WO2025051130A1 - Medicinal compound serving as neuroprotective agent and use of medicinal compound - Google Patents

Abstract

The present application relates to the field of biomedicine, and particularly relates to a medicinal compound serving as a neuroprotective agent and a use of the medicinal compound. The compound has a structure represented by formula (I), is a PSD-95 inhibitor, and can be used for preparing a drug for treating excitotoxicity-related diseases and neuropathic pain.

Description

Medicinal compounds as neuroprotectants and their use Technical Field

The present invention belongs to the field of biomedicine, and specifically relates to a medicinal compound used as a neuroprotectant and application thereof.

Background Art

N-methyl-D-aspartate receptor (NMDAR) plays an important role in the central nervous system, participating in the formation of learning and memory, the formation of synapses in the development of the central nervous system, the formation of plasticity and glutamate-mediated neurotoxicity. NMDA receptors are the main mediators of excitotoxicity (i.e., glutamate-mediated neurotoxicity), which are associated with neurodegenerative diseases and acute brain injury. They can be used as therapeutic targets for certain nervous system diseases, such as cerebral infarction, neuropathic pain, epilepsy and schizophrenia.

Postsynaptic density (PSD) is a super signaling molecule complex of the excitatory postsynaptic membrane and an important substance for the transmission function of synapses. According to the weight of the molecular weight of PSD, it can be divided into four categories: PSD-95, PSD-93, synaptic associated protein 97 (SAP-97) and SAP102.

PSD-95 is the most abundant and important scaffolding protein, mainly present in mature excitatory glutamatergic synapses. Its relative molecular mass (Mr) is 95,000. It is necessary for receptor activity and stability on the postsynaptic membrane and plays an important role in synaptic plasticity. It is the most important synaptic protein that mediates and integrates synaptic information and is also a member of the guanylate-related kinase family. PSD-95 includes three PDZ domains, one SH3 domain and one guanylate kinase-like (GK) domain, which are connected by a connecting region. PSD-95 is almost entirely located in the postsynaptic density of neurons and is involved in anchoring synaptic proteins. Its direct and indirect binding partners include neuroligins, nNOS, NMDA receptors, AMPA receptors and potassium channels.

According to the different splicing of mRNA to genes, more than 10 nNOS (neuronal nitric oxide synthase) spliceosomes are known, with a molecular weight of 160.8KD. nNOS contains two domains, the reduction domain at the C-terminus and the oxidation domain at the N-terminus. The N-terminus has two non-overlapping binding regions: (1) PDZ region: composed of 1 to 99 amino acids, involved in the formation of active nNOS dimers; (2) β-finger structure: composed of 100-300 amino acids, containing a specific amino acid sequence -ETTF-, which can bind to the PDZ of other proteins to exert its function. nNOS can be anchored to the plasma membrane or cytosolic protein through the PDZ-PDZ domain or the C-terminal PDZ reaction. PSD-95 can connect nNOS to the NMDA receptor and effectively activate nNOS through the activation of the NMDA receptor.

Stroke is characterized by the death of neurons in the ischemic area, cerebral hemorrhage area and/or trauma area. Neuronal death or damage caused by cerebral ischemia is a cascade of injury. After cerebral ischemia, tissue blood perfusion decreases, excitatory neurotransmitters increase, NMDA and AMPA receptors are activated, ion channels open, calcium ions flow in, and a large number of enzymes are activated to trigger signal cascades, resulting in multi-pathway neuronal cell damage. Although NMDA receptor antagonists effectively reduce excitotoxicity by blocking glutamate-mediated ion flux, they also block some physiologically important processes. Its downstream PSD-95 triggers a series of ischemic injuries by interacting with a variety of proteins. It is a key site of cerebral ischemic injury and a potential target for drug therapy. Therefore, the development of PSD-95 inhibitors has great medicinal significance for various excitotoxic neurotoxicity-induced nervous system injuries, including stroke. Therefore, NMDA receptor antagonists have failed in clinical trials such as stroke due to low tolerance and lack of efficacy. In contrast, specific inhibition of excitotoxicity can be obtained by using PSD-95 inhibitors to perturb the intracellular nNOS/PSD-95/NMDA receptor complex.

In addition, studies have shown that the excitatory neurotransmitter NMDA plays an important role in anxiety, epilepsy and various neurodegenerative diseases such as Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease or Huntington's disease. For example, studies have shown that excessive excitation of the central glutamatergic system can cause anxiety, and NMDA receptors (NMDARs) are responsible for the main part of glutamate excitatory neurotoxicity. The onset of epilepsy includes three different and continuous pathological and physiological processes: initiation, maintenance and extension of paroxysmal discharges, and inhibition of paroxysmal discharges. In this process, excitatory neurotransmitters such as glutamate and aspartate play an important role. In Alzheimer's disease, PSD-95 participates in the neurotoxic mechanism leading to it through the GluR6-PSD-95-MLK3 pathway. In addition, in Huntington's disease, PSD-95 is a mediator of NMDA receptor and huntingtin mutant neurotoxicity. Therefore, the development of PSD-95 inhibitors is also of great significance for the treatment, improvement and prevention of the above diseases.

Nerinetide (NA-1, TAT-NR2B9c, sequence: YGRKKRRQRRRKLSSIESDV) is a PSD-95 inhibitor that binds to the PSD-95 PDZ domain, thereby disrupting the binding of PSD-95 to NMDA receptors and neuronal nitric oxide synthase (nNOS), and reducing excitotoxicity induced by cerebral ischemia.

WO2010004003A2 describes a dimeric peptide ligand connected by a polyethylene glycol linker (PEG) that simultaneously binds to the PDZ1 and PDZ2 domains of PSD-95, and its use for treating ischemic cerebrovascular diseases. There is still a need for PSD-95 inhibitors with higher affinity to the PDZ1 and PDZ2 domains, which have improved in vivo efficacy in treating ischemic stroke and traumatic brain injury. CN103533949A discloses novel and effective inhibitors of the ternary protein complex of nNOS, PSD-95 and NMDA receptors, and pharmaceutical compositions containing such inhibitors for preventing and/or treating excitotoxin-related diseases and chronic pain symptoms in subjects, wherein AVLX-144 is a dimeric peptide drug candidate that can be used to treat acute ischemic stroke (AIS) and acute subarachnoid hemorrhage (SAH). CN105828832A discloses fatty acid derivatives of dimerization inhibitors of PSD-95. WO2022238530A1 discloses compounds capable of binding to the PDZ domain of PSD-95 and their medical uses as inhibitors of protein-protein interactions mediated by PSD-95. To meet clinical needs, more PSD-95 inhibitors still need to be developed.

Summary of the invention

The purpose of the present invention is to overcome the deficiencies of the above-mentioned prior art and provide a pharmaceutical compound as a neuroprotectant and its application.

To achieve the above object, on the one hand, the present invention provides a compound or a pharmaceutically acceptable salt thereof, wherein the structure of the compound is shown in the following general formula (I):

L ₁ is selected from

_L2 is present or absent, and when present, _L2 is selected from hydrophilic structural units;

_L3 is selected from hydrophilic structural units;

L4 _{and L5} may be independently selected from a linker, -( _CH2 )nNH-, -C(=O)( _CH2 )nNH-, -( _CH2 )nC(=O)-, -( _CH2 )n

C(＝O)NH-;

n and q can be independently selected from integers of 0 to 6; preferably, n is 1, 2 or 4; q is 0, 1 or 2;

P ₁ is selected from cell penetrating peptides (CPP);

_P2 may be selected from cell penetrating peptides (CPP) or _P2 comprises the sequence (X ⁰ ) _p X ¹ TX ² V or (X ⁰ ) _p X ¹ TX ² F or (X ⁰ ) _p X ¹ SX ² V;

_P3 is selected from the sequence (X ⁰ ) _p X ¹ TX ² V or (X ⁰ ) _p X ¹ TX ² F or (X ⁰ ) _p X ¹ SX ² V, wherein

p is 0 or 1;

X ⁰ is selected from S, T, C, Y, N or Q;

X ¹ can be selected from E, Q, A, S;

^X2 can be selected from A, T, L, V, R, Q, D, N, W.

Preferably, the CPP comprises at least 4 amino acid residues selected from arginine and/or lysine.

Preferably, the CPP can be independently selected from the following sequences: YGRKKRRQRRR, rrrqrrkkr, rrrqrrkkrGy, polyarginine consisting of 2 to 30 residues, GRKKRRQRRRPPQQ, GWTLNSAGYLLKINLKALAALAKKIL, RRLSYSRRRF, RQIKIWFQNRRMKWKK, GALFLGWLGAAGSTMGAWSQPKKKRKV, RGGRLSYSRRRFSTSTGR, KLALKLALKALKAALKLA, GALFLAFLAAALSL-MGLWSQPKKKRRV, RQIKIWFQNRRMKWKK, rqikiwfanrrmkwkk, RKKRRRESRKKRRRES, LLIILRRRIRKQAHAHSK, PLIYLRLLRGQF.

Preferably, the hydrophilic structural unit can be selected from (PEG)m, m is independently selected from integers of 0-10.

Preferably, P ₂ and P ₃ each independently comprise the sequence IETDV or LETTF or METTF or YIETDV or YIETWV or NIETDV or NIETWV.

In a second aspect, the present invention provides a compound or a pharmaceutically acceptable salt thereof, wherein the structure of the compound is shown in the following general formula (II):

_L2 is present or absent, and when present, _L2 is selected from hydrophilic structural units;

_L3 is selected from hydrophilic structural units;

L4 _{and L5} may be independently selected from a linker, -( _CH2 )nNH-, -C(=O)( _CH2 )nNH-, -( _CH2 )nC(=O)-, -(CH2)nC(=O ₎

NH-;

n and q are independently selected from integers of 0 to 6; preferably, n is 1, 2 or 4; q is 0, 1 or 2;

P ₁ is selected from cell penetrating peptides (CPP);

_P2 may be selected from cell penetrating peptides (CPP) or _P2 comprises the sequence (X ⁰ ) _p X ¹ TX ² V or (X ⁰ ) _p X ¹ TX ² F or (X ⁰ ) _p X ¹ SX ² V;

_P3 is selected from the sequence (X ⁰ ) _p X ¹ TX ² V or (X ⁰ ) _p X ¹ TX ² F or (X ⁰ ) _p X ¹ SX ² V, wherein

p is 0 or 1;

X ⁰ is selected from S, T, C, Y, N or Q;

X ¹ can be selected from E, Q, A, S;

^X2 can be selected from A, T, L, V, R, Q, D, N, W.

Preferably, the CPP comprises at least 4 amino acid residues selected from arginine and/or lysine.

Preferably, the CPP can be independently selected from the following sequences: YGRKKRRQRRR, rrrqrrkkr, rrrqrrkkrGy, polyarginine consisting of 2 to 30 residues, GRKKRRQRRRPPQQ, GWTLNSAGYLLKINLKALAALAKKIL, RRLSYSRRRF, RQIKIWFQNRRMKWKK, GALFLGWLGAAGSTMGAWSQPKKKRKV, RGGRLSYSRRRFSTSTGR, KLALKLALKALKAALKLA, GALFLAFLAAALSL-MGLWSQPKKKRRV, RQIKIWFQNRRMKWKK, rqikiwfanrrmkwkk, RKKRRRESRKKRRRES, LLIILRRRIRKQAHAHSK, PLIYLRLLRGQF.

Preferably, the hydrophilic structural unit can be selected from (PEG)m, m may be independently selected from integers of 0-10.

Preferably, P ₂ and P ₃ each independently comprise the sequence IETDV or LETTF or METTF or YIETDV or YIETWV or NIETDV or NIETWV.

In a third aspect, the present invention provides a compound or a pharmaceutically acceptable salt thereof, wherein the structure of the compound is shown in the following general formula (III):

_L2 is present or absent, and when present, _L2 is selected from hydrophilic structural units;

_L3 is selected from hydrophilic structural units;

P ₁ is selected from cell penetrating peptides (CPP);

_P2 may be selected from cell penetrating peptides (CPP) or _P2 comprises the sequence (X ⁰ ) _p X ¹ TX ² V or (X ⁰ ) _p X ¹ TX ² F or (X ⁰ ) _p X ¹ SX ² V;

_P3 is selected from the sequence (X ⁰ ) _p X ¹ TX ² V or (X ⁰ ) _p X ¹ TX ² F or (X ⁰ ) _p X ¹ SX ² V, wherein

p is 0 or 1;

X ⁰ is selected from S, T, C, Y, N or Q;

X ¹ can be selected from E, Q, A, S;

^X2 can be selected from A, T, L, V, R, Q, D, N, W. Preferably, the structure of the compound is shown in the following general formula (IV):

P ₁ is selected from cell penetrating peptides (CPP);

_P2 may be selected from cell penetrating peptides (CPP) or _P2 comprises the sequence (X ⁰ ) _p X ¹ TX ² V or (X ⁰ ) _p X ¹ TX ² F or (X ⁰ ) _p X ¹ SX ² V;

_P3 is selected from the sequence (X ⁰ ) _p X ¹ TX ² V or (X ⁰ ) _p X ¹ TX ² F or (X ⁰ ) _p X ¹ SX ² V, wherein

p is 0 or 1;

X ⁰ is selected from S, T, C, Y, N or Q;

X ¹ can be selected from E, Q, A, S;

^X2 can be selected from A, T, L, V, R, Q, D, N, W.

Preferably, _P1 can be selected from YGRKKRRQRRR, rrrqrrkkrGy.

Preferably, P ₂ and P ₃ each independently comprise the sequence IETDV or LETTF or METTF or YIETDV or YIETWV or NIETDV or NIETWV.

In a fourth aspect, the present invention provides a compound or a pharmaceutically acceptable salt thereof, wherein the structure of the compound is shown in the following general formula (V):

_L2 is present or absent, and when present, _L2 is selected from hydrophilic structural units;

_L3 is selected from hydrophilic structural units;

P ₁ is selected from cell penetrating peptides (CPP);

_P2 may be selected from cell penetrating peptides (CPP) or _P2 comprises the sequence (X ⁰ ) _p X ¹ TX ² V or (X ⁰ ) _p X ¹ TX ² F or (X ⁰ ) _p X ¹ SX ² V;

_P3 is selected from the sequence (X ⁰ ) _p X ¹ TX ² V or (X ⁰ ) _p X ¹ TX ² F or (X ⁰ ) _p X ¹ SX ² V, wherein

p is 0 or 1;

X ⁰ is selected from S, T, C, Y, N or Q;

X ¹ can be selected from E, Q, A, S;

^X2 can be selected from A, T, L, V, R, Q, D, N, W.

Preferably, the structure of the compound is shown in the following general formula (VI):

P ₁ is selected from cell penetrating peptides (CPP);

_P2 may be selected from cell penetrating peptides (CPP) or _P2 comprises the sequence (X ⁰ ) _p X ¹ TX ² V or (X ⁰ ) _p X ¹ TX ² F or (X ⁰ ) _p X ¹ SX ² V;

_P3 is selected from the sequence (X ⁰ ) _p X ¹ TX ² V or (X ⁰ ) _p X ¹ TX ² F or (X ⁰ ) _p X ¹ SX ² V, wherein

p is 0 or 1;

X ⁰ is selected from S, T, C, Y, N or Q;

X ¹ can be selected from E, Q, A, S;

^X2 can be selected from A, T, L, V, R, Q, D, N, W.

Further preferably, _P1 can be selected from YGRKKRRQRRR, rrrqrrkkrGy; _P2 can be selected from YGRKKRRQRRR, rrrqrrkkrGy.

Further preferably, P ₂ and P ₃ each independently comprise the sequence IETDV or LETTF or METTF or YIETDV or YIETWV or NIETDV or NIETWV.

In a fifth aspect, the present invention provides a compound or a pharmaceutically acceptable salt thereof, wherein the structure of the compound is shown in the following general formula (VII):

_L2 is present or absent, and when present, _L2 is selected from hydrophilic structural units;

_L3 is selected from hydrophilic structural units;

P ₁ is selected from cell penetrating peptides (CPP);

_P2 may be selected from cell penetrating peptides (CPP) or _P2 comprises the sequence (X ⁰ ) _p X ¹ TX ² V or (X ⁰ ) _p X ¹ TX ² F or (X ⁰ ) _p X ¹ SX ² V;

_P3 is selected from the sequence (X ⁰ ) _p X ¹ TX ² V or (X ⁰ ) _p X ¹ TX ² F or (X ⁰ ) _p X ¹ SX ² V, wherein

p is 0 or 1;

X ⁰ is selected from S, T, C, Y, N or Q;

X ¹ can be selected from E, Q, A, S;

^X2 can be selected from A, T, L, V, R, Q, D, N, W.

Preferably, the structure of the compound is shown in the following general formula (VIII):

P ₁ is selected from cell penetrating peptides (CPP);

_P2 may be selected from cell penetrating peptides (CPP) or _P2 comprises the sequence (X ⁰ ) _p X ¹ TX ² V or (X ⁰ ) _p X ¹ TX ² F or (X ⁰ ) _p X ¹ SX ² V;

_P3 is selected from the sequence (X ⁰ ) _p X ¹ TX ² V or (X ⁰ ) _p X ¹ TX ² F or (X ⁰ ) _p X ¹ SX ² V, wherein

p is 0 or 1;

X ⁰ is selected from S, T, C, Y, N or Q;

X ¹ can be selected from E, Q, A, S;

^X2 can be selected from A, T, L, V, R, Q, D, N, W.

Preferably, _P1 can be selected from YGRKKRRQRRR, rrrqrrkkrGy.

Preferably, P ₂ and P ₃ each independently comprise the sequence IETDV or LETTF or METTF or YIETDV or YIETWV or NIETDV or NIETWV.

As a preferred embodiment of the compound of the present invention, the compound structure can be selected from the following structures:

Furthermore, the pharmaceutically acceptable salt may be selected from trifluoroacetate, acetate, hydrochloride and phosphate.

In a sixth aspect, the present invention provides a pharmaceutical composition comprising any of the above compounds or pharmaceutically acceptable salts thereof and a pharmaceutically acceptable carrier, excipient and/or diluent.

According to the treatment and administration needs, the pharmaceutical composition may also include a pharmaceutically acceptable carrier. The carrier may be selected from diluents, excipients, fillers, adhesives, wetting agents, disintegrants, absorption promoters, surfactants, adsorption carriers, lubricants, etc., and flavoring agents, flavoring agents, etc. may be added if necessary.

The pharmaceutical composition of the present invention can be prepared into various forms such as tablets, powders, granules, capsules, oral liquids and injections. The above-mentioned various dosage forms can be prepared according to conventional methods in the pharmaceutical field. The pharmaceutical composition can be administered parenterally, intravenously, orally, subcutaneously, intraarterially, intracranially, intrathecally, intraperitoneally, topically, intranasally or intramuscularly. Intravenous administration is preferred.

In a seventh aspect, the present invention provides use of the above-mentioned compound or a pharmaceutically acceptable salt thereof or a pharmaceutical composition in the preparation of a medicament for treating, ameliorating or preventing a disease caused by abnormal PSD-95 function in an individual.

The medicine can be tablets, powders, granules, capsules, oral liquids or injections.

Furthermore, the disease caused by the above-mentioned PSD-95 dysfunction can be selected from cerebral apoplexy, stroke, neurodegenerative disease, anxiety or epilepsy, and neuropathic pain.

Furthermore, the above-mentioned stroke is selected from ischemic stroke or hemorrhagic stroke.

Furthermore, the above neurodegenerative disease is selected from Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease or Huntington's disease.

The meaning of the term "peptide" or "polypeptide" is well known to those skilled in the art. Generally, a peptide or polypeptide is two or more amino acids linked by an amide bond, which is formed by the amino group of one amino acid and the carboxyl group of the adjacent amino acid. The polypeptides described herein may contain naturally occurring amino acids or non-naturally occurring amino acids. They may be modified into analogs, derivatives, functional mimetics, pseudopeptides, and the like containing at least two amino acids. Unless it is specified that the N-terminus or C-terminus has a specific modification, a polypeptide comprising a specific amino acid sequence includes unmodified and modified amino and/or carboxyl termini, which is well known to those skilled in the art. A polypeptide of a specific amino acid sequence may include modified amino acids and/or additional amino acids, unless the N- and/or C-terminus contain modifications that prevent further addition of amino acids. Such modifications include, for example, acetylation of the N-terminus and/or amidation of the C-terminus.

The polypeptides of the present invention can be modified to form polypeptide derivatives. As is well known to those skilled in the art, various modifications can be made to the polypeptides. Typical modifications include, but are not limited to, N-terminal acetylation, C-terminal amidation, The invention also includes any well-known modifications of polypeptides. For example, polypeptide derivatives may include chemical modifications of polypeptides, such as alkylation, acylation, carbamylation, iodination or any other modifications that produce polypeptide derivatives. The modifications of polypeptides may include modified amino acids, such as hydroxyproline or carboxyglutamic acid, and may include amino acids connected by non-peptide bonds.

Furthermore, the peptides described above can optionally be derivatized (eg, acetylated, phosphorylated and/or glycosylated) to improve affinity for the inhibitor, to improve the ability of the inhibitor to be transported across the cell membrane, or to improve stability.

For other modifications of the polypeptides of the present invention, non-natural amino acids can be used to replace natural amino acids in the polypeptides, including but not limited to 2-amino fatty acids (Aad), 3-amino fatty acids (βAad), β-alanine, β-aminopropionic acid (βAla), 2-aminobutyric acid (Abu), 4-aminobutyric acid, piperidine carboxylic acid (4Abu), 6-aminohexanoic acid (Acp), 2-aminoheptanoic acid (Ahe), 2-aminoisobutyric acid (Aib), 3-aminoisobutyric acid (βAib), 2-aminopimelic acid (Apm), 2,4-diaminobutyric acid (Dbu), desmosine (Des), 2,2'- Diaminopimelate (Dpm), 2,3-diaminopropionic acid (Dpr), N-ethylglycine (EtGly), N-ethylasparagine (EtAsn), hydroxylysine (Hyl), isohydroxylysine (aHyl), 3-hydroxyproline (3Hyp), 4-hydroxyproline (4Hyp), isodesmosine (Ide), isoleucine (aIle), N-methylglycine (MeGly), N-methylisoleucine (MeIle), 6-N-methyllysine (MeLys), N-methylvaline (MeVal), norvaline (Nva), norleucine (Nle) and ornithine (Orn). Of course, all modified α-amino acids can be replaced by the corresponding β-, γ- or ω-aminocarboxylic acids.

The term "amino acid" refers to a molecule containing an amino group and a carboxyl group. Suitable amino acids include, but are not limited to, the D- and L-isomers of naturally occurring amino acids, and non-naturally occurring amino acids prepared by organic synthesis or other metabolic pathways. As used herein, the term amino acid includes, but is not limited to, α-amino acids, natural amino acids, non-natural amino acids, and amino acid analogs.

The term "naturally occurring amino acid" refers to any of the 20 L-amino acids commonly found in peptides synthesized in nature, i.e., the L-isomers of alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartic acid (Asp or D), cysteine (Cys or C), glutamic acid (Glu or E), glutamine (Glu or Q), glycine (Gly or G), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

The polypeptide of the present invention can be prepared by conventional biosynthesis or chemical synthesis methods.

The term "PDZ domain" refers to a modular protein domain of about 90 amino acids characterized by significant (e.g., at least 60%) sequence identity to the brain synaptic protein PSD-95, the Drosophila septate junction protein Discs-Large (DLG), and the epithelial tight junction protein Z01 (Z01). PDZ domains are also known as Discs-Large homology repeats ("DHRs") and GLGF repeats. PDZ domains generally show retention of a core consensus sequence (Doyle, DA, 1996, Cell 85: 1067-76). Exemplary PDZ domain-containing proteins and PDZ domain sequences are disclosed in U.S. Application No. 10/714,537.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG1 is a HPLC detection chart of compound 23 of Example 1;

FIG2 is a mass spectrum detection diagram of compound 23 in Example 1;

FIG3 is a HPLC detection chart of compound 32 in Example 2;

FIG4 is a mass spectrum detection diagram of compound 32 of Example 2;

FIG5 is a HPLC detection chart of compound 25 in Example 3;

FIG6 is a mass spectrum detection diagram of compound 25 of Example 3;

FIG7 is a HPLC detection chart of compound 26 in Example 4;

FIG8 is a mass spectrum detection diagram of compound 26 of Example 4;

FIG9 is a HPLC detection chart of compound 27 in Example 5;

FIG10 is a mass spectrum detection diagram of compound 27 in Example 5;

FIG11 is a HPLC detection chart of compound 21 in Example 6;

FIG12 is a mass spectrum detection diagram of compound 21 of Example 6;

FIG13 is a HPLC detection chart of compound 22 in Example 7;

FIG14 is a mass spectrum detection diagram of compound 22 in Example 7;

FIG15 is a HPLC detection chart of compound 31 in Example 8;

FIG16 is a mass spectrum detection diagram of compound 31 in Example 8;

FIG17 is a HPLC detection chart of compound 28 in Example 9;

FIG18 is a mass spectrum detection diagram of compound 28 in Example 9;

FIG19 is a graph showing the experimental results of mouse plasma (heparin sodium) stability in Example 5, wherein FIGA is the experimental results of compound 9, and FIGB is the experimental results of compounds 31 and 32;

FIG20 is a graph showing the experimental results of the stability of mouse plasma alteplase (heparin sodium) in Example 5, wherein FIGA is the experimental results of compound 9, and FIGB is the experimental results of compounds 31 and 32;

FIG21 is a flow chart of the drug efficacy test of the mouse tMCAO model in Test Example 6;

FIG22 is a staining image of a mouse brain section in Test Example 6;

FIG. 23 is the statistical result of embolism area in mouse brain slices of Test Example 6.

Specific embodiments

The polypeptide compound and its derivative provided by the present invention adopt a solid phase synthesis method to synthesize its linear precursor, and the crude peptide obtained after cleavage is directly purified to obtain the target compound, or the crude peptide is oxidized with DMSO to form an intramolecular disulfide bond and then purified to obtain the target compound. The synthetic carrier is 2-Chlotrityl Resin resin. During the synthesis process, 2-Chlotrityl Resin resin is firstly placed in N,N- The solid phase carrier is fully swollen in dimethylformamide (DMF), and then the solid phase carrier and the activated amino acid derivative are repeatedly condensed → washed → Fmoc protection → washed → the next round of amino acid condensation to achieve the desired length of the polypeptide chain to be synthesized. Finally, a mixed solution of trifluoroacetic acid: water: triisopropylsilane: anisyl thioether (90: 2.5: 2.5: 5, v: v: v: v) is reacted with the resin to cleave the polypeptide from the solid phase carrier, and then the solid crude product of the linear precursor is obtained after precipitation by frozen methyl tert-butyl ether. The crude linear precursor after cleavage is subjected to disulfide bond oxidation in a neutral solution to obtain the target polypeptide crude product. The solid crude product or the oxidized polypeptide crude product is purified and separated by a C18 reverse phase preparative chromatography column in a system of 0.1% trifluoroacetic acid in acetonitrile/water to obtain the pure product of the polypeptide and its derivatives.

Experimental reagents

Among them, the structural formula of Fmoc-AEEA-OH is

Example 1 Preparation of Compound 23

Step 1: Coupling of the first amino acid Fmoc-Val-OH

168 mg (0.2 mmol) of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 h. Fmoc-Val-OH (0.16 mmol) and diisopropylethylamine (DIEA, 0.64 mmol) were dissolved in 8 mL DCM and added to the resin for reaction at room temperature for 2 h. After the reaction was completed, a blocking solution (10 mL) of DCM: methanol: DIEA (85:10:5, v:v:v) was added at room temperature for 10 min for blocking. The blocked resin was washed 5 times with DCM and 5 times with DMF.

Step 2: Synthesis of linear precursor peptide chain

The linear precursor peptide chain of compound 23 is AEEA-N-I-E-T-W-V.

The resin obtained in step 1 was fully swollen in DMF for 1 h, and then the linear precursor sequence was synthesized from the second D of the carboxyl terminal to the amino terminal. Each coupling cycle was performed as follows:

· Fmoc-deprotection was performed twice with 20% piperidine/DMF (20% v/v, 10 mL), each time for 8 min.

• Rinse the resin 6-8 times with DMF until neutral pH.

Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to the resin and react at room temperature for 1 hour.

• Rinse the resin 4-6 times with DMF before coupling the next amino acid.

After the linear peptide synthesis, the resin was washed 5 times with DMF and 5 times with DCM. The resin was dried in vacuo.

Step 3: Coupling of 3-(FMOC-amino)glutaric acid

Dissolve 0.25mmol 3-(FMOC-amino)pentanedioic acid, 1mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 2mmol N,N-diisopropylethylamine (DIEA) in DMF, add to the resin obtained in step 2, react at room temperature for 1h, and react twice. After the reaction, rinse the resin with DMF 5 times and DCM 5 times.

Step 4: Synthesis of linear precursor peptide chain

The linear precursor peptide chain of compound 23 is Y-G-R-K-K-R-R-Q-R-R-R.

The resin obtained in step 3 was fully swollen in DMF for 1 h, and then the linear precursor sequence was synthesized from the carboxyl end to the amino end. Each coupling cycle was performed as follows:

· Fmoc-deprotection was performed twice with 20% piperidine/DMF (20% v/v, 10 mL), each time for 8 min.

• Rinse the resin 6-8 times with DMF until neutral pH.

Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to the resin and react at room temperature for 1 hour.

• Rinse the resin 4-6 times with DMF before coupling the next amino acid.

After the linear peptide synthesis, the resin was washed 5 times with DMF and 5 times with DCM. The resin was dried in vacuo.

Step 5: Cleavage of the linear precursor peptide chain

Freshly prepared cutting cocktail (10 mL) trifluoroacetic acid: water: triisopropylsilane: thioanisole (90:2.5:2.5:5, v:v:v:v) was added to the resin obtained in step 4 and shaken for 2 hours at room temperature. After the reaction, the reaction solution was filtered, and the resin was washed with trifluoroacetic acid, combined with the reaction solution, and precipitated with 4 times the volume of cold MTBE to obtain a crude product. The crude product was washed with MTBE 3 times and dried in a vacuum.

Step 6: Peptide purification

The crude peptide was dissolved in 20% acetonitrile aqueous solution, filtered through a 0.45um membrane, and separated using a reversed-phase high-performance liquid chromatography system. The buffers were A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). The chromatographic column was a BR-C18 (Saifen) reversed-phase column. During the purification process, the chromatograph detection wavelength was set to 230nm, the flow rate was 15mL/min, and the gradient was 30-60% acetonitrile in 40min. The product-related fractions were collected, and after HPLC identification of the purity, the fractions >95% were combined and freeze-dried to obtain the pure peptide.

Step 7: Detection and characterization methods

The purity and target molecular weight of the peptide obtained in step 6 were determined by analytical high performance liquid chromatography and liquid chromatography/mass spectrometry. The test results are shown in Figures 1 and 2.

Example 2 Preparation of Compound 32

Step 1: Coupling of the first amino acid Fmoc-Val-OH

168 mg (0.2 mmol) of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 h. Fmoc-Val-OH (0.16 mmol) and diisopropylethylamine (DIEA, 0.64 mmol) were weighed and dissolved in 8 mL of DCM and added to the resin, and reacted at room temperature for 2 h. After the reaction was completed, a blocking solution (10 mL) of DCM: methanol: DIEA (85:10:5, v:v:v) was added at room temperature for 10 min for blocking. The blocked resin was washed 5 times with DCM and 5 times with DMF.

Step 2: Synthesis of linear precursor peptide chain

The linear precursor peptide chain of compound 32 is AEEA-N-I-E-T-W-V.

The resin obtained in step 1 was fully swollen in DMF for 1 h, and then the linear precursor sequence was synthesized from the second D of the carboxyl terminal to the amino terminal. Each coupling cycle was performed as follows:

· Fmoc-deprotection was performed twice with 20% piperidine/DMF (20% v/v, 10 mL), each time for 8 min.

• Rinse the resin 6-8 times with DMF until neutral pH.

Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to the resin and react at room temperature for 1 hour.

• Rinse the resin 4-6 times with DMF before coupling the next amino acid.

After the linear peptide synthesis, the resin was washed 5 times with DMF and 5 times with DCM. The resin was dried in vacuo.

Step 3: Coupling of 3-(FMOC-amino)glutaric acid

Dissolve 0.25mmol 3-(FMOC-amino)pentanedioic acid, 1mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 2mmol N,N-diisopropylethylamine (DIEA) in DMF, add to the resin obtained in step 2, react at room temperature for 1h, and react twice. After the reaction, rinse the resin with DMF 5 times and DCM 5 times.

Step 4: Synthesis of linear precursor peptide chain

The linear precursor peptide chain of compound 32 is dR-dR-dR-dQ-dR-dR-dK-dK-dR-G-dY.

The resin obtained in step 3 was fully swollen in DMF for 1 h, and then the linear precursor sequence was synthesized from the carboxyl end to the amino end. Each coupling cycle was performed as follows:

· Fmoc-deprotection was performed twice with 20% piperidine/DMF (20% v/v, 10 mL), each time for 8 min.

• Rinse the resin 6-8 times with DMF until neutral pH.

Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to the resin and react at room temperature for 1 hour.

• Rinse the resin 4-6 times with DMF before coupling the next amino acid.

After the linear peptide synthesis, the resin was washed 5 times with DMF and 5 times with DCM. The resin was dried in vacuo.

Step 5: Cleavage of the linear precursor peptide chain

Freshly prepared cutting cocktail (10 mL) trifluoroacetic acid: water: triisopropylsilane: thioanisole (90:2.5:2.5:5, v:v:v:v) was added to the resin obtained in step 4 and shaken for 2 hours at room temperature. After the reaction, the reaction solution was filtered, and the resin was washed with trifluoroacetic acid, combined with the reaction solution, and precipitated with 4 times the volume of cold MTBE to obtain a crude product. The crude product was washed with MTBE 3 times and dried in a vacuum.

Step 6: Peptide purification

The crude peptide was dissolved in 20% acetonitrile aqueous solution, filtered through a 0.45um membrane, and separated using a reversed-phase high-performance liquid chromatography system. The buffers were A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). The chromatographic column was a BR-C18 (Saifen) reversed-phase column. During the purification process, the chromatograph detection wavelength was set to 230nm, the flow rate was 15mL/min, and the gradient was 30-60% acetonitrile in 40min. The product-related fractions were collected, and after HPLC identification of the purity, the fractions >95% were combined and freeze-dried to obtain the pure peptide.

Step 7: Detection and characterization methods

The purity and target molecular weight of the peptide obtained in step 6 were determined by analytical high performance liquid chromatography and liquid chromatography/mass spectrometry. The test results are shown in Figures 3 and 4.

Example 3 Preparation of Compound 25

Step 1: Coupling of the first amino acid Fmoc-Cys(Trt)-OH

84 mg (0.1 mmol) of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 h. Fmoc-Cys(Trt)-OH (0.08 mmol) and diisopropylethylamine (DIEA, 0.32 mmol) were weighed and dissolved in 5 mL of DCM and added to the resin. The mixture was reacted at room temperature for 2 h. After the reaction was completed, a blocking solution (10 mL) of DCM: methanol: DIEA (85:10:5, v:v:v) was added at room temperature for 10 min for blocking. The blocked resin was washed 5 times with DCM and 5 times with DMF.

Step 2: Synthesis of linear precursor peptide chain

The linear precursor peptide chain of compound 25 is C-K-M-V-T-T-F-G-I-D-V-T-T-T-Y-I-C.

The resin obtained in step 1 was fully swollen in DMF for 1 h, and then the above linear precursor sequence was synthesized from the second position I of the carboxyl terminal to the amino terminal. Each coupling cycle was performed as follows:

· Fmoc-deprotection was performed twice with 20% piperidine/DMF (20% v/v, 10 mL), each time for 8 min.

• Rinse the resin 6-8 times with DMF until neutral pH.

Dissolve 0.5 mmol Fmoc-AA and 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate in DMF Ester (HCTU) and 1 mmol 4-methylmorpholine (NMM) were added to the resin and reacted at room temperature for 1 h.

• Rinse the resin 4-6 times with DMF before coupling the next amino acid.

After the linear peptide synthesis, the resin was washed 5 times with DMF and 5 times with DCM. The resin was dried in vacuo.

Step 3: Coupling of amino acid Fmoc-Lys(Fmoc)-OH

Dissolve 0.5 mmol Fmoc-Lys(Fmoc)-OH, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to the resin obtained in step 1, and react at room temperature for 1 h. After the reaction, rinse the resin with DMF 5 times and DCM 5 times.

Step 4: Synthesis of linear precursor peptide chain

The linear precursor peptide chain of compound 25 is dR-dR-dR-dQ-dR-dR-dK-dK-dR-G-dY.

The resin obtained in step 3 was fully swollen in DMF for 1 h, and then the linear precursor sequence was synthesized from the carboxyl end to the amino end. Each coupling cycle was performed as follows:

· Fmoc-deprotection was performed twice with 20% piperidine/DMF (20% v/v, 10 mL), each time for 8 min.

• Rinse the resin 6-8 times with DMF until neutral pH.

Dissolve 1mmol Fmoc-AA, 1mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 2mmol 4-methylmorpholine (NMM) in DMF, add the resin and react at room temperature for 1h.

• Rinse the resin 4-6 times with DMF before coupling the next amino acid.

After the linear peptide synthesis, the resin was washed 5 times with DMF and 5 times with DCM. The resin was dried in vacuo.

Step 5: Cleavage of the linear precursor peptide chain

Freshly prepared cutting cocktail (10 mL) trifluoroacetic acid: water: triisopropylsilane: thioanisole (90:2.5:2.5:5, v:v:v:v) was added to the resin obtained in step 4 and shaken for 2 hours at room temperature. After the reaction, the reaction solution was filtered, and the resin was washed with trifluoroacetic acid, combined with the reaction solution, and precipitated with 4 times the volume of cold MTBE to obtain a crude product. The crude product was washed with MTBE 3 times and dried in a vacuum.

Step 6: Intramolecular disulfide bond formation

Add DMSO to the crude product obtained in step 5 to fully dissolve (the volume of DMSO is 20% of the total volume of the reaction system), prepare phosphate\guanidine hydrochloride buffer (pH=7.0, containing 50% ACN), mix 1╳PBS:6M guanidine hydrochloride (80:20, v:v) buffer with acetonitrile 1:1 to obtain phosphate\guanidine hydrochloride buffer (pH=7.0, 50% ACN), and then slowly add the polypeptide solution dropwise to the above buffer to a final concentration of 1 mg/mL, and shake at room temperature for 16 hours. LC-MS monitors the reaction results. Then proceed to purification preparation.

Step 7: Peptide purification

After filtering through a 0.45um membrane, it was separated using a reversed-phase high-performance liquid chromatography system, with buffers A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). The chromatographic column was a BR-C18 (Saifen) reversed-phase column, and during the purification process, the chromatograph detection wavelength was set to 230nm, the flow rate was 15mL/min, and the gradient was 30-60% acetonitrile in 40min. The product-related fractions were collected, and after HPLC identification of the purity, the fractions >95% were combined and freeze-dried to obtain the pure polypeptide. The liquid phase and mass spectrometry detection results are shown in Figures 5 and 6.

Example 4 Synthesis of Compound 26

Step 1: Coupling of the first amino acid Fmoc-Val-OH

168 mg (0.2 mmol) of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 h. Fmoc-Val-OH (0.16 mmol) and diisopropylethylamine (DIEA, 0.64 mmol) were weighed and dissolved in 8 mL of DCM and added to the resin. The mixture was reacted at room temperature for 2 h. After the reaction was completed, a blocking solution (10 mL) of DCM: methanol: DIEA (85:10:5, v:v:v) was added at room temperature for 10 min for blocking. The blocked resin was washed 5 times with DCM and 5 times with DMF.

Step 2: Synthesis of linear precursor peptide chain

The linear precursor peptide chain of compound 26 is AEEA-I-E-T-D-V.

The resin obtained in step 1 was fully swollen in DMF for 1 h, and then the linear precursor sequence was synthesized from the second D of the carboxyl terminal to the amino terminal. Each coupling cycle was performed as follows:

· Fmoc-deprotection was performed twice with 20% piperidine/DMF (20% v/v, 10 mL), each time for 8 min.

• Rinse the resin 6-8 times with DMF until neutral pH.

Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to the resin and react at room temperature for 1 hour.

• Rinse the resin 4-6 times with DMF before coupling the next amino acid.

After the linear peptide synthesis, the resin was washed 5 times with DMF and 5 times with DCM. The resin was dried in vacuo.

Step 3: Coupling of 3-(FMOC-amino)glutaric acid

Dissolve 0.25mmol 3-(FMOC-amino)pentanedioic acid, 1mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 2mmol N,N-diisopropylethylamine (DIEA) in DMF, add to the resin obtained in step 2, react at room temperature for 1h, and react twice. After the reaction, rinse the resin with DMF 5 times and DCM 5 times.

Step 4: Synthesis of linear precursor peptide chain

The linear precursor peptide chain of compound 26 is Y-G-R-K-K-R-R-Q-R-R-R.

The resin obtained in step 3 was fully swollen in DMF for 1 h, and then the linear precursor sequence was synthesized from the carboxyl end to the amino end. Each coupling cycle was performed as follows:

· Fmoc-deprotection was performed twice with 20% piperidine/DMF (20% v/v, 10 mL), each time for 8 min.

• Rinse the resin 6-8 times with DMF until neutral pH.

Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to the resin and react at room temperature for 1 hour.

• Rinse the resin 4-6 times with DMF before coupling the next amino acid.

After the linear peptide synthesis, the resin was washed 5 times with DMF and 5 times with DCM. The resin was dried in vacuo.

Step 5: Cleavage of the linear precursor peptide chain

Freshly prepared cutting cocktail (10 mL) trifluoroacetic acid: water: triisopropylsilane: thioanisole (90:2.5:2.5:5, v:v:v:v) was added to the resin obtained in step 4 and shaken for 2 hours at room temperature. After the reaction, the reaction solution was filtered, and the resin was washed with trifluoroacetic acid, combined with the reaction solution, and precipitated with 4 times the volume of cold MTBE to obtain a crude product. The crude product was washed with MTBE 3 times and dried in a vacuum.

Step 6: Peptide purification

The crude peptide was dissolved in 20% acetonitrile aqueous solution, filtered through a 0.45um membrane, and separated using a reversed-phase high-performance liquid chromatography system. The buffers were A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). The chromatographic column was a BR-C18 (Saifen) reversed-phase column. During the purification process, the chromatograph detection wavelength was set to 230nm, the flow rate was 15mL/min, and the gradient was 30-60% acetonitrile in 40min. The product-related fractions were collected, and after HPLC identification of the purity, the fractions >95% were combined and freeze-dried to obtain the pure peptide.

Step 7: Detection and characterization methods

The purity and target molecular weight of the peptide obtained in step 6 were determined by analytical high performance liquid chromatography and liquid chromatography/mass spectrometry. The test results are shown in Figures 7 and 8.

Example 5 Preparation of Compound 27

Step 1: Coupling of the first amino acid Fmoc-Val-OH

168 mg (0.2 mmol) of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 h. Fmoc-Val-OH (0.16 mmol) and diisopropylethylamine (DIEA, 0.64 mmol) were weighed and dissolved in 8 mL of DCM and added to the resin, and reacted at room temperature for 2 h. After the reaction was completed, a blocking solution (10 mL) of DCM: methanol: DIEA (85:10:5, v:v:v) was added at room temperature for 10 min for blocking. The blocked resin was washed 5 times with DCM and 5 times with DMF.

Step 2: Synthesis of linear precursor peptide chain

The linear precursor peptide chain of compound 27 is AEEA-I-E-T-D-V.

The resin obtained in step 1 was fully swollen in DMF for 1 h, and then the linear precursor sequence was synthesized from the second D of the carboxyl terminal to the amino terminal. Each coupling cycle was performed as follows:

· Fmoc-deprotection was performed twice with 20% piperidine/DMF (20% v/v, 10 mL), each time for 8 min.

• Rinse the resin 6-8 times with DMF until neutral pH.

Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to the resin and react at room temperature for 1 hour.

• Rinse the resin 4-6 times with DMF before coupling the next amino acid.

After the linear peptide synthesis, the resin was washed 5 times with DMF and 5 times with DCM. The resin was dried in vacuo.

Step 3: Coupling of 3-(FMOC-amino)glutaric acid

Dissolve 0.25mmol 3-(FMOC-amino)pentanedioic acid, 1mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 2mmol N,N-diisopropylethylamine (DIEA) in DMF, add to the resin obtained in step 2, react at room temperature for 1h, and react twice. After the reaction, rinse the resin with DMF 5 times and DCM 5 times.

Step 4: Synthesis of linear precursor peptide chain

The linear precursor peptide chain of compound 27 is dR-dR-dR-dQ-dR-dR-dK-dK-dR-G-dY.

The resin obtained in step 3 was fully swollen in DMF for 1 h, and then the linear precursor sequence was synthesized from the carboxyl end to the amino end. Each coupling cycle was performed as follows:

· Fmoc-deprotection was performed twice with 20% piperidine/DMF (20% v/v, 10 mL), each time for 8 min.

• Rinse the resin 6-8 times with DMF until neutral pH.

Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to the resin and react at room temperature for 1 hour.

• Rinse the resin 4-6 times with DMF before coupling the next amino acid.

After the linear peptide synthesis, the resin was washed 5 times with DMF and 5 times with DCM. The resin was dried in vacuo.

Step 5: Cleavage of the linear precursor peptide chain

Freshly prepared cutting cocktail (10 mL) trifluoroacetic acid: water: triisopropylsilane: thioanisole (90:2.5:2.5:5, v:v:v:v) was added to the resin obtained in step 4 and shaken for 2 hours at room temperature. After the reaction, the reaction solution was filtered, and the resin was washed with trifluoroacetic acid, combined with the reaction solution, and precipitated with 4 times the volume of cold MTBE to obtain a crude product. The crude product was washed with MTBE 3 times and dried in a vacuum.

Step 6: Peptide purification

The crude peptide was dissolved in 20% acetonitrile aqueous solution, filtered through a 0.45um membrane, and separated using a reversed-phase high-performance liquid chromatography system. The buffers were A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). The chromatographic column was a BR-C18 (Saifen) reversed-phase column. During the purification process, the chromatograph detection wavelength was set to 230nm, the flow rate was 15mL/min, and the gradient was 30-60% acetonitrile in 40min. The product-related fractions were collected, and after HPLC identification of the purity, the fractions >95% were combined and freeze-dried to obtain the pure peptide.

Step 7: Detection and characterization methods

The purity and target molecular weight of the peptide obtained in step 6 were determined by analytical high performance liquid chromatography and liquid chromatography/mass spectrometry. The test results are shown in Figures 9 and 10.

Example 6 Preparation of Compound 21

Step 1: Coupling of the first amino acid Fmoc-Val-OH

168 mg (0.2 mmol) of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 h. Fmoc-Val-OH (0.16 mmol) and diisopropylethylamine (DIEA, 0.64 mmol) were weighed and dissolved in 8 mL of DCM and added to the resin, and reacted at room temperature for 2 h. After the reaction was completed, a blocking solution (10 mL) of DCM: methanol: DIEA (85:10:5, v:v:v) was added at room temperature for 10 min for blocking. The blocked resin was washed 5 times with DCM and 5 times with DMF.

Step 2: Synthesis of linear precursor peptide chain

The linear precursor peptide chain of compound 21 is AEEA-Y-I-E-T-D-V.

The resin obtained in step 1 was fully swollen in DMF for 1 h, and then the linear precursor sequence was synthesized from the second D of the carboxyl terminal to the amino terminal. Each coupling cycle was performed as follows:

· Fmoc-deprotection was performed twice with 20% piperidine/DMF (20% v/v, 10 mL), each time for 8 min.

• Rinse the resin 6-8 times with DMF until neutral pH.

Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to the resin and react at room temperature for 1 hour.

• Rinse the resin 4-6 times with DMF before coupling the next amino acid.

After the linear peptide synthesis, the resin was washed 5 times with DMF and 5 times with DCM. The resin was dried in vacuo.

Step 3: Coupling of 3-(FMOC-amino)glutaric acid

Dissolve 0.25mmol 3-(FMOC-amino)pentanedioic acid, 1mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 2mmol N,N-diisopropylethylamine (DIEA) in DMF, add to the resin obtained in step 2, react at room temperature for 1h, and react twice. After the reaction, rinse the resin with DMF 5 times and DCM 5 times.

Step 4: Synthesis of linear precursor peptide chain

The linear precursor peptide chain of compound 21 is Y-G-R-K-K-R-R-Q-R-R-R.

The resin obtained in step 3 was fully swollen in DMF for 1 h, and then the linear precursor sequence was synthesized from the carboxyl end to the amino end. Each coupling cycle was performed as follows:

· Fmoc-deprotection was performed twice with 20% piperidine/DMF (20% v/v, 10 mL), each time for 8 min.

• Rinse the resin 6-8 times with DMF until neutral pH.

Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to the resin and react at room temperature for 1 hour.

• Rinse the resin 4-6 times with DMF before coupling the next amino acid.

After the linear peptide synthesis, the resin was washed 5 times with DMF and 5 times with DCM. The resin was dried in vacuo.

Step 5: Cleavage of the linear precursor peptide chain

Freshly prepared cutting cocktail (10 mL) trifluoroacetic acid: water: triisopropylsilane: thioanisole (90:2.5:2.5:5, v:v:v:v) was added to the resin obtained in step 4 and shaken for 2 hours at room temperature. After the reaction, the reaction solution was filtered, and the resin was washed with trifluoroacetic acid, combined with the reaction solution, and precipitated with 4 times the volume of cold MTBE to obtain a crude product. The crude product was washed with MTBE 3 times and dried in a vacuum.

Step 6: Peptide purification

The crude peptide was dissolved in 20% acetonitrile aqueous solution, filtered through a 0.45um membrane, and separated using a reversed-phase high-performance liquid chromatography system. The buffers were A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). The chromatographic column was a BR-C18 (Saifen) reversed-phase column. During the purification process, the chromatograph detection wavelength was set to 230nm, the flow rate was 15mL/min, and the gradient was 30-60% acetonitrile in 40min. The product-related fractions were collected, and after HPLC identification of the purity, the fractions >95% were combined and freeze-dried to obtain the pure peptide.

Step 7: Detection and characterization methods

The purity and target molecular weight of the peptide obtained in step 6 were determined by analytical high performance liquid chromatography and liquid chromatography/mass spectrometry. The test results are shown in Figures 11 and 12.

Example 7 Preparation of Compound 22

Step 1: Coupling of the first amino acid Fmoc-Val-OH

168 mg (0.2 mmol) of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 h. Fmoc-Val-OH (0.16 mmol) and diisopropylethylamine (DIEA, 0.64 mmol) were weighed and dissolved in 8 mL of DCM and added to the resin, and reacted at room temperature for 2 h. After the reaction was completed, a blocking solution (10 mL) of DCM: methanol: DIEA (85:10:5, v:v:v) was added at room temperature for 10 min for blocking. The blocked resin was washed 5 times with DCM and 5 times with DMF.

Step 2: Synthesis of linear precursor peptide chain

The linear precursor peptide chain of compound 22 is AEEA-Y-I-E-T-W-V.

The resin obtained in step 1 was fully swollen in DMF for 1 h, and then the above linear precursor sequence was synthesized from the second D of the carboxyl terminal to the amino terminal. Each coupling cycle was performed as follows:

· Fmoc-deprotection was performed twice with 20% piperidine/DMF (20% v/v, 10 mL), each time for 8 min.

• Rinse the resin 6-8 times with DMF until neutral pH.

Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to the resin and react at room temperature for 1 hour.

• Rinse the resin 4-6 times with DMF before coupling the next amino acid.

After the linear peptide synthesis, the resin was washed 5 times with DMF and 5 times with DCM. The resin was dried in vacuo.

Step 3: Coupling of 3-(FMOC-amino)glutaric acid

Dissolve 0.25mmol 3-(FMOC-amino)pentanedioic acid, 1mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 2mmol N,N-diisopropylethylamine (DIEA) in DMF, add to the resin obtained in step 2, react at room temperature for 1h, and react twice. After the reaction, rinse the resin with DMF 5 times and DCM 5 times.

Step 4: Synthesis of linear precursor peptide chain

The linear precursor peptide chain of compound 22 is Y-G-R-K-K-R-R-Q-R-R-R.

The resin obtained in step 3 was fully swollen in DMF for 1 h, and then the linear precursor sequence was synthesized from the carboxyl end to the amino end. Each coupling cycle was performed as follows:

· Fmoc-deprotection was performed twice with 20% piperidine/DMF (20% v/v, 10 mL), each time for 8 min.

• Rinse the resin 6-8 times with DMF until neutral pH.

Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to the resin and react at room temperature for 1 hour.

• Rinse the resin 4-6 times with DMF before coupling the next amino acid.

After the linear peptide synthesis, the resin was washed 5 times with DMF and 5 times with DCM. The resin was dried in vacuo.

Step 5: Cleavage of the linear precursor peptide chain

Freshly prepared cutting cocktail (10 mL) trifluoroacetic acid: water: triisopropylsilane: thioanisole (90:2.5:2.5:5, v:v:v:v) was added to the resin obtained in step 4 and shaken for 2 hours at room temperature. After the reaction, the reaction solution was filtered, and the resin was washed with trifluoroacetic acid, combined with the reaction solution, and precipitated with 4 times the volume of cold MTBE to obtain a crude product. The crude product was washed with MTBE 3 times and dried in a vacuum.

Step 6: Peptide purification

The crude peptide was dissolved in 20% acetonitrile aqueous solution, filtered through a 0.45um membrane, and separated using a reversed-phase high-performance liquid chromatography system. The buffers were A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). The chromatographic column was a BR-C18 (Saifen) reversed-phase column. During the purification process, the chromatograph detection wavelength was set to 230nm, the flow rate was 15mL/min, and the gradient was 30-60% acetonitrile in 40min. The product-related fractions were collected, and after HPLC identification of the purity, the fractions >95% were combined and freeze-dried to obtain the pure peptide.

Step 7: Detection and characterization methods

The purity and target molecular weight of the peptide obtained in step 6 were determined by analytical high performance liquid chromatography and liquid chromatography/mass spectrometry. The test results are shown in Figures 13 and 14.

Example 8 Preparation of Compound 31

Step 1: Coupling of the first amino acid Fmoc-Val-OH

168 mg (0.2 mmol) of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 h. Fmoc-Val-OH (0.16 mmol) and diisopropylethylamine (DIEA, 0.64 mmol) were weighed and dissolved in 8 mL of DCM and added to the resin, and reacted at room temperature for 2 h. After the reaction was completed, a blocking solution (10 mL) of DCM: methanol: DIEA (85:10:5, v:v:v) was added at room temperature for 10 min for blocking. The blocked resin was washed 5 times with DCM and 5 times with DMF.

Step 2: Synthesis of linear precursor peptide chain

The linear precursor peptide chain of compound 31 is AEEA-Y-I-E-T-W-V.

The resin obtained in step 1 was fully swollen in DMF for 1 h, and then the above linear precursor sequence was synthesized from the second D of the carboxyl terminal to the amino terminal. Each coupling cycle was performed as follows:

· Fmoc-deprotection was performed twice with 20% piperidine/DMF (20% v/v, 10 mL), each time for 8 min.

• Rinse the resin 6-8 times with DMF until neutral pH.

Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to the resin and react at room temperature for 1 hour.

• Rinse the resin 4-6 times with DMF before coupling the next amino acid.

After the linear peptide synthesis, the resin was washed 5 times with DMF and 5 times with DCM. The resin was dried in vacuo.

Step 3: Coupling of 3-(FMOC-amino)glutaric acid

Dissolve 0.25mmol 3-(FMOC-amino)pentanedioic acid, 1mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 2mmol N,N-diisopropylethylamine (DIEA) in DMF, add to the resin obtained in step 2, react at room temperature for 1h, and react twice. After the reaction, rinse the resin with DMF 5 times and DCM 5 times.

Step 4: Synthesis of linear precursor peptide chain

The linear precursor peptide chain of compound 31 is dR-dR-dR-dQ-dR-dR-dK-dK-dR-G-dY.

The resin obtained in step 3 was fully swollen in DMF for 1 h, and then the linear precursor sequence was synthesized from the carboxyl end to the amino end. Each coupling cycle was performed as follows:

· Fmoc-deprotection was performed twice with 20% piperidine/DMF (20% v/v, 10 mL), each time for 8 min.

• Rinse the resin 6-8 times with DMF until neutral pH.

Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to the resin and react at room temperature for 1 hour.

• Rinse the resin 4-6 times with DMF before coupling the next amino acid.

After the linear peptide synthesis, the resin was washed 5 times with DMF and 5 times with DCM. The resin was dried in vacuo.

Step 5: Cleavage of the linear precursor peptide chain

Freshly prepared cutting cocktail (10 mL) trifluoroacetic acid: water: triisopropylsilane: thioanisole (90:2.5:2.5:5, v:v:v:v) was added to the resin obtained in step 4 and shaken for 2 hours at room temperature. After the reaction, the reaction solution was filtered, and the resin was washed with trifluoroacetic acid, combined with the reaction solution, and precipitated with 4 times the volume of cold MTBE to obtain a crude product. The crude product was washed with MTBE 3 times and dried in a vacuum.

Step 6: Peptide purification

The crude peptide was dissolved in 20% acetonitrile aqueous solution, filtered through a 0.45um membrane, and separated using a reversed-phase high-performance liquid chromatography system. The buffers were A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). The chromatographic column was a BR-C18 (Saifen) reversed-phase column. During the purification process, the chromatograph detection wavelength was set to 230nm, the flow rate was 15mL/min, and the gradient was 30-60% acetonitrile in 40min. The product-related fractions were collected, and after HPLC identification of the purity, the fractions >95% were combined and freeze-dried to obtain the pure peptide.

Step 7: Detection and characterization methods

The purity and target molecular weight of the peptide obtained in step 6 were determined by analytical high performance liquid chromatography and liquid chromatography/mass spectrometry. The test results are shown in Figures 15 and 16.

Example 9 Preparation of Compound 28

Step 1: Coupling of the first amino acid Fmoc-Cys(Trt)-OH

84 mg (0.1 mmol) of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 h. Fmoc-Cys(Trt)-OH (0.08 mmol) and diisopropylethylamine (DIEA, 0.32 mmol) were weighed and dissolved in 5 mL of DCM and added to the resin. The mixture was reacted at room temperature for 2 h. After the reaction was completed, a blocking solution (10 mL) of DCM: methanol: DIEA (85:10:5, v:v:v) was added at room temperature for 10 min for blocking. The blocked resin was washed 5 times with DCM and 5 times with DMF.

Step 2: Synthesis of linear precursor peptide chain

The linear precursor peptide chain of compound 28 is AEEA-C-K-M-V-T-T-F-G-I-D-V-T-T-T-Y-I-C.

The resin obtained in step 1 was fully swollen in DMF for 1 h, and then the above linear precursor sequence was synthesized from the second position I of the carboxyl terminal to the amino terminal. Each coupling cycle was performed as follows:

· Fmoc-deprotection was performed twice with 20% piperidine/DMF (20% v/v, 10 mL), each time for 8 min.

• Rinse the resin 6-8 times with DMF until neutral pH.

Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to the resin and react at room temperature for 1 hour.

• Rinse the resin 4-6 times with DMF before coupling the next amino acid.

After the linear peptide synthesis, the resin was washed 5 times with DMF and 5 times with DCM. The resin was dried in vacuo.

Step 3: Coupling of amino acid Fmoc-Lys(Fmoc)-OH

Dissolve 0.5 mmol Fmoc-Lys(Fmoc)-OH, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to the resin obtained in step 1, and react at room temperature for 1 h. After the reaction, rinse the resin with DMF 5 times and DCM 5 times.

Step 4: Synthesis of linear precursor peptide chain

The linear precursor peptide chain of compound 28 is dR-dR-dR-dQ-dR-dR-dK-dK-dR-G-dY.

The resin obtained in step 3 was fully swollen in DMF for 1 h, and then the linear precursor sequence was synthesized from the carboxyl end to the amino end. Each coupling cycle was performed as follows:

· Fmoc-deprotection was performed twice with 20% piperidine/DMF (20% v/v, 10 mL), each time for 8 min.

• Rinse the resin 6-8 times with DMF until neutral pH.

Dissolve 1mmol Fmoc-AA, 1mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 2mmol 4-methylmorpholine (NMM) in DMF, add the resin and react at room temperature for 1h.

• Rinse the resin 4-6 times with DMF before coupling the next amino acid.

After the linear peptide synthesis, the resin was washed 5 times with DMF and 5 times with DCM. The resin was dried in vacuo.

Step 5: Cleavage of the linear precursor peptide chain

Freshly prepared cutting cocktail (10 mL) trifluoroacetic acid: water: triisopropylsilane: thioanisole (90:2.5:2.5:5, v:v:v:v) was added to the resin obtained in step 4 and shaken for 2 hours at room temperature. After the reaction, the reaction solution was filtered, and the resin was washed with trifluoroacetic acid, combined with the reaction solution, and precipitated with 4 times the volume of cold MTBE to obtain a crude product. The crude product was washed with MTBE 3 times and dried in a vacuum.

Step 6: Intramolecular disulfide bond formation

Add DMSO to the crude product obtained in step 5 to fully dissolve (the volume of DMSO is 20% of the total volume of the reaction system), prepare phosphate\guanidine hydrochloride buffer (pH=7.0, containing 50% ACN), mix 1╳PBS:6M guanidine hydrochloride (80:20, v:v) buffer with acetonitrile 1:1 to obtain phosphate\guanidine hydrochloride buffer (pH=7.0, 50% ACN), and then slowly add the polypeptide solution dropwise to the above buffer to a final concentration of 1 mg/mL, and shake at room temperature for 16 hours. LC-MS monitors the reaction results, and purification and preparation are directly carried out after the reaction is completed.

Step 7: Peptide purification

After filtering through a 0.45um membrane, it was separated using a reversed-phase high-performance liquid chromatography system, with buffers A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). The chromatographic column was a BR-C18 (Saifen) reversed-phase column, and during the purification process, the chromatograph detection wavelength was set to 230nm, the flow rate was 15mL/min, and the gradient was 30-60% acetonitrile in 40min. The product-related fractions were collected, and after HPLC identification of the purity, the fractions >95% were combined and freeze-dried to obtain the pure polypeptide. The liquid phase and mass spectrometry detection results are shown in Figures 17 and 18.

Example 10 Synthesis of Compound 1

Step 1: Coupling of the first amino acid Fmoc-Val-OH

168 mg (0.2 mmol) of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 h. Fmoc-Val-OH (0.16 mmol) and diisopropylethylamine (DIEA, 0.64 mmol) were weighed and dissolved in 8 mL of DCM and added to the resin. The mixture was reacted at room temperature for 2 h. After the reaction was completed, a blocking solution (10 mL) of DCM: methanol: DIEA (85:10:5, v:v:v) was added at room temperature for 10 min for blocking. The blocked resin was washed 5 times with DCM and 5 times with DMF.

Step 2: Synthesis of linear precursor peptide chain

The linear precursor peptide chain of compound 1 is AEEA-I-E-T-D-V.

The resin obtained in step 1 was fully swollen in DMF for 1 h, and then the above linear precursor sequence was synthesized from the second D of the carboxyl terminal to the amino terminal. Each coupling cycle was performed as follows:

· Fmoc-deprotection was performed twice with 20% piperidine/DMF (20% v/v, 10 mL), each time for 8 min.

• Rinse the resin 6-8 times with DMF until neutral pH.

Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to the resin and react at room temperature for 1 hour.

• Rinse the resin 4-6 times with DMF before coupling the next amino acid.

After the linear peptide synthesis, the resin was washed 5 times with DMF and 5 times with DCM. The resin was dried in vacuo.

Step 3: Coupling of 3-(FMOC-amino)malonic acid

Dissolve 0.25mmol 3-(FMOC-amino)malonic acid, 1mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 2mmol N,N-diisopropylethylamine (DIEA) in DMF, add to the resin obtained in step 2, react at room temperature for 1h, and react twice. After the reaction, rinse the resin with DMF 5 times and DCM 5 times.

Step 4: Synthesis of linear precursor peptide chain

The linear precursor peptide chain of compound 1 is Y-G-R-K-K-R-R-Q-R-R-R.

The resin obtained in step 3 was fully swollen in DMF for 1 h, and then the linear precursor sequence was synthesized from the carboxyl end to the amino end. Each coupling cycle was performed as follows:

· Fmoc-deprotection was performed twice with 20% piperidine/DMF (20% v/v, 10 mL), each time for 8 min.

• Rinse the resin 6-8 times with DMF until neutral pH.

Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to the resin and react at room temperature for 1 hour.

• Rinse the resin 4-6 times with DMF before coupling the next amino acid.

After the linear peptide synthesis, the resin was washed 5 times with DMF and 5 times with DCM. The resin was dried in vacuo.

Step 5: Cleavage of the linear precursor peptide chain

Freshly prepared cutting cocktail (10 mL) trifluoroacetic acid: water: triisopropylsilane: thioanisole (90:2.5:2.5:5, v:v:v:v) was added to the resin obtained in step 4 and shaken for 2 hours at room temperature. After the reaction, the reaction solution was filtered, and the resin was washed with trifluoroacetic acid, combined with the reaction solution, and precipitated with 4 times the volume of cold MTBE to obtain a crude product. The crude product was washed with MTBE 3 times and dried in a vacuum.

Step 6: Peptide purification

The crude peptide was dissolved in 20% acetonitrile aqueous solution, filtered through a 0.45um membrane, and separated using a reversed-phase high-performance liquid chromatography system. The buffers were A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). The chromatographic column was a BR-C18 (Saifen) reversed-phase column. During the purification process, the chromatograph detection wavelength was set to 230nm, the flow rate was 15mL/min, and the gradient was 30-60% acetonitrile in 40min. The product-related fractions were collected, and after HPLC identification of the purity, the fractions >95% were combined and freeze-dried to obtain the pure peptide.

Step 7: Detection and characterization methods

The purified peptide obtained in step 6 was subjected to analytical HPLC and LC/MS to determine purity and target molecular weight.

Example 11 Synthesis of Compound 33

Step 1: Coupling of the first amino acid Fmoc-Val-OH

168 mg (0.2 mmol) of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 h. Fmoc-Val-OH (0.16 mmol) and diisopropylethylamine (DIEA, 0.64 mmol) were weighed and dissolved in 8 mL of DCM and added to the resin. The mixture was reacted at room temperature for 2 h. After the reaction was completed, a blocking solution (10 mL) of DCM: methanol: DIEA (85:10:5, v:v:v) was added at room temperature for 10 min for blocking. The blocked resin was washed 5 times with DCM and 5 times with DMF.

Step 2: Synthesis of linear precursor peptide chain

The linear precursor peptide chain of compound 26 is AEEA-I-E-T-D-V.

The resin obtained in step 1 was fully swollen in DMF for 1 h, and then the linear precursor sequence was synthesized from the second D of the carboxyl terminal to the amino terminal. Each coupling cycle was performed as follows:

· Fmoc-deprotection was performed twice with 20% piperidine/DMF (20% v/v, 10 mL), each time for 8 min.

• Rinse the resin 6-8 times with DMF until neutral pH.

Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to the resin and react at room temperature for 1 hour.

• Rinse the resin 4-6 times with DMF before coupling the next amino acid.

After the linear peptide synthesis, the resin was washed 5 times with DMF and 5 times with DCM. The resin was dried in vacuo.

Step 3: Coupling of 3-(FMOC-amino)glutaric acid

Dissolve 0.25mmol 3-(FMOC-amino)pentanedioic acid, 1mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 2mmol N,N-diisopropylethylamine (DIEA) in DMF, add to the resin obtained in step 2, react at room temperature for 1h, and react twice. After the reaction, rinse the resin with DMF 5 times and DCM 5 times.

Step 4: Synthesis of linear precursor peptide chain

The linear precursor peptide chain of compound 26 is R-R-L-S-Y-S-R-R-R-F.

The resin obtained in step 3 was fully swollen in DMF for 1 h, and then the linear precursor sequence was synthesized from the carboxyl end to the amino end. Each coupling cycle was performed as follows:

· Fmoc-deprotection was performed twice with 20% piperidine/DMF (20% v/v, 10 mL), each time for 8 min.

• Rinse the resin 6-8 times with DMF until neutral pH.

Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to the resin and react at room temperature for 1 hour.

• Rinse the resin 4-6 times with DMF before coupling the next amino acid.

After the linear peptide synthesis, the resin was washed 5 times with DMF and 5 times with DCM. The resin was dried in vacuo.

Step 5: Cleavage of the linear precursor peptide chain

Freshly prepared cutting cocktail (10 mL) trifluoroacetic acid: water: triisopropylsilane: thioanisole (90:2.5:2.5:5, v:v:v:v) was added to the resin obtained in step 4 and shaken for 2 hours at room temperature. After the reaction, the reaction solution was filtered, and the resin was washed with trifluoroacetic acid, combined with the reaction solution, and precipitated with 4 times the volume of cold MTBE to obtain a crude product. The crude product was washed with MTBE 3 times and dried in a vacuum.

Step 6: Peptide purification

The crude peptide was dissolved in 20% acetonitrile aqueous solution, filtered through a 0.45um membrane, and separated using a reversed-phase high-performance liquid chromatography system. The buffers were A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). The chromatographic column was a BR-C18 (Saifen) reversed-phase column. During the purification process, the chromatograph detection wavelength was set to 230nm, the flow rate was 15mL/min, and the gradient was 30-60% acetonitrile in 40min. The product-related fractions were collected, and after HPLC identification of the purity, the fractions >95% were combined and freeze-dried to obtain the pure peptide.

Step 7: Detection and characterization methods

The purified peptide obtained in step 6 was subjected to analytical HPLC and LC/MS to determine purity and target molecular weight.

Other compounds of the present invention can be synthesized by referring to the synthesis methods of the above examples.

Test Example 1 ELISA test compound The compound inhibits the binding of Tat-NR2B9c-B to cMyc-PSD95 alpha 1-392

(1) Experimental materials:

(2) Experimental steps:

The inhibitory effect of the compounds on Tat-NR2B9c-B binding to cMyc-PSD95alpha 1-392 was tested by ELISA (enzyme-linked immunosorbent assay). cMyc-PSD95 alpha 1-392 was coated on a 384-well plate (greiner 781097) at 0.37 μg/mL, 25 μL/well. After blocking the ELSIA plate coated with cMyc-PSD95 alpha 1-392, the compounds were diluted to 0-10 μM (from 10 μM down to 3-fold gradient dilution for a total of 8 concentrations). The workstation was used to transfer 12.5 μL/well of the compound to the 384-well plate and centrifuged to remove bubbles. Then, an 8-well pipette (10-100 μL) was used to transfer 12.5 μL/well of 12 nM Tat-NR2B9c-B, centrifuged to remove bubbles, and incubated at 37°C for 1 hour. Discard the liquid in the well and add 80 μL of pH = 7.4 Wash the plate 3-5 times with 1×TBST washing solution, 3-5 minutes each time. Add 25μL/well of 1:10000 diluted Streptavidin HRP to the test plate, centrifuge to remove bubbles, and incubate in a 37℃ incubator for 1h. Wash the plate again and control the test plate to dry, add 25μl TMB colorimetric solution to each well, and continue to incubate in the incubator at 37℃ for 30min. Finally, add 25μl stop solution (1M HCL) to each well to terminate the reaction. Use the microplate reader Cytation5 to read the absorbance at 450nm, and calculate the IC ₅₀ of the compound inhibiting the binding of Tat-NR2B9c-B to cMyc-PSD95 alpha 1-392.

The experimental results are shown in Table 1. The compounds of the present invention can inhibit the binding of Tat-NR2B9c-B to cMyc-PSD95 alpha 1-392 and have a good inhibitory effect. The affinity of the polypeptide of the present invention to cMyc-PSD95 alpha 1-392 is stronger than that of Tat-NR2B9c-B.

Table 1 Results of compounds inhibiting the binding of Tat-NR2B9c-B to cMyc-PSD95 alpha 1-392

Test Example 2 FRET test compounds inhibit the binding of cMyc-PSD95 alpha 1-392 to Tat-NR2B9C-B

(1) Experimental materials:

(2) Experimental steps:

The FRET (fluorescence resonance energy transfer) method was used to test the inhibitory effect of the compounds on the binding of cMyc-PSD95 alpha 1-392 to Tat-NR2B9C-B. The compounds were diluted in multiples to 0-10 μM (3-fold gradient dilution from 10 μM down, a total of 8 concentrations). Use an 8-well gun (1-10 μL) to transfer 4 μL/well of 4nM cMyc-PSD95 alpha 1-392 to a 384-well white plate (Thermo 264706), and centrifuge briefly to remove bubbles. Use the workstation to transfer 4 μL/well of the compound to a 384-well plate, and centrifuge briefly to remove bubbles. Use the 8-well gun again to transfer 4 μL/well of 20nM Tat-NR2B9c-B to a 384-well white plate, and centrifuge briefly. Remove bubbles. Mix the prepared 0.02μg/mL EU-steptavidin with 1ug/mL Mouse Anti-C-MYC IgG SureLightAPC in a 1:1 ratio. Use an 8-well pipette to transfer 8μL/well of EU-APC premix to a 384-well white plate and centrifuge to remove bubbles. Incubate at room temperature in the dark for 2 hours and use a microplate reader cytation5 to read the emission fluorescence values at 320nM excitation 620nm and 665nm.

The experimental results are shown in Table 2, where control group 1 is Control group 2 The preparation method thereof is described in patent CN103533949A.

Table 2 Results of compounds inhibiting the binding of cMyc-PSD95 alpha 1-392 to Tat-NR2B9C-B

The experimental results are shown in Table 2. The compounds of the present invention can inhibit the binding of cMyc-PSD95 alpha 1-392 and Tat-NR2B9C-B and have a good inhibitory effect.

Test Example 3 ELISA test compounds inhibit the binding of biotin-nNOS1-299 to cMyc-PSD95 alpha 1-392

(1) Experimental materials:

(2) Experimental steps:

The ELISA method was used to test the compounds for the binding of biotin-nNOS1-299 to cMyc-PSD95 cMyc-PSD95 alpha 1-392 was coated on a 384-well plate (greiner 781097) at 10 μg/mL, 25 μL/well. After blocking the ELSIA plate coated with cMyc-PSD95 alpha 1-392, the compound was diluted to 0-10 μM (8 concentrations in 3-fold gradient dilution from 10 μM). The compound was transferred to the 384-well plate at 12.5 μL/well using the workstation and centrifuged to remove bubbles. Then, 12.5 μL/well of 10 μg/mL 743 biotin-nNOS 1-299 was pipetted using an 8-well pipette (10-100 μL), and the mixture was centrifuged to remove bubbles and incubated at 37°C for 1 hour. Discard the liquid in the wells, add 80μl 1×TBST washing solution with pH=7.4 and wash the plate 3-5 times, 3-5min each time. Drain the test plate and add 25μL/well of Streptavidin HRP diluted 1:10000, centrifuge to remove bubbles and incubate in a 37℃ incubator for 1h. Wash the plate again and drain the test plate, add 25μLTMB colorimetric solution to each well, and continue to incubate in the incubator at 37℃ for 30min. Finally, add 25μL stop solution (1M HCL) to each well to terminate the reaction. Use the microplate reader Cytation5 to read the absorbance at 450nm and calculate the IC ₅₀ of the compound inhibiting the binding of biotin-nNOS1-299 to cMyc-PSD95 alpha 1-392.

Table 3 Results of compounds inhibiting the binding of biotin-nNOS 1-299 to cMyc-PSD95 alpha 1-392

The experimental results are shown in Table 3. The compounds of the present invention can inhibit the binding of biotin-nNOS1-299 and cMyc-PSD95 alpha1-392 and have a good inhibitory effect.

Test Example 4 FP test compound inhibits the binding of 6H-PSD95 alpha 61-249 and 5FAM-N-bis-(PEG2-IETAV)2/5-FAM-NPEG4(IETAV)2

(1) Experimental materials:

(2) Experimental steps:

The FP (fluorescence polarization immunoassay) method was used to test the inhibitory effect of the compounds on the binding of 6H-PSD95 alpha 61-249 to 5FAM-N-bis-(PEG2-IETAV)2/5-FAM-NPEG4(IETAV)2. The compounds were diluted to 0-10 μM (from 10 μM down to 3-fold gradient dilution for a total of 8 concentrations). Use an 8-well pipette (1-10 μl) to transfer 5 μL/well of 5nM 6H-PSD95alpha 61-249 to a 384-well plate (Corning 3575) and centrifuge briefly to remove bubbles. Use a workstation to transfer 5 μL/well of the compound to a 384-well plate and centrifuge briefly to remove bubbles. Use an 8-well pipette to transfer 5 μL/well of 0.5 nM 5FAM-N-bis-(PEG2-IETAV) ₂ /5-FAM-NPEG4(IETAV) ₂ to a 384-well plate, centrifuge briefly to remove bubbles, incubate at room temperature in the dark for 2 h, and read the values using an ELISA reader Cytation5 FP module.

Among them, the preparation method of 5-FAM-NPEG ₄ (IETAV) ₂ is as described in patent CN103533949A.

Table 4 Compounds inhibiting the binding of 6H-PSD95 alpha 61-249 and 5FAM-N-bis-(PEG2-IETAV)2/5-FAM-NPEG4(IETAV)2

The experimental results are shown in Table 4. The compounds of the present invention can inhibit the binding of 6H-PSD95 alpha 61-249 and 5FAM-N-bis-(PEG2-IETAV)2/5-FAM-NPEG4(IETAV)2, and have a good inhibitory effect. The affinity of the polypeptide of the present invention to 6H-PSD95 alpha 61-249 is stronger than that of FAM-N-bis-(PEG2-IETAV)2/5-FAM-NPEG4(IETAV)2.

Test Example 5 Mouse plasma stability

1. Mouse plasma (heparin sodium) stability

1.1 Experimental Materials:

Drug: Compound of the present invention.

Experimental related reagents: nainitide (Tat-NR2B9C/NA-1) (purchased from Nanjing GenScript Biotechnology Co., Ltd.); methanol (purchased from Sigma); formic acid (purchased from Aladdin); DMSO (dimethyl sulfoxide) (purchased from Aladdin); mouse plasma (Slack) (self-collected).

1.2 Experimental methods:

Positive sample preparation: Take nainitide and dissolve it in DMSO to 100 times the final concentration of 1mM, and place it at -20℃ for use. Negative sample preparation: Test sample preparation: Take the peptide to be tested and dissolve it in DMSO or other organic solvents to 100 times the final concentration of 1mM, and place it at -20℃ for use. Plasma thawing: Take out (number of samples * 2.1) mL of plasma from the -80℃ refrigerator and quickly melt it in a 37℃ water bath. Prepare MIX (mixture): Take 693μL of plasma and add it to a 1.5mL EP tube, 3 parallel samples at each time point, and prepare 3 tubes of MIX. Then add 7μL of the sample to be tested to each tube to make the final concentration a detectable concentration or an in vivo drug concentration of 10μM. Oscillate on a vortex oscillator for 30s, divide 100μL each according to the time gradient, and incubate, and operate on ice throughout the process. Incubation: Incubate in a 37°C water bath at 0min, 15min, 30min, 60min, 90min, and 120min. Termination: After incubation, add 4 times the volume of 0.1% formic acid 75% acetonitrile water precipitation agent. Mixing: Oscillate on a vortex oscillator for 30s. Centrifugation: Centrifuge at 4°C, 13000r/min for 10min. Take the supernatant, transfer to a small injection tube, and send to LC-MS/MS for analysis.

1.3 Experimental results:

A dot-line graph with the ordinate being the original drug remaining rate (%) and the abscissa being time, as shown in FIG19 , shows a trend of sample degradation in in vitro plasma over time, and the results of sample stability are obtained.

The half-life T _1/2 of the drug in plasma was calculated according to the first-order kinetic formula to obtain the elimination rate constant (Ke), and the T1/2 (min) of the compound in plasma was further calculated according to the formula.
T _1/2 = -0.693/Ke

By comparing the original drug residual rate (%), the original drug residual rate (%) of compound 9, compound 31, and compound 32 is 89.2%, 88.91%, and 97.03%, respectively, and the compounds of the present invention have good _2h plasma stability.

2. Stability of Alteplase (Heparin Sodium) in Mouse Plasma

2.1 Experimental Materials:

Drug: Compound of the present invention.

Experimental related reagents: nainitide (Tat-NR2B9C/NA-1) (purchased from Nanjing GenScript Biotechnology Co., Ltd.); methanol (purchased from Sigma); formic acid (purchased from Aladdin); DMSO (dimethyl sulfoxide) (purchased from Aladdin); mouse plasma (Slack) (self-collected); alteplase for injection (Aitongli).

2.2 Experimental methods:

Positive sample preparation: Take nainitide and dissolve it in DMSO to 100 times the final concentration of 1mM, and place it at -20℃ for use. Test sample preparation: Take the peptide to be tested and dissolve it in DMSO or other organic solvents to 100 times the final concentration of 1mM, and place it at -20℃ for use. Plasma thawing: Take out plasma (number of samples * 2.1)mL from the -80℃ refrigerator and quickly thaw it in a 37℃ water bath. Prepare a mixed solution: Take (98μL×(6 time points)+1)686μL plasma (heparin sodium) and add it to a 1.5mL EP tube. At least 3 parallel samples are prepared for each time point, and 1 tube of mixed solution is prepared. Add 7μL of alteplase to each tube to make the final concentration of 9μg/mL. Add 7μL of the sample to be tested to each tube to make the final concentration of 10μM. Oscillate on a vortex oscillator for 30s, divide 100μL into each according to the time gradient, and incubate, and operate on ice throughout the process. Incubation: Incubate in a 37°C water bath at 0min, 5min, 15min, 30min, 45min, and 60min. Termination: After incubation, add 4 times the volume of 0.1% formic acid 75% acetonitrile aqueous solution as a precipitant. Mixing: Shake on a vortex oscillator for 30s. Centrifugation: Centrifuge at 4°C, 13000r/min for 10min. Take the supernatant, transfer to a sample tube, and send to LC-MS/MS for analysis.

2.3 Experimental results:

The vertical axis is the original drug remaining rate (%), and the horizontal axis is the time point line graph, as shown in Figure 20, we can see that a The degradation trend of the sample in in vitro plasma is analyzed over time to obtain the stability results of the sample.

Calculate the half-life T1/2 of the drug in plasma according to the first-order kinetic formula to obtain the elimination rate constant (Ke), and further use the formula to obtain the T1/2 (min) of the compound in plasma.
T _1/2 = -0.693/Ke

Experimental results: By comparing the original drug residual rate (%), the original drug residual rates (%) of compound 9, compound 31, and compound 32 are 91.8%, 75.3%, and 100.7%, respectively, and the compounds of the present invention have good 1h plasma stability. The T _1/2 of each compound is shown in Table 5.

Table 5 T _1/2 results of each compound

The compound of the invention still has good stability in human plasma (heparin sodium) and human plasma alteplase (heparin sodium).

In summary, the stability of the compounds of the present invention in plasma is greatly improved compared with Tat-NR2B9C and AVLX-144.

Test Example 6: Mouse efficacy test

1. Experimental Materials

Drug: Compound of the present invention.

Related reagents and consumables: 1mL insulin needle, isoflurane, TTC, low-adsorption EP tube, normal saline, 6cm culture dish, microsurgical scissors, microsurgical forceps, absorbable suture, thread plug (Guangzhou Jialing), artery clamp, cotton thread, glucose, and penicillin.

Experimental animals: C57 mice, 100, male, 7-8 weeks, 20-25 g, purchased from Hunan Anshengmei Pharmaceutical Research Institute Co., Ltd.

2. Experimental methods:

2.1 Experimental preparation: The day before the experiment, the necks of mice were depilated with depilatory cream. The mice were grouped using random grouping software.

2.2 Anesthesia: Fix the mice under a stereomicroscope and use isoflurane inhalation anesthesia.

2.3 After the anesthesia is stable, the neck skin of the mouse is disinfected, the anterior neck skin of the mouse is cut along the midline, and the chest muscles are further separated and divided to expose the common carotid artery and vagus nerve, and then gradually expose the common carotid artery bifurcation, external carotid artery and internal carotid artery. Separate the common carotid artery, clamp it with an artery clamp at the proximal end and tie a slipknot with cotton thread, separate the internal carotid artery, clamp it with an artery clamp. Separate the external carotid artery of the rat and ligate it. Make a small cut at the distal end between the two knots of the common carotid artery, with the small cut obliquely pointing to the distal end, and carefully insert a professional thread plug into the lumen of the common carotid artery along the edge of the blood vessel. The thread plug enters the common carotid artery and gently tightens the slipknot.

2.4 Transfer the suture from the bifurcation to the internal carotid artery, loosen the artery clamp at the internal carotid artery, and continue to go deeper into the brain until it reaches the starting section of the middle cerebral artery and finally makes the suture mark at the bifurcation of the common carotid artery. Tighten the slipknot of the common carotid artery to prevent the suture from moving. Remove the artery clamp at the proximal end of the common carotid artery. Cut the excess suture and thread ends short, and suture the skin.

2.5 30 minutes after embolization, the test substance and placebo were administered to the tail vein of the mice. The drug was randomly taken by a person who was not involved in the operation, and the person injecting the drug was unaware of the drug information.

2.6 After surgery, penicillin and glucose were injected into the model mice and the mice were monitored until they woke up. 24 hours after embolization, the neurological function of the animals was graded according to the Longa score method. The standards are as follows:

0 points: no neurological symptoms;

1 point: the forelimb on the contralateral side of surgery is flexed and close to the chest wall;

2 points: the animal rotates or circles to the opposite side when moving freely;

3 points: the animal falls to the opposite side when moving freely;

4 points: flaccid paralysis, no spontaneous movement of the limbs.

2.7 Observe the condition of mice, record the survival rate and score 24h and 48h after embolization.

2.8 Mice in each group were killed 48 hours after embolization, and the brains were removed intact, washed with saline, dried with filter paper, and weighed.

2.9 Freeze the whole brain and make coronal sections at the optic chiasm and 2 mm before and after it. Use 2% TTC solution to stain at 37°C in the dark for 30 minutes, then place the brain slices in order, from small to large and from top to bottom, with the cortex facing up and the infarct area on the right. Place them neatly and take pictures.

2.10 Use software to calculate the cerebral infarction area and total area.

Calculate the cerebral infarction rate: infarction percentage (%) = pale area/total area × 100%.

2.11. Animals that have the following conditions are excluded from the statistics.

Exclusion criteria: 1. Animals with no behavioral manifestations 24 hours after embolization. 2. Animals with subarachnoid hemorrhage during brain removal. 3. Animals that died during surgery or died without drug administration after surgery.

3. Experimental results

30 minutes after MCAO modeling, mice were randomly double-blindly administered and saline solution. After administration, behavioral scores were performed on each group of experimental mice, and mice with scores of 2-3 points were selected for inclusion in the group. Mice without behavioral scores were excluded from the statistics. The experimental process is shown in Figure 21. After the experimental endpoint, the mouse brain was taken out and its slices were stained as shown in Figure 22. The staining results showed that the embolism area of each administration group was smaller than that of the saline control group. After staining, the cerebral embolism area of each group was counted, and the statistical results are shown in Figure 23. The statistical results of the cerebral embolism area showed that the embolism area of each group after administration was reduced compared with the saline control group. The compound of the present invention has a stronger efficacy than AVLX-144, among which the compound 32 administration group had the highest percentage of embolism area reduction.

The preferred embodiments of the present invention have been specifically described above, but the present invention is not limited to the described embodiments. Those skilled in the art may make various equivalent modifications or substitutions without violating the spirit of the present invention. These equivalent modifications or substitutions are all included in the scope defined by the claims of this application.

Claims (28)

A compound or a pharmaceutically acceptable salt thereof, wherein the structure of the compound is shown in the following general formula (I):
L ₁ is selected from _L2 is present or absent, and when present, _L2 is selected from hydrophilic structural units; _L3 is selected from hydrophilic structural units; L ₄ and L ₅ may be independently selected from a linking bond, -(CH ₂ )nNH-, -C(=O)(CH ₂ )nNH-, -(CH ₂ )nC(=O)-, -(CH ₂ )nC(=O)NH-; n and q can be independently selected from integers of 0 to 6; P ₁ is selected from cell penetrating peptides; _P2 is selected from a cell penetrating peptide or _P2 comprises the sequence (X ⁰ ) _p X ¹ TX ² V or (X ⁰ ) _p X ¹ TX ² F or (X ⁰ ) _p X ¹ SX ² V; _P3 is selected from the sequence (X ⁰ ) _p X ¹ TX ² V or (X ⁰ ) _p X ¹ TX ² F or (X ⁰ ) _p X ¹ SX ² V, wherein p is 0 or 1; X ⁰ is selected from S, T, C, Y, N or Q; X ¹ is selected from E, Q, A, S; ^X2 is selected from A, T, L, V, R, Q, D, N, W. According to the compound or pharmaceutically acceptable salt thereof of claim 1, the cell penetrating peptide (CPP) comprises at least 4 amino acid residues selected from arginine and/or lysine. According to the compound according to claim 2 or a pharmaceutically acceptable salt thereof, the cell-penetrating peptide (CPP) can be independently selected from the following sequences: YGRKKRRQRRR, rrrqrrkkr, rrrqrrkkrGy, polyarginine consisting of 2 to 30 residues, GRKKRRQRRRPPQQ, RKKRRRESRKKRRRES, GWTLNSAGYLLKINLKALAALAKKIL, RRLSYSRRRF, RQIKIWFQNRRMKWKK, GALFLGWLGAAGSTMGAWSQPKKKRKV, RGGRLSYSRRRFSTSTGR, KLALKLALKALKAALKLA, GALFLAFLAAALSLMGLWSQPKKKRRV, RQIKIWFQNRRMKWKK, rqikiwfanrrmkwkk, LLIILRRRIRKQAHAHSK, PLIYLRLLRGQF. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein the hydrophilic structural unit is selected from (PEG) _m , m is independently selected from integers of 0-10. According to the compound or pharmaceutically acceptable salt thereof of claim 1, P ₂ and P ₃ each independently comprise the sequence IETDV or LETTF or METTF or YIETDV or YIETWV or NIETDV or NIETWV. A compound or a pharmaceutically acceptable salt thereof, wherein the structure of the compound is shown in the following general formula (II):
_L2 is present or absent, and when present, _L2 is selected from hydrophilic structural units; _L3 is selected from hydrophilic structural units; L ₄ and L ₅ may be independently selected from a linking bond, -(CH ₂ )nNH-, -C(=O)(CH ₂ )nNH-, -(CH ₂ )nC(=O), -(CH ₂ )nC(=O)NH-; n and q are independently selected from integers of 0 to 6; P ₁ is selected from cell penetrating peptides; _P2 is selected from a cell penetrating peptide or _P2 comprises the sequence (X ⁰ ) _p X ¹ TX ² V or (X ⁰ ) _p X ¹ TX ² F or (X ⁰ ) _p X ¹ SX ² V; _P3 is selected from the sequence (X ⁰ ) _p X ¹ TX ² V or (X ⁰ ) _p X ¹ TX ² F or (X ⁰ ) _p X ¹ SX ² V, wherein p is 0 or 1; X ⁰ is selected from S, T, C, Y, N or Q; X ¹ is selected from E, Q, A, S; ^X2 is selected from A, T, L, V, R, Q, D, N, W. The compound according to claim 6 or a pharmaceutically acceptable salt thereof, wherein the cell-penetrating peptide comprises at least 4 amino acid residues selected from arginine and/or lysine. According to the compound of claim 7 or a pharmaceutically acceptable salt thereof, the cell-penetrating peptide can be independently selected from the following sequences: YGRKKRRQRRR, rrrqrrkkr, rrrqrrkkrGy, polyarginine consisting of 2 to 30 residues, GRKKRRQRRRPPQQ, GWTLNSAGYLLKINLKALAALAKKIL, RRLSYSRRRF, RQIKIWFQNRRMKWKK, GALFLGWLGAAGSTMGAWSQPKKKRKV, RGGRLSYSRRRFSTSTGR, KLALKLALKALKAALKLA, GALFLAFLAAAALSL -MGLWSQPKKKRRV, RQIKIWFQNRRMKWKK, rqikiwfanrrmkwkk, RKKRRRESRKKRRRES, LLIILRRRIRKQAHAHSK, PLIYLRLLRGQF. The compound according to claim 6 or a pharmaceutically acceptable salt thereof, wherein the hydrophilic structural unit is selected from (PEG) m, m may be independently selected from integers of 0-10. According to the compound or pharmaceutically acceptable salt thereof of claim 6, P ₂ and P ₃ each independently comprise the sequence IETDV or LETTF or METTF or YIETDV or YIETWV or NIETDV or NIETWV. A compound or a pharmaceutically acceptable salt thereof, wherein the structure of the compound is shown in the following general formula (III):
_L2 is present or absent, and when present, _L2 is selected from hydrophilic structural units; _L3 is selected from hydrophilic structural units; P ₁ is selected from cell penetrating peptides; _P2 is selected from a cell penetrating peptide or _P2 comprises the sequence (X ⁰ ) _p X ¹ TX ² V or (X ⁰ ) _p X ¹ TX ² F or (X ⁰ ) _p X ¹ SX ² V; _P3 is selected from the sequence (X ⁰ ) _p X ¹ TX ² V or (X ⁰ ) _p X ¹ TX ² F or (X ⁰ ) _p X ¹ SX ² V, wherein p is 0 or 1; X ⁰ is selected from S, T, C, Y, N or Q; X ¹ is selected from E, Q, A, S; ^X2 is selected from A, T, L, V, R, Q, D, N, W. The compound according to claim 11 or a pharmaceutically acceptable salt thereof, wherein the structure of the compound is shown in the following general formula (IV):
P ₁ is selected from cell penetrating peptides (CPP); _P2 is selected from a cell penetrating peptide (CPP) or _P2 comprises the sequence (X ⁰ ) _p X ¹ TX ² V or (X ⁰ ) _p X ¹ TX ² F or (X ⁰ ) _p X ¹ SX ² V; _P3 is selected from the sequence (X ⁰ ) _p X ¹ TX ² V or (X ⁰ ) _p X ¹ TX ² F or (X ⁰ ) _p X ¹ SX ² V, wherein p is 0 or 1; X ⁰ is selected from S, T, C, Y, N or Q; X ¹ is selected from E, Q, A, S; ^X2 is selected from A, T, L, V, R, Q, D, N, W. The compound according to claim 10 or 11 or a pharmaceutically acceptable salt thereof, wherein _P1 is selected from YGRKKRRQRRR, rrrqrrkkrGy. According to the compound or pharmaceutically acceptable salt thereof of claim 10 or 11, P ₂ and P ₃ each independently comprise the sequence IETDV or LETTF or METTF or YIETDV or YIETWV or NIETDV or NIETWV. A compound or a pharmaceutically acceptable salt thereof, wherein the structure of the compound is shown in the following general formula (V):
_L2 is present or absent, and when present, _L2 is selected from hydrophilic structural units; _L3 is selected from hydrophilic structural units; P ₁ is selected from cell penetrating peptides; _P2 is selected from a cell penetrating peptide or _P2 comprises the sequence (X ⁰ ) _p X ¹ TX ² V or (X ⁰ ) _p X ¹ TX ² F or (X ⁰ ) _p X ¹ SX ² V; _P3 is selected from the sequence (X ⁰ ) _p X ¹ TX ² V or (X ⁰ ) _p X ¹ TX ² F or (X ⁰ ) _p X ¹ SX ² V, wherein p is 0 or 1; X ⁰ is selected from S, T, C, Y, N or Q; X ¹ is selected from E, Q, A, S; ^X2 is selected from A, T, L, V, R, Q, D, N, W. The compound according to claim 15 or a pharmaceutically acceptable salt thereof, wherein the structure of the compound is shown in the following general formula (VI):
P ₁ is selected from cell penetrating peptides; _P2 is selected from a cell penetrating peptide or _P2 comprises the sequence (X ⁰ ) _p X ¹ TX ² V or (X ⁰ ) _p X ¹ TX ² F or (X ⁰ ) _p X ¹ SX ² V; _P3 is selected from the sequence (X ⁰ ) _p X ¹ TX ² V or (X ⁰ ) _p X ¹ TX ² F or (X ⁰ ) _p X ¹ SX ² V, wherein p is 0 or 1; X ⁰ is selected from S, T, C, Y, N or Q; X ¹ is selected from E, Q, A, S; ^X2 is selected from A, T, L, V, R, Q, D, N, W. According to the compound or pharmaceutically acceptable salt thereof of claim 15 or 16, _P1 is selected from YGRKKRRQRRR, rrrqrrkkrGy; _P2 is selected from YGRKKRRQRRR, rrrqrrkkrGy. According to the compound or pharmaceutically acceptable salt thereof according to claim 15 or 16, P ₂ and P ₃ each independently comprise the sequence IETDV or LETTF or METTF or YIETDV or YIETWV or NIETDV or NIETWV. A compound or a pharmaceutically acceptable salt thereof, wherein the structure of the compound is shown in the following general formula (VII):
_L2 is present or absent, and when present, _L2 is selected from hydrophilic structural units; _L3 is selected from hydrophilic structural units; P ₁ is selected from cell penetrating peptides; _P2 is selected from a cell penetrating peptide or _P2 comprises the sequence (X ⁰ ) _p X ¹ TX ² V or (X ⁰ ) _p X ¹ TX ² F or (X ⁰ ) _p X ¹ SX ² V; _P3 is selected from the sequence (X ⁰ ) _p X ¹ TX ² V or (X ⁰ ) _p X ¹ TX ² F or (X ⁰ ) _p X ¹ SX ² V, wherein p is 0 or 1; X ⁰ is selected from S, T, C, Y, N or Q; X ¹ is selected from E, Q, A, S; ^X2 is selected from A, T, L, V, R, Q, D, N, W. The compound according to claim 19 or a pharmaceutically acceptable salt thereof, wherein the structure of the compound is shown in the following general formula (VIII):
P ₁ is selected from cell penetrating peptides; _P2 is selected from a cell penetrating peptide or _P2 comprises the sequence (X ⁰ ) _p X ¹ TX ² V or (X ⁰ ) _p X ¹ TX ² F or (X ⁰ ) _p X ¹ SX ² V; _P3 is selected from the sequence (X ⁰ ) _p X ¹ TX ² V or (X ⁰ ) _p X ¹ TX ² F or (X ⁰ ) _p X ¹ SX ² V, wherein p is 0 or 1; X ⁰ is selected from S, T, C, Y, N or Q; X ¹ is selected from E, Q, A, S; ^X2 is selected from A, T, L, V, R, Q, D, N, W. The compound according to claim 19 or 20 or a pharmaceutically acceptable salt thereof, wherein _P1 is selected from YGRKKRRQRRR, rrrqrrkkrGy. According to the compound or pharmaceutically acceptable salt thereof of claim 19 or 20, P ₂ and P ₃ each independently comprise the sequence IETDV or LETTF or METTF or YIETDV or YIETWV or NIETDV or NIETWV. A compound or a pharmaceutically acceptable salt thereof, wherein the compound structure is selected from the following structures:





A pharmaceutical composition comprising the compound according to any one of claims 1 to 23 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier. Use of the compound or pharmaceutically acceptable salt thereof according to any one of claims 1 to 23 or the pharmaceutical composition according to claim 24 in the preparation of a medicament for treating, ameliorating or preventing a disease caused by abnormal PSD-95 function in an individual. The use according to claim 25, wherein the disease caused by the abnormal function of PSD-95 is selected from cerebral infarction, stroke, neurodegenerative disease, anxiety or epilepsy, and neuropathic pain. The use according to claim 26, wherein the stroke is selected from ischemic stroke or hemorrhagic stroke. The use according to claim 26, wherein the neurodegenerative disease is selected from Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease or Huntington's disease.