CN-122011100-A - N-terminal palmitoylation/C-terminal ethylamino double-modified LMN-NKA polypeptide derivative, pharmaceutically acceptable salt thereof and preparation method thereof

CN122011100ACN 122011100 ACN122011100 ACN 122011100ACN-122011100-A

Abstract

The invention relates to an N-palmitoyl/C-terminal ethylamino double-modified LMN-NKA polypeptide derivative, a pharmaceutically acceptable salt thereof and a preparation method thereof, wherein the chemical name of the LMN-NKA polypeptide derivative is N-palmitoyl-L-aspartic acid-L-lysine-L-phenylalanine-L-valine-glycine-N-methyl-L-leucine-L-norleucine-ethylamine, and the structural formula is Pal-Asp-Lys-Phe-Val-Gly-N-Me-Leu-Nle-NHCH 2 CH 3 . The double modification strategy can specifically retain the NK 2 receptor binding capacity of the neurokinin A core active fragment, cooperatively promote enzymolysis stability, fat solubility and in-vivo metabolic stability, remarkably improve transmembrane efficiency and realize the accurate balance of water solubility and fat solubility. The polypeptide derivative and the salt thereof are particularly suitable for preparing long-acting therapeutic drugs for 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. The N-terminal palmitoylation/C-terminal ethylamino double-modified LMN-NKA polypeptide derivative and pharmaceutically acceptable salt thereof are characterized in that the LMN-NKA polypeptide derivative is N-palmitoyl-aspartic acid-lysine-phenylalanine-valine-glycine-N-methyl-leucine-norleucine-ethylamine, and the structural general formula of the LMN-NKA polypeptide derivative is C 15 H 31 CO-Asp-Lys-Phe-Val-Gly-N-Me-Leu-Nle-NHCH 2 CH 3 .
2. The N-terminal palmitoylation/C-terminal ethylamino double-modified LMN-NKA polypeptide derivative and pharmaceutically acceptable salt thereof according to claim 1, wherein only the α -amino group of aspartic acid is substituted by palmitoyl group and the epsilon-amino group of lysine and other amino acid side chains are unmodified; And/or, the carboxyl group of norleucine forms-CONHCH 2 CH 3 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 palmitoylation/C-terminal ethylamino double-modified LMN-NKA polypeptide derivative and pharmaceutically acceptable salt thereof according to claim 1, wherein the LMN-NKA polypeptide derivative has a log p value of 3.92±0.09, an isoelectric point of 8.5±0.2, a specific optical rotation [ α ] 25 D = -42.3 ° ±0.3 ° (c=1.0, methanol), a melting point of 245.7 to 247.5 ℃ and a moisture content of 0.5% or less; And/or, the ESI-MS measured molecular ion peak m/z of the LMN-NKA polypeptide derivative is 1042.45-1042.57 Da; and/or the enzymolysis half-life of the LMN-NKA polypeptide derivative under the conditions of 37 ℃ and trypsin (enzyme: peptide=1:50, w/w) is more than or equal to 10.0h, the residual rate after 4h incubation in human plasma is more than or equal to 90%, the NK 2 receptor binding Ki value is less than or equal to 0.80nM, and the selectivity to the NK 1 、NK 3 receptor is more than or equal to 110 times.
4. The method of preparing an LMN-NKA polypeptide derivative according to any one of claims 1 to 3, wherein the combination of N-terminal directed palmitoylation and C-terminal ethylamine techniques is performed by a solid phase synthesis method, comprising the steps of: (1) Providing a solid phase reaction column loaded with 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, carrying out directional palmitoylation modification on the N end of the LMN-NKA polypeptide to obtain N end palmitoylation LMN-NKA polypeptide; (4) In the solid-phase reaction column, carrying out amination reaction on the N-terminal palmitoylation LMN-NKA polypeptide and ethylamine to obtain an N-terminal palmitoylation/C-terminal ethylamino double-modified LMN-NKA polypeptide derivative; (5) And cutting and separating the N-terminal palmitoylation/C-terminal ethylamino-double-modified LMN-NKA polypeptide derivative from the 2-chlorotrityl chloride resin by adopting a cutting reagent, and performing aftertreatment to obtain the purified N-terminal palmitoylation/C-terminal ethylamino-double-modified LMN-NKA polypeptide derivative.
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 palmitoyl chloride and DIPEA is added to the solid phase reaction column, the N-terminal palmitoyl LMN-NKA polypeptide is obtained by reaction, preferably the palmitoyl chloride content is 3-5 mmol, and/or the DIPEA content is preferably 3-5 mmol, and/or the volume ratio of the DMF to the DCM is 1 (1-3), and/or the reaction temperature is preferably 20-30 ℃, and/or the reaction time is preferably 3-5 h, and/or the palmitoyl chloride preparation method is characterized in that the palmitoyl acid and the thionyl chloride are subjected to reflux reaction in the presence of a catalyst, and the obtained reaction liquid is subjected to reduced pressure distillation and collected to obtain 160-162 ℃/2kPa fraction, namely the palmitoyl chloride, preferably the molar ratio of the palmitoyl acid to the thionyl chloride is 1 (1.1-1.5), and/or the reaction temperature is preferably 20-30 ℃ and/or the reaction time is preferably 3-5 h, and/or the reflux reaction time is preferably 80-3 h.
6. The method for producing an LMN-NKA polypeptide derivative according to claim 4, wherein in step (4), a mixed solution of ethylamine and DMF is added to the solid phase reaction column to carry out the reaction, after the reaction is completed, the resin is drained, the resin is washed with DMF and DCM, preferably the volume fraction of ethylamine in the mixed solution of ethylamine and DMF is 25% -35%, and/or the reaction is preferably carried out at 20-30 ℃, and/or the reaction time is preferably 3-5 h.
7. The preparation method of the LMN-NKA polypeptide derivative according to claim 4, wherein in the step (5), the cutting reagent is a mixed solution of TFA, TIS and H 2 O (0.8-1.2) according to the volume ratio of (36-40), and/or the cutting reagent is added into the solid phase reaction column to react for 3-4 at 20-30 ℃, the filtrate is collected by filtration, the filtrate is subjected to post-treatment, preferably the post-treatment comprises adding the filtrate into glacial diethyl ether precooled to-20 ℃, the crude peptide is collected by centrifugation, the crude peptide is dissolved by adopting a TFA aqueous solution, and then a white powder pure product is obtained by freeze-drying after reverse phase HPLC purification, preferably the TFA aqueous solution is subjected to gradient elution by adopting a C18 column, the mobile phase A is 0.08-0.12% aqueous solution, the mobile phase B is 0.08-0.08% aqueous solution, the mobile phase B is 0.08-0.45% TFA, the gradient elution is 0.25-25 m 2-0.98 nm, the gradient elution is carried out for 0.25-25 m, the target purity is more than or equal to 0.98 nm, and the conditions of the gradient elution is more than or equal to 0.40-25 nm; and/or in the 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 into a solid phase reaction column, then DMF is added for soaking, and DCM is used for washing the resin after pumping; 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. 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. 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. 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, rheumatoid arthritis, neuropathic pain, inflammatory bowel disease, obesity.

Description

N-terminal palmitoylation/C-terminal ethylamino double-modified LMN-NKA polypeptide derivative, 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-palmitoylation/C-terminal ethylamino double-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 only solve the problem of N-terminal enzymolysis, C-terminal is still easy to degrade, the enzymolysis stability is improved to a limited extent (half-life is less than or equal to 3.5 h), the C-terminal esterification modification of Chinese patent CN110590128A leads to the reduction of NK 2 receptor binding activity by more than 40%, the prior art （Zhang Y, et al. C-terminal amination of peptides: synthesis and biological activity [J]. Journal of Peptide Research, 2021, 77 (4): e3365） also reports that the modification (such as palmitoylation) of N-terminal long-chain fatty acid can improve the fat solubility and N-terminal stability, but the enzymolysis problem of C-terminal free carboxyl is not solved, and the defects of poor modification selectivity, rapid reduction of water solubility (generally less than or equal to 5 mg/mL) and the like are overcome. Meanwhile, double modification of LMN-NKA sequences faces special technical problems that the reaction compatibility of ① C-terminal ethylamino and N-terminal palmitoylation is poor, palmitoyl chloride hydrolysis or peptide chain degradation is easy to occur under conventional amination conditions, ② C-terminal modification is incomplete, hydrophobicity of products after the double modification of ③ is further enhanced, separation difficulty of impurities and incompletely aminated products is greatly improved due to Lys side chain side modification, and purity requirements are difficult to meet by conventional purification processes. Therefore, a double-modification strategy capable of cooperatively solving the problem of double enzymolysis of the N end and the C end of the LMN-NKA fragment is developed, the cooperative optimization of activity-stability-water solubility-bioavailability is realized, a preparation process with high selectivity and high yield is established, the technical bottleneck of the existing single-modification strategy is broken through, and the method has important technical breakthrough significance and application value for promoting the clinical transformation of NK 2 receptor targeted drugs. Disclosure of Invention The invention aims to solve the problem of providing an N-terminal palmitoylation/C-terminal ethylamino double-modified LMN-NKA polypeptide derivative, a pharmaceutically acceptable salt thereof and a preparation method thereof. The invention provides an N-palmitoyl/C-terminal ethylamino double-modified LMN-NKA polypeptide derivative and pharmaceutically acceptable salt thereof, wherein the chemical name of the LMN-NKA polypeptide derivative is N-palmitoyl-aspartic acid-lysine-phenylalanine-valine-glycine-N-methyl-leucine-norleucine-ethylamine, and the structural general formula of the LMN-NKA-NHCH 2CH3 is C 15H31CO-Asp-Lys-Phe-Val-Gly-N-Me-Leu-Nle-NHCH2CH3. In particular, only the α -amino group of the aspartic acid is substituted with palmitoyl groups (Pal-, i.e., C 15H31 CO-) and the epsilon-ami