stability is insufficient, long-chain water solubility is extremely poor, and accurate modification of medium-long chain (C12) lacks mature process support. Therefore, a medium-long chain modification strategy in the N end of the LMN-NKA sequence is developed, the problem of enzymolysis of the N end of the LMN-NKA fragment is solved in a targeted manner, the cooperative optimization of activity, stability, water solubility and bioavailability is realized, a high-selectivity and high-yield synthesis process is established, the technical bottlenecks of insufficient stability of short chain modification and poor water solubility of long chain modification in the prior art are broken through, and the method has important technical breakthrough significance and application value for pushing the low-cost and multi-dosage form clinical transformation of NK 2 receptor targeted drugs. Disclosure of Invention The invention aims to solve the problem of providing an N-end lauroyl modified LMN-NKA polypeptide derivative, a pharmaceutically acceptable salt thereof and a preparation method thereof. The invention provides an N-end lauroyl modified LMN-NKA polypeptide derivative and a pharmaceutically acceptable salt thereof, wherein the chemical name of the LMN-NKA polypeptide derivative is N-lauroyl-aspartic acid-lysine-phenylalanine-valine-glycine-N-methyl-leucine-norleucine, and the structural general formula is C 11H23 CO-Asp-Lys-Phe-Val-Gly-N-Me-Leu-Nle (Lau-LMN-NKA for short). In particular, only the α -amino group of the aspartic acid is replaced by lauroyl (Lau-, i.e. C 11H23 CO-) groups, the remaining amino acids being un