Medicinal compound serving as neuroprotective agent and use of medicinal compound

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 neuroprotective agents and their use Technical Field

[0001] The present invention belongs to the field of biomedicine, and in particular relates to a medicinal compound used as a neuroprotectant and its application. Background Art

[0002] The N-methyl-D-aspartate receptor (NMDAR) plays a crucial role in the central nervous system, participating in learning and memory formation, synaptic formation during central nervous system development, plasticity, and glutamate-mediated neurotoxicity. NMDARs are the primary mediators of excitotoxicity (i.e., glutamate-mediated neurotoxicity), which is associated with neurodegenerative diseases and acute brain injury. They may serve as therapeutic targets for certain neurological diseases, such as cerebral infarction, neuropathic pain, epilepsy, and schizophrenia.

[0003] The postsynaptic density (PSD) is a complex of super-signaling molecules at the postsynaptic membrane of excitatory neurons, crucial for synaptic transmission. PSDs can be divided into four categories based on their molecular weight: PSD-95, PSD-93, synaptophysin-associated protein 97 (SAP-97), and SAP102.

[0004] PSD-95 is the most abundant and important scaffolding protein, primarily found in mature excitatory glutamatergic synapses. With a relative molecular mass (Mr) of 95,000, it is essential for receptor activity and stability at the postsynaptic membrane and plays a crucial role in synaptic plasticity. It is the most important synaptic protein mediating and integrating synaptic information and a member of the guanylate-related kinase family. PSD-95 consists of three PDZ domains, an SH3 domain, and a guanylate kinase-like (GK) domain, connected by a linker region. PSD-95 is almost exclusively localized to the postsynaptic density of neurons and participates in anchoring synaptic proteins. Its direct and indirect binding partners include neuroligins, nNOS, NMDA receptors, AMPA receptors, and potassium channels.

[0005] Based on the different splicing of mRNA genes, more than 10 nNOS (neuronal nitric oxide synthase) spliceosomes are currently known, with a molecular weight of 160.8 kD. nNOS contains two structural domains: a reducing domain at the C-terminus and an oxidative 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 proteins through the PDZ-PDZ domain or the C-terminal PDZ reaction. PSD-95 can connect nNOS to the NMDA receptor, effectively activating nNOS through the activation of the NMDA receptor.

[0006] Stroke is characterized by neuronal cell death in areas of local ischemia, hemorrhage, and / or trauma. Neuronal death or damage caused by cerebral ischemia is a cascade of injury. Following cerebral ischemia, tissue blood perfusion decreases, excitatory neurotransmitters increase, and NMDA and AMPA receptors activate, leading to ion channel opening, calcium influx, and the activation of numerous enzymes, triggering signaling cascades and causing multi-pathway neuronal damage. Although NMDA receptor antagonists effectively reduce excitotoxicity by blocking glutamate-mediated ion flux, they also inhibit several physiologically important processes. PSD-95, a downstream receptor, interacts with multiple proteins to trigger a series of ischemic insults. It is a key site of cerebral ischemic injury and a potential target for drug therapy. Therefore, the development of PSD-95 inhibitors holds great therapeutic potential for treating various neurological disorders caused by excitotoxicity, including stroke. However, NMDA receptor antagonists have failed in clinical trials for treatments such as stroke due to poor tolerability and lack of efficacy. In contrast, specific inhibition of excitotoxicity can be achieved by perturbing the intracellular nNOS / PSD-95 / NMDA receptor complex using PSD-95 inhibitors.

[0007] Furthermore, research has shown that the excitatory neurotransmitter NMDA plays a crucial role in anxiety, epilepsy, and various neurodegenerative diseases such as Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, and Huntington's disease. For example, studies have shown that overexcitation of the central glutamatergic system can trigger anxiety, and that NMDA receptors (NMDARs) are responsible for a major portion of glutamate excitotoxicity. Epilepsy seizures involve three distinct and sequential pathophysiological processes: initiation, maintenance and propagation of paroxysmal discharges, and inhibition of paroxysmal discharges. Excitatory neurotransmitters such as glutamate and aspartate play a crucial role in these processes. In Alzheimer's disease, PSD-95 is implicated in the neurotoxic mechanism through the GluR6-PSD-95-MLK3 pathway. Furthermore, in Huntington's disease, PSD-95 mediates the neurotoxicity of NMDA receptors and huntingtin mutants. Therefore, the development of PSD-95 inhibitors is of great significance for the treatment, amelioration, and prevention of these diseases.

[0008] 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.

[0009] 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 cerebral vascular disease. There is still a need for PSD-95 inhibitors with higher affinity for 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 receptor, 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 PSD-95 dimerization inhibitors. WO2022238530A1 discloses compounds that can bind to the PDZ domain of PSD-95 and their medical uses as inhibitors of PSD-95-mediated protein-protein interactions. To meet clinical needs, more PSD-95 inhibitors are still needed.

[0010] Summary of the Invention

[0011] The purpose of the present invention is to overcome the deficiencies of the above-mentioned prior art and provide a pharmaceutical compound as a neuroprotective agent and its application.

[0012] To achieve the above object, in one aspect, the present invention provides a compound or a pharmaceutically acceptable salt thereof, the structure of the compound is shown in the following general formula (I):

[0013] L1 is selected from

[0014] L2 is present or absent, and when present, L2 is selected from hydrophilic structural units;

[0015] L3 is selected from hydrophilic structural units;

[0016] L4 and L5 can be independently selected from a linker, -(CH2)nNH-, -C(=O)(CH2)nNH-, -(CH2)nC(=O)-, -(CH2)n

[0017] C(=O)NH-;

[0018] 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;

[0019] P1 is selected from cell-penetrating peptides (CPPs);

[0020] P2 can be selected from cell penetrating peptides (CPP) or P2 comprising 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;

[0021] 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, where

[0022] p is 0 or 1;

[0023] X 0 Selected from S, T, C, Y, N or Q;

[0024] X 1 Can be selected from E, Q, A, S;

[0025] X 2 Can be selected from A, T, L, V, R, Q, D, N, W.

[0026] Preferably, the CPP comprises at least 4 amino acid residues selected from arginine and / or lysine.

[0027] 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.

[0028] Preferably, the hydrophilic structural unit can be selected from (PEG)m, m is independently selected from integers of 0-10.

[0029] Preferably, said P2 and P3 each independently comprise the sequence IETDV or LETTF or METTF or YIETDV or YIETWV or NIETDV or NIETWV.

[0030] 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):

[0031] L2 is present or absent, and when present, L2 is selected from hydrophilic structural units;

[0032] L3 is selected from hydrophilic structural units;

[0033] L4 and L5 can be independently selected from a linker, -(CH2)nNH-, -C(=O)(CH2)nNH-, -(CH2)nC(=O)-, -(CH2)nC(=O)

[0034] NH-;

[0035] 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;

[0036] P1 is selected from cell-penetrating peptides (CPPs);

[0037] P2 can be selected from cell penetrating peptides (CPP) or P2 comprising 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;

[0038] 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, where

[0039] p is 0 or 1;

[0040] X 0 Selected from S, T, C, Y, N or Q;

[0041] X 1 Can be selected from E, Q, A, S;

[0042] X 2 Can be selected from A, T, L, V, R, Q, D, N, W.

[0043] Preferably, the CPP comprises at least 4 amino acid residues selected from arginine and / or lysine.

[0044] Preferably, the CPP is 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.

[0045] Preferably, the hydrophilic structural unit can be selected from (PEG)m, m can be independently selected from integers of 0-10.

[0046] Preferably, said P2 and P3 each independently comprise the sequence IETDV or LETTF or METTF or YIETDV or YIETWV or NIETDV or NIETWV.

[0047] 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):

[0048] L2 is present or absent, and when present, L2 is selected from hydrophilic structural units;

[0049] L3 is selected from hydrophilic structural units;

[0050] P1 is selected from cell-penetrating peptides (CPPs);

[0051] P2 can be selected from cell penetrating peptides (CPP) or P2 comprising 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;

[0052] P3 is selected from the sequence (X0 ) p X 1 TX 2 V or (X 0 ) p X 1 TX 2 F or (X 0 ) p X 1 SX 2 V, where

[0053] p is 0 or 1;

[0054] X 0 Selected from S, T, C, Y, N or Q;

[0055] X 1 Can be selected from E, Q, A, S;

[0056] X 2 It can be selected from A, T, L, V, R, Q, D, N, and W. Preferably, the structure of the compound is shown in the following general formula (IV):

[0057] P1 is selected from cell-penetrating peptides (CPPs);

[0058] P2 can be selected from cell penetrating peptides (CPP) or P2 comprising 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;

[0059] 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, where

[0060] p is 0 or 1;

[0061] X 0 Selected from S, T, C, Y, N or Q;

[0062] X 1 Can be selected from E, Q, A, S;

[0063] X 2 Can be selected from A, T, L, V, R, Q, D, N, W.

[0064] Preferably, P1 can be selected from YGRKKRRQRRR, rrrqrrkkrGy.

[0065] Preferably, said P2 and P3 each independently comprise the sequence IETDV or LETTF or METTF or YIETDV or YIETWV or NIETDV or NIETWV.

[0066] 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):

[0067] L2 is present or absent, and when present, L2 is selected from hydrophilic structural units;

[0068] L3 is selected from hydrophilic structural units;

[0069] P1 is selected from cell-penetrating peptides (CPPs);

[0070] P2 can be selected from cell penetrating peptides (CPP) or P2 comprising 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;

[0071] 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, where

[0072] p is 0 or 1;

[0073] X 0Selected from S, T, C, Y, N or Q;

[0074] X 1 Can be selected from E, Q, A, S;

[0075] X 2 Can be selected from A, T, L, V, R, Q, D, N, W.

[0076] Preferably, the structure of the compound is shown in the following general formula (VI):

[0077] P1 is selected from cell-penetrating peptides (CPPs);

[0078] P2 can be selected from cell penetrating peptides (CPP) or P2 comprising 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;

[0079] 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, where

[0080] p is 0 or 1;

[0081] X 0 Selected from S, T, C, Y, N or Q;

[0082] X 1 Can be selected from E, Q, A, S;

[0083] X 2 Can be selected from A, T, L, V, R, Q, D, N, W.

[0084] Further preferably, P1 can be selected from YGRKKRRQRRR, rrrqrrkkrGy; P2 can be selected from YGRKKRRQRRR, rrrqrrkkrGy.

[0085] Further preferably, said P2 and P3 each independently comprise the sequence IETDV or LETTF or METTF or YIETDV or YIETWV or NIETDV or NIETWV.

[0086] 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):

[0087] L2 is present or absent, and when present, L2 is selected from hydrophilic structural units;

[0088] L3 is selected from hydrophilic structural units;

[0089] P1 is selected from cell-penetrating peptides (CPPs);

[0090] P2 can be selected from cell penetrating peptides (CPP) or P2 comprising 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;

[0091] 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, where

[0092] p is 0 or 1;

[0093] X 0 Selected from S, T, C, Y, N or Q;

[0094] X 1 Can be selected from E, Q, A, S;

[0095] X 2 Can be selected from A, T, L, V, R, Q, D, N, W.

[0096] Preferably, the structure of the compound is shown in the following general formula (VIII):

[0097] P1 is selected from cell-penetrating peptides (CPPs);

[0098] P2 can be selected from cell penetrating peptides (CPP) or P2 comprising 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;

[0099] 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, where

[0100] p is 0 or 1;

[0101] X 0 Selected from S, T, C, Y, N or Q;

[0102] X 1 Can be selected from E, Q, A, S;

[0103] X 2 Can be selected from A, T, L, V, R, Q, D, N, W.

[0104] Preferably, P1 can be selected from YGRKKRRQRRR, rrrqrrkkrGy.

[0105] Preferably, said P2 and P3 each independently comprise the sequence IETDV or LETTF or METTF or YIETDV or YIETWV or NIETDV or NIETWV.

[0106] As a preferred embodiment of the compound of the present invention, the compound structure can be selected from the following structures:

[0107] Furthermore, the pharmaceutically acceptable salt may be selected from trifluoroacetate, acetate, hydrochloride and phosphate.

[0108] In a sixth aspect, the present invention provides a pharmaceutical composition comprising any of the above-mentioned compounds or pharmaceutically acceptable salts thereof and a pharmaceutically acceptable carrier, excipient and / or diluent.

[0109] Depending on the treatment and administration needs, the pharmaceutical composition may further include a pharmaceutically acceptable carrier. The carrier may be selected from diluents, excipients, fillers, binders, wetting agents, disintegrants, absorption enhancers, surfactants, adsorption carriers, lubricants, etc., and flavoring agents and flavoring agents may be added as needed.

[0110] The pharmaceutical compositions of the present invention can be prepared in various forms, including tablets, powders, granules, capsules, oral solutions, and injections. These various dosage forms can be prepared according to conventional methods in the pharmaceutical field. Administration of the pharmaceutical compositions can be parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. Intravenous administration is preferred.

[0111] In a seventh aspect, the present invention provides use of the above-mentioned compound or a pharmaceutically acceptable salt or pharmaceutical composition thereof in the preparation of a medicament for treating, ameliorating or preventing a disease caused by PSD-95 dysfunction in an individual.

[0112] The medicine can be in the form of tablets, powders, granules, capsules, oral solutions or injections.

[0113] Furthermore, the disease caused by the abnormal function of PSD-95 can be selected from cerebral apoplexy, stroke, neurodegenerative disease, anxiety or epilepsy, and neuropathic pain.

[0114] Furthermore, the above-mentioned stroke is selected from ischemic stroke or hemorrhagic stroke.

[0115] Furthermore, the neurodegenerative disease is selected from Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease or Huntington's disease.

[0116] The terms "peptide" or "polypeptide" have meanings 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 to form analogs, derivatives, functional mimetics, pseudopeptides, and the like, comprising at least two amino acids. Unless a specific modification is indicated at the N-terminus or C-terminus, a polypeptide comprising a specific amino acid sequence includes both unmodified and modified amino and / or carboxyl termini, as is well known to those skilled in the art. A polypeptide having a specific amino acid sequence may include modified amino acids and / or additional amino acids, unless the N- and / or C-termini contain modifications that prevent the addition of further amino acids. Such modifications include, for example, acetylation of the N-terminus and / or amidation of the C-terminus.

[0117] 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 polypeptides. Typical modifications include, but are not limited to, N-terminal acetylation, C-terminal amidation, D-amino acid substitution, non-natural amino acid substitution, fatty acid modification, or a combination of the above modifications. The present invention includes any well-known modification of polypeptides. For example, polypeptide derivatives can include chemical modifications to the polypeptide, such as alkylation, acylation, carbamylation, iodination, or any other modification that produces a polypeptide derivative. Modifications of the polypeptide can include modified amino acids, for example, hydroxyproline or carboxyglutamate, and can include amino acids connected by non-peptide bonds.

[0118] Furthermore, the peptides described above can optionally be derivatized (eg, acetylated, phosphorylated, and / or glycosylated) to improve affinity for the inhibitor, improve the ability of the inhibitor to be transported across the cell membrane, or improve stability.

[0119] For other modifications of the polypeptides of the present invention, non-natural amino acids can be used to replace the natural amino acids in the polypeptides. Non-natural amino acids include but are 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.

[0120] 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, as well as 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.

[0121] 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).

[0122] The polypeptide of the present invention can be prepared by conventional biosynthesis or chemical synthesis methods.

[0123] The term "PDZ domain" refers to a modular protein domain of approximately 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 exhibit 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

[0124] FIG1 is a graph showing the HPLC detection of compound 23 in Example 1;

[0125] FIG2 is a mass spectrometry diagram of compound 23 in Example 1;

[0126] FIG3 is a HPLC detection chart of compound 32 in Example 2;

[0127] FIG4 is a mass spectrometry diagram of compound 32 in Example 2;

[0128] FIG5 is a graph showing the HPLC detection of compound 25 from Example 3;

[0129] FIG6 is a mass spectrometry diagram of compound 25 in Example 3;

[0130] FIG7 is a HPLC detection chart of compound 26 in Example 4;

[0131] FIG8 is a mass spectrometry diagram of compound 26 in Example 4;

[0132] FIG9 is a graph showing HPLC detection of compound 27 in Example 5;

[0133] FIG10 is a mass spectrometry diagram of compound 27 in Example 5;

[0134] Figure 11 is a HPLC test chart of compound 21 in Example 6;

[0135] FIG12 is a mass spectrometry diagram of compound 21 in Example 6;

[0136] FIG13 is a HPLC detection chart of compound 22 in Example 7;

[0137] FIG14 is a mass spectrometry diagram of compound 22 in Example 7;

[0138] Figure 15 is a HPLC test chart of compound 31 in Example 8;

[0139] FIG16 is a mass spectrometry diagram of compound 31 in Example 8;

[0140] Figure 17 is a HPLC detection chart of compound 28 in Example 9;

[0141] Figure 18 is a mass spectrometry diagram of compound 28 from Example 9;

[0142] Figure 19 is a graph showing the experimental results of mouse plasma (heparin sodium) stability in Example 5, wherein Figure A shows the experimental results for compound 9, and Figure B shows the experimental results for compounds 31 and 32;

[0143] FIG20 is a graph showing the experimental results of the stability of mouse plasma alteplase (heparin sodium) in Example 5, wherein FIGA shows the experimental results of compound 9, and FIGB shows the experimental results of compounds 31 and 32;

[0144] FIG21 is a flow chart of the efficacy test in the mouse tMCAO model according to Test Example 6;

[0145] Figure 22 is a staining image of a mouse brain section in Test Example 6;

[0146] FIG23 shows the statistical results of embolism area in mouse brain slices in Test Example 6. Specific embodiments

[0147] The polypeptide compounds and derivatives provided herein are synthesized using a solid-phase synthesis method to obtain a linear precursor. After cleavage, the crude peptide is directly purified to obtain the target compound, or the crude peptide is oxidized with DMSO to form intramolecular disulfide bonds and then purified to obtain the target compound. The synthesis support is 2-Chlotrityl Resin resin. During the synthesis process, the 2-Chlotrityl Resin resin is first fully swollen in N,N-dimethylformamide (DMF). The solid support is then repeatedly condensed with an activated amino acid derivative, washed, deprotected with Fmoc, washed, and then subjected to another round of amino acid condensation to achieve the desired polypeptide chain length. Finally, the peptide is cleaved from the solid support by reacting with a mixture of trifluoroacetic acid: water: triisopropylsilane: thioanisole (90:2.5:2.5:5, v:v:v:v). The crude linear precursor solid is then precipitated with chilled methyl tert-butyl ether to obtain the crude target polypeptide. The cleaved crude linear precursor is then subjected to disulfide bond oxidation in a neutral solution. The solid crude product or the oxidized polypeptide crude product is purified and separated by a C18 reverse phase preparative chromatography column in a 0.1% trifluoroacetic acid acetonitrile / water system to obtain pure products of the polypeptide and its derivatives.

[0148] Experimental reagents

[0149] Among them, the structural formula of Fmoc-AEEA-OH is

[0150] Example 1 Preparation of Compound 23

[0151] Step 1: Coupling of the first amino acid Fmoc-Val-OH

[0152] 168 mg (0.2 mmol) of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 hour. 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 allowed to react at room temperature for 2 hours. After completion of the reaction, a blocking solution (10 mL) of DCM:methanol:DIEA (85:10:5, v:v:v) was added and blocked at room temperature for 10 minutes. The blocked resin was washed five times with DCM and five times with DMF.

[0153] Step 2: Synthesis of linear precursor peptide chains

[0154] The linear precursor peptide chain of compound 23 is AEEA-NIETWV.

[0155] The resin obtained in step 1 was fully swollen in DMF for 1 hour. Then, the linear precursor sequence was synthesized from the second D at the carboxyl end to the amino end. Each coupling cycle was performed as follows:

[0156] • Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), each time for 8 min.

[0157] • Rinse the resin 6-8 times with DMF until the pH is neutral.

[0158] 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.

[0159] • Rinse the resin 4-6 times with DMF before coupling the next amino acid.

[0160] After the linear peptide synthesis, the resin was washed with DMF 5 times and DCM 5 times. The resin was dried in vacuo.

[0161] Step 3: Coupling of 3-(FMOC-amino)glutaric acid

[0162] Dissolve 0.25 mmol of 3-(FMOC-amino)pentanedioic acid, 1 mmol of 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), and 2 mmol of N,N-diisopropylethylamine (DIEA) in DMF. Add the mixture to the resin obtained in step 2 and allow to react at room temperature for 1 hour, twice. After the reaction, rinse the resin five times with DMF and five times with DCM.

[0163] Step 4: Synthesis of linear precursor peptide chain

[0164] The linear precursor peptide chain of compound 23 is YGRKKRRQRRR.

[0165] The resin obtained in step 3 was fully swelled in DMF for 1 hour, and then the linear precursor sequence was synthesized from the carboxyl end to the amino end. Each coupling cycle was performed as follows:

[0166] • Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), each time for 8 min.

[0167] • Rinse the resin 6-8 times with DMF until the pH is neutral.

[0168] 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.

[0169] • Rinse the resin 4-6 times with DMF before coupling the next amino acid.

[0170] After the linear peptide synthesis, the resin was washed with DMF 5 times and DCM 5 times. The resin was dried in vacuo.

[0171] Step 5: Cleavage of the linear precursor peptide chain

[0172] Add 10 mL of freshly prepared cleavage cocktail (trifluoroacetic acid: water: triisopropylsilane: thioanisole (90:2.5:2.5:5, v:v:v:v) to the resin obtained in step 4 and shake at room temperature for 2 hours. After the reaction, filter the reaction solution, wash the resin with trifluoroacetic acid, combine it with the reaction solution, and precipitate with 4 volumes of cold MTBE to obtain a crude product. Wash the crude product three times with MTBE and dry it in a vacuum.

[0173] Step 6: Peptide purification

[0174] The crude peptide was dissolved in 20% acetonitrile in water, filtered through a 0.45 μm membrane, and separated using a reversed-phase HPLC system with buffers A (0.1% trifluoroacetic acid in water) and B (0.1% trifluoroacetic acid in acetonitrile). The chromatographic column was a BR-C18 (Saifen) reversed-phase column. During purification, the detection wavelength was set at 230 nm, the flow rate was 15 mL / min, and the gradient was 30-60% acetonitrile in 40 minutes. Product-related fractions were collected, and after HPLC analysis of purity, fractions exceeding 95% were combined and lyophilized to obtain the pure peptide.

[0175] Step 7: Detection and Characterization Methods

[0176] The purified peptide obtained in step 6 was analyzed by analytical HPLC and liquid chromatography / mass spectrometry to determine purity and target molecular weight. The test results are shown in Figures 1 and 2.

[0177] Example 2 Preparation of Compound 32

[0178] Step 1: Coupling of the first amino acid Fmoc-Val-OH

[0179] 168 mg (0.2 mmol) of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 hour. 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 allowed to react at room temperature for 2 hours. After completion of the reaction, a blocking solution (10 mL) of DCM:methanol:DIEA (85:10:5, v:v:v) was added and blocked at room temperature for 10 minutes. The blocked resin was washed five times with DCM and five times with DMF.

[0180] Step 2: Synthesis of linear precursor peptide chains

[0181] The linear precursor peptide chain of compound 32 is AEEA-NIETWV.

[0182] The resin obtained in step 1 was fully swollen in DMF for 1 hour. Then, the linear precursor sequence was synthesized from the second D at the carboxyl end to the amino end. Each coupling cycle was performed as follows:

[0183] • Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), each time for 8 min.

[0184] • Rinse the resin 6-8 times with DMF until the pH is neutral.

[0185] 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.

[0186] • Rinse the resin 4-6 times with DMF before coupling the next amino acid.

[0187] After the linear peptide synthesis, the resin was washed with DMF 5 times and DCM 5 times. The resin was dried in vacuo.

[0188] Step 3: Coupling of 3-(FMOC-amino)glutaric acid

[0189] Dissolve 0.25 mmol of 3-(FMOC-amino)pentanedioic acid, 1 mmol of 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), and 2 mmol of N,N-diisopropylethylamine (DIEA) in DMF. Add the mixture to the resin obtained in step 2 and allow to react at room temperature for 1 hour, twice. After the reaction, rinse the resin five times with DMF and five times with DCM.

[0190] Step 4: Synthesis of linear precursor peptide chain

[0191] The linear precursor peptide chain of compound 32 is dR-dR-dR-dQ-dR-dR-dK-dK-dR-G-dY.

[0192] The resin obtained in step 3 was fully swelled in DMF for 1 hour, and then the linear precursor sequence was synthesized from the carboxyl end to the amino end. Each coupling cycle was performed as follows:

[0193] • Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), each time for 8 min.

[0194] • Rinse the resin 6-8 times with DMF until the pH is neutral.

[0195] 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.

[0196] • Rinse the resin 4-6 times with DMF before coupling the next amino acid.

[0197] After the linear peptide synthesis, the resin was washed with DMF 5 times and DCM 5 times. The resin was dried in vacuo.

[0198] Step 5: Cleavage of the linear precursor peptide chain

[0199] Add 10 mL of freshly prepared cleavage cocktail (trifluoroacetic acid: water: triisopropylsilane: thioanisole (90:2.5:2.5:5, v:v:v:v) to the resin obtained in step 4 and shake at room temperature for 2 hours. After the reaction, filter the reaction solution, wash the resin with trifluoroacetic acid, combine it with the reaction solution, and precipitate with 4 volumes of cold MTBE to obtain a crude product. Wash the crude product three times with MTBE and dry it in a vacuum.

[0200] Step 6: Peptide purification

[0201] The crude peptide was dissolved in 20% acetonitrile in water, filtered through a 0.45 μm membrane, and separated using a reversed-phase HPLC system with buffers A (0.1% trifluoroacetic acid in water) and B (0.1% trifluoroacetic acid in acetonitrile). The chromatographic column was a BR-C18 (Saifen) reversed-phase column. During purification, the detection wavelength was set at 230 nm, the flow rate was 15 mL / min, and the gradient was 30-60% acetonitrile in 40 minutes. Product-related fractions were collected, and after HPLC analysis of purity, fractions exceeding 95% were combined and lyophilized to obtain the pure peptide.

[0202] Step 7: Detection and Characterization Methods

[0203] The purified peptide obtained in step 6 was analyzed by analytical HPLC and liquid chromatography / mass spectrometry to determine purity and target molecular weight. The test results are shown in Figures 3 and 4.

[0204] Example 3 Preparation of Compound 25

[0205] Step 1: Coupling of the first amino acid Fmoc-Cys(Trt)-OH

[0206] 84 mg (0.1 mmol) of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 hour. Fmoc-Cys(Trt)-OH (0.08 mmol) and diisopropylethylamine (DIEA, 0.32 mmol) were weighed and dissolved in 5 mL of DCM. The mixture was added to the resin and allowed to react at room temperature for 2 hours. After the reaction, a blocking solution (10 mL) of DCM:methanol:DIEA (85:10:5, v:v:v) was added and blocked at room temperature for 10 minutes. The blocked resin was washed five times with DCM and five times with DMF.

[0207] Step 2: Synthesis of linear precursor peptide chains

[0208] The linear precursor peptide chain of compound 25 is CKMVTTFGIDVTTTYIC.

[0209] The resin obtained in step 1 was fully swollen in DMF for 1 hour, and then the linear precursor sequence was synthesized from the second position of the carboxyl end to the amino end. Each coupling cycle was performed as follows:

[0210] • Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), each time for 8 min.

[0211] • Rinse the resin 6-8 times with DMF until the pH is neutral.

[0212] 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.

[0213] • Rinse the resin 4-6 times with DMF before coupling the next amino acid.

[0214] After the linear peptide synthesis, the resin was washed with DMF 5 times and DCM 5 times. The resin was dried in vacuo.

[0215] Step 3: Coupling of amino acid Fmoc-Lys(Fmoc)-OH

[0216] Dissolve 0.5 mmol of Fmoc-Lys(Fmoc)-OH, 0.5 mmol of 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), and 1 mmol of 4-methylmorpholine (NMM) in DMF and add to the resin obtained in step 1. Allow to react at room temperature for 1 hour. After the reaction, rinse the resin five times with DMF and five times with DCM.

[0217] Step 4: Synthesis of linear precursor peptide chain

[0218] The linear precursor peptide chain of compound 25 is dR-dR-dR-dQ-dR-dR-dK-dK-dR-G-dY.

[0219] The resin obtained in step 3 was fully swelled in DMF for 1 hour, and then the linear precursor sequence was synthesized from the carboxyl end to the amino end. Each coupling cycle was performed as follows:

[0220] • Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), each time for 8 min.

[0221] • Rinse the resin 6-8 times with DMF until the pH is neutral.

[0222] Dissolve 1 mmol of Fmoc-AA, 1 mmol of 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 2 mmol of 4-methylmorpholine (NMM) in DMF, add to the resin and react at room temperature for 1 hour.

[0223] • Rinse the resin 4-6 times with DMF before coupling the next amino acid.

[0224] After the linear peptide synthesis, the resin was washed with DMF 5 times and DCM 5 times. The resin was dried in vacuo.

[0225] Step 5: Cleavage of the linear precursor peptide chain

[0226] Add 10 mL of freshly prepared cleavage cocktail (trifluoroacetic acid: water: triisopropylsilane: thioanisole (90:2.5:2.5:5, v:v:v:v) to the resin obtained in step 4 and shake at room temperature for 2 hours. After the reaction, filter the reaction solution, wash the resin with trifluoroacetic acid, combine it with the reaction solution, and precipitate with 4 volumes of cold MTBE to obtain a crude product. Wash the crude product three times with MTBE and dry it in a vacuum.

[0227] Step 6: Intramolecular disulfide bond formation

[0228] Dissolve the crude product from step 5 in DMSO (20% of the total reaction volume). Prepare a phosphate / guanidine hydrochloride buffer (pH 7.0, containing 50% ACN) by mixing 1 / PBS:6M guanidine hydrochloride (80:20, v:v) with acetonitrile in a 1:1 ratio. Slowly add the peptide solution dropwise to this buffer to a final concentration of 1 mg / mL. Shake at room temperature for 16 hours. Monitor the reaction by LC-MS. Purify the product immediately after completion.

[0229] Step 7: Peptide purification

[0230] After filtration through a 0.45 μm membrane, the product was separated using a reversed-phase HPLC system using buffers A (0.1% trifluoroacetic acid in water) and B (0.1% trifluoroacetic acid in acetonitrile). The chromatographic column was a BR-C18 (Saifen) reversed-phase column. During purification, the detection wavelength was set at 230 nm, the flow rate was 15 mL / min, and the gradient was 30-60% acetonitrile over 40 minutes. Product-related fractions were collected, and after HPLC analysis of purity, fractions exceeding 95% were combined and lyophilized to obtain the pure peptide. The results of liquid chromatography and mass spectrometry are shown in Figures 5 and 6.

[0231] Example 4 Synthesis of Compound 26

[0232] Step 1: Coupling of the first amino acid Fmoc-Val-OH

[0233] 168 mg (0.2 mmol) of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 hour. 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 allowed to react at room temperature for 2 hours. After the reaction, a blocking solution (10 mL) of DCM:methanol:DIEA (85:10:5, v:v:v) was added and blocked at room temperature for 10 minutes. The blocked resin was washed five times with DCM and five times with DMF.

[0234] Step 2: Synthesis of linear precursor peptide chains

[0235] The linear precursor peptide chain AEEA-IETDV of compound 26.

[0236] The resin obtained in step 1 was fully swollen in DMF for 1 hour. Then, the linear precursor sequence was synthesized from the second D at the carboxyl end to the amino end. Each coupling cycle was performed as follows:

[0237] • Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), each time for 8 min.

[0238] • Rinse the resin 6-8 times with DMF until the pH is neutral.

[0239] 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.

[0240] • Rinse the resin 4-6 times with DMF before coupling the next amino acid.

[0241] After the linear peptide synthesis, the resin was washed with DMF 5 times and DCM 5 times. The resin was dried in vacuo.

[0242] Step 3: Coupling of 3-(FMOC-amino)glutaric acid

[0243] Dissolve 0.25 mmol of 3-(FMOC-amino)pentanedioic acid, 1 mmol of 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), and 2 mmol of N,N-diisopropylethylamine (DIEA) in DMF. Add the mixture to the resin obtained in step 2 and allow to react at room temperature for 1 hour, twice. After the reaction, rinse the resin five times with DMF and five times with DCM.

[0244] Step 4: Synthesis of linear precursor peptide chain

[0245] The linear precursor peptide chain of compound 26 is YGRKKRRQRRR.

[0246] The resin obtained in step 3 was fully swelled in DMF for 1 hour, and then the linear precursor sequence was synthesized from the carboxyl end to the amino end. Each coupling cycle was performed as follows:

[0247] • Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), each time for 8 min.

[0248] • Rinse the resin 6-8 times with DMF until the pH is neutral.

[0249] 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.

[0250] • Rinse the resin 4-6 times with DMF before coupling the next amino acid.

[0251] After the linear peptide synthesis, the resin was washed with DMF 5 times and DCM 5 times. The resin was dried in vacuo.

[0252] Step 5: Cleavage of the linear precursor peptide chain

[0253] Add 10 mL of freshly prepared cleavage cocktail (trifluoroacetic acid: water: triisopropylsilane: thioanisole (90:2.5:2.5:5, v:v:v:v) to the resin obtained in step 4 and shake at room temperature for 2 hours. After the reaction, filter the reaction solution, wash the resin with trifluoroacetic acid, combine it with the reaction solution, and precipitate with 4 volumes of cold MTBE to obtain a crude product. Wash the crude product three times with MTBE and dry it in a vacuum.

[0254] Step 6: Peptide purification

[0255] The crude peptide was dissolved in 20% acetonitrile in water, filtered through a 0.45 μm membrane, and separated using a reversed-phase HPLC system with buffers A (0.1% trifluoroacetic acid in water) and B (0.1% trifluoroacetic acid in acetonitrile). The chromatographic column was a BR-C18 (Saifen) reversed-phase column. During purification, the detection wavelength was set at 230 nm, the flow rate was 15 mL / min, and the gradient was 30-60% acetonitrile in 40 minutes. Product-related fractions were collected, and after HPLC analysis of purity, fractions exceeding 95% were combined and lyophilized to obtain the pure peptide.

[0256] Step 7: Detection and Characterization Methods

[0257] The purified peptide obtained in step 6 was analyzed by analytical HPLC and liquid chromatography / mass spectrometry to determine purity and target molecular weight. The test results are shown in Figures 7 and 8.

[0258] Example 5 Preparation of Compound 27

[0259] Step 1: Coupling of the first amino acid Fmoc-Val-OH

[0260] 168 mg (0.2 mmol) of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 hour. 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 allowed to react at room temperature for 2 hours. After completion of the reaction, a blocking solution (10 mL) of DCM:methanol:DIEA (85:10:5, v:v:v) was added and blocked at room temperature for 10 minutes. The blocked resin was washed five times with DCM and five times with DMF.

[0261] Step 2: Synthesis of linear precursor peptide chains

[0262] The linear precursor peptide chain AEEA-IETDV of compound 27.

[0263] The resin obtained in step 1 was fully swollen in DMF for 1 hour. Then, the linear precursor sequence was synthesized from the second D at the carboxyl end to the amino end. Each coupling cycle was performed as follows:

[0264] • Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), each time for 8 min.

[0265] • Rinse the resin 6-8 times with DMF until the pH is neutral.

[0266] 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.

[0267] • Rinse the resin 4-6 times with DMF before coupling the next amino acid.

[0268] After the linear peptide synthesis, the resin was washed with DMF 5 times and DCM 5 times. The resin was dried in vacuo.

[0269] Step 3: Coupling of 3-(FMOC-amino)glutaric acid

[0270] Dissolve 0.25 mmol of 3-(FMOC-amino)pentanedioic acid, 1 mmol of 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), and 2 mmol of N,N-diisopropylethylamine (DIEA) in DMF. Add the mixture to the resin obtained in step 2 and allow to react at room temperature for 1 hour, twice. After the reaction, rinse the resin five times with DMF and five times with DCM.

[0271] Step 4: Synthesis of linear precursor peptide chain

[0272] The linear precursor peptide chain of compound 27 is dR-dR-dR-dQ-dR-dR-dK-dK-dR-G-dY.

[0273] The resin obtained in step 3 was fully swelled in DMF for 1 hour, and then the linear precursor sequence was synthesized from the carboxyl end to the amino end. Each coupling cycle was performed as follows:

[0274] • Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), each time for 8 min.

[0275] • Rinse the resin 6-8 times with DMF until the pH is neutral.

[0276] 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.

[0277] • Rinse the resin 4-6 times with DMF before coupling the next amino acid.

[0278] After the linear peptide synthesis, the resin was washed with DMF 5 times and DCM 5 times. The resin was dried in vacuo.

[0279] Step 5: Cleavage of the linear precursor peptide chain

[0280] Add 10 mL of freshly prepared cleavage cocktail (trifluoroacetic acid: water: triisopropylsilane: thioanisole (90:2.5:2.5:5, v:v:v:v) to the resin obtained in step 4 and shake at room temperature for 2 hours. After the reaction, filter the reaction solution, wash the resin with trifluoroacetic acid, combine it with the reaction solution, and precipitate with 4 volumes of cold MTBE to obtain a crude product. Wash the crude product three times with MTBE and dry it in a vacuum.

[0281] Step 6: Peptide purification

[0282] The crude peptide was dissolved in 20% acetonitrile in water, filtered through a 0.45 μm membrane, and separated using a reversed-phase HPLC system with buffers A (0.1% trifluoroacetic acid in water) and B (0.1% trifluoroacetic acid in acetonitrile). The chromatographic column was a BR-C18 (Saifen) reversed-phase column. During purification, the detection wavelength was set at 230 nm, the flow rate was 15 mL / min, and the gradient was 30-60% acetonitrile in 40 minutes. Product-related fractions were collected, and after HPLC analysis of purity, fractions exceeding 95% were combined and lyophilized to obtain the pure peptide.

[0283] Step 7: Detection and Characterization Methods

[0284] The purified peptide obtained in step 6 was analyzed by analytical HPLC and liquid chromatography / mass spectrometry to determine purity and target molecular weight. The test results are shown in Figures 9 and 10.

[0285] Example 6 Preparation of Compound 21

[0286] Step 1: Coupling of the first amino acid Fmoc-Val-OH

[0287] 168 mg (0.2 mmol) of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 hour. 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 allowed to react at room temperature for 2 hours. After completion of the reaction, a blocking solution (10 mL) of DCM:methanol:DIEA (85:10:5, v:v:v) was added and blocked at room temperature for 10 minutes. The blocked resin was washed five times with DCM and five times with DMF.

[0288] Step 2: Synthesis of linear precursor peptide chains

[0289] The linear precursor peptide chain of compound 21 is AEEA-YIETDV.

[0290] The resin obtained in step 1 was fully swollen in DMF for 1 hour. Then, the linear precursor sequence was synthesized from the second D at the carboxyl end to the amino end. Each coupling cycle was performed as follows:

[0291] • Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), each time for 8 min.

[0292] • Rinse the resin 6-8 times with DMF until the pH is neutral.

[0293] 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.

[0294] • Rinse the resin 4-6 times with DMF before coupling the next amino acid.

[0295] After the linear peptide synthesis, the resin was washed with DMF 5 times and DCM 5 times. The resin was dried in vacuo.

[0296] Step 3: Coupling of 3-(FMOC-amino)glutaric acid

[0297] Dissolve 0.25 mmol of 3-(FMOC-amino)pentanedioic acid, 1 mmol of 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), and 2 mmol of N,N-diisopropylethylamine (DIEA) in DMF. Add the mixture to the resin obtained in step 2 and allow to react at room temperature for 1 hour, twice. After the reaction, rinse the resin five times with DMF and five times with DCM.

[0298] Step 4: Synthesis of linear precursor peptide chain

[0299] The linear precursor peptide chain of compound 21 is YGRKKRRQRRR.

[0300] The resin obtained in step 3 was fully swelled in DMF for 1 hour, and then the linear precursor sequence was synthesized from the carboxyl end to the amino end. Each coupling cycle was performed as follows:

[0301] • Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), each time for 8 min.

[0302] • Rinse the resin 6-8 times with DMF until the pH is neutral.

[0303] 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.

[0304] • Rinse the resin 4-6 times with DMF before coupling the next amino acid.

[0305] After the linear peptide synthesis, the resin was washed with DMF 5 times and DCM 5 times. The resin was dried in vacuo.

[0306] Step 5: Cleavage of the linear precursor peptide chain

[0307] Add 10 mL of freshly prepared cleavage cocktail (trifluoroacetic acid: water: triisopropylsilane: thioanisole (90:2.5:2.5:5, v:v:v:v) to the resin obtained in step 4 and shake at room temperature for 2 hours. After the reaction, filter the reaction solution, wash the resin with trifluoroacetic acid, combine it with the reaction solution, and precipitate with 4 volumes of cold MTBE to obtain a crude product. Wash the crude product three times with MTBE and dry it in a vacuum.

[0308] Step 6: Peptide purification

[0309] The crude peptide was dissolved in 20% acetonitrile in water, filtered through a 0.45 μm membrane, and separated using a reversed-phase HPLC system with buffers A (0.1% trifluoroacetic acid in water) and B (0.1% trifluoroacetic acid in acetonitrile). The chromatographic column was a BR-C18 (Saifen) reversed-phase column. During purification, the detection wavelength was set at 230 nm, the flow rate was 15 mL / min, and the gradient was 30-60% acetonitrile in 40 minutes. Product-related fractions were collected, and after HPLC analysis of purity, fractions exceeding 95% were combined and lyophilized to obtain the pure peptide.

[0310] Step 7: Detection and Characterization Methods

[0311] The purified peptide obtained in step 6 was analyzed by analytical HPLC and liquid chromatography / mass spectrometry to determine purity and target molecular weight. The test results are shown in Figures 11 and 12.

[0312] Example 7 Preparation of Compound 22

[0313] Step 1: Coupling of the first amino acid Fmoc-Val-OH

[0314] 168 mg (0.2 mmol) of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 hour. 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 allowed to react at room temperature for 2 hours. After completion of the reaction, a blocking solution (10 mL) of DCM:methanol:DIEA (85:10:5, v:v:v) was added and blocked at room temperature for 10 minutes. The blocked resin was washed five times with DCM and five times with DMF.

[0315] Step 2: Synthesis of linear precursor peptide chains

[0316] The linear precursor peptide chain of compound 22 is AEEA-YIETWV.

[0317] The resin obtained in step 1 was fully swollen in DMF for 1 hour. Then, the linear precursor sequence was synthesized from the second D at the carboxyl end to the amino end. Each coupling cycle was performed as follows:

[0318] • Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), each time for 8 min.

[0319] • Rinse the resin 6-8 times with DMF until the pH is neutral.

[0320] 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.

[0321] • Rinse the resin 4-6 times with DMF before coupling the next amino acid.

[0322] After the linear peptide synthesis, the resin was washed with DMF 5 times and DCM 5 times. The resin was dried in vacuo.

[0323] Step 3: Coupling of 3-(FMOC-amino)glutaric acid

[0324] Dissolve 0.25 mmol of 3-(FMOC-amino)pentanedioic acid, 1 mmol of 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), and 2 mmol of N,N-diisopropylethylamine (DIEA) in DMF. Add the mixture to the resin obtained in step 2 and allow to react at room temperature for 1 hour, twice. After the reaction, rinse the resin five times with DMF and five times with DCM.

[0325] Step 4: Synthesis of linear precursor peptide chain

[0326] The linear precursor peptide chain of compound 22 is YGRKKRRQRRR.

[0327] The resin obtained in step 3 was fully swelled in DMF for 1 hour, and then the linear precursor sequence was synthesized from the carboxyl end to the amino end. Each coupling cycle was performed as follows:

[0328] • Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), each time for 8 min.

[0329] • Rinse the resin 6-8 times with DMF until the pH is neutral.

[0330] 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.

[0331] • Rinse the resin 4-6 times with DMF before coupling the next amino acid.

[0332] After the linear peptide synthesis, the resin was washed with DMF 5 times and DCM 5 times. The resin was dried in vacuo.

[0333] Step 5: Cleavage of the linear precursor peptide chain

[0334] Add 10 mL of freshly prepared cleavage cocktail (trifluoroacetic acid: water: triisopropylsilane: thioanisole (90:2.5:2.5:5, v:v:v:v) to the resin obtained in step 4 and shake at room temperature for 2 hours. After the reaction, filter the reaction solution, wash the resin with trifluoroacetic acid, combine it with the reaction solution, and precipitate with 4 volumes of cold MTBE to obtain a crude product. Wash the crude product three times with MTBE and dry it in a vacuum.

[0335] Step 6: Peptide purification

[0336] The crude peptide was dissolved in 20% acetonitrile in water, filtered through a 0.45 μm membrane, and separated using a reversed-phase HPLC system with buffers A (0.1% trifluoroacetic acid in water) and B (0.1% trifluoroacetic acid in acetonitrile). The chromatographic column was a BR-C18 (Saifen) reversed-phase column. During purification, the detection wavelength was set at 230 nm, the flow rate was 15 mL / min, and the gradient was 30-60% acetonitrile in 40 minutes. Product-related fractions were collected, and after HPLC analysis of purity, fractions exceeding 95% were combined and lyophilized to obtain the pure peptide.

[0337] Step 7: Detection and Characterization Methods

[0338] The purified peptide obtained in step 6 was analyzed by analytical HPLC and liquid chromatography / mass spectrometry to determine purity and target molecular weight. The test results are shown in Figures 13 and 14.

[0339] Example 8 Preparation of Compound 31

[0340] Step 1: Coupling of the first amino acid Fmoc-Val-OH

[0341] 168 mg (0.2 mmol) of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 hour. 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 allowed to react at room temperature for 2 hours. After completion of the reaction, a blocking solution (10 mL) of DCM:methanol:DIEA (85:10:5, v:v:v) was added and blocked at room temperature for 10 minutes. The blocked resin was washed five times with DCM and five times with DMF.

[0342] Step 2: Synthesis of linear precursor peptide chains

[0343] The linear precursor peptide chain of compound 31 is AEEA-YIETWV.

[0344] The resin obtained in step 1 was fully swollen in DMF for 1 hour. Then, the linear precursor sequence was synthesized from the second D at the carboxyl end to the amino end. Each coupling cycle was performed as follows:

[0345] • Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), each time for 8 min.

[0346] • Rinse the resin 6-8 times with DMF until the pH is neutral.

[0347] 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.

[0348] • Rinse the resin 4-6 times with DMF before coupling the next amino acid.

[0349] After the linear peptide synthesis, the resin was washed with DMF 5 times and DCM 5 times. The resin was dried in vacuo.

[0350] Step 3: Coupling of 3-(FMOC-amino)glutaric acid

[0351] Dissolve 0.25 mmol of 3-(FMOC-amino)pentanedioic acid, 1 mmol of 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), and 2 mmol of N,N-diisopropylethylamine (DIEA) in DMF. Add the mixture to the resin obtained in step 2 and allow to react at room temperature for 1 hour, twice. After the reaction, rinse the resin five times with DMF and five times with DCM.

[0352] Step 4: Synthesis of linear precursor peptide chain

[0353] The linear precursor peptide chain of compound 31 is dR-dR-dR-dQ-dR-dR-dK-dK-dR-G-dY.

[0354] The resin obtained in step 3 was fully swelled in DMF for 1 hour, and then the linear precursor sequence was synthesized from the carboxyl end to the amino end. Each coupling cycle was performed as follows:

[0355] • Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), each time for 8 min.

[0356] • Rinse the resin 6-8 times with DMF until the pH is neutral.

[0357] 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.

[0358] • Rinse the resin 4-6 times with DMF before coupling the next amino acid.

[0359] After the linear peptide synthesis, the resin was washed with DMF 5 times and DCM 5 times. The resin was dried in vacuo.

[0360] Step 5: Cleavage of the linear precursor peptide chain

[0361] Add 10 mL of freshly prepared cleavage cocktail (trifluoroacetic acid: water: triisopropylsilane: thioanisole (90:2.5:2.5:5, v:v:v:v) to the resin obtained in step 4 and shake at room temperature for 2 hours. After the reaction, filter the reaction solution, wash the resin with trifluoroacetic acid, combine it with the reaction solution, and precipitate with 4 volumes of cold MTBE to obtain a crude product. Wash the crude product three times with MTBE and dry it in a vacuum.

[0362] Step 6: Peptide purification

[0363] The crude peptide was dissolved in 20% acetonitrile in water, filtered through a 0.45 μm membrane, and separated using a reversed-phase HPLC system with buffers A (0.1% trifluoroacetic acid in water) and B (0.1% trifluoroacetic acid in acetonitrile). The chromatographic column was a BR-C18 (Saifen) reversed-phase column. During purification, the detection wavelength was set at 230 nm, the flow rate was 15 mL / min, and the gradient was 30-60% acetonitrile in 40 minutes. Product-related fractions were collected, and after HPLC analysis of purity, fractions exceeding 95% were combined and lyophilized to obtain the pure peptide.

[0364] Step 7: Detection and Characterization Methods

[0365] The purified peptide obtained in step 6 was analyzed by analytical HPLC and liquid chromatography / mass spectrometry to determine purity and target molecular weight. The test results are shown in Figures 15 and 16.

[0366] Example 9 Preparation of Compound 28

[0367] Step 1: Coupling of the first amino acid Fmoc-Cys(Trt)-OH

[0368] 84 mg (0.1 mmol) of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 hour. Fmoc-Cys(Trt)-OH (0.08 mmol) and diisopropylethylamine (DIEA, 0.32 mmol) were weighed and dissolved in 5 mL of DCM. The mixture was added to the resin and allowed to react at room temperature for 2 hours. After the reaction, a blocking solution (10 mL) of DCM:methanol:DIEA (85:10:5, v:v:v) was added and blocked at room temperature for 10 minutes. The blocked resin was washed five times with DCM and five times with DMF.

[0369] Step 2: Synthesis of linear precursor peptide chains

[0370] The linear precursor peptide chain of compound 28 is AEEA-CKMVTTFGIDVTTTYIC.

[0371] The resin obtained in step 1 was fully swollen in DMF for 1 hour, and then the linear precursor sequence was synthesized from the second position of the carboxyl end to the amino end. Each coupling cycle was performed as follows:

[0372] • Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), each time for 8 min.

[0373] • Rinse the resin 6-8 times with DMF until the pH is neutral.

[0374] 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.

[0375] • Rinse the resin 4-6 times with DMF before coupling the next amino acid.

[0376] After the linear peptide synthesis, the resin was washed with DMF 5 times and DCM 5 times. The resin was dried in vacuo.

[0377] Step 3: Coupling of amino acid Fmoc-Lys(Fmoc)-OH

[0378] Dissolve 0.5 mmol of Fmoc-Lys(Fmoc)-OH, 0.5 mmol of 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), and 1 mmol of 4-methylmorpholine (NMM) in DMF and add to the resin obtained in step 1. Allow to react at room temperature for 1 hour. After the reaction, rinse the resin five times with DMF and five times with DCM.

[0379] Step 4: Synthesis of linear precursor peptide chain

[0380] The linear precursor peptide chain of compound 28 is dR-dR-dR-dQ-dR-dR-dK-dK-dR-G-dY.

[0381] The resin obtained in step 3 was fully swelled in DMF for 1 hour, and then the linear precursor sequence was synthesized from the carboxyl end to the amino end. Each coupling cycle was performed as follows:

[0382] • Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), each time for 8 min.

[0383] • Rinse the resin 6-8 times with DMF until the pH is neutral.

[0384] Dissolve 1 mmol of Fmoc-AA, 1 mmol of 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU) and 2 mmol of 4-methylmorpholine (NMM) in DMF, add to the resin and react at room temperature for 1 hour.

[0385] • Rinse the resin 4-6 times with DMF before coupling the next amino acid.

[0386] After the linear peptide synthesis, the resin was washed with DMF 5 times and DCM 5 times. The resin was dried in vacuo.

[0387] Step 5: Cleavage of the linear precursor peptide chain

[0388] Add 10 mL of freshly prepared cleavage cocktail (trifluoroacetic acid: water: triisopropylsilane: thioanisole (90:2.5:2.5:5, v:v:v:v) to the resin obtained in step 4 and shake at room temperature for 2 hours. After the reaction, filter the reaction solution, wash the resin with trifluoroacetic acid, combine it with the reaction solution, and precipitate with 4 volumes of cold MTBE to obtain a crude product. Wash the crude product three times with MTBE and dry it in a vacuum.

[0389] Step 6: Intramolecular disulfide bond formation

[0390] Dissolve the crude product from step 5 in DMSO (20% of the total reaction volume). Prepare a phosphate / guanidine hydrochloride buffer (pH 7.0, containing 50% ACN) by mixing 1 / PBS:6M guanidine hydrochloride (80:20, v:v) with acetonitrile in a 1:1 ratio. Slowly add the peptide solution dropwise to this buffer to a final concentration of 1 mg / mL. Shake at room temperature for 16 hours. Monitor the reaction by LC-MS. Purify the product immediately after completion.

[0391] Step 7: Peptide purification

[0392] After filtration through a 0.45 μm membrane, the product was separated using a reverse-phase HPLC system using buffers A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). The chromatographic column was a BR-C18 (Saifen) reverse-phase column. During purification, the chromatograph detection wavelength was set at 230 nm, the flow rate was 15 mL / min, and the gradient was 30-60% acetonitrile in 40 minutes. Product-related fractions were collected, and after HPLC analysis of purity, fractions with >95% purity were combined and lyophilized to obtain the pure peptide. The results of liquid chromatography and mass spectrometry are shown in Figures 17 and 18.

[0393] Example 10 Synthesis of Compound 1

[0394] Step 1: Coupling of the first amino acid Fmoc-Val-OH

[0395] 168 mg (0.2 mmol) of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 hour. 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 allowed to react at room temperature for 2 hours. After the reaction, a blocking solution (10 mL) of DCM:methanol:DIEA (85:10:5, v:v:v) was added and blocked at room temperature for 10 minutes. The blocked resin was washed five times with DCM and five times with DMF.

[0396] Step 2: Synthesis of linear precursor peptide chains

[0397] The linear precursor peptide chain AEEA-IETDV of compound 1.

[0398] The resin obtained in step 1 was fully swollen in DMF for 1 hour. Then, the linear precursor sequence was synthesized from the second D at the carboxyl end to the amino end. Each coupling cycle was performed as follows:

[0399] • Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), each time for 8 min.

[0400] • Rinse the resin 6-8 times with DMF until the pH is neutral.

[0401] 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.

[0402] • Rinse the resin 4-6 times with DMF before coupling the next amino acid.

[0403] After the linear peptide synthesis, the resin was washed with DMF 5 times and DCM 5 times. The resin was dried in vacuo.

[0404] Step 3: Coupling of 3-(FMOC-amino)malonic acid

[0405] Dissolve 0.25 mmol of 3-(FMOC-amino)malonic acid, 1 mmol of 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), and 2 mmol of N,N-diisopropylethylamine (DIEA) in DMF and add to the resin obtained in step 2. React at room temperature for 1 hour, twice. After the reaction, rinse the resin five times with DMF and five times with DCM.

[0406] Step 4: Synthesis of linear precursor peptide chain

[0407] The linear precursor peptide chain of compound 1 is YGRKKRRQRRR.

[0408] The resin obtained in step 3 was fully swelled in DMF for 1 hour, and then the linear precursor sequence was synthesized from the carboxyl end to the amino end. Each coupling cycle was performed as follows:

[0409] • Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), each time for 8 min.

[0410] • Rinse the resin 6-8 times with DMF until the pH is neutral.

[0411] 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.

[0412] • Rinse the resin 4-6 times with DMF before coupling the next amino acid.

[0413] After the linear peptide synthesis, the resin was washed with DMF 5 times and DCM 5 times. The resin was dried in vacuo.

[0414] Step 5: Cleavage of the linear precursor peptide chain

[0415] Add 10 mL of freshly prepared cleavage cocktail (trifluoroacetic acid: water: triisopropylsilane: thioanisole (90:2.5:2.5:5, v:v:v:v) to the resin obtained in step 4 and shake at room temperature for 2 hours. After the reaction, filter the reaction solution, wash the resin with trifluoroacetic acid, combine it with the reaction solution, and precipitate with 4 volumes of cold MTBE to obtain a crude product. Wash the crude product three times with MTBE and dry it in a vacuum.

[0416] Step 6: Peptide purification

[0417] The crude peptide was dissolved in 20% acetonitrile in water, filtered through a 0.45 μm membrane, and separated using a reversed-phase HPLC system with buffers A (0.1% trifluoroacetic acid in water) and B (0.1% trifluoroacetic acid in acetonitrile). The chromatographic column was a BR-C18 (Saifen) reversed-phase column. During purification, the detection wavelength was set at 230 nm, the flow rate was 15 mL / min, and the gradient was 30-60% acetonitrile in 40 minutes. Product-related fractions were collected, and after HPLC analysis of purity, fractions exceeding 95% were combined and lyophilized to obtain the pure peptide.

[0418] Step 7: Detection and Characterization Methods

[0419] The purified peptide obtained in step 6 was subjected to analytical HPLC and LC / MS to determine its purity and target molecular weight.

[0420] Example 11 Synthesis of Compound 33

[0421] Step 1: Coupling of the first amino acid Fmoc-Val-OH

[0422] 168 mg (0.2 mmol) of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 hour. 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 allowed to react at room temperature for 2 hours. After the reaction, a blocking solution (10 mL) of DCM:methanol:DIEA (85:10:5, v:v:v) was added and blocked at room temperature for 10 minutes. The blocked resin was washed five times with DCM and five times with DMF.

[0423] Step 2: Synthesis of linear precursor peptide chains

[0424] The linear precursor peptide chain AEEA-IETDV of compound 26.

[0425] The resin obtained in step 1 was fully swollen in DMF for 1 hour. Then, the linear precursor sequence was synthesized from the second D at the carboxyl end to the amino end. Each coupling cycle was performed as follows:

[0426] • Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), each time for 8 min.

[0427] • Rinse the resin 6-8 times with DMF until the pH is neutral.

[0428] 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.

[0429] • Rinse the resin 4-6 times with DMF before coupling the next amino acid.

[0430] After the linear peptide synthesis, the resin was washed with DMF 5 times and DCM 5 times. The resin was dried in vacuo.

[0431] Step 3: Coupling of 3-(FMOC-amino)glutaric acid

[0432] Dissolve 0.25 mmol of 3-(FMOC-amino)pentanedioic acid, 1 mmol of 6-chlorobenzotriazole-1,1,3,3-tetramethyluronium hexafluorophosphate (HCTU), and 2 mmol of N,N-diisopropylethylamine (DIEA) in DMF. Add the mixture to the resin obtained in step 2 and allow to react at room temperature for 1 hour, twice. After the reaction, rinse the resin five times with DMF and five times with DCM.

[0433] Step 4: Synthesis of linear precursor peptide chain

[0434] The linear precursor peptide chain of compound 26 is RRLSYSRRRF.

[0435] The resin obtained in step 3 was fully swelled in DMF for 1 hour, and then the linear precursor sequence was synthesized from the carboxyl end to the amino end. Each coupling cycle was performed as follows:

[0436] • Fmoc-deprotection was performed twice with 20% piperidine / DMF (20% v / v, 10 mL), each time for 8 min.

[0437] • Rinse the resin 6-8 times with DMF until the pH is neutral.

[0438] 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.

[0439] • Rinse the resin 4-6 times with DMF before coupling the next amino acid.

[0440] After the linear peptide synthesis, the resin was washed with DMF 5 times and DCM 5 times. The resin was dried in vacuo.

[0441] Step 5: Cleavage of the linear precursor peptide chain

[0442] Add 10 mL of freshly prepared cleavage cocktail (trifluoroacetic acid: water: triisopropylsilane: thioanisole (90:2.5:2.5:5, v:v:v:v) to the resin obtained in step 4 and shake at room temperature for 2 hours. After the reaction, filter the reaction solution, wash the resin with trifluoroacetic acid, combine it with the reaction solution, and precipitate with 4 volumes of cold MTBE to obtain a crude product. Wash the crude product three times with MTBE and dry it in a vacuum.

[0443] Step 6: Peptide purification

[0444] The crude peptide was dissolved in 20% acetonitrile in water, filtered through a 0.45 μm membrane, and separated using a reversed-phase HPLC system with buffers A (0.1% trifluoroacetic acid in water) and B (0.1% trifluoroacetic acid in acetonitrile). The chromatographic column was a BR-C18 (Saifen) reversed-phase column. During purification, the detection wavelength was set at 230 nm, the flow rate was 15 mL / min, and the gradient was 30-60% acetonitrile in 40 minutes. Product-related fractions were collected, and after HPLC analysis of purity, fractions exceeding 95% were combined and lyophilized to obtain the pure peptide.

[0445] Step 7: Detection and Characterization Methods

[0446] The purified peptide obtained in step 6 was subjected to analytical HPLC and LC / MS to determine its purity and target molecular weight.

[0447] Other compounds of the present invention can be synthesized by referring to the synthesis methods of the above examples.

[0448] Test Example 1 ELISA Test Compounds Inhibit the Binding of Tat-NR2B9c-B to cMyc-PSD95 alpha 1-392

[0449] (1) Experimental materials:

[0450] (2) Experimental steps:

[0451] Compounds were tested for their inhibitory effects on Tat-NR2B9c-B binding to cMyc-PSD95alpha 1-392 using an ELISA (enzyme-linked immunosorbent assay). cMyc-PSD95alpha 1-392 was coated at 0.37 μg / mL, 25 μL / well, on a 384-well plate (greiner 781097). After blocking the cMyc-PSD95alpha 1-392-coated ELISA plate, compounds were serially diluted to 0-10 μM (from 10 μM down in a 3-fold series, totaling eight concentrations). Using a workstation, 12.5 μL / well of compound was transferred to the 384-well plate and centrifuged briefly to remove air bubbles. Then, using an 8-well pipette (10-100 μL), 12.5 μL / well of 12 nM Tat-NR2B9c-B was pipetted. After centrifugation to remove air bubbles, the plate was incubated at 37°C for 1 hour. Discard the liquid in the wells, add 80 μL of 1×TBST washing solution with a pH of 7.4 and wash the plate 3-5 times, each time for 3 to 5 minutes. Control the test plate to dryness and add 25 μL / well of 1:10000 diluted Streptavidin HRP. Centrifuge briefly to remove bubbles and incubate in a 37°C incubator for 1 hour. After washing and drying the test plate again, add 25 μl of TMB colorimetric solution to each well and continue to incubate in the incubator at 37°C for 30 minutes. Finally, add 25 μl of stop solution (1M HCL) to each well to terminate the reaction. Use the enzyme reader Cytation5 to read the absorbance at 450 nm and calculate the IC value of the compound inhibiting the binding of Tat-NR2B9c-B to cMyc-PSD95 alpha 1-392. 50 .

[0452] 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.

[0453] Table 1 Results of compounds inhibiting the binding of Tat-NR2B9c-B to cMyc-PSD95 alpha 1-392

[0454] Test Example 2 FRET Test Compound Inhibits Binding of cMyc-PSD95 alpha 1-392 to Tat-NR2B9C-B

[0455] (1) Experimental materials:

[0456] (2) Experimental steps:

[0457] Compounds were tested for their inhibitory activity against cMyc-PSD95 alpha 1-392 binding to Tat-NR2B9C-B using FRET (fluorescence resonance energy transfer). Compounds were serially diluted to 0-10 μM (8 concentrations, 3-fold serial dilutions from 10 μM downward). Using an 8-well pipette (1-10 μL), 4 μL / well of 4 nM cMyc-PSD95 alpha 1-392 was transferred to a 384-well plate (Thermo 264706) and briefly centrifuged to remove air bubbles. Using a workstation, 4 μL / well of compound was transferred to a 384-well plate and briefly centrifuged to remove air bubbles. Again, using an 8-well pipette, 4 μL / well of 20 nM Tat-NR2B9c-B was transferred to a 384-well plate and briefly centrifuged to remove air bubbles. Mix 0.02 μg / mL EU-steptavidin with 1 μg / mL Mouse Anti-C-MYC IgG SureLight APC in a 1:1 ratio. Use an 8-well pipette to transfer 8 μL / well of the EU-APC premix to a 384-well plate. Centrifuge briefly to remove bubbles. Incubate at room temperature in the dark for 2 hours. Read the fluorescence at 620 nm and 665 nm using a Cytation 5 microplate reader with 320 nM excitation.

[0458] The experimental results are shown in Table 2, where the control group 1 is Control group 2 The preparation method is described in patent CN103533949A.

[0459] Table 2 Results of compounds inhibiting the binding of cMyc-PSD95 alpha 1-392 to Tat-NR2B9C-B

[0460] The experimental results are shown in Table 2. The compounds of the present invention can inhibit the binding of cMyc-PSD95 alpha 1-392 to Tat-NR2B9C-B and have a good inhibitory effect.

[0461] Test Example 3 ELISA test compound inhibits the binding of biotin-nNOS1-299 to cMyc-PSD95 alpha 1-392

[0462] (1) Experimental materials:

[0463] (2) Experimental steps:

[0464] Compounds were tested for their inhibitory effects on the binding of biotin-nNOS1-299 to cMyc-PSD95 alpha 1-392 using an ELISA (enzyme-linked immunosorbent assay). cMyc-PSD95 alpha 1-392 was coated at 10 μg / mL, 25 μL / well, on a 384-well plate (greiner 781097). After blocking the cMyc-PSD95 alpha 1-392-coated ELISA plate, compounds were serially diluted to 0-10 μM (from 10 μM down in a 3-fold series, totaling 8 concentrations). Using a workstation, 12.5 μL / well of compound was transferred to the 384-well plate and briefly centrifuged to remove air bubbles. Then, using an 8-well pipette (10-100 μL), pipette 12.5 μL / well of 10 μg / mL 743 biotin-nNOS 1-299. Centrifuge briefly to remove air bubbles, and incubate at 37°C for 1 hour. Discard any remaining liquid and wash the plate 3-5 times with 80 μL of 1× TBST (pH 7.4) for 3-5 minutes each wash. Drain the plate and add 25 μL / well of a 1:10,000 dilution of Streptavidin HRP. Centrifuge briefly to remove air bubbles, and incubate at 37°C for 1 hour. Wash and drain the plate again, then add 25 μL of TMB colorimetric solution to each well and incubate at 37°C for another 30 minutes. Finally, add 25 μL of stop solution (1 M HCl) to each well to terminate the reaction. The absorbance at 450 nm was read using a Cytation5 microplate reader, and the IC value of the compound for inhibiting the binding of biotin-nNOS1-299 to cMyc-PSD95 alpha 1-392 was calculated. 50 .

[0465] Table 3 Results of compounds inhibiting the binding of biotin-nNOS 1-299 to cMyc-PSD95 alpha 1-392

[0466] The experimental results are shown in Table 3. The compounds of the present invention can inhibit the binding of biotin-nNOS1-299 to cMyc-PSD95 alpha1-392 and have a good inhibitory effect.

[0467] Test Example 4 FP Test Compound Inhibits Binding of 6H-PSD95 alpha 61-249 and 5FAM-N-bis-(PEG2-IETAV)2 / 5-FAM-NPEG4(IETAV)2

[0468] (1) Experimental materials:

[0469] (2) Experimental steps:

[0470] Compounds were tested for their inhibitory activity against the binding of 6H-PSD95 alpha 61-249 to 5FAM-N-bis-(PEG2-IETAV)2 / 5-FAM-NPEG4(IETAV)2 using a fluorescence polarization immunoassay (FP) assay. Compounds were serially diluted to 0-10 μM (8 concentrations, 3-fold serial dilutions from 10 μM downward). An 8-well pipette (1-10 μl) was used to pipette 5 μL / well of 5 nM 6H-PSD95 alpha 61-249 into a 384-well plate (Corning 3575) and briefly centrifuged to remove air bubbles. Using a workstation, 5 μL / well of compound was transferred to a 384-well plate and briefly centrifuged to remove air bubbles. Again, an 8-well pipette was used to pipette 5 μL / well of 0.5 nM 5FAM-N-bis-(PEG2-IETAV)2 / 5-FAM-NPEG4(IETAV)2 into the 384-well plate and briefly centrifuged to remove air bubbles. Incubate at room temperature in the dark for 2 h, and read the values ​​using the cytation5 FP module of the microplate reader.

[0471] Among them, the preparation method of 5-FAM-NPEG4(IETAV)2 is as described in patent CN103533949A.

[0472] Table 4 Inhibition of the binding of 6H-PSD95 alpha 61-249 and 5FAM-N-bis-(PEG2-IETAV)2 / 5-FAM-NPEG4(IETAV)2 by compounds

[0473] As shown in Table 4, the compounds of the present invention were able to inhibit the binding of 6H-PSD95 alpha 61-249 to 5FAM-N-bis-(PEG2-IETAV)2 / 5-FAM-NPEG4(IETAV)2 with a good inhibitory effect. The peptides of the present invention had a stronger affinity for 6H-PSD95 alpha 61-249 than for FAM-N-bis-(PEG2-IETAV)2 / 5-FAM-NPEG4(IETAV)2.

[0474] Test Example 5 Mouse Plasma Stability

[0475] 1. Mouse plasma (heparin sodium) stability

[0476] 1.1 Experimental Materials:

[0477] Drug: Compound of the present invention.

[0478] 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).

[0479] 1.2 Experimental methods:

[0480] Positive sample preparation: Take nainitide and dissolve it in DMSO to a final concentration of 100 times 1mM, and place it at -20℃ for later use. Negative sample preparation: Test sample preparation: Take the peptide to be tested and dissolve it in DMSO or other organic solvents to a final concentration of 100 times 1mM, and place it at -20℃ for later 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 MIX (mixture): Take 693μL of plasma and add it to a 1.5mL EP tube. Prepare 3 parallel samples for 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 into each aliquot according to the time gradient, and incubate. Operate on ice throughout the process. Incubation: Incubate in a 37°C water bath at six time points: 0 min, 15 min, 30 min, 60 min, 90 min, and 120 min. Termination: After incubation, add 4 volumes of 0.1% formic acid in 75% acetonitrile water as a precipitant. Mix: Vortex on a vortex mixer for 30 seconds. Centrifugation: Centrifuge at 4°C, 13,000 rpm for 10 min. Transfer the supernatant to a vial for analysis by LC-MS / MS.

[0481] 1.3 Experimental results:

[0482] A dot-line graph with the ordinate representing the remaining rate of the original drug (%) and the abscissa representing time is shown in FIG19 . It can be seen that as time changes, the degradation trend of the sample in in vitro plasma changes, and the results of the sample stability are obtained.

[0483] Calculate the half-life of the drug in plasma T 1 / 2 , calculated according to the first-order kinetic formula, the elimination rate constant (Ke) was obtained, and further according to the formula, the T1 / 2 (min) of the compound in plasma was obtained. 1 / 2 =-0.693 / Ke

[0484] By comparing the residual rate (%) of the original drug, the residual rate (%) of compound 9, compound 31 and compound 32 are 89.2%, 88.91% and 97.03% respectively, and the compounds of the present invention have good 2h plasma stability. 1 / 2 As shown in Table 5.

[0485] 2. Stability of Alteplase (Heparin Sodium) in Mouse Plasma

[0486] 2.1 Experimental Materials

[0487] Drug: Compound of the present invention.

[0488] 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).

[0489] 2.2 Experimental methods:

[0490] Positive sample preparation: Dissolve nainitide in DMSO to a final concentration of 100 times 1 mM and store at -20°C until ready for use. Test sample preparation: Dissolve the peptide to be tested in DMSO or other organic solvent to a final concentration of 100 times 1 mM and store at -20°C until ready for use. Plasma thawing: Remove (number of samples * 2.1) mL of plasma from the -80°C freezer and rapidly thaw in a 37°C water bath. Prepare a homogenate: Add (98 μL × (6 time points) + 1) 686 μL of plasma (heparin sodium) to a 1.5 mL EP tube. Prepare at least 3 replicates for each time point and prepare 1 tube of homogenate. Add 7 μL of alteplase to each tube to a final concentration of 9 μg / mL. Add 7 μL of the sample to be tested to each tube to a final concentration of 10 μM. Vortex on a vortex shaker for 30 seconds, aliquot 100 μL at a time gradient, and incubate. Operate on ice throughout. Incubation: Incubate in a 37°C water bath for six time points: 0 min, 5 min, 15 min, 30 min, 45 min, and 60 min. Termination: After incubation, add 4 volumes of 0.1% formic acid in 75% acetonitrile aqueous solution as a precipitant. Mix: Vortex on a vortex shaker for 30 seconds. Centrifugation: Centrifuge at 4°C, 13,000 rpm for 10 min. Remove the supernatant, transfer to a vial, and analyze by LC-MS / MS.

[0491] 2.3 Experimental results:

[0492] A dot-line graph with the ordinate representing the remaining rate of the original drug (%) and the abscissa representing time is shown in FIG20 . It can be seen that the degradation trend of the sample in in vitro plasma changes with time, and the results of the sample stability are obtained.

[0493] 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 calculate the T1 / 2 (min) of the compound in plasma according to the formula. 1 / 2=-0.693 / Ke

[0494] Experimental results: By comparing the residual rate (%) of the original drug, the residual rate (%) of compound 9, compound 31 and compound 32 were 91.8%, 75.3% and 100.7% respectively. The plasma stability of the compounds of the present invention was good for 1h. 1 / 2 As shown in Table 5.

[0495] Table 5 Compound T 1 / 2 result

[0496] The compound of the present invention still has good stability in human plasma (heparin sodium) and human plasma alteplase (heparin sodium).

[0497] In summary, the stability of the compounds of the present invention in plasma is greatly improved compared with Tat-NR2B9C and AVLX-144.

[0498] Test Example 6: Mouse Efficacy Test

[0499] 1. Experimental Materials

[0500] Drug: Compound of the present invention.

[0501] Related reagents and consumables: 1 mL insulin needle, isoflurane, TTC, low-adsorption EP tube, normal saline, 6 cm culture dish, microsurgical scissors, microsurgical forceps, absorbable suture, suture plug (Guangzhou Jialing), artery clamp, cotton thread, glucose, penicillin.

[0502] Experimental animals: 100 C57 mice, male, 7-8 weeks old, weighing 20-25 g, purchased from Hunan Anshengmei Pharmaceutical Research Institute Co., Ltd.

[0503] 2. Experimental methods:

[0504] 2.1 Experimental Preparation: The day before the experiment, the necks of mice were depilated with depilatory cream. Mice were randomly grouped using random grouping software.

[0505] 2.2 Anesthesia: Fix the mice under a stereomicroscope and use isoflurane inhalation anesthesia.

[0506] 2.3 After anesthesia is stable, disinfect the mouse's neck skin. Cut the mouse's anterior neck skin along the midline, and further separate and divide the chest muscles to expose the common carotid artery and vagus nerve. Then gradually expose the common carotid artery bifurcation, external carotid artery, and internal carotid artery. Isolate the common carotid artery, clamp it with an artery clamp at the proximal end, and tie a slipknot with cotton thread. Isolate the internal carotid artery and clamp it with an artery clamp. Isolate the rat's external carotid artery and ligate it. Make a small incision at the distal end between the two knots of the common carotid artery, with the incision pointing obliquely to the distal end. Use a professional suture to carefully insert it into the lumen of the common carotid artery along the edge of the blood vessel. The suture enters the common carotid artery and gently tighten the slipknot.

[0507] 2.4 Transfer the suture from the bifurcation into the internal carotid artery. Loosen the internal carotid artery clamp and continue advancing deeper into the brain until it reaches the origin of the middle cerebral artery, ultimately marking the suture at the common carotid bifurcation. Tighten the common carotid artery slipknot to prevent the suture from moving. Remove the proximal common carotid artery clamp. Trim any excess suture and thread, and suture the skin.

[0508] 2.5 30 minutes after embolization, the test substance and placebo were administered via the tail vein of the mice. The drug injections were randomly administered by a person not involved in the surgery and the person injecting the drug was unaware of the drug information.

[0509] 2.6 After surgery, the model mice were injected with penicillin and glucose and monitored until they regained consciousness. 24 hours after embolization, the neurological function of the animals was graded according to the Longa score. The criteria are as follows:

[0510] 0 points: no neurological symptoms;

[0511] 1 point: the forelimb on the contralateral side of surgery is flexed and close to the chest wall;

[0512] 2 points: The animal rotates or circles toward the opposite side when moving freely;

[0513] 3 points: the animal falls to the opposite side when moving freely;

[0514] 4 points: flaccid paralysis, no spontaneous movement of limbs.

[0515] 2.7 Observe the condition of mice, record the survival rate and score 24h and 48h after embolization.

[0516] 2.8 Mice in each group were killed 48 h after embolization, and the brains were removed intact, washed with normal saline, dried with filter paper, and weighed.

[0517] 2.9 Freeze the whole brain and make coronal sections at the optic chiasm and 2 mm in front and behind it. Stain with 2% TTC solution at 37°C in the dark for 30 minutes. Arrange the brain slices in order from small to large and from top to bottom, with the cortex facing up and the infarcted area on the right. After neatly arranging, photograph them.

[0518] 2.10 Use software to calculate the cerebral infarct area and total area.

[0519] Calculate the cerebral infarction rate: infarction percentage (%) = pale area / total area × 100%.

[0520] 2.11. Animals that have the following conditions will be excluded from the statistics.

[0521] Exclusion criteria: 1. Animals with no behavioral signs 24 hours after embolization. 2. Animals with subarachnoid hemorrhage during brain removal. 3. Animals that died during surgery or died without receiving medication after surgery.

[0522] 3. Experimental Results

[0523] 30 minutes after MCAO modeling, mice were randomly double-blindly administered with dapoxetine and saline. After administration, behavioral scores were performed on the experimental mice in each group. Mice with scores of 2-3 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 brains were removed and the sections were stained as shown in Figure 22. The staining results showed that the embolic area in each administration group was smaller than that in the saline control group. After staining, the cerebral embolic area of ​​each group was statistically analyzed, and the statistical results are shown in Figure 23. The statistical results of the cerebral embolic area showed that the embolic 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 decrease in embolic area.

[0524] 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 departing from the spirit of the present invention. These equivalent modifications or substitutions are all within the scope defined by the claims of this application.

Claims

1. A compound or a pharmaceutically acceptable salt thereof, wherein the structure of the compound is shown in the following general formula (I): L1 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 can be independently selected from a linker, -(CH2)nNH-, -C(=O)(CH2)nNH-, -(CH2)nC(=O)-, -(CH2)nC(=O)NH-; n and q can be independently selected from integers of 0 to 6; P1 is selected from cell penetrating peptides; P2 is selected from a cell penetrating peptide or P2 comprises a 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, where p is 0 or 1; X 0 Selected from S, T, C, Y, N or Q; X 1 Selected from E, Q, A, S; X 2 Selected from A, T, L, V, R, Q, D, N, W. 2 . The compound according to claim 1 , or a pharmaceutically acceptable salt thereof, wherein the cell-penetrating peptide (CPP) comprises at least 4 amino acid residues selected from arginine and / or lysine.

3. The compound according to claim 2 or a pharmaceutically acceptable salt thereof, wherein the cell-penetrating peptide (CPP) is 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, and PLIYLRLLRGQF.

4. 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.

5. The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein P2 and P3 each independently comprise the sequence IETDV or LETTF or METTF or YIETDV or YIETWV or NIETDV or NIETWV.

6. 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 can 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; P1 is selected from cell penetrating peptides; P2 is selected from a cell penetrating peptide or P2 comprises a 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, where p is 0 or 1; X 0 Selected from S, T, C, Y, N or Q; X 1 Selected from E, Q, A, S; X 2 Selected from A, T, L, V, R, Q, D, N, W.

7. 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.

8. The compound according to claim 7 or a pharmaceutically acceptable salt thereof, wherein the cell-penetrating peptide is 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, and PLIYLRLLRGQF.

9. 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.

10. The compound according to claim 6 or a pharmaceutically acceptable salt thereof, wherein P2 and P3 each independently comprise the sequence IETDV or LETTF or METTF or YIETDV or YIETWV or NIETDV or NIETWV.

11. A compound or a pharmaceutically acceptable salt thereof, wherein the compound has a structure as 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; P1 is selected from cell penetrating peptides; P2 is selected from a cell penetrating peptide or P2 comprises a 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, where p is 0 or 1; X 0 Selected from S, T, C, Y, N or Q; X 1 Selected from E, Q, A, S; X 2 Selected from A, T, L, V, R, Q, D, N, W.

12. 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): P1 is selected from cell penetrating peptides (CPP); P2 is selected from a cell penetrating peptide (CPP) or P2 comprises a 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, where p is 0 or 1; X 0 Selected from S, T, C, Y, N or Q; X 1 Selected from E, Q, A, S; X 2 Selected from A, T, L, V, R, Q, D, N, W.

13. The compound according to claim 10 or 11 or a pharmaceutically acceptable salt thereof, wherein P1 is selected from YGRKKRRQRRR, rrrqrrkkrGy.

14. The compound according to claim 10 or 11 or a pharmaceutically acceptable salt thereof, wherein P2 and P3 each independently comprise the sequence IETDV or LETTF or METTF or YIETDV or YIETWV or NIETDV or NIETWV.

15. A compound or a pharmaceutically acceptable salt thereof, wherein the compound has a structure as 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; P1 is selected from cell penetrating peptides; P2 is selected from a cell penetrating peptide or P2 comprises a 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, where p is 0 or 1; X 0 Selected from S, T, C, Y, N or Q; X 1 Selected from E, Q, A, S; X 2 Selected from A, T, L, V, R, Q, D, N, W.

16. 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): P1 is selected from cell penetrating peptides; P2 is selected from a cell penetrating peptide or P2 comprises a 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, where p is 0 or 1; X 0 Selected from S, T, C, Y, N or Q; X 1 Selected from E, Q, A, S; X 2 Selected from A, T, L, V, R, Q, D, N, W.

17. The compound according to claim 15 or 16 or a pharmaceutically acceptable salt thereof, wherein P1 is selected from YGRKKRRQRRR, rrrqrrkkrGy; and P2 is selected from YGRKKRRQRRR, rrrqrrkkrGy.

18. The compound according to claim 15 or 16 or a pharmaceutically acceptable salt thereof, wherein P2 and P3 each independently comprise the sequence IETDV or LETTF or METTF or YIETDV or YIETWV or NIETDV or NIETWV.

19. A compound or a pharmaceutically acceptable salt thereof, wherein the compound has a structure as 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; P1 is selected from cell penetrating peptides; P2 is selected from a cell penetrating peptide or P2 comprises a 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, where p is 0 or 1; X 0 Selected from S, T, C, Y, N or Q; X 1 Selected from E, Q, A, S; X 2 Selected from A, T, L, V, R, Q, D, N, W.

20. 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): P1 is selected from cell penetrating peptides; P2 is selected from a cell penetrating peptide or P2 comprises a 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, where p is 0 or 1; X 0 Selected from S, T, C, Y, N or Q; X 1 Selected from E, Q, A, S; X 2 Selected from A, T, L, V, R, Q, D, N, W.

21. The compound according to claim 19 or 20, or a pharmaceutically acceptable salt thereof, wherein P1 is selected from YGRKKRRQRRR, rrrqrrkkrGy.

22. The compound according to claim 19 or 20 or a pharmaceutically acceptable salt thereof, wherein P2 and P3 each independently comprise the sequence IETDV or LETTF or METTF or YIETDV or YIETWV or NIETDV or NIETWV.

23. A compound or a pharmaceutically acceptable salt thereof, wherein the compound structure is selected from the following structures:

24. 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.

25. 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.

26. 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.

27. The use according to claim 26, wherein the stroke is selected from ischemic stroke or hemorrhagic stroke.

28. 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.

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