Pharmaceutical compounds as neuroprotective agents and uses thereof

Abstract

The application belongs to the field of biological medicine, and particularly relates to a medicinal compound used as a neuroprotective agent and application thereof. The compound has a structure shown in formula (I), is a PSD-95 inhibitor, and can be used for preparing a medicine for treating an excitotoxicity-related disease and neuropathic pain.

Description

Technical Field

[0001] This invention belongs to the field of biomedicine, specifically relating to a pharmaceutical compound as a neuroprotective agent and its application. Background Technology

[0002] N-methyl-D-aspartate receptors (NMDARs) play a crucial role in the central nervous system, participating in the formation of learning and memory, synapse formation during central nervous system development, plasticity development, and glutamate-mediated neurotoxicity. NMDA receptors are major mediators of excitotoxicity (i.e., glutamate-mediated neurotoxicity), and are associated with neurodegenerative diseases and acute brain injury. They can serve as therapeutic targets for certain neurological disorders, such as cerebral infarction, neuropathic pain, epilepsy, and schizophrenia.

[0003] Postsynaptic density (PSD) is a supersignaling molecule complex on the excitatory postsynaptic membrane and is an important substance for synaptic transmission. Based on the molecular weight of PSD, it can be divided into four categories: PSD-95, PSD-93, synaptic-associated protein 97 (SAP-97), and SAP102.

[0004] PSD-95 is the most abundant and important scaffold 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 on the postsynaptic membrane, playing a crucial role in synaptic plasticity. It is the most important synaptic protein mediating and integrating synaptic information and is also a member of the guanylate-associated kinase family. PSD-95 comprises three PDZ domains, one SH3 domain, and one guanylate kinase-like (GK) domain, connected by a connective tissue. PSD-95 is almost entirely located in the postsynaptic density region of neurons and participates in anchoring synaptic proteins. Its direct and indirect binding partners include neuroconnectins, nNOS, NMDA receptors, AMPA receptors, and potassium channels.

[0005] Based on the different splicing of genes by mRNA, more than 10 nNOS (Neuronal nitric oxide synthase) splice variants are currently known, with a molecular weight of 160.8 KD. nNOS contains two structural domains: a C-terminal reduction domain and an N-terminal oxidation domain. The N-terminus has two non-overlapping binding regions: (1) the PDZ region: composed of 1 to 99 amino acids, which participates in the formation of active nNOS dimers; (2) the β-finger structure: composed of 100 to 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 cytosol proteins through the PDZ-PDZ domain or the C-terminal PDZ reaction. PSD-95 can link nNOS to the NMDA receptor, thereby effectively activating nNOS through the activation of the NMDA receptor.

[0006] Stroke is characterized by neuronal death in areas of localized ischemia, hemorrhage, and / or trauma. Neuronal death or damage caused by cerebral ischemia is a cascade of damage. Following ischemia, decreased tissue blood perfusion leads to increased excitatory neurotransmitters, activating NMDA and AMPA receptors, causing ion channel opening, calcium ion influx, and activation of numerous enzymes, triggering a signaling cascade that results in multi-pathway neuronal damage. While NMDA receptor antagonists effectively reduce excitotoxicity by blocking glutamate-mediated ion flux, they also inhibit some physiologically important processes. PSD-95, a downstream receptor, interacts with various proteins, inducing a series of ischemic injuries and is a key site of cerebral ischemia-related damage, as well as a potential target for drug therapy. Therefore, the development of PSD-95 inhibitors is of great pharmacological significance for neurological injuries caused by various excitotoxic neurotoxicities, including stroke. Consequently, NMDA receptor antagonists have failed in clinical trials for conditions such as stroke due to low 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 a PSD-95 inhibitor.

[0007] Furthermore, studies have 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, research indicates that excessive excitation of the central glutamatergic system can trigger anxiety, and the NMDA receptor (NMDAR) is responsible for the main part of glutamate excitatory neurotoxicity. Epilepsy involves three distinct but continuous pathophysiological processes: initiation, maintenance and spread of paroxysmal discharges, and inhibition of paroxysmal discharges. Excitatory neurotransmitters such as glutamate and aspartate play important roles in these processes. In Alzheimer's disease, PSD-95 participates in the neurotoxic mechanism through the GluR6-PSD-95-MLK3 pathway. Moreover, in Huntington's disease, PSD-95 is a mediator of NMDA receptor and huntingtin mutant neurotoxicity. Therefore, the development of PSD-95 inhibitors is also of great significance for the treatment, improvement, and prevention of 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 dimeropeptide ligand linked by a polyethylene glycol linker (PEG) that simultaneously binds to the PDZ1 and PDZ2 domains of PSD-95, and its use in the treatment of ischemic cerebrovascular diseases. There is still a need for PSD-95 inhibitors with higher affinity for the PDZ1 and PDZ2 domains, which would have improved in vivo efficacy in the treatment of ischemic stroke and traumatic brain injury. CN103533949A discloses a novel and potent inhibitor of a ternary protein complex of nNOS, PSD-95, and NMDA receptors, and a pharmaceutical composition containing this inhibitor for the prevention and / or treatment of excitotoxic-related diseases and chronic pain symptoms in subjects, wherein AVLX-144 is a dimeropeptide candidate for the treatment of acute ischemic stroke (AIS) and acute subarachnoid hemorrhage (SAH). CN105828832A discloses a fatty acid derivative of a dimer inhibitor of PSD-95. WO2022238530A1 discloses compounds capable of binding to the PDZ domain of PSD-95 and their medical use as inhibitors of PSD-95-mediated protein-protein interactions. To meet clinical needs, further development of more PSD-95 inhibitors is still required. Summary of the Invention

[0010] The purpose of this invention is to overcome the shortcomings of the prior art and provide a pharmaceutical compound as a neuroprotective agent and its application.

[0011] To achieve the above objectives, in one respect, the present invention provides a compound or a pharmaceutically acceptable salt thereof, the structure of which is shown in the following general formula (I).

[0012]

[0013] L1 is selected from

[0014] L2 may or may not exist; if it exists, L2 is selected from hydrophilic structural units.

[0015] L3 is selected from hydrophilic structural units;

[0016] L4 and L5 can be independently selected from the following: linker bond, -(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 from 0 to 6; preferably, n is 1, 2 or 4; q is 0, 1 or 2.

[0019] P1 is selected from cell-penetrating peptide (CPP);

[0020] P2 can be selected from cell-penetrating peptide (CPP) or P2 containing 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 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,

[0021] p is 0 or 1;

[0022] X0 Selected from S, T, C, Y, N, or Q;

[0023] X 1 Optional from E, Q, A, S;

[0024] X 2 It can be selected from A, T, L, V, R, Q, D, N, W.

[0025] Preferably, the CPP contains at least four amino acid residues selected from arginine and / or lysine.

[0026] 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

[0027] -MGLWSQPKKKRRV, RQIKIWFQNRRMKWKK, rqikiwfanrrmkwkk, RKKRRRESRKKRRRES, LLIILRRRIRKQAHAHSK, PLIYLRLLRGQF.

[0028] Preferably, the hydrophilic structural unit can be selected from (PEG)m,

[0029] m is independently selected from integers from 0 to 10.

[0030] Preferably, P2 and P3 each independently contain the sequence IEDTDV, LETTF, METTF, YIETDV, YIETWV, NIETDV, or NIETDV.

[0031] Secondly, the present invention provides a compound or a pharmaceutically acceptable salt thereof, the structure of which is shown in general formula (II) below.

[0032]

[0033] L2 may or may not exist; if it exists, L2 is selected from hydrophilic structural units.

[0034] L3 is selected from hydrophilic structural units;

[0035] L4 and L5 can be independently selected from the following: linking bond, -(CH2)nNH-, -C(=O)(CH2)nNH-, -(CH2)n C(=O)-, -(CH2)n C(=O).

[0036] NH-;

[0037] n and q are independently selected from integers from 0 to 6; preferably, n is 1, 2 or 4; and q is 0, 1 or 2.

[0038] P1 is selected from cell-penetrating peptide (CPP);

[0039] P2 can be selected from cell-penetrating peptide (CPP) or P2 containing 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 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,

[0040] p is 0 or 1;

[0041] X 0 Selected from S, T, C, Y, N, or Q;

[0042] X 1 Optional from E, Q, A, S;

[0043] X 2 It can be selected from A, T, L, V, R, Q, D, N, W.

[0044] Preferably, the CPP contains at least four amino acid residues selected from arginine and / or lysine.

[0045] 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

[0046] -MGLWSQPKKKRRV, RQIKIWFQNRRMKWKK, rqikiwfanrrmkwkk, RKKRRRESRKKRRRES, LLIILRRRIRKQAHAHSK, PLIYLRLLRGQF.

[0047] Preferably, the hydrophilic structural unit can be selected from (PEG)m,

[0048] m can be independently selected from integers from 0 to 10.

[0049] Preferably, P2 and P3 each independently contain the sequence IEDTDV, LETTF, METTF, YIETDV, YIETWV, NIETDV, or NIETDV.

[0050] Thirdly, the present invention provides a compound or a pharmaceutically acceptable salt thereof, the structure of which is shown in general formula (III) below.

[0051]

[0052] L2 may or may not exist; if it exists, L2 is selected from hydrophilic structural units.

[0053] L3 is selected from hydrophilic structural units;

[0054] P1 is selected from cell-penetrating peptide (CPP);

[0055] P2 can be selected from cell-penetrating peptide (CPP) or P2 containing sequence (X). 0 ) p X 1 TX 2 V or (X) 0 ) p X 1 TX 2 F or (X) 0 ) pX 1 SX 2 V; P3 is selected from 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,

[0056] p is 0 or 1;

[0057] X 0 Selected from S, T, C, Y, N, or Q;

[0058] X 1 Optional from E, Q, A, S;

[0059] X 2 The components 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).

[0060]

[0061] P1 is selected from cell-penetrating peptide (CPP);

[0062] P2 can be selected from cell-penetrating peptide (CPP) or P2 containing 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 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,

[0063] p is 0 or 1;

[0064] X 0 Selected from S, T, C, Y, N, or Q;

[0065] X 1 Optional from E, Q, A, S;

[0066] X 2 It can be selected from A, T, L, V, R, Q, D, N, W.

[0067] Preferably, P1 can be selected from YGRKKRRQRRR or rrrqrrkkrGy.

[0068] Preferably, P2 and P3 each independently contain the sequence IEDTDV, LETTF, METTF, YIETDV, YIETWV, NIETDV, or NIETDV.

[0069] Fourthly, the present invention provides a compound or a pharmaceutically acceptable salt thereof, the structure of which is shown in the following general formula (V).

[0070]

[0071] L2 may or may not exist; if it exists, L2 is selected from hydrophilic structural units.

[0072] L3 is selected from hydrophilic structural units;

[0073] P1 is selected from cell-penetrating peptide (CPP);

[0074] P2 can be selected from cell-penetrating peptide (CPP) or P2 containing 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;

[0075] P3 is selected from 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,

[0076] p is 0 or 1;

[0077] X 0 Selected from S, T, C, Y, N, or Q;

[0078] X 1 Optional from E, Q, A, S;

[0079] X 2 It can be selected from A, T, L, V, R, Q, D, N, W.

[0080] Preferably, the structure of the compound is shown in the following general formula (VI).

[0081]

[0082] P1 is selected from cell-penetrating peptide (CPP);

[0083] P2 can be selected from cell-penetrating peptide (CPP) or P2 containing 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 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,

[0084] p is 0 or 1;

[0085] X 0 Selected from S, T, C, Y, N, or Q;

[0086] X 1 Optional from E, Q, A, S;

[0087] X 2 It can be selected from A, T, L, V, R, Q, D, N, W.

[0088] More preferably, P1 can be selected from YGRKKRRQRRR, rrrqrrkkrGy; P2 can be selected from YGRKKRRQRRR, rrrqrrkkrGy.

[0089] More preferably, P2 and P3 each independently contain the sequence IEDTDV, LETTF, METTF, YIETDV, YIETWV, NIETDV, or NIETDV.

[0090] Fifthly, the present invention provides a compound or a pharmaceutically acceptable salt thereof, the structure of which is shown in general formula (VII).

[0091]

[0092] L2 may or may not exist; if it exists, L2 is selected from hydrophilic structural units.

[0093] L3 is selected from hydrophilic structural units;

[0094] P1 is selected from cell-penetrating peptide (CPP);

[0095] P2 can be selected from cell-penetrating peptide (CPP) or P2 containing 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;

[0096] P3 is selected from 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,

[0097] p is 0 or 1;

[0098] X 0 Selected from S, T, C, Y, N, or Q;

[0099] X 1 Optional from E, Q, A, S;

[0100] X 2 It can be selected from A, T, L, V, R, Q, D, N, W.

[0101] Preferably, the structure of the compound is shown in the following general formula (VIII).

[0102]

[0103] P1 is selected from cell-penetrating peptide (CPP);

[0104] P2 can be selected from cell-penetrating peptide (CPP) or P2 containing 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;

[0105] P3 is selected from 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,

[0106] p is 0 or 1;

[0107] X 0 Selected from S, T, C, Y, N, or Q;

[0108] X 1 Optional from E, Q, A, S;

[0109] X 2 It can be selected from A, T, L, V, R, Q, D, N, W.

[0110] Preferably, P1 can be selected from YGRKKRRQRRR or rrrqrrkkrGy.

[0111] Preferably, P2 and P3 each independently contain the sequence IEDTDV, LETTF, METTF, YIETDV, YIETWV, NIETDV, or NIETDV.

[0112] As a preferred embodiment of the compound described in this invention, the compound structure may be selected from the following structures:

[0113]

[0114]

[0115]

[0116]

[0117]

[0118]

[0119]

[0120] Furthermore, the pharmaceutically acceptable salt may be selected from trifluoroacetate, acetate, hydrochloride and phosphate.

[0121] In a sixth aspect, the present invention provides a pharmaceutical composition comprising any of the above-described compounds or pharmaceutically acceptable salts thereof, and pharmaceutically acceptable carriers, excipients, and / or diluents.

[0122] Depending on the therapeutic and administration requirements, the pharmaceutical composition may also include a pharmaceutically acceptable carrier. The carrier may be selected from diluents, excipients, fillers, binders, humectants, disintegrants, absorption enhancers, surfactants, adsorbents, lubricants, etc., and flavoring agents, tasters, etc., may be added if necessary.

[0123] The pharmaceutical compositions of the present invention can be formulated into various forms, including tablets, powders, granules, capsules, oral solutions, and injections. All of the above dosage forms can be prepared according to conventional methods in the pharmaceutical field. The pharmaceutical compositions can be administered parenterally, intravenously, orally, subcutaneously, intraarterially, intracranially, intrathecally, intraperitoneally, locally, intranasally, or intramuscularly. Intravenous administration is preferred.

[0124] In a seventh aspect, the present invention provides the use of the above-described compounds or pharmaceutically acceptable salts or pharmaceutical compositions thereof in the preparation of medicaments for treating, improving or preventing diseases caused by PSD-95 dysfunction in an individual.

[0125] The drug may be in the form of tablets, powders, granules, capsules, oral liquids, or injections.

[0126] Furthermore, the diseases caused by the above-mentioned PSD-95 dysfunction can be selected from stroke, neurodegenerative diseases, anxiety or epilepsy, and neuropathic pain.

[0127] Furthermore, the aforementioned stroke is selected from ischemic stroke or hemorrhagic stroke.

[0128] Furthermore, the aforementioned neurodegenerative diseases are selected from Alzheimer's disease, amyotrophic lateral sclerosis, Parkinson's disease, or Huntington's disease.

[0129] The meaning of the terms "peptide" or "polypeptide" is well known to those skilled in the art. Generally, a peptide or polypeptide is two or more amino acids linked by an amide bond, which is formed by the amino group of one amino acid and the carboxyl group of an adjacent amino acid. Polypeptides described herein may contain naturally occurring or non-naturally occurring amino acids. They can be modified into analogs, derivatives, functional mimics, pseudopeptides, and other compounds containing at least two amino acids. Unless a specific modification is specified at the N-terminus or C-terminus, a polypeptide containing a particular amino acid sequence includes both unmodified and modified amino and / or carboxyl terminals, as is well known to those skilled in the art. A polypeptide with a particular amino acid sequence may include modified amino acids and / or additional amino acids unless the N- and / or C-terminus contain modifications that prevent the further addition of amino acids. Such modifications include, for example, acetylation of the N-terminus and / or amidation of the C-terminus.

[0130] The peptides of this invention can be modified to form peptide derivatives. As is well known to those skilled in the art, various modifications can be made to the peptides. 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 combinations of the above modifications. This invention includes any well-known modification of peptides. For example, peptide derivatives may include chemical modifications to the peptide, such as alkylation, acylation, carbamylation, iodination, or any other modification that produces peptide derivatives. The modification of the peptide may include modified amino acids, such as hydroxyproline or carboxyglutamic acid, and may include amino acids linked by non-peptide bonds.

[0131] In addition, the peptides described above can be optionally derivatized (e.g., acetylated, phosphorylated, and / or glycosylated) to enhance affinity for inhibitors, enhance the ability of inhibitors to be transported across cell membranes, or enhance stability.

[0132] 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), desmokinin (Des), 2,2'- Diaminopimelic acid (Dpm), 2,3-diaminopropionic acid (Dpr), N-ethylglycine (EtGly), N-ethylasparagine (EtAsn), hydroxylysine (Hyl), isohydroxylysine (aHyl), 3-hydroxyproline (3Hyp), 4-hydroxyproline (4Hyp), isodesin (Ide), isoleucine (aIle), N-methylglycine (MeGly), N-methylisoleucine (MeIle), 6-N-methyllysine (MeLys), N-methylvaline (MeVal), n-valine (Nva), n-leucine (Nle), and ornithine (Orn). Of course, all modified α-amino acids can be replaced by the corresponding β-, γ-, or ω-aminocarboxylic acids.

[0133] The term "amino acid" refers to a molecule containing both an amino and a carboxyl group. Suitable amino acids include, but are not limited to, D- and L-isomers of naturally occurring amino acids, as well as non-natural amino acids prepared through 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.

[0134] The term "naturally occurring amino acid" refers to any one of the 20 L-amino acids commonly found in peptides synthesized in nature, namely, 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).

[0135] The polypeptides of the present invention can be prepared by conventional biosynthesis or chemical synthesis methods.

[0136] 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 with the brain synaptic protein PSD-95, the Drosophila septal connexin Discs-Large (DLG), and the epithelial tight junction protein Z01 (Z01). PDZ domains are also referred to as Discs-Large homologous repeats ("DHRs") and GLGF repeats. PDZ domains typically exhibit a preserved core concordance sequence (Doyle, DA, 1996, Cell 85:1067-76). Exemplary proteins containing PDZ domains and PDZ domain sequences are disclosed in U.S. Patent Application No. 10 / 714,537. Attached Figure Description

[0137] Figure 1 This is the HPLC detection chromatogram of compound 23 in Example 1;

[0138] Figure 2 This is the mass spectrometry result of compound 23 from Example 1;

[0139] Figure 3 This is the HPLC detection chromatogram of compound 32 in Example 2;

[0140] Figure 4 This is the mass spectrometry result of compound 32 in Example 2;

[0141] Figure 5 This is the HPLC detection chromatogram of compound 25 in Example 3;

[0142] Figure 6 This is the mass spectrometry result of compound 25 in Example 3;

[0143] Figure 7 This is the HPLC detection chromatogram of compound 26 in Example 4;

[0144] Figure 8 This is the mass spectrometry result of compound 26 in Example 4;

[0145] Figure 9 This is the HPLC detection chromatogram of compound 27 in Example 5;

[0146] Figure 10 This is the mass spectrometry result of compound 27 in Example 5;

[0147] Figure 11 This is the HPLC detection chromatogram of compound 21 in Example 6;

[0148] Figure 12 This is the mass spectrometry result of compound 21 in Example 6;

[0149] Figure 13 This is the HPLC detection chromatogram of compound 22 in Example 7;

[0150] Figure 14 This is the mass spectrometry result of compound 22 in Example 7;

[0151] Figure 15 This is the HPLC detection chromatogram of compound 31 in Example 8;

[0152] Figure 16 This is the mass spectrometry result of compound 31 in Example 8;

[0153] Figure 17 This is the HPLC detection chromatogram of compound 28 in Example 9;

[0154] Figure 18 This is the mass spectrometry result of compound 28 in Example 9;

[0155] Figure 19 Figure A shows the results of the mouse plasma (heparin sodium) stability test in Example 5. Figure B shows the results of compound 9 and compounds 31 and 32.

[0156] Figure 20 Figure 5 shows the results of the mouse plasma alteplase (heparin sodium) stability experiment. Figure A shows the experimental results of compound 9, and Figure B shows the experimental results of compounds 31 and 32.

[0157] Figure 21 Procedure for efficacy testing of the mouse tMCAO model (Example 6);

[0158] Figure 22 Stained images of brain slices from test case 6 mouse;

[0159] Figure 23 The results show the statistical results of the embolism area in the brain slices of test case 6 mice. Specific Implementation

[0160] The polypeptide compounds and their derivatives provided by this invention employ a solid-phase synthesis method to synthesize their linear precursors. After cleavage, the crude peptides are directly purified to obtain the target compounds, or the crude peptides are oxidized with DMSO to form intramolecular disulfide bonds and then purified to obtain the target compounds. 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). Then, the solid support is repeatedly condensed with the activated amino acid derivatives → washing → deprotection of Fmoc → washing → the next round of amino acid condensation to achieve the desired polypeptide chain length. Finally, the polypeptide is cleaved from the solid support by reacting the resin with a mixed solution of trifluoroacetic acid:water:triisopropylsilane:aniline sulfide (90:2.5:2.5:5, v:v:v:v). After precipitation with frozen methyl tert-butyl ether, a solid crude linear precursor is obtained. The cleaved crude linear precursor is then subjected to disulfide bond oxidation in a neutral solution to obtain the crude target polypeptide. The crude solid product or the crude oxidized peptide product was purified and separated by a C18 reversed-phase preparative chromatography column in a system of 0.1% trifluoroacetic acid in acetonitrile / water to obtain pure peptides and their derivatives.

[0161] Experimental reagents

[0162]

[0163]

[0164] The structural formula of Fmoc-AEEA-OH is as follows: Example 1 Preparation of Compound 23

[0165]

[0166] Step 1: Couple the first amino acid Fmoc-Val-OH

[0167] 168 mg (0.2 mmol) of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 h. Fmoc-Val-OH (0.16 mmol) and diisopropylethylamine (DIEA, 0.64 mmol) were dissolved in 8 mL of DCM and added to the resin. The reaction was carried out at room temperature for 2 h. After the reaction was complete, blocking buffer (10 mL) of DCM:methanol:DIEA (85:10:5, v:v:v) was added, and the mixture was blocked at room temperature for 10 min. The blocked resin was then washed 5 times with DCM and 5 times with DMF.

[0168] Step 2: Synthesis of linear precursor peptide chains

[0169] The linear precursor peptide chain of compound 23 is AEEA-NIETWV.

[0170] The resin obtained in step 1 was fully swollen in DMF for 1 hour, and then synthesized in the order of the above linear precursor sequence from the second D at the carboxyl terminus to the amino terminus. Each coupling cycle was performed as follows:

[0171] • Perform Fmoc-deprotection twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time.

[0172] Rinse the resin with DMF 6-8 times until neutral pH is reached.

[0173] • Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to resin and react at room temperature for 1 h.

[0174] Rinse the resin with DMF 4-6 times before coupling the next amino acid.

[0175] After synthesis of the linear peptides, the resin was washed five times with DMF and five times with DCM. The resin was then dried under vacuum.

[0176] Step 3: Couple 3-(FMOC-amino)glutaric acid

[0177] Dissolve 0.25 mmol of 3-(FMOC-amino)glutaric acid, 1 mmol of 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU), and 2 mmol of N,N-diisopropylethylamine (DIEA) in DMF, add the solution to the resin obtained in step 2, and react at room temperature for 1 h. Repeat the reaction twice. After the reaction, rinse the resin 5 times with DMF and 5 times with DCM.

[0178] Step 4: Synthesis of linear precursor peptide chains

[0179] The linear precursor peptide chain of compound 23 is YGRKKRRQRRR.

[0180] The resin obtained in step 3 was fully swollen in DMF for 1 hour, and then synthesized in the order from the carboxyl terminus to the amino terminus according to the above linear precursor sequence. Each coupling cycle was performed as follows:

[0181] • Perform Fmoc-deprotection twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time.

[0182] Rinse the resin with DMF 6-8 times until neutral pH is reached.

[0183] • Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to resin and react at room temperature for 1 h.

[0184] Rinse the resin with DMF 4-6 times before coupling the next amino acid.

[0185] After synthesis of the linear peptides, the resin was washed five times with DMF and five times with DCM. The resin was then dried under vacuum.

[0186] Step 5: Cleavage of the linear precursor peptide chain

[0187] Add 10 mL of freshly prepared cut cocktail (trifluoroacetic acid:water:triisopropylsilane:aniline sulfide) (90:2.5:2.5:5, v:v:v:v) to the resin obtained in step 4, and react with shaking at room temperature for 2 hours. After the reaction is complete, filter the reaction solution, wash the resin with trifluoroacetic acid, combine the washed resin with the reaction solution, and precipitate with 4 times the volume of cold MTBE to obtain the crude product. Wash the crude product 3 times with MTBE and dry it under vacuum.

[0188] Step 6: Preparation of Peptides

[0189] The crude peptide was dissolved in a 20% acetonitrile aqueous solution, filtered through a 0.45 μm membrane, and then separated using a reversed-phase high-performance liquid chromatography (RP-HPLC) system. The buffer solutions were A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). A BR-C18 (Saifen) reversed-phase column was used. During purification, the detection wavelength was set to 230 nm, the flow rate to 15 mL / min, and the gradient to 30-60% acetonitrile for 40 min. The relevant fractions were collected, and after HPLC purity assessment, fractions >95% were combined, lyophilized, and the purified peptide was obtained.

[0190] Step 7: Detection and Characterization Methods

[0191] The purity and target molecular weight of the peptide obtained in step 6 were determined by analytical high-performance liquid chromatography (HPLC) and liquid chromatography / mass spectrometry (LC / MS). The results are shown below. Figure 1 , Figure 2 .

[0192] Example 2 Preparation of compound 32

[0193]

[0194] Step 1: Couple the first amino acid Fmoc-Val-OH

[0195] 168 mg (0.2 mmol) of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 h. Fmoc-Val-OH (0.16 mmol) and diisopropylethylamine (DIEA, 0.64 mmol) were dissolved in 8 mL of DCM and added to the resin. The reaction was carried out at room temperature for 2 h. After the reaction was complete, blocking buffer (10 mL) of DCM:methanol:DIEA (85:10:5, v:v:v) was added, and the mixture was blocked at room temperature for 10 min. The blocked resin was then washed 5 times with DCM and 5 times with DMF.

[0196] Step 2: Synthesis of linear precursor peptide chains

[0197] The linear precursor peptide chain of compound 32 is AEEA-NIETWV.

[0198] The resin obtained in step 1 was fully swollen in DMF for 1 hour, and then synthesized in the order of the above linear precursor sequence from the second D at the carboxyl terminus to the amino terminus. Each coupling cycle was performed as follows:

[0199] • Perform Fmoc-deprotection twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time.

[0200] Rinse the resin with DMF 6-8 times until neutral pH is reached.

[0201] • Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to resin and react at room temperature for 1 h.

[0202] Rinse the resin with DMF 4-6 times before coupling the next amino acid.

[0203] After synthesis of the linear peptides, the resin was washed five times with DMF and five times with DCM. The resin was then dried under vacuum.

[0204] Step 3: Couple 3-(FMOC-amino)glutaric acid

[0205] Dissolve 0.25 mmol of 3-(FMOC-amino)glutaric acid, 1 mmol of 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU), and 2 mmol of N,N-diisopropylethylamine (DIEA) in DMF, add the solution to the resin obtained in step 2, and react at room temperature for 1 h. Repeat the reaction twice. After the reaction, rinse the resin 5 times with DMF and 5 times with DCM.

[0206] Step 4: Synthesis of linear precursor peptide chains

[0207] The linear precursor peptide chain of compound 32 is dR-dR-dR-dQ-dR-dR-dK-dK-dR-G-dY.

[0208] The resin obtained in step 3 was fully swollen in DMF for 1 hour, and then synthesized in the order from the carboxyl terminus to the amino terminus according to the above linear precursor sequence. Each coupling cycle was performed as follows:

[0209] • Perform Fmoc-deprotection twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time.

[0210] Rinse the resin with DMF 6-8 times until neutral pH is reached.

[0211] • Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to resin and react at room temperature for 1 h.

[0212] Rinse the resin with DMF 4-6 times before coupling the next amino acid.

[0213] After synthesis of the linear peptides, the resin was washed five times with DMF and five times with DCM. The resin was then dried under vacuum.

[0214] Step 5: Cleavage of the linear precursor peptide chain

[0215] Add 10 mL of freshly prepared cut cocktail (trifluoroacetic acid:water:triisopropylsilane:aniline sulfide) (90:2.5:2.5:5, v:v:v:v) to the resin obtained in step 4, and react with shaking at room temperature for 2 hours. After the reaction is complete, filter the reaction solution, wash the resin with trifluoroacetic acid, combine the washed resin with the reaction solution, and precipitate with 4 times the volume of cold MTBE to obtain the crude product. Wash the crude product 3 times with MTBE and dry it under vacuum.

[0216] Step 6: Preparation of Peptides

[0217] The crude peptide was dissolved in a 20% acetonitrile aqueous solution, filtered through a 0.45 μm membrane, and then separated using a reversed-phase high-performance liquid chromatography (RP-HPLC) system. The buffer solutions were A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). A BR-C18 (Saifen) reversed-phase column was used. During purification, the detection wavelength was set to 230 nm, the flow rate to 15 mL / min, and the gradient to 30-60% acetonitrile for 40 min. The relevant fractions were collected, and after HPLC purity assessment, fractions >95% were combined, lyophilized, and the purified peptide was obtained.

[0218] Step 7: Detection and Characterization Methods

[0219] The purity and target molecular weight of the peptide obtained in step 6 were determined by analytical high-performance liquid chromatography (HPLC) and liquid chromatography / mass spectrometry (LC / MS). The results are shown below. Figure 3 , Figure 4 .

[0220] Example 3 Preparation of Compound 25

[0221]

[0222] Step 1: Couple the first amino acid Fmoc-Cys(Trt)-OH

[0223] 84 mg (0.1 mmol) of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 h. Fmoc-Cys(Trt)-OH (0.08 mmol) and diisopropylethylamine (DIEA, 0.32 mmol) were weighed, dissolved in 5 mL of DCM, and added to the resin. The reaction was carried out at room temperature for 2 h. After the reaction was complete, blocking buffer (10 mL) of DCM:methanol:DIEA (85:10:5, v:v:v) was added, and the mixture was blocked at room temperature for 10 min. The blocked resin was washed 5 times with DCM and 5 times with DMF.

[0224] Step 2: Synthesis of linear precursor peptide chains

[0225] The linear precursor peptide chain of compound 25 is CKMVTTFGIDVTTTYIC.

[0226] The resin obtained in step 1 was fully swollen in DMF for 1 hour, and then synthesized in the order of the above linear precursor sequence from the second I position at the carboxyl terminus to the amino terminus. Each coupling cycle was performed as follows:

[0227] • Perform Fmoc-deprotection twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time.

[0228] Rinse the resin with DMF 6-8 times until neutral pH is reached.

[0229] • Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to resin and react at room temperature for 1 h.

[0230] Rinse the resin with DMF 4-6 times before coupling the next amino acid.

[0231] After synthesis of the linear peptides, the resin was washed five times with DMF and five times with DCM. The resin was then dried under vacuum.

[0232] Step 3: Couple amino acid Fmoc-Lys(Fmoc)-OH

[0233] Dissolve 0.5 mmol of Fmoc-Lys(Fmoc)-OH, 0.5 mmol of 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU), and 1 mmol of 4-methylmorpholine (NMM) in DMF, add the solution to the resin obtained in step 1, and react at room temperature for 1 h. After the reaction, rinse the resin 5 times with DMF and 5 times with DCM.

[0234] Step 4: Synthesis of linear precursor peptide chains

[0235] The linear precursor peptide chain of compound 25 is dR-dR-dR-dQ-dR-dR-dK-dK-dR-G-dY.

[0236] The resin obtained in step 3 was fully swollen in DMF for 1 hour, and then synthesized in the order from the carboxyl terminus to the amino terminus according to the above linear precursor sequence. Each coupling cycle was performed as follows:

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

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

[0239] • Dissolve 1 mmol Fmoc-AA, 1 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU) and 2 mmol 4-methylmorpholine (NMM) in DMF, add to resin and react at room temperature for 1 h.

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

[0241] After synthesis of the linear peptides, the resin was washed five times with DMF and five times with DCM. The resin was then dried under vacuum.

[0242] Step 5: Cleavage of the linear precursor peptide chain

[0243] Add 10 mL of freshly prepared cut cocktail (trifluoroacetic acid:water:triisopropylsilane:aniline sulfide) (90:2.5:2.5:5, v:v:v:v) to the resin obtained in step 4, and react with shaking at room temperature for 2 hours. After the reaction is complete, filter the reaction solution, wash the resin with trifluoroacetic acid, combine the washed resin with the reaction solution, and precipitate with 4 times the volume of cold MTBE to obtain the crude product. Wash the crude product 3 times with MTBE and dry it under vacuum.

[0244] Step 6: Intramolecular disulfide bond formation

[0245] The crude product obtained in step 5 was dissolved in DMSO (DMSO volume was 20% of the total reaction volume) to prepare a phosphate / guanidine hydrochloride buffer (pH = 7.0, containing 50% ACN). A 1:1 mixture of 1×PBS:6M guanidine hydrochloride (80:20, v:v) buffer and acetonitrile was added to obtain the phosphate / guanidine hydrochloride buffer (pH = 7.0, 50% ACN). The peptide solution was then slowly added dropwise to the above buffer to a final concentration of 1 mg / mL. The mixture was incubated at room temperature with shaking for 16 hours. The reaction results were monitored by LC-MS. After the reaction was completed, purification was performed directly.

[0246] Step 7: Preparation of Peptides

[0247] After filtration through a 0.45 μm membrane, separation was performed using a reversed-phase high-performance liquid chromatography (RP-HPLC) system. The buffer solutions were A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). A BR-C18 (Saifen) reversed-phase column was used. During purification, the chromatographic detection wavelength was set to 230 nm, the flow rate to 15 mL / min, and the gradient to 30-60% acetonitrile for 40 min. Relevant fractions were collected, and after HPLC purity identification, fractions >95% were combined, lyophilized, and the pure peptide was obtained. HPLC and mass spectrometry results are shown below. Figure 5 , Figure 6 .

[0248] Example 4 Synthesis of Compound 26

[0249]

[0250] Step 1: Couple the first amino acid Fmoc-Val-OH

[0251] 168 mg (0.2 mmol) of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 h. Fmoc-Val-OH (0.16 mmol) and diisopropylethylamine (DIEA, 0.64 mmol) were weighed, dissolved in 8 mL of DCM, and added to the resin. The reaction was carried out at room temperature for 2 h. After the reaction was complete, blocking buffer (10 mL) of DCM:methanol:DIEA (85:10:5, v:v:v) was added, and the mixture was blocked at room temperature for 10 min. The blocked resin was then washed 5 times with DCM and 5 times with DMF.

[0252] Step 2: Synthesis of linear precursor peptide chains

[0253] The linear precursor peptide chain of compound 26 is AEEA-IETDV.

[0254] The resin obtained in step 1 was fully swollen in DMF for 1 hour, and then synthesized in the order of the above linear precursor sequence from the second D at the carboxyl terminus to the amino terminus. Each coupling cycle was performed as follows:

[0255] • Perform Fmoc-deprotection twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time.

[0256] Rinse the resin with DMF 6-8 times until neutral pH is reached.

[0257] • Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to resin and react at room temperature for 1 h.

[0258] Rinse the resin with DMF 4-6 times before coupling the next amino acid.

[0259] After synthesis of the linear peptides, the resin was washed five times with DMF and five times with DCM. The resin was then dried under vacuum.

[0260] Step 3: Couple 3-(FMOC-amino)glutaric acid

[0261] Dissolve 0.25 mmol of 3-(FMOC-amino)glutaric acid, 1 mmol of 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU), and 2 mmol of N,N-diisopropylethylamine (DIEA) in DMF, add the solution to the resin obtained in step 2, and react at room temperature for 1 h. Repeat the reaction twice. After the reaction, rinse the resin 5 times with DMF and 5 times with DCM.

[0262] Step 4: Synthesis of linear precursor peptide chains

[0263] The linear precursor peptide chain of compound 26 is YGRKKRRQRRR.

[0264] The resin obtained in step 3 was fully swollen in DMF for 1 hour, and then synthesized in the order from the carboxyl terminus to the amino terminus according to the above linear precursor sequence. Each coupling cycle was performed as follows:

[0265] • Perform Fmoc-deprotection twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time.

[0266] Rinse the resin with DMF 6-8 times until neutral pH is reached.

[0267] • Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to resin and react at room temperature for 1 h.

[0268] Rinse the resin with DMF 4-6 times before coupling the next amino acid.

[0269] After synthesis of the linear peptides, the resin was washed five times with DMF and five times with DCM. The resin was then dried under vacuum.

[0270] Step 5: Cleavage of the linear precursor peptide chain

[0271] Add 10 mL of freshly prepared cut cocktail (trifluoroacetic acid:water:triisopropylsilane:aniline sulfide) (90:2.5:2.5:5, v:v:v:v) to the resin obtained in step 4, and react with shaking at room temperature for 2 hours. After the reaction is complete, filter the reaction solution, wash the resin with trifluoroacetic acid, combine the washed resin with the reaction solution, and precipitate with 4 times the volume of cold MTBE to obtain the crude product. Wash the crude product 3 times with MTBE and dry it under vacuum.

[0272] Step 6: Preparation of Peptides

[0273] The crude peptide was dissolved in a 20% acetonitrile aqueous solution, filtered through a 0.45 μm membrane, and then separated using a reversed-phase high-performance liquid chromatography (RP-HPLC) system. The buffer solutions were A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). A BR-C18 (Saifen) reversed-phase column was used. During purification, the detection wavelength was set to 230 nm, the flow rate to 15 mL / min, and the gradient to 30-60% acetonitrile for 40 min. The relevant fractions were collected, and after HPLC purity assessment, fractions >95% were combined, lyophilized, and the purified peptide was obtained.

[0274] Step 7: Detection and Characterization Methods

[0275] The purity and target molecular weight of the peptide obtained in step 6 were determined by analytical high-performance liquid chromatography (HPLC) and liquid chromatography / mass spectrometry (LC / MS). The results are shown below. Figure 7 , Figure 8 .

[0276] Example 5 Preparation of Compound 27

[0277]

[0278] Step 1: Couple the first amino acid Fmoc-Val-OH

[0279] 168 mg (0.2 mmol) of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 h. Fmoc-Val-OH (0.16 mmol) and diisopropylethylamine (DIEA, 0.64 mmol) were dissolved in 8 mL of DCM and added to the resin. The reaction was carried out at room temperature for 2 h. After the reaction was complete, blocking buffer (10 mL) of DCM:methanol:DIEA (85:10:5, v:v:v) was added, and the mixture was blocked at room temperature for 10 min. The blocked resin was then washed 5 times with DCM and 5 times with DMF.

[0280] Step 2: Synthesis of linear precursor peptide chains

[0281] The linear precursor peptide chain of compound 27 is AEEA-IETDV.

[0282] The resin obtained in step 1 was fully swollen in DMF for 1 hour, and then synthesized in the order of the above linear precursor sequence from the second D at the carboxyl terminus to the amino terminus. Each coupling cycle was performed as follows:

[0283] • Perform Fmoc-deprotection twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time.

[0284] Rinse the resin with DMF 6-8 times until neutral pH is reached.

[0285] • Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to resin and react at room temperature for 1 h.

[0286] Rinse the resin with DMF 4-6 times before coupling the next amino acid.

[0287] After synthesis of the linear peptides, the resin was washed five times with DMF and five times with DCM. The resin was then dried under vacuum.

[0288] Step 3: Couple 3-(FMOC-amino)glutaric acid

[0289] Dissolve 0.25 mmol of 3-(FMOC-amino)glutaric acid, 1 mmol of 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU), and 2 mmol of N,N-diisopropylethylamine (DIEA) in DMF, add the solution to the resin obtained in step 2, and react at room temperature for 1 h. Repeat the reaction twice. After the reaction, rinse the resin 5 times with DMF and 5 times with DCM.

[0290] Step 4: Synthesis of linear precursor peptide chains

[0291] The linear precursor peptide chain of compound 27 is dR-dR-dR-dQ-dR-dR-dK-dK-dR-G-dY.

[0292] The resin obtained in step 3 was fully swollen in DMF for 1 hour, and then synthesized in the order from the carboxyl terminus to the amino terminus according to the above linear precursor sequence. Each coupling cycle was performed as follows:

[0293] • Perform Fmoc-deprotection twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time.

[0294] Rinse the resin with DMF 6-8 times until neutral pH is reached.

[0295] • Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to resin and react at room temperature for 1 h.

[0296] Rinse the resin with DMF 4-6 times before coupling the next amino acid.

[0297] After synthesis of the linear peptides, the resin was washed five times with DMF and five times with DCM. The resin was then dried under vacuum.

[0298] Step 5: Cleavage of the linear precursor peptide chain

[0299] Add 10 mL of freshly prepared cut cocktail (trifluoroacetic acid:water:triisopropylsilane:aniline sulfide) (90:2.5:2.5:5, v:v:v:v) to the resin obtained in step 4, and react with shaking at room temperature for 2 hours. After the reaction is complete, filter the reaction solution, wash the resin with trifluoroacetic acid, combine the washed resin with the reaction solution, and precipitate with 4 times the volume of cold MTBE to obtain the crude product. Wash the crude product 3 times with MTBE and dry it under vacuum.

[0300] Step 6: Preparation of Peptides

[0301] The crude peptide was dissolved in a 20% acetonitrile aqueous solution, filtered through a 0.45 μm membrane, and then separated using a reversed-phase high-performance liquid chromatography (RP-HPLC) system. The buffer solutions were A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). A BR-C18 (Saifen) reversed-phase column was used. During purification, the detection wavelength was set to 230 nm, the flow rate to 15 mL / min, and the gradient to 30-60% acetonitrile for 40 min. The relevant fractions were collected, and after HPLC purity assessment, fractions >95% were combined, lyophilized, and the purified peptide was obtained.

[0302] Step 7: Detection and Characterization Methods

[0303] The purity and target molecular weight of the peptide obtained in step 6 were determined by analytical high-performance liquid chromatography (HPLC) and liquid chromatography / mass spectrometry (LC / MS). The results are shown below. Figure 9 , Figure 10 .

[0304] Example 6 Preparation of Compound 21

[0305]

[0306] Step 1: Couple the first amino acid Fmoc-Val-OH

[0307] 168 mg (0.2 mmol) of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 h. Fmoc-Val-OH (0.16 mmol) and diisopropylethylamine (DIEA, 0.64 mmol) were dissolved in 8 mL of DCM and added to the resin. The reaction was carried out at room temperature for 2 h. After the reaction was complete, blocking buffer (10 mL) of DCM:methanol:DIEA (85:10:5, v:v:v) was added, and the mixture was blocked at room temperature for 10 min. The blocked resin was then washed 5 times with DCM and 5 times with DMF.

[0308] Step 2: Synthesis of linear precursor peptide chains

[0309] The linear precursor peptide chain of compound 21 is AEEA-YIETDV.

[0310] The resin obtained in step 1 was fully swollen in DMF for 1 hour, and then synthesized in the order of the above linear precursor sequence from the second D at the carboxyl terminus to the amino terminus. Each coupling cycle was performed as follows:

[0311] • Perform Fmoc-deprotection twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time.

[0312] Rinse the resin with DMF 6-8 times until neutral pH is reached.

[0313] • Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to resin and react at room temperature for 1 h.

[0314] Rinse the resin with DMF 4-6 times before coupling the next amino acid.

[0315] After synthesis of the linear peptides, the resin was washed five times with DMF and five times with DCM. The resin was then dried under vacuum.

[0316] Step 3: Couple 3-(FMOC-amino)glutaric acid

[0317] Dissolve 0.25 mmol of 3-(FMOC-amino)glutaric acid, 1 mmol of 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU), and 2 mmol of N,N-diisopropylethylamine (DIEA) in DMF, add the solution to the resin obtained in step 2, and react at room temperature for 1 h. Repeat the reaction twice. After the reaction, rinse the resin 5 times with DMF and 5 times with DCM.

[0318] Step 4: Synthesis of linear precursor peptide chains

[0319] The linear precursor peptide chain of compound 21 is YGRKKRRQRRR.

[0320] The resin obtained in step 3 was fully swollen in DMF for 1 hour, and then synthesized in the order from the carboxyl terminus to the amino terminus according to the above linear precursor sequence. Each coupling cycle was performed as follows:

[0321] • Perform Fmoc-deprotection twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time.

[0322] Rinse the resin with DMF 6-8 times until neutral pH is reached.

[0323] • Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to resin and react at room temperature for 1 h.

[0324] Rinse the resin with DMF 4-6 times before coupling the next amino acid.

[0325] After synthesis of the linear peptides, the resin was washed five times with DMF and five times with DCM. The resin was then dried under vacuum.

[0326] Step 5: Cleavage of the linear precursor peptide chain

[0327] Add 10 mL of freshly prepared cut cocktail (trifluoroacetic acid:water:triisopropylsilane:aniline sulfide) (90:2.5:2.5:5, v:v:v:v) to the resin obtained in step 4, and react with shaking at room temperature for 2 hours. After the reaction is complete, filter the reaction solution, wash the resin with trifluoroacetic acid, combine the washed resin with the reaction solution, and precipitate with 4 times the volume of cold MTBE to obtain the crude product. Wash the crude product 3 times with MTBE and dry it under vacuum.

[0328] Step 6: Preparation of Peptides

[0329] The crude peptide was dissolved in a 20% acetonitrile aqueous solution, filtered through a 0.45 μm membrane, and then separated using a reversed-phase high-performance liquid chromatography (RP-HPLC) system. The buffer solutions were A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). A BR-C18 (Saifen) reversed-phase column was used. During purification, the detection wavelength was set to 230 nm, the flow rate to 15 mL / min, and the gradient to 30-60% acetonitrile for 40 min. The relevant fractions were collected, and after HPLC purity assessment, fractions >95% were combined, lyophilized, and the purified peptide was obtained.

[0330] Step 7: Detection and Characterization Methods

[0331] The purity and target molecular weight of the peptide obtained in step 6 were determined by analytical high-performance liquid chromatography (HPLC) and liquid chromatography / mass spectrometry (LC / MS). The results are shown below. Figure 11 , Figure 12 .

[0332] Example 7 Preparation of Compound 22

[0333]

[0334] Step 1: Couple the first amino acid Fmoc-Val-OH

[0335] 168 mg (0.2 mmol) of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 h. Fmoc-Val-OH (0.16 mmol) and diisopropylethylamine (DIEA, 0.64 mmol) were dissolved in 8 mL of DCM and added to the resin. The reaction was carried out at room temperature for 2 h. After the reaction was complete, blocking buffer (10 mL) of DCM:methanol:DIEA (85:10:5, v:v:v) was added, and the mixture was blocked at room temperature for 10 min. The blocked resin was then washed 5 times with DCM and 5 times with DMF.

[0336] Step 2: Synthesis of linear precursor peptide chains

[0337] The linear precursor peptide chain of compound 22 is AEEA-YIETWV.

[0338] The resin obtained in step 1 was fully swollen in DMF for 1 hour, and then synthesized in the order of the above linear precursor sequence from the second D at the carboxyl terminus to the amino terminus. Each coupling cycle was performed as follows:

[0339] • Perform Fmoc-deprotection twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time.

[0340] Rinse the resin with DMF 6-8 times until neutral pH is reached.

[0341] • Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to resin and react at room temperature for 1 h.

[0342] Rinse the resin with DMF 4-6 times before coupling the next amino acid.

[0343] After synthesis of the linear peptides, the resin was washed five times with DMF and five times with DCM. The resin was then dried under vacuum.

[0344] Step 3: Couple 3-(FMOC-amino)glutaric acid

[0345] Dissolve 0.25 mmol of 3-(FMOC-amino)glutaric acid, 1 mmol of 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU), and 2 mmol of N,N-diisopropylethylamine (DIEA) in DMF, add the solution to the resin obtained in step 2, and react at room temperature for 1 h. Repeat the reaction twice. After the reaction, rinse the resin 5 times with DMF and 5 times with DCM.

[0346] Step 4: Synthesis of linear precursor peptide chains

[0347] The linear precursor peptide chain of compound 22 is YGRKKRRQRRR.

[0348] The resin obtained in step 3 was fully swollen in DMF for 1 hour, and then synthesized in the order from the carboxyl terminus to the amino terminus according to the above linear precursor sequence. Each coupling cycle was performed as follows:

[0349] • Perform Fmoc-deprotection twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time.

[0350] Rinse the resin with DMF 6-8 times until neutral pH is reached.

[0351] • Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to resin and react at room temperature for 1 h.

[0352] Rinse the resin with DMF 4-6 times before coupling the next amino acid.

[0353] After synthesis of the linear peptides, the resin was washed five times with DMF and five times with DCM. The resin was then dried under vacuum.

[0354] Step 5: Cleavage of the linear precursor peptide chain

[0355] Add 10 mL of freshly prepared cut cocktail (trifluoroacetic acid:water:triisopropylsilane:aniline sulfide) (90:2.5:2.5:5, v:v:v:v) to the resin obtained in step 4, and react with shaking at room temperature for 2 hours. After the reaction is complete, filter the reaction solution, wash the resin with trifluoroacetic acid, combine the washed resin with the reaction solution, and precipitate with 4 times the volume of cold MTBE to obtain the crude product. Wash the crude product 3 times with MTBE and dry it under vacuum.

[0356] Step 6: Preparation of Peptides

[0357] The crude peptide was dissolved in a 20% acetonitrile aqueous solution, filtered through a 0.45 μm membrane, and then separated using a reversed-phase high-performance liquid chromatography (RP-HPLC) system. The buffer solutions were A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). A BR-C18 (Saifen) reversed-phase column was used. During purification, the detection wavelength was set to 230 nm, the flow rate to 15 mL / min, and the gradient to 30-60% acetonitrile for 40 min. The relevant fractions were collected, and after HPLC purity assessment, fractions >95% were combined, lyophilized, and the purified peptide was obtained.

[0358] Step 7: Detection and Characterization Methods

[0359] The purity and target molecular weight of the peptide obtained in step 6 were determined by analytical high-performance liquid chromatography (HPLC) and liquid chromatography / mass spectrometry (LC / MS). The results are shown below. Figure 13 , Figure 14 .

[0360] Example 8 Preparation of Compound 31

[0361]

[0362] Step 1: Couple the first amino acid Fmoc-Val-OH

[0363] 168 mg (0.2 mmol) of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 h. Fmoc-Val-OH (0.16 mmol) and diisopropylethylamine (DIEA, 0.64 mmol) were dissolved in 8 mL of DCM and added to the resin. The reaction was carried out at room temperature for 2 h. After the reaction was complete, blocking buffer (10 mL) of DCM:methanol:DIEA (85:10:5, v:v:v) was added, and the mixture was blocked at room temperature for 10 min. The blocked resin was then washed 5 times with DCM and 5 times with DMF.

[0364] Step 2: Synthesis of linear precursor peptide chains

[0365] The linear precursor peptide chain of compound 31 is AEEA-YIETWV.

[0366] The resin obtained in step 1 was fully swollen in DMF for 1 hour, and then synthesized in the order of the above linear precursor sequence from the second D at the carboxyl terminus to the amino terminus. Each coupling cycle was performed as follows:

[0367] • Perform Fmoc-deprotection twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time.

[0368] Rinse the resin with DMF 6-8 times until neutral pH is reached.

[0369] • Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to resin and react at room temperature for 1 h.

[0370] Rinse the resin with DMF 4-6 times before coupling the next amino acid.

[0371] After synthesis of the linear peptides, the resin was washed five times with DMF and five times with DCM. The resin was then dried under vacuum.

[0372] Step 3: Couple 3-(FMOC-amino)glutaric acid

[0373] Dissolve 0.25 mmol of 3-(FMOC-amino)glutaric acid, 1 mmol of 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU), and 2 mmol of N,N-diisopropylethylamine (DIEA) in DMF, add the solution to the resin obtained in step 2, and react at room temperature for 1 h. Repeat the reaction twice. After the reaction, rinse the resin 5 times with DMF and 5 times with DCM.

[0374] Step 4: Synthesis of linear precursor peptide chains

[0375] The linear precursor peptide chain of compound 31 is dR-dR-dR-dQ-dR-dR-dK-dK-dR-G-dY.

[0376] The resin obtained in step 3 was fully swollen in DMF for 1 hour, and then synthesized in the order from the carboxyl terminus to the amino terminus according to the above linear precursor sequence. Each coupling cycle was performed as follows:

[0377] • Perform Fmoc-deprotection twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time.

[0378] Rinse the resin with DMF 6-8 times until neutral pH is reached.

[0379] • Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to resin and react at room temperature for 1 h.

[0380] Rinse the resin with DMF 4-6 times before coupling the next amino acid.

[0381] After synthesis of the linear peptides, the resin was washed five times with DMF and five times with DCM. The resin was then dried under vacuum.

[0382] Step 5: Cleavage of the linear precursor peptide chain

[0383] Add 10 mL of freshly prepared cut cocktail (trifluoroacetic acid:water:triisopropylsilane:aniline sulfide) (90:2.5:2.5:5, v:v:v:v) to the resin obtained in step 4, and react with shaking at room temperature for 2 hours. After the reaction is complete, filter the reaction solution, wash the resin with trifluoroacetic acid, combine the washed resin with the reaction solution, and precipitate with 4 times the volume of cold MTBE to obtain the crude product. Wash the crude product 3 times with MTBE and dry it under vacuum.

[0384] Step 6: Preparation of Peptides

[0385] The crude peptide was dissolved in a 20% acetonitrile aqueous solution, filtered through a 0.45 μm membrane, and then separated using a reversed-phase high-performance liquid chromatography (RP-HPLC) system. The buffer solutions were A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). A BR-C18 (Saifen) reversed-phase column was used. During purification, the detection wavelength was set to 230 nm, the flow rate to 15 mL / min, and the gradient to 30-60% acetonitrile for 40 min. The relevant fractions were collected, and after HPLC purity assessment, fractions >95% were combined, lyophilized, and the purified peptide was obtained.

[0386] Step 7: Detection and Characterization Methods

[0387] The purity and target molecular weight of the peptide obtained in step 6 were determined by analytical high-performance liquid chromatography (HPLC) and liquid chromatography / mass spectrometry (LC / MS). The results are shown below. Figure 15 , Figure 16 .

[0388] Example 9 Preparation of Compound 28

[0389]

[0390] Step 1: Couple the first amino acid Fmoc-Cys(Trt)-OH

[0391] 84 mg (0.1 mmol) of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 h. Fmoc-Cys(Trt)-OH (0.08 mmol) and diisopropylethylamine (DIEA, 0.32 mmol) were weighed, dissolved in 5 mL of DCM, and added to the resin. The reaction was carried out at room temperature for 2 h. After the reaction was complete, blocking buffer (10 mL) of DCM:methanol:DIEA (85:10:5, v:v:v) was added, and the mixture was blocked at room temperature for 10 min. The blocked resin was washed 5 times with DCM and 5 times with DMF.

[0392] Step 2: Synthesis of linear precursor peptide chains

[0393] The linear precursor peptide chain of compound 28 is AEEA-CKMVTTFGIDVTTTYIC.

[0394] The resin obtained in step 1 was fully swollen in DMF for 1 hour, and then synthesized in the order of the above linear precursor sequence from the second I position at the carboxyl terminus to the amino terminus. Each coupling cycle was performed as follows:

[0395] • Perform Fmoc-deprotection twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time.

[0396] Rinse the resin with DMF 6-8 times until neutral pH is reached.

[0397] • Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to resin and react at room temperature for 1 h.

[0398] Rinse the resin with DMF 4-6 times before coupling the next amino acid.

[0399] After synthesis of the linear peptides, the resin was washed five times with DMF and five times with DCM. The resin was then dried under vacuum.

[0400] Step 3: Couple amino acid Fmoc-Lys(Fmoc)-OH

[0401] Dissolve 0.5 mmol of Fmoc-Lys(Fmoc)-OH, 0.5 mmol of 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU), and 1 mmol of 4-methylmorpholine (NMM) in DMF, add the solution to the resin obtained in step 1, and react at room temperature for 1 h. After the reaction, rinse the resin 5 times with DMF and 5 times with DCM.

[0402] Step 4: Synthesis of linear precursor peptide chains

[0403] The linear precursor peptide chain of compound 28 is dR-dR-dR-dQ-dR-dR-dK-dK-dR-G-dY.

[0404] The resin obtained in step 3 was fully swollen in DMF for 1 hour, and then synthesized in the order from the carboxyl terminus to the amino terminus according to the above linear precursor sequence. Each coupling cycle was performed as follows:

[0405] • Perform Fmoc-deprotection twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time.

[0406] Rinse the resin with DMF 6-8 times until neutral pH is reached.

[0407] • Dissolve 1 mmol Fmoc-AA, 1 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU) and 2 mmol 4-methylmorpholine (NMM) in DMF, add to resin and react at room temperature for 1 h.

[0408] Rinse the resin with DMF 4-6 times before coupling the next amino acid.

[0409] After synthesis of the linear peptides, the resin was washed five times with DMF and five times with DCM. The resin was then dried under vacuum.

[0410] Step 5: Cleavage of the linear precursor peptide chain

[0411] Add 10 mL of freshly prepared cut cocktail (trifluoroacetic acid:water:triisopropylsilane:aniline sulfide) (90:2.5:2.5:5, v:v:v:v) to the resin obtained in step 4, and react with shaking at room temperature for 2 hours. After the reaction is complete, filter the reaction solution, wash the resin with trifluoroacetic acid, combine the washed resin with the reaction solution, and precipitate with 4 times the volume of cold MTBE to obtain the crude product. Wash the crude product 3 times with MTBE and dry it under vacuum.

[0412] Step 6: Intramolecular disulfide bond formation

[0413] The crude product obtained in step 5 was dissolved in DMSO (DMSO volume was 20% of the total reaction volume) to prepare a phosphate / guanidine hydrochloride buffer (pH = 7.0, containing 50% ACN). A 1:1 mixture of 1×PBS:6M guanidine hydrochloride (80:20, v:v) buffer and acetonitrile was added to obtain the phosphate / guanidine hydrochloride buffer (pH = 7.0, 50% ACN). The peptide solution was then slowly added dropwise to the above buffer to a final concentration of 1 mg / mL. The mixture was incubated at room temperature with shaking for 16 hours. The reaction results were monitored by LC-MS. After the reaction was completed, purification was performed directly.

[0414] Step 7: Preparation of Peptides

[0415] After filtration through a 0.45 μm membrane, separation was performed using a reversed-phase high-performance liquid chromatography (RP-HPLC) system. The buffer solutions were A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). A BR-C18 (Saifen) reversed-phase column was used. During purification, the chromatographic detection wavelength was set to 230 nm, the flow rate to 15 mL / min, and the gradient to 30-60% acetonitrile for 40 min. Relevant fractions were collected, and after HPLC purity identification, fractions >95% were combined, lyophilized, and the pure peptide was obtained. HPLC and mass spectrometry results are shown below. Figure 17 , Figure 18 .

[0416] Example 10 Synthesis of Compound 1

[0417]

[0418] Step 1: Couple the first amino acid Fmoc-Val-OH

[0419] 168 mg (0.2 mmol) of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 h. Fmoc-Val-OH (0.16 mmol) and diisopropylethylamine (DIEA, 0.64 mmol) were weighed, dissolved in 8 mL of DCM, and added to the resin. The reaction was carried out at room temperature for 2 h. After the reaction was complete, blocking buffer (10 mL) of DCM:methanol:DIEA (85:10:5, v:v:v) was added, and the mixture was blocked at room temperature for 10 min. The blocked resin was then washed 5 times with DCM and 5 times with DMF.

[0420] Step 2: Synthesis of linear precursor peptide chains

[0421] The linear precursor peptide chain of compound 1 is AEEA-IETDV.

[0422] The resin obtained in step 1 was fully swollen in DMF for 1 hour, and then synthesized in the order of the above linear precursor sequence from the second D at the carboxyl terminus to the amino terminus. Each coupling cycle was performed as follows:

[0423] • Perform Fmoc-deprotection twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time.

[0424] Rinse the resin with DMF 6-8 times until neutral pH is reached.

[0425] • Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to resin and react at room temperature for 1 h.

[0426] Rinse the resin with DMF 4-6 times before coupling the next amino acid.

[0427] After synthesis of the linear peptides, the resin was washed five times with DMF and five times with DCM. The resin was then dried under vacuum.

[0428] Step 3: Couple 3-(FMOC-amino)malonic acid

[0429] Dissolve 0.25 mmol of 3-(FMOC-amino)malonic acid, 1 mmol of 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU), and 2 mmol of N,N-diisopropylethylamine (DIEA) in DMF, add the solution to the resin obtained in step 2, and react at room temperature for 1 h. Repeat the reaction twice. After the reaction, rinse the resin 5 times with DMF and 5 times with DCM.

[0430] Step 4: Synthesis of linear precursor peptide chains

[0431] The linear precursor peptide chain of compound 1 is YGRKKRRQRRR.

[0432] The resin obtained in step 3 was fully swollen in DMF for 1 hour, and then synthesized in the order from the carboxyl terminus to the amino terminus according to the above linear precursor sequence. Each coupling cycle was performed as follows:

[0433] • Perform Fmoc-deprotection twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time.

[0434] Rinse the resin with DMF 6-8 times until neutral pH is reached.

[0435] • Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to resin and react at room temperature for 1 h.

[0436] Rinse the resin with DMF 4-6 times before coupling the next amino acid.

[0437] After synthesis of the linear peptides, the resin was washed five times with DMF and five times with DCM. The resin was then dried under vacuum.

[0438] Step 5: Cleavage of the linear precursor peptide chain

[0439] Add 10 mL of freshly prepared cut cocktail (trifluoroacetic acid:water:triisopropylsilane:aniline sulfide) (90:2.5:2.5:5, v:v:v:v) to the resin obtained in step 4, and react with shaking at room temperature for 2 hours. After the reaction is complete, filter the reaction solution, wash the resin with trifluoroacetic acid, combine the washed resin with the reaction solution, and precipitate with 4 times the volume of cold MTBE to obtain the crude product. Wash the crude product 3 times with MTBE and dry it under vacuum.

[0440] Step 6: Preparation of Peptides

[0441] The crude peptide was dissolved in a 20% acetonitrile aqueous solution, filtered through a 0.45 μm membrane, and then separated using a reversed-phase high-performance liquid chromatography (RP-HPLC) system. The buffer solutions were A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). A BR-C18 (Saifen) reversed-phase column was used. During purification, the detection wavelength was set to 230 nm, the flow rate to 15 mL / min, and the gradient to 30-60% acetonitrile for 40 min. The relevant fractions were collected, and after HPLC purity assessment, fractions >95% were combined, lyophilized, and the purified peptide was obtained.

[0442] Step 7: Detection and Characterization Methods

[0443] The purity and target molecular weight of the peptide obtained in step 6 were determined by analytical high performance liquid chromatography and liquid chromatography / mass spectrometry.

[0444] Example 11 Synthesis of Compound 33

[0445]

[0446] Step 1: Couple the first amino acid Fmoc-Val-OH

[0447] 168 mg (0.2 mmol) of 2-Chlorotrityl chloride resin was fully swollen in DCM for 1 h. Fmoc-Val-OH (0.16 mmol) and diisopropylethylamine (DIEA, 0.64 mmol) were weighed, dissolved in 8 mL of DCM, and added to the resin. The reaction was carried out at room temperature for 2 h. After the reaction was complete, blocking buffer (10 mL) of DCM:methanol:DIEA (85:10:5, v:v:v) was added, and the mixture was blocked at room temperature for 10 min. The blocked resin was then washed 5 times with DCM and 5 times with DMF.

[0448] Step 2: Synthesis of linear precursor peptide chains

[0449] The linear precursor peptide chain of compound 26 is AEEA-IETDV.

[0450] The resin obtained in step 1 was fully swollen in DMF for 1 hour, and then synthesized in the order of the above linear precursor sequence from the second D at the carboxyl terminus to the amino terminus. Each coupling cycle was performed as follows:

[0451] • Perform Fmoc-deprotection twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time.

[0452] Rinse the resin with DMF 6-8 times until neutral pH is reached.

[0453] • Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to resin and react at room temperature for 1 h.

[0454] Rinse the resin with DMF 4-6 times before coupling the next amino acid.

[0455] After synthesis of the linear peptides, the resin was washed five times with DMF and five times with DCM. The resin was then dried under vacuum.

[0456] Step 3: Couple 3-(FMOC-amino)glutaric acid

[0457] Dissolve 0.25 mmol of 3-(FMOC-amino)glutaric acid, 1 mmol of 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU), and 2 mmol of N,N-diisopropylethylamine (DIEA) in DMF, add the solution to the resin obtained in step 2, and react at room temperature for 1 h. Repeat the reaction twice. After the reaction, rinse the resin 5 times with DMF and 5 times with DCM.

[0458] Step 4: Synthesis of linear precursor peptide chains

[0459] The linear precursor peptide chain of compound 26 is RRLSYSRRRF.

[0460] The resin obtained in step 3 was fully swollen in DMF for 1 hour, and then synthesized in the order from the carboxyl terminus to the amino terminus according to the above linear precursor sequence. Each coupling cycle was performed as follows:

[0461] • Perform Fmoc-deprotection twice with 20% piperidine / DMF (20% v / v, 10 mL), 8 min each time.

[0462] Rinse the resin with DMF 6-8 times until neutral pH is reached.

[0463] • Dissolve 0.5 mmol Fmoc-AA, 0.5 mmol 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU) and 1 mmol 4-methylmorpholine (NMM) in DMF, add to resin and react at room temperature for 1 h.

[0464] Rinse the resin with DMF 4-6 times before coupling the next amino acid.

[0465] After synthesis of the linear peptides, the resin was washed five times with DMF and five times with DCM. The resin was then dried under vacuum.

[0466] Step 5: Cleavage of the linear precursor peptide chain

[0467] Add 10 mL of freshly prepared cut cocktail (trifluoroacetic acid:water:triisopropylsilane:aniline sulfide) (90:2.5:2.5:5, v:v:v:v) to the resin obtained in step 4, and react with shaking at room temperature for 2 hours. After the reaction is complete, filter the reaction solution, wash the resin with trifluoroacetic acid, combine the washed resin with the reaction solution, and precipitate with 4 times the volume of cold MTBE to obtain the crude product. Wash the crude product 3 times with MTBE and dry it under vacuum.

[0468] Step 6: Preparation of Peptides

[0469] The crude peptide was dissolved in a 20% acetonitrile aqueous solution, filtered through a 0.45 μm membrane, and then separated using a reversed-phase high-performance liquid chromatography (RP-HPLC) system. The buffer solutions were A (0.1% trifluoroacetic acid, aqueous solution) and B (0.1% trifluoroacetic acid, acetonitrile). A BR-C18 (Saifen) reversed-phase column was used. During purification, the detection wavelength was set to 230 nm, the flow rate to 15 mL / min, and the gradient to 30-60% acetonitrile for 40 min. The relevant fractions were collected, and after HPLC purity assessment, fractions >95% were combined, lyophilized, and the purified peptide was obtained.

[0470] Step 7: Detection and Characterization Methods

[0471] The purity and target molecular weight of the peptide obtained in step 6 were determined by analytical high performance liquid chromatography and liquid chromatography / mass spectrometry.

[0472] Other compounds of the present invention can be synthesized by referring to the synthesis method of the above embodiments.

[0473] Test Example 1 ELISA test compound The compound inhibits the binding of Tat-NR2B9c-B to cMyc-PSD95 alpha 1-392 (1) Experimental materials:

[0474]

[0475] (2) Experimental steps:

[0476] The inhibitory effect of the compound on the binding of Tat-NR2B9c-B to cMyc-PSD95alpha 1-392 was tested using ELISA (enzyme-linked immunosorbent assay). cMyc-PSD95 alpha 1-392 was coated at 0.37 μg / mL, 25 μL / well onto a 384-well plate (Greiner 781097). After blocking the cMyc-PSD95 alpha 1-392-coated ELISA plate, the compound was serially diluted to 0–10 μM (8 concentrations in 3-fold serial dilutions from 10 μM downwards). 12.5 μL / well of the compound was transferred to the 384-well plate using a workstation, and the plate was briefly centrifuged to remove air bubbles. Then, 12.5 μL / well of 12 nM Tat-NR2B9c-B was pipetted using an 8-well multipipe (10–100 μL), briefly centrifuged to remove air bubbles, and incubated at 37°C for 1 h. Discard the liquid in the wells, add 80 μL of 1×TBST washing buffer (pH=7.4) and wash the plate 3-5 times, 3-5 min each time. After drying the plate, add 25 μL / well of Streptavidin HRP diluted 1:10000, briefly centrifuge to remove air bubbles, and incubate at 37°C for 1 h. Wash the plate again and dry it completely. Add 25 μL of TMB chromogenic solution to each well and continue incubating at 37°C for 30 min. Finally, add 25 μL of stop solution (1 MHCL) to each well to terminate the reaction. Use a Cytation 5 microplate reader to read the absorbance at 450 nm and calculate the IC50 of the compound inhibiting the binding of Tat-NR2B9c-B to cMyc-PSD95 alpha 1-392. 50 .

[0477] The experimental results are shown in Table 1. The compound of the present invention can inhibit the binding of Tat-NR2B9c-B to cMyc-PSD95 alpha1-392 and has a good inhibitory effect. The peptide of the present invention has a stronger affinity for cMyc-PSD95 alpha1-392 than Tat-NR2B9c-B.

[0478] Table 1 Results of the compounds inhibiting the binding of Tat-NR2B9c-B to cMyc-PSD95 alpha 1-392

[0479] 3 0.003 4 0.0022 5 0.001 6 0.0019 7 0.003 8 0.0022 26 0.03

[0480] Test Example 2: FRET test compound inhibits the binding of cMyc-PSD95 alpha 1-392 to Tat-NR2B9C-B.

[0481] (1) Experimental materials:

[0482]

[0483] (2) Experimental steps:

[0484] The inhibitory effect of the compound on the binding of cMyc-PSD95 alpha 1-392 to Tat-NR2B9C-B was tested using FRET (fluorescence resonance energy transfer) method. The compound was serially diluted to 0–10 μM (8 concentrations in total, 3-fold serial dilutions from 10 μM downwards). Using an 8-well multipipe (1–10 μL), 4 μL / well of 4 nM cMyc-PSD95 alpha 1-392 was pipetted into a 384-well white plate (Thermo 264706) and briefly centrifuged to remove air bubbles. 4 μL / well of the compound was then transferred to a 384-well plate using a workstation and briefly centrifuged to remove air bubbles. 4 μL / well of 20 nM Tat-NR2B9c-B was again pipetted into a 384-well white plate using an 8-well multipipe and briefly centrifuged to remove air bubbles. The prepared 0.02 μg / mL EU-steptavidin was mixed with 1 μg / mL Mouse Anti-C-MYC IgG SureLightAPC at a 1:1 ratio. Pipe 8 μL / well of EU-APC premixed solution into a 384-well white plate using an 8-well pipette, and briefly centrifuge to remove air bubbles. Incubate at room temperature in the dark for 2 hours, and read the emission fluorescence values ​​at 620 nm and 665 nm using a Cytation 5 microplate reader after excitation at 320 nM.

[0485] The experimental results are shown in Table 2, where control group 1 is... Control group 2 was Its preparation method is as described in patent CN103533949A.

[0486] Table 2 Results of the compounds inhibiting the binding of cMyc-PSD95 alpha 1-392 to Tat-NR2B9C-B

[0487] Control group 1 0.07 Control group 2 0.1277 2 0.13 4 0.19 5 0.09 9 0.056 26 0.008 31 0.009 32 0.020

[0488] 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.

[0489] Test Example 3: ELISA test showed that the compound inhibited the binding of biotin-nNOS1-299 to cMyc-PSD95 alpha 1-392.

[0490] (1) Experimental materials:

[0491]

[0492] (2) Experimental steps:

[0493] The inhibitory effect of the compound on the binding of biotin-nNOS1-299 to cMyc-PSD95alpha 1-392 was tested using ELISA (enzyme-linked immunosorbent assay). cMyc-PSD95 alpha 1-392 was coated at 10 μg / mL, 25 μL / well onto 384-well plates (Greiner 781097). After blocking the cMyc-PSD95 alpha 1-392-coated ELISA plates, the compound was serially diluted to 0–10 μM (8 concentrations in 3-fold serial dilutions from 10 μM downwards). 12.5 μL / well of the compound was transferred to the 384-well plate using a workstation, and the plate was briefly centrifuged to remove air bubbles. Then, 12.5 μL / well of 10 μg / mL 743 biotin-nNOS1-299 was pipetted using an 8-well multipipe (10–100 μL), briefly centrifuged to remove air bubbles, and incubated at 37°C for 1 h. Discard the liquid in the wells, add 80 μl of 1×TBST washing buffer (pH=7.4) and wash the plate 3-5 times, 3-5 min each time. After drying the plate, add 25 μl / well of Streptavidin HRP diluted 1:10000, briefly centrifuge to remove air bubbles, and incubate at 37°C for 1 h. Wash the plate again and dry it completely. Add 25 μl of TMB chromogenic solution to each well and continue incubating at 37°C for 30 min. Finally, add 25 μl of stop solution (1 MHCL) to each well to terminate the reaction. Use a Cytation 5 microplate reader to read the absorbance at 450 nm and calculate the IC50 of the compound inhibiting the binding of biotin-nNOS1-299 to cMyc-PSD95 alpha 1-392. 50 .

[0494] Table 3 shows the results of the compounds inhibiting the binding of biotin-nNOS1-299 and cMyc-PSD95 alpha 1-392.

[0495] Control group 2 0.01994 9 0.029 21 0.035 22 0.023 23 0.021 26 0.02 31 0.010 32 0.010

[0496] The experimental results are shown in Table 3. The compounds of the present invention can inhibit the binding of biotin-nNOS1-299 and cMyc-PSD95alpha1-392 and have a good inhibitory effect.

[0497] Test Example 4FP: The test compound inhibited the binding of 6H-PSD95 alpha 61-249 and 5FAM-N-bis-(PEG2-IETAV)2 / 5-FAM-NPEG4(IETAV)2.

[0498] (1) Experimental materials:

[0499]

[0500] (2) Experimental steps:

[0501] The inhibitory effect of the compound on the binding of 6H-PSD95 alpha 61-249 to 5FAM-N-bis-(PEG2-IETAV)2 / 5-FAM-NPEG4(IETAV)2 was tested using FP (fluorescence polarization immunoassay). The compound was serially diluted to 0–10 μM (eight concentrations in three-fold gradients from 10 μM downwards). Using an 8-well pipette (1–10 μL), 5 μL / well of 5 nM 6H-PSD95 alpha 61-249 was pipetted into a 384-well plate (Corning 3575), and the mixture was briefly centrifuged to remove air bubbles. Then, using a workstation, 5 μL / well of the compound was transferred into the 384-well plate, and the mixture was briefly centrifuged to remove air bubbles. Finally, using an 8-well pipette, 5 μL / well of 0.5 nM 5FAM-N-bis-(PEG2-IETAV)2 / 5-FAM-NPEG4(IETAV)2 was pipetted into the 384-well plate, and the mixture was briefly centrifuged to remove air bubbles. Incubate at room temperature in the dark for 2 hours, and read the values ​​using the Cytation5 FP module of a microplate reader.

[0502] The preparation method of 5-FAM-NPEG4(IETAV)2 is as described in patent CN103533949A.

[0503] Table 4 shows the results of the compounds inhibiting the binding of 6H-PSD95 alpha 61-249 and 5FAM-N-bis-(PEG2-IETAV)2 / 5-FAM-NPEG4(IETAV)2.

[0504] Control group 1 0.02333 Control group 2 0.02391 3 0.02 9 0.055 21 0.015 22 0.012 23 0.011 26 0.01 31 0.018 32 0.023

[0505] The experimental results are shown in Table 4. The compounds of this invention can inhibit the binding of 6H-PSD95 alpha 61-249 and 5FAM-N-bis-(PEG2-IETAV)2 / 5-FAM-NPEG4(IETAV)2 with good inhibitory effects. The peptides of this invention have a stronger affinity for 6H-PSD95 alpha 61-249 than for FAM-N-bis-(PEG2-IETAV)2 / 5-FAM-NPEG4(IETAV)2.

[0506] Test Example 5: Mouse plasma stability

[0507] 1. Stability of mouse plasma (heparin sodium)

[0508] 1.1 Experimental Materials:

[0509] Drug: The compound of this invention.

[0510] Experimental reagents: Neptipeptide (Tat-NR2B9C / NA-1) (purchased from Nanjing Genscript Biotech Co., Ltd.); methanol (purchased from Sigma); formic acid (purchased from Aladdin); DMSO (dimethyl sulfoxide) (purchased from Aladdin); mouse plasma (Slack) (self-collected).

[0511] 1.2 Experimental Methods:

[0512] Positive sample preparation: Dissolve nifedipine in DMSO to a final concentration of 100 times (1 mM) and store at -20℃. Negative sample preparation: Dissolve the test peptide in DMSO or other organic solvent to a final concentration of 100 times (1 mM) and store at -20℃. Plasma thawing: Remove plasma (sample number * 2.1) mL from the -80℃ freezer and thaw rapidly in a 37℃ water bath. MIX preparation: Add 693 μL of plasma to a 1.5 mL EP tube, with 3 parallel samples at each time point, preparing 3 MIX tubes. Add 7 μL of the test sample to each tube to achieve a final concentration of detectable concentration or in vivo drug concentration of 10 μM. Vortex for 30 s, aliquot 100 μL at each time point, and incubate on ice throughout. Incubation: Incubate in a 37℃ water bath at six time points: 0 min, 15 min, 30 min, 60 min, 90 min, and 120 min. Termination of reaction: After incubation, add 4 volumes of 0.1% formic acid in a 75% acetonitrile aqueous precipitant. Mixing: Vortex for 30 s. Centrifugation: Centrifuge at 13000 rpm for 10 min at 4℃. Collect the supernatant, transfer it to a sample tube, and analyze by LC-MS / MS.

[0513] 1.3 Experimental Results:

[0514] A line graph with the vertical axis representing the remaining percentage of original drug (%) and the horizontal axis representing time, such as... Figure 19 As shown, we can see the trend of sample degradation in in vitro plasma over time, thus obtaining the results of sample stability.

[0515]

[0516] Calculate the half-life T of the drug in plasma 1 / 2 The elimination rate constant (Ke) was calculated using the first-order kinetic formula, and the T1 / 2 (min) of the compound in plasma was further determined using the formula.

[0517] T / 2 = -0.693 / Ke

[0518] By comparing the residual rate (%) of the original drug, the residual rates (%) of compounds 9, 31, and 32 were 89.2%, 88.91%, and 97.03%, respectively, indicating that the compounds of this invention exhibit good plasma stability over 2 hours. The T values ​​for each compound... 1 / 2 As shown in Table 5. 2. Stability of mouse plasma alteplase (heparin sodium)

[0519] 2.1 Experimental Materials:

[0520] Drug: The compound of this invention.

[0521] Experimental reagents: Neptipeptide (Tat-NR2B9C / NA-1) (purchased from Nanjing Genscript Biotech 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 (Aitol).

[0522] 2.2 Experimental Methods:

[0523] Positive sample preparation: Dissolve neptin in DMSO to a final concentration of 1mM (100 times the final concentration) and store at -20℃. Test sample preparation: Dissolve the test peptide in DMSO or other organic solvent to a final concentration of 1mM (100 times the final concentration) and store at -20℃. Plasma thawing: Remove plasma (sample number * 2.1) mL from the -80℃ freezer and thaw rapidly in a 37℃ water bath. Preparation of mixing buffer: Add (98 μL × (6 time points) + 1) 686 μL of plasma (heparin sodium) to a 1.5 mL EP tube. Prepare one mixing buffer for at least three replicates at each time point. Add 7 μL of alteplase to each tube to a final concentration of 9 μg / mL. Add 7 μL of the test sample to each tube to a final concentration of 10 μM. Vortex for 30 s, aliquot into 100 μL portions according to the time gradient, and incubate on ice throughout. Incubation: Incubate in a 37℃ water bath at six time points: 0 min, 5 min, 15 min, 30 min, 45 min, and 60 min. Termination of reaction: After incubation, add 4 volumes of 0.1% formic acid in 75% acetonitrile aqueous solution as a precipitant. Mixing: Vortex for 30 s. Centrifugation: Centrifuge at 13000 rpm for 10 min at 4℃. Collect the supernatant, transfer it to a sample tube, and analyze by LC-MS / MS.

[0524] 2.3 Experimental Results:

[0525] A line graph with the vertical axis representing the remaining percentage of original drug (%) and the horizontal axis representing time, such as... Figure 20 As shown, we can see the trend of sample degradation in in vitro plasma over time, thus obtaining the results of sample stability.

[0526]

[0527] The half-life T1 / 2 of the drug in plasma was calculated, and the elimination rate constant (Ke) was obtained by calculating according to the first-order kinetic formula. Then, the T1 / 2 (min) of the compound in plasma was obtained according to the formula.

[0528] T / 2 = -0.693 / Ke

[0529] Experimental results: By comparing the residual rate (%) of the original drug, the residual rates (%) of compounds 9, 31, and 32 were 91.8%, 75.3%, and 100.7%, respectively. The compounds of this invention exhibit good plasma stability over 1 hour. (T values ​​for each compound are not provided in the original text.) 1 / 2 As shown in Table 5.

[0530] Table 5. T for each compound 1 / 2 result

[0531] 9 >120 >120 31 >120 >125.6 32 >120 >120 Tat-NR2B9C 83.36 31.3 AVLX-144 46.72 58.6

[0532] The compounds of this invention still exhibit good stability in human plasma (heparin sodium) and human plasma alteplase (heparin sodium).

[0533] In summary, the stability of the compounds of this invention in plasma is significantly improved compared to Tat-NR2B9C and AVLX-144.

[0534] Test Example 6: Drug Efficacy Test in Mice

[0535] 1. Experimental Materials

[0536] Drug: The compound of this invention.

[0537] Related reagents and consumables: 1mL insulin injection, isoflurane, TTC, low-absorption EP tube, physiological saline, 6cm culture dish, microsurgical scissors, microsurgical forceps, absorbable sutures, suture plugs (Guangzhou Jialing), arterial clamps, cotton thread, glucose, penicillin.

[0538] Experimental animals: 100 male C57 mice, 7-8 weeks old, 20-25g, purchased from Hunan Anshengmei Pharmaceutical Research Institute Co., Ltd.

[0539] 2. Experimental Methods:

[0540] 2.1 Experimental Preparation: Hair removal cream was used to remove hair from the necks of mice the day before the experiment. Mice were then randomly assigned to groups using randomization software.

[0541] 2.2 Anesthesia: Mice were fixed under a stereomicroscope and anesthetized by inhalation with isoflurane.

[0542] 2.3 After the anesthesia has stabilized, disinfect the skin of the mouse's neck. Make an incision along the midline in the anterior neck skin, and further separate and divide the pectoral muscles to expose the common carotid artery and vagus nerve. Then, gradually expose the bifurcation of the common carotid artery, the external carotid artery, and the internal carotid artery. Separate the common carotid artery, clamp it proximally with an arterial clamp, and tie a slipknot with cotton thread. Separate the internal carotid artery and clamp it with an arterial clamp. Separate the external carotid artery and ligate it. Make a small incision distal to the two ligatures in the common carotid artery, with the incision obliquely pointing towards the distal end. Carefully insert a professional suture along the edge of the vessel into the lumen of the common carotid artery. Once the suture is inside the common carotid artery, gently tighten the slipknot.

[0543] 2.4 Transfer the suture plug from the bifurcation into the internal carotid artery, release the arterial clamp at the internal carotid artery, and continue deeper into the brain until reaching the origin of the middle cerebral artery, ultimately marking the suture plug at the bifurcation of the common carotid artery. Tighten the common carotid artery slipknot to prevent suture plug movement. Remove the arterial clamp from the proximal end of the common carotid artery. Trim any excess suture plug and suture ends, and suture the skin.

[0544] 2.5 Thirty minutes after embolization, the test drug and placebo were administered to the tail vein of mice. The drugs were randomly selected by personnel not involved in the surgery, and the personnel administering the injections were unaware of the drug information.

[0545] 2.6 Postoperatively, the model mice were injected with penicillin and glucose, and monitored until the mice regained consciousness. 24 hours after embolization, the neurological function of the animals was graded according to the Longascore scoring method, with the following criteria:

[0546] 0 points: No neurological symptoms;

[0547] 1 point: The contralateral forelimb is flexed and closely pressed against the chest wall;

[0548] 2 points: When the animal is moving freely, it turns or circles to the opposite side;

[0549] 3 points: When animals move freely, they tend to lean to the opposite side;

[0550] 4 points; flaccid paralysis, with no spontaneous movement of the limbs.

[0551] 2.7 Observe the mouse status, record the survival rate and score 24h and 48h after embolization.

[0552] 2.8 Mice in each group were sacrificed 48 hours after embolization, and their brains were removed intact. The brains were washed with physiological saline, dried with filter paper, and weighed.

[0553] 2.9 The whole brain was frozen, and coronal sections were prepared at the optic chiasm and 2 mm anterior and posterior. After staining with 2% TTC solution at 37°C in the dark for 30 minutes, the brain slices were arranged in order, from smallest to largest and from top to bottom, with the cortex facing upwards and the infarcted area on the right side. The slices were then photographed after being arranged neatly.

[0554] 2.10 Use software to calculate the area of ​​cerebral infarction and the total area.

[0555] Calculate the cerebral infarction rate: Percentage of infarction (%) = Area of ​​pallid area / Total area × 100%.

[0556] 2.11. Animals exhibiting the following conditions are excluded from the statistics.

[0557] Exclusion criteria: 1. Animals showing no behavioral changes within 24 hours of embolization. 2. Animals with subarachnoid hemorrhage during brain harvesting. 3. Animals that died during surgery or died without receiving medication post-surgery.

[0558] 3. Experimental Results

[0559] Thirty minutes after MCAO modeling in mice, mice were randomly and double-blindly administered either medication or saline. After administration, behavioral scores were assessed in each group. Mice with scores of 2-3 were included in the group, while mice without behavioral scores were excluded. The experimental procedure is as follows: Figure 21 After the experiment ended, the mouse brain was removed, and its sections were stained. Figure 22 The staining results showed that the embolic area in each treatment group was smaller than that in the saline control group. The cerebral embolic area was statistically analyzed after staining, and the results are shown below. Figure 23 The statistical results of cerebral embolism area showed that the embolism area in each group decreased compared with the saline control group after drug administration. The efficacy of the compound of this invention was stronger than that of AVLX-144, and the percentage decrease in embolism area was the highest in the compound 32 administration group.

[0560] The preferred embodiments of the present invention have been described in detail above, but the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention, and these equivalent modifications or substitutions are all included within the scope defined by the claims of this application.

Claims

1. A compound or a pharmaceutically acceptable salt thereof, characterized in that, The structure of the compound is as follows: 31、 32。 2. A pharmaceutical composition, characterized in that, The pharmaceutical composition comprises the compound of claim 1 or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

3. Use of the compound of claim 1 or a pharmaceutically acceptable salt thereof or the pharmaceutical composition of claim 2 in the preparation of a medicament for treating or improving diseases caused by PSD-95 dysfunction in an individual, wherein the diseases caused by PSD-95 dysfunction are selected from stroke.

4. The use according to claim 3, characterized in that, The stroke is selected from ischemic stroke or hemorrhagic stroke.

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