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CN-122011099-A - N-terminal acetylation modified LMN-NKA polypeptide derivative or pharmaceutically acceptable salt thereof and preparation method thereof

CN122011099ACN 122011099 ACN122011099 ACN 122011099ACN-122011099-A

Abstract

The invention relates to an N-terminal acetylation modified LMN-NKA polypeptide derivative or pharmaceutically acceptable salt thereof and a preparation method thereof, wherein the chemical name of the LMN-NKA polypeptide derivative is N-acetyl-L-aspartic acid-L-lysine-L-phenylalanine-L-valine-glycine-N-methyl-L-leucine-L-norleucine amide, and the structural general formula is Ac-Asp-Lys-Phe-Val-Gly-N-Me-Leu-Nle-NH 2 . The modification strategy of the invention only carries out acetylation modification on the free amino at the N end of the parent structure, the natural amidation is reserved at the C end, the rest amino acid sequence and configuration are completely unchanged, the spatial conformation of the receptor binding site of the parent structure is not changed, meanwhile, the polarity of the polypeptide is reduced by closing the free amino, the recognition and hydrolysis of aminopeptidase in the body are reduced, the enzymolysis stability is obviously improved, the modification process is simple, and no additional impurity is introduced. The LMN-NKA polypeptide derivative or the pharmaceutically acceptable salt thereof is particularly suitable for preparing the therapeutic drugs for the related diseases of NK 2 receptors.

Inventors

  • ZHANG NAN
  • HU MINGHAI
  • 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 acetylation modified LMN-NKA polypeptide derivative or pharmaceutically acceptable salt thereof, which is characterized in that the chemical name of the LMN-NKA polypeptide derivative is N-acetyl-L-aspartic acid-L-lysine-L-phenylalanine-L-valine-glycine-N-methyl-L-leucine-L-norleucine amide, and the structural general formula is Ac-Asp-Lys-Phe-Val-Gly-N-Me-Leu-Nle-NH 2 .
  2. 2. The LMN-NKA polypeptide derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein the LMN-NKA polypeptide derivative has only the free a-amino group of the L-aspartic acid at the N-terminus substituted with acetyl; And/or the amino acid sequence is L-aspartic acid, L-lysine, L-phenylalanine, L-valine, glycine, N-methyl-L-leucine, L-norleucine in sequence; and/or all amino acids except the glycine are in the L-configuration; and/or, the carboxyl of the L-norleucine at the C end forms an amide bond without other terminal modifications; And/or the pharmaceutically acceptable salt is selected from one or more of hydrochloride, acetate, phosphate and sulfate.
  3. 3. The LMN-NKA polypeptide derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein the molecular ion peak m/z measured by ESI-MS of the LMN-NKA polypeptide derivative is 883.01-883.11 da.
  4. 4. The LMN-NKA polypeptide derivative or a pharmaceutically acceptable salt thereof according to claim 1, wherein the enzymatic half-life of the LMN-NKA polypeptide derivative is ∈4.0h or more and ∈6h or less under conditions of 37 ℃ trypsin (enzyme: peptide=1:50, w/w).
  5. 5. The method of producing an LMN-NKA polypeptide derivative according to any one of claims 1 to 4, wherein the technique of N-terminal acetylation directed modification is carried out by solid phase synthesis, comprising the steps of: (1) Providing a solid phase reaction column loaded with RINK AMIDE MBHA resin and loading an initial amino acid Nle onto the RINK AMIDE MBHA 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 acetylation modification on the N end of the LMN-NKA polypeptide to obtain an N-end acetylation modified LMN-NKA polypeptide; (4) And cutting and separating the N-terminal acetylation modified LMN-NKA polypeptide derivative from the RINK AMIDE MBHA resin by adopting a cutting reagent, and obtaining the purified N-terminal acetylation modified LMN-NKA polypeptide derivative through post-treatment.
  6. 6. The method for producing an LMN-NKA polypeptide derivative according to claim 5, wherein in the step (1), the RINK AMIDE MBHA resin has a substitution degree of 0.5 to 0.8mmol/g; and/or, in the step (1), adding DMF into the solid phase reaction column loaded with RINK AMIDE MBHA resin, soaking, draining, washing with DCM, adding DCM solution containing Fmoc-Nle-OH and DIPEA, stirring at 20-30 ℃, adding a mixed solution of methanol and DIPEA, stirring at 20-30 ℃, draining, and washing with DMF; And/or in the step (2), the coupling operation method comprises the steps of adding a DMF solution containing Fmoc-protected amino acid, HBTU and HOBt into the solid phase reaction column, stirring and reacting at 20-30 ℃, and detecting the reaction end point by using ninhydrin; And/or, in the step (3), after removing Fmoc protecting groups of Asp, adding DMF solution containing acetyl anhydride into the solid phase reaction column, stirring at 20-30 ℃ for reaction, washing with DMF and DCM respectively, and detecting the reaction end point by ninhydrin; And/or in the step (4), the cutting reagent is a mixed solution of TFA, TIS and H 2 O according to the volume ratio of (36-40): (0.8-1.2): 1; And/or, in the step (4), adding the cutting reagent into the solid phase reaction column, reacting at 20-30 ℃, filtering and collecting filtrate, and performing post-treatment on the filtrate, wherein the post-treatment preferably comprises adding the filtrate into pre-cooled glacial diethyl ether, centrifuging and collecting precipitate to obtain crude peptide, dissolving the crude peptide by using TFA aqueous solution, purifying by reversed phase HPLC, and freeze-drying to obtain white powder pure product; and/or in the step (4), the cutting time is 3-5 h.
  7. 7. The method for producing an LMN-NKA polypeptide derivative according to claim 6, wherein in the step (2), the molar ratio of the Fmoc-protecting amino acid to the RINK AMIDE MBHA resin is 3:0.8-1.2; and/or in the step (2), the DMF solution containing Fmoc-protected amino acid, HBTU and HOBt further contains DIPEA to adjust the pH to 8.0-8.5, preferably, wherein the molar ratio of the HBTU, the HOBt and the DIPEA is 1:0.8-1.2:1.6-2.4; And/or in the step (2), the coupling temperature is controlled to be 20-25 ℃; And/or, in step (3), adding a solution of piperidine in DMF to the solid phase reaction column to remove the Fmoc protecting group of Asp; And/or, in the step (3), the molar ratio of the acetyl anhydride to the RINK AMIDE MBHA resin is 3.5:0.8-1.2; And/or, in the step (3), the using amount of the acetyl anhydride is 1.1-1.5 times of the substitution degree of RINK AMIDE MBHA resin; And/or, in the step (3), DIPEA is further added into the DMF solution containing the acetyl anhydride to adjust the pH to 8.0-8.5; And/or, in the step (4), a C18 column is adopted for purification, gradient elution is carried out by using a mobile phase A and a mobile phase B, wherein the mobile phase A is TFA water with the mass and volume percentage of 0.08% -0.12%, the mobile phase B is TFA acetonitrile with the mass and volume percentage of 0.08% -0.12%, the gradient elution condition is that the temperature rising rate of the mobile phase B is controlled to be 1.8% -2.2%/min, the flow rate is 0.8% -1.5 mL/min, the detection wavelength is 220nm, the target peak with the purity of more than or equal to 98.5% is collected, and the temperature rising rate of the mobile phase B is preferably controlled to be 1.8% -2.2%/min; and/or in the step (4), the yield of the freeze-dried product is 85% -90%.
  8. 8. The method for preparing a pharmaceutically acceptable salt of an LMN-NKA polypeptide derivative according to any one of claims 1 to 4, wherein the LMN-NKA polypeptide derivative is dissolved in methanol, a methanolic hydrochloric acid solution is added to a ph=2 to 3, the mixture is stirred and concentrated under reduced pressure, and the pharmaceutically acceptable salt is obtained by recrystallization from an ether, preferably for a period of 1 to 2 hours, and/or the ether is preferably diethyl ether.
  9. 9. The use of the LMN-NKA polypeptide derivative and pharmaceutically acceptable salts thereof as claimed in any one of claims 1 to 4 in the manufacture of a medicament for the treatment of diseases associated with the NK 2 receptor.
  10. 10. The use according to claim 9, wherein said treatment of NK 2 receptor-related disorders comprises inflammation, abnormal nerve signaling and obesity.

Description

N-terminal acetylation modified LMN-NKA polypeptide derivative or pharmaceutically acceptable salt thereof and preparation method thereof Technical Field The invention belongs to the field of polypeptide pharmaceutical chemistry, and in particular relates to an N-terminal acetylation modified LMN-NKA polypeptide derivative or pharmaceutically acceptable salt thereof and a preparation method. Background Neurokinin A (NKA) is an important neuropeptide, and a 4-10-bit fragment (Asp-Lys-Phe-Val-Gly-N-Me-Leu-Nle-NH 2) of the neurokinin A is taken as a core active region, can be specifically combined with a neurokinin receptor (NK 2 receptor), and plays a key role in physiological processes such as inflammatory regulation, smooth muscle contraction, nerve signal transmission, energy metabolism control and the like. However, the natural polypeptide fragment has the defects of poor enzymolysis stability, short in vivo half-life, low bioavailability caused by high polarity and the like, and the clinical application of the natural polypeptide fragment is limited. In the prior art, N-terminal modification of polypeptides is a classical strategy to ameliorate the above-mentioned drawbacks. For example, patent WO2019123456A1 discloses N-terminal alkylated modified neuropeptide derivatives, but the modification results in a 30% decrease in receptor binding activity or more, and non-patent document Journal of PEPTIDE SCIENCE, volume 28 2022 reports N-terminal benzoylated NKA analogs, which have improved stability but complex synthesis process, require multiple steps of pre-modification, and have a product purity of 98% or less. Furthermore, the schemes of C-terminal modification or amino acid sequence substitution, such as patent CN110590128a, tend to alter the spatial conformation of the receptor binding site, resulting in loss of biological activity. In recent years, polypeptide medicine research and development become a hot spot in the field of new medicines, more than 80 polypeptide medicines are sold in batches, and more than 170 polypeptide medicines enter a clinical test stage, wherein the non-natural modified polypeptide has great attention due to excellent proteolytic stability and pharmacokinetic properties. However, aiming at modification of LMN-NKA (4-10 bits) fragments, the prior art has not found a scheme of 'only N-terminal acetylation without changing core sequence and C-terminal amidation', and the modification strategy not only maintains receptor binding activity, but also can improve enzymolysis stability by reducing N-terminal polarity, thereby solving the clinical application bottleneck of natural fragments. Therefore, the LMN-NKA derivative which does not change the core sequence of the parent structure and the modification of the C end, improves the stability only through the accurate modification of the N end and has simple and controllable synthesis process is developed, and has important clinical application value and industrialization prospect. Disclosure of Invention The invention aims to provide an N-terminal acetylation modified LMN-NKA polypeptide derivative or pharmaceutically acceptable salt thereof and a preparation method thereof. The invention provides an N-terminal acetylation modified LMN-NKA polypeptide derivative or pharmaceutically acceptable salt thereof, wherein the chemical name of the LMN-NKA polypeptide derivative is N-acetyl-L-aspartic acid-L-lysine-L-phenylalanine-L-valine-glycine-N-methyl-L-leucine-L-norleucine amide, and the structural general formula is Ac-Asp-Lys-Phe-Val-Gly-N-Me-Leu-Nle-NH 2. Specifically, the LMN-NKA polypeptide derivative has only the free alpha-amino group of the L-aspartic acid (Asp) at the N-terminal thereof replaced by acetyl (Ac-) and the epsilon-amino group of lysine (Lys) and other amino acid side chains have no modification. Specifically, the amino acid sequence is L-aspartic acid (Asp), L-lysine (Lys), L-phenylalanine (Phe), L-valine (Val), glycine (Gly), N-methyl-L-leucine (N-Me-Leu) and L-norleucine (Nle) in sequence, and the sequence is not replaceable. Specifically, all other amino acids except the glycine are in the L-configuration, and specifically glycine is achiral and does not distinguish between specific configurations. Specifically, the carboxyl group of L-norleucine (Nle) at the C end forms an amide bond (-NH 2) with the amino group, and no other terminal modification exists. In some embodiments, the pharmaceutically acceptable salt is selected from one or more of hydrochloride, acetate, phosphate, sulfate. Specifically, ESI-MS measured molecular ion peak m/z of the LMN-NKA polypeptide derivative is 883.01-883.11 Da, and theoretical molecular weight is 883.06Da. Specifically, the enzymolysis half-life of the LMN-NKA polypeptide derivative under the condition of 37 ℃ and trypsin (enzyme: peptide=1:50, w/w) is more than or equal to 4.0h and less than or equal to 6h, such as 4h, 4.5h, 5h, 5.5h or 6h. The second aspect of the present