WO2019006692A1

WO2019006692A1 - Compound for treating, ameliorating, or preventing disease related to nervous system and use thereof

Info

Publication number: WO2019006692A1
Application number: PCT/CN2017/091794
Authority: WO
Inventors: 韩化敏; 田雨佳
Original assignee: 拜西欧斯（北京）生物技术有限公司
Priority date: 2017-07-05
Filing date: 2017-07-05
Publication date: 2019-01-10
Also published as: CN110799547A; CN110799547B

Abstract

The present application provides a compound having a structure shown in general formula (I): R1-S1-YEKL-S2-R2 (I). The present application also provides a use of the compound.

Description

Compounds for treating, ameliorating or preventing a nervous system related disorder and uses thereof

Technical field

This application relates generally to the field of biomedicine. In particular, the present application provides compounds and uses thereof for the treatment, amelioration or prevention of disorders associated with the nervous system.

Background of the invention

The cascade of pathways activated by the reaction of neurotransmitters with chemical receptors is associated with a variety of physiological activities in humans. The manifestations of nervous system-related diseases caused by abnormal pathways are diverse and seriously endanger people's health status and quality of life.

Stroke is a common acute cerebrovascular disease in middle-aged and elderly people, and tends to be younger. It is one of the three diseases (cancer, cardiovascular disease, diabetes) that are the most harmful to humans in the world today. According to statistics, China has nearly 3 million deaths from cerebrovascular diseases each year, 4 to 5 times higher than that of European and American countries, 3.5 times that of Japan, and even higher than that of developing countries such as Thailand and India; the incidence rate rises at an annual rate of 8.7%. The recurrence rate was over 30%, and the recurrence rate was 54% within 5 years; 75% of stroke survivors lost their ability to work and 40% were disabled.

Stroke can be broadly divided into two categories, namely ischemic stroke and hemorrhagic stroke, of which ischemic stroke accounts for 85% of the total number of stroke patients. At present, the therapeutic drugs for ischemic stroke are mainly divided into the following categories: vasodilators (such as dipyridamole), drugs that improve microcirculation, expand blood volume (such as low molecular dextran, etc.), drugs that dissolve thrombus (such as urine). Kinase, etc., anticoagulant therapy, drugs that prevent platelet aggregation (such as aspirin), traditional Chinese medicine, neuroprotective agents, etc., but because most of these drugs have side effects, potential risks, or ineffective effects, so research The pathogenesis of stroke and drug development for its mechanism have important social significance for the prevention and treatment of cerebrovascular disease.

Stroke is characterized by neuronal cell death in areas of ischemia, cerebral hemorrhage, and/or traumatic areas. Neuronal death or injury caused by cerebral ischemia is a process of injury cascade. After cerebral ischemia, blood perfusion decreases, excitatory neurotransmitters increase, and NMDA and AMPA receptors are activated, causing ion channel opening, calcium ion Inflow, activation of a large number of enzymes triggers a signal cascade that causes multiple pathways of neuronal damage. Its downstream post-synaptic density 95 protein (PSD-95) triggers a series of ischemic injuries through interaction with various proteins, is a key site for cerebral ischemic injury, and is also a potential target for drug therapy. Therefore, the development of PSD-95 inhibitors has great medicinal significance for neurological damage caused by various excitatory neurotoxicity including stroke.

In addition, studies have shown that the excitatory neurotransmitter NMDA is involved in anxiety, epilepsy, and various neurodegenerative diseases such as Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, or Huntington's disease. Both play an important role. For example, studies have shown that excessive excitation of the central glutamatergic system can cause anxiety, while the NMDA receptor (NMDAR) is responsible for a major part of glutamate excitotoxicity. The onset of epilepsy includes three different and continuous pathophysiological processes, including initiation, maintenance and expansion of episodic discharge, and inhibition of episodes. During this process, excitatory neurotransmitters such as glutamate and aspartate play an important role. . In Alzheimer's disease, PSD-95 is involved in the neurotoxic mechanism that leads to it through the GluR6-PSD-95-MLK3 pathway. In addition, in Huntington's disease, PSD-95 is a mediator of neurotoxicity of NMDA receptors and huntingtin mutants. Therefore, the development of PSD-95 inhibitors is also important for the treatment, improvement and prevention of the above diseases.

Summary of invention

In a first aspect, the present application provides a compound having the structure of the formula (I) or a pharmaceutically acceptable salt thereof,

R ¹ -S ¹ -YEKL-S ² -R ² (I)

among them

R ^{1 is} selected from the group consisting of hydrogen, pyroglutamic acid residue, C _1-18 alkyl group, C _3-18 cycloalkyl group, C _1-6 heterocyclic group, R ³ C(O)- or -NR ⁴ R ⁵ , Wherein R ^{3 is} independently selected from C _1-18 alkyl, C _3-18 cycloalkyl, C _1-6 heterocyclyl, and R ⁴ and R ⁵ are each independently selected from hydrogen, C _1-6 alkyl, C _3-6 cycloalkyl and C _1-6 heterocyclic;

S ¹ is an amino acid sequence of an internalization peptide;

S ² is the amino acid sequence of LDTEI or a functional variant thereof;

R ^{2 is} selected from the group consisting of -OH and -NR ⁶ R ⁷ , wherein R ⁶ and R ⁷ are each independently selected from the group consisting of hydrogen, C _1-6 alkyl, C _3-6 cycloalkyl, and C _1-6 heterocyclic.

In some embodiments, R ¹ is hydrogen or acetyl (Ac).

In some embodiments, R ² is -OH or -NH ₂ .

In some embodiments, S ^{1 is} selected from the group consisting of YGRKKRRQRRR (SEQ ID NO: 1), polyarginine of 2 to 30 residues, GRKKRRQRRRPPQQ (SEQ ID NO: 2), RQIKIWFQNRRMKWKK (SEQ ID NO: 3), GWTLNSAGYLLKINLKALAALAKKIL (SEQ ID NO: 4), GALFLAFLAAALSLMGLWSQPKKKRRV (SEQ ID NO: 5), RGGRLSYSRRRFSTSTGR (SEQ ID NO: 6), RRLSYSRRRF (SEQ ID NO: 7), KLALKLALKALKAALKLA (SEQ ID NO: 8) , GALFLGWLGAAGSTMGAWSQPKKKRKV (SEQ ID NO: 9).

In some embodiments, the amino acid sequence of S ¹ is YGRKKRRQRRR.

In some embodiments, functional variants of LDTEI include, but are not limited to, any of the modifications described below or any combination thereof:

L (leucine) is substituted with L or D isoleucine, β-leucine, β-leucine, norleucine, tert-leucine, iso- leucine, valine, --cyclopropylalanine, β-cyclopentylalanine, β-cyclohexylalanine, 2-amino-5-methylhexanoic acid, iso-proline, N-alkyl-leucine , N-alkyl-isoleucine, N-alkyl-β-leucine, N-alkyl-β-leucine, N-alkyl-norleucine, N-alkyl-tert Leucine, N-alkyl-isoisoleucine or N-alkyl-proline;

D (aspartic acid) is substituted with L or D type glutamic acid, asparagine, glutamine, N-alkyl aspartic acid, N-alkyl glutamic acid, N-alkyl aspartate Amide or N-alkyl glutamine;

T (threonine) is substituted with L or D type serine, N-alkylthreonine or N-alkylserine;

E (glutamic acid) is substituted with L or D type aspartic acid, asparagine, glutamine, N-alkyl aspartic acid, N-alkyl glutamic acid, N-alkyl aspartate Amide or N-alkyl glutamine;

I (isoleucine) is substituted with L or D leucine, β-leucine, β-leucine, norleucine, uncle Leucine, iso- leucine, valine, β-cyclopropylalanine, β-cyclopentylalanine, β-cyclohexylalanine, 2-amino-5-methylhexanoic acid ,isoproline, N-alkyl-leucine, N-alkyl-isoleucine, N-alkyl-β-leucine, N-alkyl-β-leucine, N- Alkyl-norleucine, N-alkyl-tert-leucine, N-alkyl-isoisoleucine or N-alkyl-valine.

In some embodiments, the alkyl group in the N-alkyl group is a _C1-10 alkyl group or a _C3-10 cycloalkyl group.

In some embodiments, the alkyl group in the N-alkyl group is a C _1-6 alkyl group or a C _3-6 cycloalkyl group.

In some embodiments, the alkyl group in the N-alkyl group is a C _1-4 alkyl group or a C _3-4 cycloalkyl group.

In some embodiments, the alkyl group in the N-alkyl group is a methyl group.

In some embodiments, a functional variant of LDTEI is a variant in which one or more amino acids in LDTEI are substituted with the corresponding D-amino acid.

In some embodiments, the functional variant of LDTEI is selected from the group consisting of: (D)-Leu-DTEI, L-(D)-Asp-TEI, LD-(D)-Thr-EI, LDT-(D)-Glu -I and LDTE-(D)-Ile.

In some embodiments, the compound has a structure selected from the group consisting of:

Ac-YGRKKRRQRRRYEKLLDTEI (SEQ ID NO: 10),

YGRKKRRQRRRYEKLLDTEI-NH ₂ (SEQ ID NO: 11,

YGRKKRRQRRRYEKL-(D)-Leu-DTEI (SEQ ID NO: 12),

YGRKKRRQRRRYEKLL-(D)-Asp-TEI (SEQ ID NO: 13),

YGRKKRRQRRRYEKLLD-(D)-Thr-EI (SEQ ID NO: 14),

YGRKKRRQRRRYEKLLDT-(D)-Glu-I (SEQ ID NO: 15),

YGRKKRRQRRRYEKLLDTE-(D)-Ile(SEQ ID NO: 16), or

YGRKKRRQRRRYEKLLDTEI (SEQ ID NO: 17).

In some embodiments, the pharmaceutically acceptable salt is selected from the group consisting of trifluoroacetate, acetate, hydrochloride, and phosphate.

In a second aspect, the present application provides a pharmaceutical composition comprising the compound of the first aspect, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient and/or diluent.

In some embodiments, the pharmaceutical composition is a pre-lyophilized formulation, preferably comprising histidine and trehalose.

In some embodiments, the pharmaceutical composition is a lyophilized formulation, preferably prepared by lyophilizing the pre-lyophilized formulation described above.

In some embodiments, the pharmaceutical composition is a reconstituted formulation, preferably prepared by combining the lyophilized formulation described above with an aqueous solution.

In some embodiments, the pharmaceutical composition is for treating, ameliorating or preventing a nervous system injury, a disease or pain associated with nervous system damage, a neurodegenerative disease, anxiety or epilepsy in an individual.

In some embodiments, the pharmaceutical composition is used as a neuroprotective agent.

In a third aspect, the present application provides a method of treating, ameliorating or preventing a nervous system injury, a neurological damage-related disease or pain, a neurodegenerative disease, anxiety or epilepsy in an individual, the method comprising administering to a subject in need thereof A compound according to one aspect or the pharmaceutical composition of the second aspect.

In a fourth aspect, the present application provides the compound of the first aspect, or a pharmaceutically acceptable salt thereof, or a second party The pharmaceutical composition described in the preparation of a medicament for treating, ameliorating or preventing a nervous system injury, a nervous system damage-related disease or pain, a neurodegenerative disease, anxiety or epilepsy in an individual or in preparing a neuroprotective agent Use in.

In some embodiments of the second to fourth aspects, the nervous system damage is a nervous system damage caused by excitatory neurotoxicity.

In some embodiments of the second to fourth aspects, the neurological damage caused by excitotoxicity comprises an ischemic or traumatic injury selected from the group consisting of stroke, spinal cord injury, brain or spinal cord, central nervous system (CNS) Neuronal damage, including acute CNS injury, ischemic stroke, or spinal cord injury, as well as hypoxia, ischemia, mechanical injury, and neurodegenerative diseases, anxiety, epilepsy, and stroke-induced damage.

In some embodiments of the second to fourth aspects, the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, or Huntington's disease.

In some embodiments of the second to fourth aspects, the nervous system injury or pain is located in the peripheral nervous system or the central nervous system.

In some embodiments of the second to fourth aspects, the neurological damage-related disease is stroke.

In some embodiments of the second to fourth aspects, the stroke is selected from the group consisting of ischemic stroke, hemorrhagic stroke, and hemorrhagic stroke converted from ischemic stroke.

In some embodiments of the second to fourth aspects, the stroke is an ischemic stroke.

In some embodiments of the second to fourth aspects, the individual is a mammal, such as a non-primate or primate, such as a human.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 shows the Pull-down assay to detect the interaction of the chimeric peptide No. 8 with the PDZ1/2 domain. M represents the molecular weight marker of the protein; Lane 1 is the His+PDZ1/2+8 chimeric peptide; Lane 2 is the single chimeric peptide No. 8; Lane 3 is the His+8 chimeric peptide; Lane 4 is His+PDZ1/ 2. The elution band shown in lane 1 contains both the chimeric peptide No. 8 and PDZ1/2, confirming that the chimeric peptide No. 8 is capable of binding to the PDZ1/2 domain.

Detailed description of the invention

The inventors of the present application conducted intensive studies on peptides that are capable of reducing the damaging effects of at least a portion of NMDAR-excitatory neurotoxicity-mediated neurological disorders. Without wishing to be bound by any theory, it is believed that such peptides function, at least in part, by inhibiting the interaction between NMDAR and postsynaptic density 95 protein (PSD-95) (i.e., PSD-95 inhibitors). On this basis, the inventors of the present application have intensively considered various therapeutic targets of diseases related to nervous system, and carried out the design and screening of polypeptide neuroprotective agents through pharmacological and pharmacological experiments in vitro and in vivo. A polypeptide sequence having desirable properties.

definition

Unless otherwise indicated, the terms used in this application have the meaning commonly understood by those skilled in the art.

The one-letter or three-letter abbreviations used in the present application for amino acids follow international conventions.

In the general formula (I) of the present application, S ¹ , YEKL and S ² represent a peptide sequence in which a single-letter abbreviation or a three-letter abbreviation is used to represent an amino acid residue. For example, YEKL in the general formula (I) and LDTEI in the S ² are all understood to represent peptides consisting of one-letter abbreviations of amino acid residues.

In the general formula (I) of the present application, R ¹ , R ² and the like represent chemical modifications, the meaning of which is generally understood by those skilled in the art, unless otherwise indicated.

For amino acid residues present in the peptide sequences of the present application, unless otherwise indicated, it is to be understood as an L-form residue, ie, a form in which the native state is present. In some descriptions involving D-type amino acids, it is specified in the form of a (D)-amino acid three letter abbreviation. For example, (D)-Leu-DTEI means that the first leucine (L) is D type, DTEI is L type, and so on.

The term "alkyl" refers to a fully saturated aliphatic hydrocarbon group. C _1-18 alkyl refers to a straight or branched alkyl group having from 1 to 18 carbon atoms in the chain, including C ₁ , C ₂ , C ₃ , C ₄ , C ₅ , C ₆ , C ₇ , C ₈ , C ₉ , C ₁₀ , C ₁₁ , C ₁₂ , C ₁₃ , C ₁₄ , C ₁₅ , C ₁₆ , C ₁₇ , C ₁₈ alkyl. The C _1-6 alkyl group means a straight or branched alkyl group having 1 to 6 carbon atoms in the chain, and includes C ₁ , C ₂ , C ₃ , C ₄ , C ₅ , C ₆ alkyl groups. Examples of alkyl groups include, but are not limited to, methyl, ethyl (Et), n-propyl, isopropyl, butyl, isobutyl, sec-butyl, t-butyl ^(t Bu), pentyl, isopentyl , tert-amyl, hexyl, isohexyl.

The term "cycloalkyl" refers to a saturated or partially saturated carbocyclic ring of a monocyclic, fused polycyclic, bridged monocyclic, bridged polycyclic, spiro or spiro polycyclic carbocyclic ring having at least 3 ring atoms per carbocyclic ring. . C _3-18 cycloalkyl means that each carbocyclic ring has 3 to 18 ring atoms (for example, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17 ,18 pcs). C _3-6 cycloalkyl means that each carbocyclic ring has 3 to 6 ring atoms (for example, 3, 4, 5, 6). Illustrative examples of cycloalkyl groups include the following entities in the form of suitable bonding moieties:

"Heterocyclyl" means saturated or partially saturated and each ring structure has at least 3 ring atoms selected from carbon atoms and up to three heteroatoms or fused multiples selected from nitrogen, oxygen and sulfur. Ring-ring structure, bridge polycyclic ring structure or spiro polycyclic ring structure. The C _1-6 heterocyclic group means that each ring structure has 1 to 6 carbon atoms as a ring atom. Illustrative examples of heterocyclic groups include the following entities in the form of suitably bonded moieties:

The term "PDZ domain" refers to a modular protein domain of approximately 90 amino acids characterized by a synaptic protein PSD-95, a Drosophila-separating connexin, a Discs-Large (DLG), and an epithelial tight junction protein Z01. (Z01) has significant (eg, at least 60%) sequence identity. The PDZ domain is also known as Discs-Large homology repeats ("DHRs") and GLGF repeats. The PDZ domain typically displays a retained core consensus sequence (Doyle, D.A., 1996, Cell 85: 1067-76). Exemplary PDZ domain-containing proteins and PDZ domain sequences are disclosed in U.S. Patent Application Serial No. 10/714,537.

The term "NMDA receptor" or "NMDAR" refers to a membrane associated protein known to interact with NMDA. These receptors can be human or non-human (eg, mice, rats, rabbits, monkeys, etc.).

The term "specifically binds" refers to a bond between two molecules (eg, a ligand and a receptor) characterized by one molecule (ligand) and another specific molecule in the presence of many other different molecules ( The ability of a receptor to bind, ie, the ability to display a preferential binding of one molecule to another in a heterogeneous mixture of molecules. Specific binding of the ligand to the receptor is also demonstrated as follows: When an excess of unlabeled ligand is present, the binding of the detectably labeled ligand to the receptor is reduced (i.e., binding competition assay).

The term "functional variant" refers to a variant having the same or similar biological function and properties as the parent. As a non-limiting example, a "functional variant" can be obtained by performing one or more substitutions (eg, conservative substitutions or D-type amino acid residue substitutions) in the parent.

The term "lyophilization" relates to a process by which the material to be dried is first frozen and then biochemically removed under vacuum to remove ice or frozen solvent.

Statistically significant means a p value of <0.05, preferably <0.01, most preferably <0.001.

The words "including", "comprising" and "comprising" are intended to mean "including, but not limited to", and are not intended to exclude other parts, additions, components or steps.

R ¹ -S ¹ -YEKL-S ² -R ² (I)

among them

S ¹ is an amino acid sequence of an internalization peptide;

S ² is the amino acid sequence of LDTEI or a functional variant thereof;

In the general formula (I), S ¹ , YEKL and S ² represent three peptide sequences, and R ¹ and R ² represent different chemical modifications which may be present. S ¹ is an internalization peptide sequence whose function is to promote uptake and absorption of active peptides bound thereto by cells. YEKL+S ² is understood to be a reactive peptide moiety and plays a key role in the biological activity of the compounds of formula (I).

According to prior studies, some active peptides that inhibit the interaction between NMDAR and PSD-95 are NMDAR-based structures. For example, NMDAR2B has GenBank ID 4099612 and the C-terminal 20 amino acids are FNGSSNGHVYEKLSSLESDV and PL motif ESDV. Some of the existing active peptides have selected a partial amino acid sequence at the C-terminus of NMDAR2B, thereby producing competitive inhibition of PSD-95 with NMDAR2B. Studies have suggested that the ESDV or LESDV segments in the above peptides play an important role in inhibiting the interaction between NMDAR and PSD-95 proteins. The inventors of the present application obtained the peptide sequence YEKLLDTEI by analysis and verification, which has a C-terminal amino acid composition with respect to the above-mentioned NMDAR2B, does not contain two residues of SS after KL, and increases the N-terminal direction of YEKL with respect to the PL motif. The amino acid sequence, the inventors of the present application confirmed that this sequence enhances the interaction of the active peptide with the PDZ1/2 domain. The C-terminal LDTEI can be varied relative to the YEKL motif and is not expected to affect the activity of the active peptide or possibly increase its activity. Based on this, the inventors of the present application have developed a compound having a structure represented by the general formula (I).

Without being bound by any particular theory, certain chemical modifications may be made to the N-terminus and/or C-terminus of a biological peptide molecule, or substitution of one or more sites may be performed with a D-form amino acid residue, possibly maintaining biological activity. At the same time, the metabolic stability and bioavailability of the biopeptide molecule are improved, so that the compound of the structure represented by the general formula (I) is expected to have the same or similar biological activity, and may also be in other properties than biological activity. Advantages.

In some specific embodiments, R ¹ is hydrogen or acetyl.

In some specific embodiments, R ² is -OH or -NH ₂ .

The "internalized peptide" represented by S ¹ may also be referred to as a penetrating peptide, and is widely used in the field of protein medicine, and its function is to promote the uptake and absorption of active peptides bound thereto by cells. The internalization peptide and the active peptide represented by YEKL+S ² can form a chimeric peptide, and the internalization peptide can promote the uptake of the chimeric peptide by the cell. It will be understood by those skilled in the art that the purpose of chimerizing the active peptide and the internalization peptide is mainly to make the active peptide better reach the target of the action. Therefore, the internalization peptide suitable for the present application is not limited to a specific species, as long as The purpose of transmembrane and internalization can be achieved. It will also be understood by those skilled in the art that since the target of action of the active peptide is mainly located inside the neuronal cell, it is preferred that the internalization peptide which is specifically adapted to the neuronal cell. In some embodiments, S ^{1 is} selected from the group consisting of YGRKKRRQRRR (SEQ ID NO: 1), polyarginine of 2 to 30 residues, GRKKRRQRRRPPQQ (SEQ ID NO: 2), RQIKIWFQNRRMKWKK (SEQ ID NO: 3), GWTLNSAGYLLKINLKALAALAKKIL (SEQ ID NO: 4), GALFLAFLAAALSLMGLWSQPKKKRRV (SEQ ID NO: 5), RGGRLSYSRRRFSTSTGR (SEQ ID NO: 6), RRLSYSRRRF (SEQ ID NO: 7), KLALKLALKALKAALKLA (SEQ ID NO: 8), GALFLGWLGAAGSTMGAWSQPKKKRKV (SEQ ID NO: 9). In some embodiments, the amino acid sequence of S ¹ is YGRKKRRQRRR.

It should be appreciated that the peptide YEKL + S ¹ and S ² between the active peptide represented may be connected by an amide bond to form a fusion peptide, but may also be joined by other suitable means, for example chemically bonded. Further, the active peptide represented by the internalization peptide S ¹ and YEKL+S ² may be linked by other linking arms, such as hydrophilic linking arms of 1-6 hydrophilic amino acid residues, polyethylene glycol, polyamide, and the like.

In some embodiments, a functional variant of LDTEI comprises a variant obtained by substituting one or more residues therein, the substitutions may comprise any of the following or any combination thereof:

I (isoleucine) is substituted with L or D leucine, β-leucine, β-leucine, norleucine, tert-leucine, iso- leucine, valine, --cyclopropylalanine, β-cyclopentylalanine, β-cyclohexylalanine, 2-amino-5-methylhexanoic acid, iso-proline, N-alkyl-leucine , N-alkyl-isoleucine, N-alkyl-β-leucine, N-alkyl-β-leucine, N-alkyl-norleucine, N-alkyl-tert Leucine, N-alkyl-isoisoleucine or N-alkyl-valine.

In some embodiments, the alkyl group in the N-alkyl group is a methyl group.

In some embodiments, a functional variant of LDTEI comprises a variant obtained by one or more residues thereof following one or more of the following substitutions:

L (leucine) is substituted with L or D isoleucine, β-leucine, β-leucine, norleucine, tert-leucine, iso- leucine, valine, --cyclopropylalanine, β-cyclopentylalanine, β-cyclohexylalanine, 2-amino-5-methylhexanoic acid, iso-proline, N-methyl-leucine , N-methyl-isoleucine, N-methyl-β-leucine, N-methyl-β-leucine, N-methyl-norleucine, N-methyl-tert Leucine, N-methyl-discolor Or N-methyl-proline;

D (aspartic acid) is substituted with L or D type glutamic acid, asparagine, glutamine, N-methyl aspartic acid, N-methyl glutamic acid, N-methyl aspartate Amide or N-methyl glutamine;

T (threonine) is substituted with L or D type serine, N-methylthreonine or N-methylserine;

E (glutamic acid) is substituted with L or D type aspartic acid, asparagine, glutamine, N-methyl aspartic acid, N-methyl glutamic acid, N-methyl aspartate Amide or N-methyl glutamine;

I (isoleucine) is substituted with L or D leucine, β-leucine, β-leucine, norleucine, tert-leucine, iso- leucine, valine, --cyclopropylalanine, β-cyclopentylalanine, β-cyclohexylalanine, 2-amino-5-methylhexanoic acid, iso-proline, N-methyl-leucine , N-methyl-isoleucine, N-methyl-β-leucine, N-methyl-β-leucine, N-methyl-norleucine, N-methyl-tert Leucine, N-methyl-isoisoleucine or N-methyl-proline.

In some embodiments, the functional variants disclosed herein further comprise the same as at least 60%, 70%, 75%, 80%, 85%, 90%, 95%, or even higher than the peptides mentioned above. Sexual amino acid sequence. It is known in the art that "identity" between two proteins is determined by aligning the sequence of a second protein substituted with the amino acid sequence of one protein and its conserved amino acid. The degree of identity between the two proteins is determined using computer algorithms and methods well known to those skilled in the art. The identity between two amino acid sequences is preferably determined by using the BLASTP algorithm.

In some embodiments, the functional variants disclosed herein include substitutions, deletions, additions and/or insertions having one or more amino acid residues compared to LDTEI, which are distinguished from the specific peptides disclosed above.

As noted above, a functional variant can be distinguished from a particular peptide disclosed above by one or more substitutions, deletions, additions, and/or insertions. These variants may be naturally occurring or may be synthetically produced, for example, by modifying one or more of the above-described peptide sequences disclosed herein and evaluating them according to any of a variety of techniques well known in the art as described herein. Biological activity.

In some embodiments, the compound of formula (I) has a structure selected from the group consisting of:

Ac-YGRKKRRQRRRYEKLLDTEI (SEQ ID NO: 10),

YGRKKRRQRRRYEKLLDTEI-NH ₂ (SEQ ID NO: 11,

YGRKKRRQRRRYEKL-(D)-Leu-DTEI (SEQ ID NO: 12),

YGRKKRRQRRRYEKLL-(D)-Asp-TEI (SEQ ID NO: 13),

YGRKKRRQRRRYEKLLD-(D)-Thr-EI (SEQ ID NO: 14),

YGRKKRRQRRRYEKLLDT-(D)-Glu-I (SEQ ID NO: 15),

YGRKKRRQRRRYEKLLDTE-(D)-Ile(SEQ ID NO: 16), or

YGRKKRRQRRRYEKLLDTEI (SEQ ID NO: 17).

As a non-limiting exemplary method, the R ¹ group can be introduced directly at the time of solid phase synthesis. For example, after the last amino acid of the N-terminus is attached to the solid support, the N-terminal protecting group of the amino acid is removed, and the corresponding modifier having the R ¹ group is added, which condenses with the amino group of the N-terminal amino acid to form an amide bond or other Chemical bond. For example, the introduction of an acetyl group can be carried out by adding acetic acid after deprotecting the N-terminal protecting group, which condenses with the amino group of the N-terminal amino acid to form an amide bond.

As a non-limiting exemplary method, the introduction of the R ² group can be carried out by solid-phase synthesis of the polypeptide sequence, and the polypeptide sequence is cleaved from the resin using an amino cleavage agent having an R ² group to obtain a band. There are different C-terminally modified polypeptide sequences. Wherein, when R ² is NH ₂ , Rink amide resin can be used as a carrier, and solid phase synthesis can be directly carried out by cleavage with trifluoroacetic acid.

Without wishing to be bound by any theory, it may be desirable to improve certain undesirable physicochemical or biopharmaceutical properties of the drug, such as altering the solubility or dissolution of the drug, by combining a molecule or ion that is oppositely charged with the drug to form a salt with the drug. , reduce hygroscopicity, improve stability, change the melting point, and the like. The final determination of the ideal salt form requires a balance between physicochemical properties and biopharmaceutical properties. The choice of the pharmaceutically acceptable salt form of the drug should give priority to the following requirements: solubility, wettability, stability to environmental factors under different conditions. The pharmaceutically acceptable salts of the compounds of the present application may be in any suitable pharmaceutically acceptable salt form. In some embodiments, the pharmaceutically acceptable salt of the compound is a trifluoroacetate salt. In some embodiments, the pharmaceutically acceptable salt of the compound is an acetate salt. In some embodiments, the pharmaceutically acceptable salt of the compound is the hydrochloride salt. In some embodiments, the pharmaceutically acceptable salt of the compound is a phosphate. In some embodiments, the pharmaceutically acceptable salt of the compound is an acetate or hydrochloride salt.

In some embodiments, the compounds of the present application can be administered in the form of a pharmaceutical composition. The pharmaceutical compositions can be made by conventional methods of mixing, dissolving, granulating, tableting, milling, emulsifying, encapsulating, capturing or lyophilizing. The pharmaceutical composition can be formulated in a conventional manner using one or more physiologically acceptable carriers, diluents, excipients or auxiliaries which facilitate processing the compounds of the present application into pharmaceutically acceptable formulations. Proper formulation depends on the route of administration chosen.

In some embodiments, administration can be parenteral, intravenous, oral, subcutaneous, intraarterial, intracranial, intrathecal, intraperitoneal, topical, intranasal, or intramuscular. It is preferably administered intravenously.

In some embodiments, the pharmaceutical composition for parenteral administration is preferably sterile and substantially isotonic. For injection, the compounds of the present application can be formulated into aqueous solutions, preferably in physiologically compatible buffers such as Hank's solution, Ringer's solution, or physiological saline or acetate buffer (to alleviate discomfort at the site of injection) . The solution may contain formulas such as suspending, stabilizing and/or dispersing agents.

Alternatively, the compounds of the present application may be in the form of a powder for constitution with a suitable vehicle, such as sterile non-pyrogenic water, prior to use.

For transmucosal administration, penetrants appropriate to the barrier to be penetrated are used in the formulation. This route of administration can be used to deliver a compound to the nasal cavity or for sublingual administration.

In some embodiments, for oral administration, a compound of the present application, or a pharmaceutically acceptable salt thereof, can be formulated with a pharmaceutically acceptable carrier into tablets, pills, lozenges, capsules, liquids, gels, Syrup, slurry, Suspensions, etc., for oral ingestion by the patient being treated. For oral solid formulations such as powders, capsules and tablets, suitable excipients include fillers such as sugars such as lactose, sucrose, mannitol and sorbitol; cellulose preparations such as corn starch, wheat starch, rice starch, Potato starch, gelatin, tragacanth, methylcellulose, carboxypropylmethylcellulose, sodium carboxymethylcellulose and/or povidone (PVP); granulating agents and binders. If necessary, a disintegrating agent such as crosslinked polyvinylpyrrolidone, agar, or alginic acid or a salt thereof such as sodium alginate may be added. If desired, the solid dosage form can be sugar coated or enteric coated using standard techniques. For oral liquid preparations such as suspensions, elixirs and solutions, suitable carriers, excipients or diluents include water, glycerol, oil, alcohol. Further, a flavoring agent, a preservative, a coloring agent, or the like may be added.

In addition to the formulations previously described, the compounds of the present application, or pharmaceutically acceptable salts thereof, may also be formulated as a depot preparation. Such long acting formulations can be administered by implantation (for example subcutaneously or intramuscularly) or by intramuscular injection. Thus, for example, the compound can be formulated with a suitable polymeric or hydrophobic material (for example, as an emulsion in an acceptable oil) or an ion exchange resin, or as a sparingly soluble derivative, for example, formulated as a slightly soluble solution. Salt.

Alternatively, other drug delivery systems can be used. Chimeric peptides can be delivered using liposomes and emulsions. Certain organic solvents such as dimethyl sulfoxide can also be used. Additionally, the compound can be delivered using a sustained release system, such as a semipermeable matrix of a solid polymer containing a therapeutic agent.

Depending on its chemical nature, sustained release capsules release the chimeric peptide for several weeks up to over 100 days. Other strategies for protein stabilization can be used depending on the chemical nature and biostability of the therapeutic agent.

The compound of the first aspect of the present application, or a pharmaceutically acceptable salt thereof, can be prepared in the form of a lyophilized preparation. In some embodiments, the application provides a lyophilized formulation. The lyophilized preparation is prepared from a pre-lyophilized preparation by lyophilization comprising at least an active ingredient, a buffer, a filler and water, wherein the active ingredient is a compound of the present application or a pharmaceutically acceptable salt thereof. In some embodiments, a preferred buffer is histidine. Other buffers are selected from the group consisting of succinate, citrate, gluconate, acetate, phosphate, and Tris. Fillers provide structure for the lyophilized compound. In some embodiments, the filler is selected from the group consisting of mannitol, trehalose, dextran-40, glycine, lactose, sorbitol, and sucrose, and the like, with trehalose being preferred. In some embodiments, the lyophilized formulation of the present application comprises a compound described above, or a pharmaceutically acceptable salt thereof, and histidine and trehalose.

The lyophilized formulation can be reconstituted by rehydrating the lyophilized formulation with a solution to a solution of microparticles that are invisible to the naked eye. In some embodiments, the application provides a reconstituted formulation prepared by combining a lyophilized formulation with an aqueous solution. In some embodiments, the aqueous solution is water for injection. In some embodiments, the aqueous solution is physiological saline.

The compounds of the present application, or pharmaceutically acceptable salts thereof, are used in an amount effective to achieve the intended purpose (e.g., to reduce the damaging effects of damaging stroke and related conditions). A therapeutically effective amount means that in a patient (or animal model population) treated with a compound of the present application, relative to a central nervous system injury in a control population of a patient (or animal model) not treated with a compound of the present application The amount of compound that significantly reduces the damage caused by stroke. If an individual treated patient achieves a better output than a mean output in a comparable patient control population that is not treated by the methods disclosed herein (as determined by infarct volume or disability index), then the amount is also considered to be It is therapeutically effective. If the individual being treated shows 2 or fewer disability in the Rankin scale and The amount shown in the Barthel scale is 75 or more, and the amount is also considered to be a therapeutically effective amount. The dose is also considered therapeutically effective if the population of treated patients shows a significant improvement (ie less disability) score distribution on the disability scale compared to comparable untreated populations, see Lees et al. N Engl J Med 2006; 354: 588-600. A therapeutically effective regimen represents a combination of a therapeutically effective dose and the frequency of administration required to achieve the above intended purpose.

In some embodiments, a preferred dosage range for the pharmaceutical composition comprises administering from 0.001 to 20 [mu]mol of the compound of the present application per kg of patient body weight, optionally 0.03 to 3 [mu]mol of the compound of the present application per kg of patient body weight, including any values therebetween. Or a range between any two values. In some methods, 0.1-20 [mu]mol of the compound of the present application is administered per kg patient body weight over 6 hours. In some methods, 0.1 to 10 μmol of the compound of the present application is administered per kg of patient body weight within 6 hours, more preferably about 0.3 μmol of the compound of the present application per kg of patient body weight within 6 hours. In other instances, the dosage range is from 0.005 to 0.5 [mu]mol of the pharmaceutical composition of the present application per kg patient body weight. The different surface area: mass ratio can be compensated by dividing by 6.2, while the dose per kg of body weight is converted from rat to human. A suitable dose of the compound of the present application for humans in grams may be 0.01 to 100 mg/kg of patient body weight, or more preferably 0.01 to 30 mg/kg of patient body weight or 0.01 to 10 mg/kg of patient body weight, or 0.01 to 1 mg/kg. Patient weight, including any value in between or a range between any two values.

In some embodiments, the amount of a compound of the present application, or a pharmaceutically acceptable salt thereof, is administered depending on the subject being treated, the weight of the subject, the severity of the pain, the mode of administration, and the modulation of the prescribing physician. The treatment can be repeated when the symptoms are detectable or even undetectable. Treatment can be provided alone or in combination with other drugs.

In some embodiments, a therapeutically effective dose of a compound of the present application, or a pharmaceutically acceptable salt thereof, is capable of providing a therapeutic benefit without causing significant toxicity. The toxicity of the chimeric peptide can be determined in cell cultures or experimental animals by standard pharmaceutical procedures, for example by measuring LD50 (a dose that kills 50% of the population) or LD100 (a dose that kills 100% of the population). The dose ratio between toxic and therapeutic effects is the therapeutic index. Compounds of the present application which exhibit a high therapeutic index are preferred (see, for example, Fingl et al, 1975, In: The Pharmacological Basis of Therapeutics, Chapter 1, page 1).

In some embodiments, the nervous system damage is a nervous system damage caused by excitotoxicity.

In some embodiments, neurological damage caused by excitotoxicity comprises an episode of stroke, spinal cord injury, ischemic or traumatic injury of the brain or spinal cord, damage to central nervous system (CNS) neurons, including acute CNS Injury, ischemic stroke or spinal cord injury, as well as hypoxia, ischemia, mechanical injury and neurodegenerative diseases, anxiety, epilepsy, stroke-induced damage.

In some embodiments, the pharmaceutical composition is for treating, ameliorating or preventing neurological damage caused by ischemic stroke or ischemic stroke. In some embodiments, the pharmaceutical composition is for treating, ameliorating or preventing neurological damage caused by hemorrhagic stroke or hemorrhagic stroke. In some embodiments, the pharmaceutical composition is for treating, ameliorating or preventing hemorrhagic stroke converted from ischemic stroke or is transformed by ischemic stroke Nervous system damage caused by hemorrhagic stroke.

Stroke is a condition caused by impaired blood flow in the CNS. Possible causes include embolism, bleeding, and thrombosis. Some neuronal cells die immediately due to impaired blood flow. These cells release their component molecules (including glutamate), which then activate the NMDA receptor, which increases intracellular calcium levels and intracellular enzyme levels, resulting in more neuronal cell death ( Excitatory neurotoxicity cascade amplification). The death of the CNS organization is called infarction. The infarct volume (i.e., the volume of neuronal cells in the brain caused by stroke) can be used as an indicator of the extent of pathological damage caused by stroke. Symptomatic effects depend both on the infarct volume and on where the infarct is located in the brain. Disability index can be used as a measure of symptomatic damage, such as the Rankin Stroke Outcome Scale (Rankin, Scott MedJ; 2:200-15 (1957)) and the Barthel Index (Barthel Index). The Rankin scale is based on a direct assessment of the patient's global condition as follows:

0 is completely symptom free.

1 Despite symptoms but no significant disability; able to perform all daily work and activities.

2 minor disability; unable to perform all previous activities, but able to take care of their own affairs

No need for help.

3 A moderate disability that requires some help, but is able to walk without help.

4 moderate to severe disability, unable to walk without help, and unable to take photos without help

Take care of your own body needs.

5 severe disability; bedridden, incontinence, and need for lasting care and attention.

The Barthel Index is based on a series of questions about the patient's ability to perform 10 basic activities of daily living, which scores between 0 and 100, with lower scores indicating more disability (Mahoney et al., Maryland State Medical Journal) 14:56-61 (1965).

Alternatively, stroke severity/output can be measured using the NIH Stroke Scale, available on the World Wide Web at ninds.nih.gov/doctors/NIH_Stroke_Scale_Booklet.pdf. The scale is based on the patient's ability to perform 11 sets of functions, including assessing the patient's level of consciousness, movement, feel, and language function.

Ischemic stroke more clearly indicates a type of stroke caused by blockage of blood flow to the brain. The potential for such blockages is most commonly the occurrence of fatty deposits along the walls of blood vessels. This condition is called atherosclerosis. These fat deposits can cause two types of obstruction. Cerebral thrombosis refers to a thrombus (blood clot) that is produced in the obstructed portion of a blood vessel. "Cerebral embolism" usually refers to various emboli in the blood (such as a wall thrombus in the heart, atherosclerotic plaque, fat, tumor cells, fibrocartilage or air, etc.) blocked by blood flow into the cerebral artery. Blood vessels, when the collateral circulation can not be compensated, cause ischemic necrosis of brain tissue in the blood supply area of the artery, and focal neurological deficit occurs. The second important cause of embolism is an irregular heartbeat called arterial fibrillation. It causes a condition in which a blood clot can form in the heart, move and transfer to the brain. Other potential causes of ischemic stroke are hemorrhage, thrombosis, arterial or venous severing, cardiac arrest, shock from any cause (including bleeding), and iatrogenic causes, such as cerebral blood vessels or blood vessels leading to the brain. Direct surgical injury or cardiac surgery. Ischemic stroke constitutes approximately 83% of all stroke cases.

Several other neurological disorders can also cause neuronal death through NDMAR-mediated excitotoxicity. These conditions include neurodegenerative diseases, anxiety, epilepsy, hypoxia, trauma to the CNS unrelated to stroke such as traumatic brain injury and spinal cord injury. Accordingly, in some embodiments, the pharmaceutical composition is for treating, ameliorating or preventing a neurodegenerative disease, anxiety or epilepsy, wherein the neurodegenerative disease comprises Alzheimer's disease, amyotrophic lateral sclerosis ( ALS), Parkinson's disease or Huntington's disease.

In some embodiments, the compounds of the present application are effective to reduce the severity of cerebral infarction caused by cerebral ischemia.

In a third aspect, the present application provides a method of treating, ameliorating or preventing a nervous system injury, a neurological damage-related disease or pain, a neurodegenerative disease, anxiety or epilepsy in an individual, the method comprising administering to a subject in need thereof A compound according to one aspect, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to the second aspect.

In some embodiments, in some embodiments, the nervous system injury is a neurological damage caused by excitatory neurotoxicity, wherein the injury or pain is located in the peripheral nervous system or the central nervous system. In some embodiments, neurological damage caused by excitatory neurotoxicity comprises an episode of stroke or spinal cord injury, ischemic or traumatic injury of the brain or spinal cord, and damage to central nervous system (CNS) neurons, including acute CNS. Injury, ischemic stroke or spinal cord injury, as well as hypoxia, ischemia, mechanical injury and neurodegenerative diseases, anxiety, epilepsy, stroke-induced damage.

In some embodiments, the neurodegenerative disease includes Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease, or Huntington's disease.

In some embodiments, the disease is a nervous system injury caused by ischemic stroke or ischemic stroke. In some embodiments, the disease is a neurological injury caused by a hemorrhagic stroke or a hemorrhagic stroke. In some embodiments, the disease is a neurological injury caused by a hemorrhagic stroke converted from ischemic stroke or a hemorrhagic stroke converted from ischemic stroke.

In a fourth aspect, the present application provides the compound of the first aspect, or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition of the second aspect, for the preparation, for treating, ameliorating or preventing neurological damage, nervous system in an individual A drug for injury-related diseases or pain, neurodegenerative diseases, anxiety or epilepsy or use in the preparation of a neuroprotective agent.

In some embodiments, the nervous system injury is a neurological injury caused by excitotoxicity, wherein the injury or pain is located in the peripheral nervous system or the central nervous system. In some embodiments, neurological damage caused by excitatory neurotoxicity comprises an episode of stroke or spinal cord injury, ischemic or traumatic injury of the brain or spinal cord, and damage to central nervous system (CNS) neurons, including acute CNS. Injury, ischemic stroke or spinal cord injury, as well as hypoxia, ischemia, mechanical injury and neurodegenerative diseases, anxiety, epilepsy, stroke-induced damage.

In some embodiments, the disease is a nervous system injury caused by ischemic stroke or ischemic stroke. In some embodiments, the disease is a neurological damage caused by a hemorrhagic stroke or a hemorrhagic stroke hurt. In some embodiments, the disease is a neurological injury caused by a hemorrhagic stroke converted from ischemic stroke or a hemorrhagic stroke converted from ischemic stroke.

As used herein, "individual" refers to an animal comprising birds, reptiles, and mammals. In some embodiments, the animal is a mammal, including primates and non-primates, such as humans, chimpanzees, cows, horses, pigs, sheep, goats, dogs, cats, and such as rats and mice. Rodents.

It is to be understood that the foregoing detailed description is only to be understood as Those skilled in the art will be able to make various modifications and variations to the described embodiments.

Example

The following examples are provided to illustrate some embodiments of the present application without any limitation of purpose or nature.

Example 1: Screening of active peptide molecules

Based on the reported results, the Tat transmembrane peptide YGRKKRRQRRR (SEQ ID NO: 1) was selected and ligated to a different number of amino acids to form a peptide library. The chimeric peptide molecules in the peptide library were respectively interacted with the PDZ1/2 domain expressed and purified in vitro, and the polypeptide was initially screened according to the strength of the interaction force.

The immobilized molecule (ligand) is PDZ1/2 protein, molecular weight: ~20kD, concentration: 2mg/ml; molecular phase of mobile phase (analyte): polypeptide to be screened, molecular weight: ~2kD, concentration: 10mg/ml. The CM5 chip was used for fixation using a Biacore 3000 instrument. The running buffer was PBS + 0.005% Tween 20. Fixation was carried out using an amino coupling method. The concentration of the ligand was 10 μg/ml. The fixing buffer was 10 mM sodium acetate, pH 4.0. Fixed amount: 1400 RU, fixed to flow cells 2. The flow rate used was 10 μl/ml and the ligand was injected for 1 minute. 10 mM Gly at pH 2.0 + 2.5 was used as a regenerant, and regeneration was carried out at a flow rate of 30 μl/min. The injection time is 30s.

Kinetic analysis was performed using the following conditions: control channel: flow cell 1; electrophoresis buffer was PBS; concentration gradient was 6.25n, 12.5n, 25n, 50n, 100n, 200n, 400nM using Kinetic Analysis Wizard mode; injection time It was 1 minute; the dissociation time was 2 min; the flow rate was 30 μl/min.

The data was fitted using the Biaevaluation 4.1 software. The quasi-sum model is a 1:1 binding model. The dissociation constant KD value is inversely proportional to the force.

By screening, 8 chimeric peptides with strong interaction ability with PDZ1/2 domain were obtained, and the structure was as follows. The interaction between the chimeric peptide No. 8 (SEQ ID NO: 17) and the PDZ1/2 domain was observed. The strongest:

1.Ac-YGRKKRRQRRRYEKLLDTEI (SEQ ID NO: 10),

2. YGRKKRRQRRRYEKLLDTEI-NH ₂ (SEQ ID NO: 11,

3. YGRKKRRQRRRYEKL-(D)-Leu-DTEI (SEQ ID NO: 12),

4. YGRKKRRQRRRYEKLL-(D)-Asp-TEI (SEQ ID NO: 13),

5. YGRKKRRQRRRYEKLLD-(D)-Thr-EI (SEQ ID NO: 14),

6. YGRKKRRQRRRYEKLLDT-(D)-Glu-I (SEQ ID NO: 15),

7. YGRKKRRQRRRYEKLLDTE-(D)-Ile(SEQ ID NO: 16), or

8. YGRKKRRQRRRYEKLLDTEI (SEQ ID NO: 17).

In order to directly compare with similar chimeric peptides in the reported studies, the control chimeric peptide NA-1 was introduced with the following sequence:

NA-1: YGRKKRRQRRRKLSSIESDV (SEQ ID NO: 18)

Furthermore, by comparing the structural differences between the series of peptides and NA-1, a chimeric peptide YE-NA-1 having two residues of YE added to the N-terminus of the active peptide of the chimeric peptide NA-1 was introduced, and the sequence was as follows:

YE-NA-1: YGRKKRRQRRRYEKLSSIESDV (SEQ ID NO: 19)

The chimeric peptides NA-1, YE-NA-1 and the chimeric peptide No. 8 were simultaneously subjected to the above-described test for interaction with the PDZ1/2 domain, and the results are shown in Table 1 below:

Table 1. Detection of interaction between three chimeric peptides and PDZ1/2 domain

嵌合肽Chimeric peptide	NA-1NA-1	YE-NA-1YE-NA-1	8号嵌合肽Chimeric peptide No. 8
KD(M)KD(M)	7.53E-087.53E-08	5.44E-085.44E-08	2.99E-082.99E-08

As shown in Table 1, the chimeric peptides YE-NA-1 and No. 8 chimeric peptides interacted more strongly with the PDZ1/2 domain than the control chimeric peptide NA-1, and the chimeric peptide No. 8 The effect is better. Therefore, according to the inventors' hypothesis, the additional YE two amino acid residues at the N-terminus of the active peptide have a certain potentiating effect on the interaction of the polypeptide with the PDZ1/2 domain. In addition, the chimeric peptide No. 8 reduced two less hydrophobic serines (SS) relative to the carboxy terminus of YE-NA-1, which, according to the inventors' hypothesis, may further increase the polypeptide and the PDZ1/2 domain. Interaction.

Example 2: Pull-down assay to detect the interaction of chimeric peptide No. 8 with PDZ1/2 domain

To demonstrate that this series of peptides can interact with the PDZ1/2 domain, a chimeric peptide No. 8 was selected as a representative for the Pull-down assay.

The column was equilibrated with 100 μl of His beads and 1 ml of MCAC-0 buffer for 5 min. Concussion at 4 °C. The mixture was centrifuged at 5000 g for 1 minute at 4 ° C, and the supernatant was discarded. 1 mg of PDZ1/2 protein was added to the mixture and made up to 1 ml with buffer. The mixture was spun for 1 hour at 4 °C. The mixture was centrifuged at 5000 g for 1 minute at 4 ° C, and the supernatant was discarded. Wash 3 times with 1 ml of MCAC-0 buffer for 5 minutes each time (at 4 ° C, shake wash). 1 mg of chimeric peptide No. 8 was added to the mixture and made up to 1 ml with buffer. The mixture was spun for 2 hours at 4 °C. The mixture was centrifuged at 5000 g for 1 minute at 4 ° C, and the supernatant was discarded. Wash with 3 ml of lysate 3 times for 5 minutes each time (at 4 ° C, shake wash). 20 μl of MCAC-300 was added after washing. After centrifugation, the eluate was taken for SDS-PAGE detection. The experimental results are shown in Figure 1.

As shown in Figure 1, both the chimeric peptide No. 8 and the PDZ1/2 domain were included in the elution band of the chimeric peptide No. 8, thereby confirming that the chimeric peptide No. 8 was able to bind to the PDZ1/2 domain.

Example 3: Synthesis of chimeric peptides

The inventors designed and synthesized the eight peptides selected by the solid phase synthesis method. The specific synthesis methods are as follows:

The chimeric peptide is prepared by a solid phase synthesis method. Synthesis of

chimeric peptides

3, 4, 5, 6, 7 and 8 using Wang resin and Fmoc-protection strategy, using DCC/HOBT or BOP/DIEA as condensation reagent, piperidine/DMF as deprotection reagent, after completion of reaction The peptide was cleaved from the resin with trifluoroacetic acid, wherein the D configuration residue was introduced depending on the position during the synthesis of the

chimeric peptides

3, 4, 5, 6, and 7. The synthetic step of the chimeric peptide No. 1 is substantially the same as the above, except that after the last amino acid Y condensation at the N-terminus completes the deprotection, 10% acetic acid is added for acetylation, and then the peptide chain is cleaved from the resin. The synthesis of chimeric peptide No. 2 was carried out using Rink amide resin as a carrier, and the other steps were similar to those of the chimeric peptide No. 3-8.

Example 4: Therapeutic effect of chimeric peptide on rat MCAO model

In this example, the therapeutic effects of the two representative chimeric peptides prepared in Example 3 were tested on a rat MCAO model.

MCAO model method:

The preparation of the focal cerebral ischemia-reperfusion model was based on the reversible middle cerebral artery occlusion (MCAO) suture method proposed by Longa and improved according to the anatomical structure of the rat brain to prepare a focal cerebral ischemia-reperfusion model. % chloral hydrate 0.3ml/kg abdominal anesthesia, cervical midline incision, exposure of common carotid artery (CCA), external carotid artery (ECA) and pterygopalatine, 0.26mm monofilament nylon fishing line head 0.5cm with paraffin, Marked at a distance of 20mm, all rats were inserted through the right CCA incision, and the pterygopalatine artery was temporarily clamped to prevent mis-insertion. The length of the suture was about 18-20 mm from the CCA bifurcation, depending on the animal's weight, embolization right The middle cerebral artery is then sutured, and the end of the suture is fixed to the skin. After the ischemia reached 2 hours, the suture was carefully taken out to form a reperfusion. The sham operation was just not inserted into the nylon fishing line, and the rest of the steps were the same as the surgery group. The body temperature was maintained at (37 ± 0.5) °C during ischemia and 2 h after reperfusion. The success of the model is that the left limb is paralyzed after anesthesia in rats, and the standing is unstable. When turning the tail, turning to one side is the criterion for successful model.

Laboratory animals and materials

Animals: Adult SD rats (Vittalia), SPF grade, body weight 220-250 g, male.

Instruments and medicines: 1 line scissors, 2 eye surgery scissors, 4 bends, 4#, 5# surgical sutures, 6×17 triangular needles, 0.26 mm diameter bolts, and needle holders. EnbiP sodium chloride injection (NBP) (Shijiazhuang Group Enbi Pharmaceutical Co., Ltd.), chloral hydrate, furosemide (20mg / support), gentamicin sulfate (80mg / support), cotton swab, medical Tray, etc.

Experimental grouping

The experimental sub-model group (untreated group), the positive drug group (NBP) and the chimeric peptide-administered group (chimeric peptides No. 7 and No. 8 in Example 1). At 1 h after ischemia, physiological saline, 2.5 mg/kg of the positive drug Enbip, and 10 mg/kg of each peptide were administered to each group via tail vein injection.

Infarct volume calculation

After the score, the rats were killed by decapitation. The brain tissue was quickly placed in a refrigerator at -20 °C. After 10 minutes, the brain was placed in a room temperature environment. The brain was placed in a rat brain slice mold, and the olfactory bulb was removed. The cerebellum and the low brain stem were analyzed according to the map. The lesions were cut at a distance of 2 mm and cut into five knives, and cut into six consecutive continuous coronal slices. Then, the brain slices were quickly placed in a 5 ml solution containing 2% TTC, and incubated at 37 ° C for 30 minutes in the dark, and the brain slices were flipped once every 5 minutes. After TTC staining, the normal tissue was rose red, and the infarcted tissue was unstained and white. Each group of brain slices was arranged neatly, photographed and saved, and processed by image analysis system software and counted. The infarct area of each brain slice was calculated, multiplied by the thickness of each slice of brain 2 mm, and the infarct area of each brain slice of each animal was multiplied by The thickness is added, which is the volume of cerebral infarction. Volume is expressed as a percentage of the brain's hemisphere to eliminate the effects of cerebral edema. The experimental results are shown in Table 2.

Table 2: In vivo pharmacodynamics and metabolic evaluation of peptides

As shown in the results of Table 2, both chimeric peptides No. 7 and No. 8 significantly reduced the volume of cerebral infarction caused by cerebral ischemia-reperfusion.

All publications and patent documents cited in this specification are hereby incorporated by reference in their entirety in their entirety in their entirety herein Various changes and substitutions of equivalents may be made to the various embodiments disclosed herein without departing from the true scope and scope of the disclosure. Any feature, step or embodiment of an embodiment of the present disclosure can be used in combination with any other feature, step or embodiment, unless otherwise stated in the context.

Claims

a compound having the structure of the formula (I) or a pharmaceutically acceptable salt thereof,

R 1 -S 1 -YEKL-S 2 -R 2 (I)

among them

R 1 is selected from the group consisting of hydrogen, pyroglutamic acid residue, C 1-18 alkyl group, C 3-18 cycloalkyl group, C 1-6 heterocyclic group, R 3 C(O)- or -NR 4 R 5 , Wherein R 3 is independently selected from C 1-18 alkyl, C 3-18 cycloalkyl, C 1-6 heterocyclyl, and R 4 and R 5 are each independently selected from hydrogen, C 1-6 alkyl, C 3-6 cycloalkyl and C 1-6 heterocyclic;

S 1 is an amino acid sequence of an internalization peptide;

S 2 is the amino acid sequence of LDTEI or a functional variant thereof;

R 2 is selected from the group consisting of -OH and -NR 6 R 7 , wherein R 6 and R 7 are each independently selected from the group consisting of hydrogen, C 1-6 alkyl, C 3-6 cycloalkyl, and C 1-6 heterocyclic.
A compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein R 1 is hydrogen or acetyl (Ac).
The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein R 2 is -OH or -NH 2 .
The compound according to claim 1 or a pharmaceutically acceptable salt thereof, wherein S 1 is selected from the group consisting of YGRKKRRQRRR (SEQ ID NO: 1), polyarginine of 2 to 30 residues, GRKKRRQRRRPPQQ ( SEQ ID NO: 2), RQIKIWFQNRRMKWKK (SEQ ID NO: 3), GWTLNSAGYLLKINLKALAALAKKIL (SEQ ID NO: 4), GALFLAFLAAALSLMGLWSQPKKKRRV (SEQ ID NO: 5), RGGRLSYSRRRFSTSTGR (SEQ ID NO: 6), RRLSYSRRRF (SEQ ID NO: 7) ), KLALKLALKALKAALKLA (SEQ ID NO: 8), GALFLGWLGAAGSTMGAWSQPKKKRKV (SEQ ID NO: 9).
The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the amino acid sequence of S 1 is YGRKKRRQRRR.
The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the functional variant of LDTEI is a variant obtained by subjecting one or more residues thereof to any one or more of the following substitutions:

L (leucine) is substituted with L or D isoleucine, β-leucine, β-leucine, norleucine, tert-leucine, iso- leucine, valine, --cyclopropylalanine, β-cyclopentylalanine, β-cyclohexylalanine, 2-amino-5-methylhexanoic acid, iso-proline, N-alkyl-leucine , N-alkyl-isoleucine, N-alkyl-β-leucine, N-alkyl-β-leucine, N-alkyl-norleucine, N-alkyl-tert Leucine, N-alkyl-isoisoleucine or N-alkyl-proline;

D (aspartic acid) is substituted with L or D type glutamic acid, asparagine, glutamine, N-alkyl aspartic acid, N-alkyl glutamic acid, N-alkyl aspartate Amide or N-alkyl glutamine;

T (threonine) is substituted with L or D type serine, N-alkylthreonine or N-alkylserine;

E (glutamic acid) is substituted with L or D type aspartic acid, asparagine, glutamine, N-alkyl aspartic acid, N-alkyl glutamic acid, N-alkyl aspartate Amide or N-alkyl glutamine;

I (isoleucine) is substituted with L or D leucine, β-leucine, β-leucine, norleucine, tert-leucine, iso- leucine, valine, --cyclopropylalanine, β-cyclopentylalanine, β-cyclohexylalanine, 2-amino-5-methylhexanoic acid, isovaline, N-alkyl-leucine, N-alkyl-isoleucine, N-alkyl-β-leucine, N-ane Benzyl-leucine, N-alkyl-norleucine, N-alkyl-tert-leucine, N-alkyl-isoisoleucine or N-alkyl-proline;

Wherein the alkyl group in the N-alkyl group is preferably a C 1-10 alkyl group or a C 3-10 cycloalkyl group, more preferably a C 1-6 alkyl group or a C 3-6 cycloalkyl group, more preferably C. 1-4 alkyl or C 3-4 cycloalkyl, most preferably methyl.
The compound of claim 6 or a pharmaceutically acceptable salt thereof, wherein the functional variant of LDTEI is a variant obtained by subjecting one or more residues thereof to any one or more of the following substitutions:

L (leucine) is substituted with L or D isoleucine, β-leucine, β-leucine, norleucine, tert-leucine, iso- leucine, valine, --cyclopropylalanine, β-cyclopentylalanine, β-cyclohexylalanine, 2-amino-5-methylhexanoic acid, iso-proline, N-methyl-leucine , N-methyl-isoleucine, N-methyl-β-leucine, N-methyl-β-leucine, N-methyl-norleucine, N-methyl-tert Leucine, N-methyl-isoisoleucine or N-methyl-proline;

D (aspartic acid) is substituted with L or D type glutamic acid, asparagine, glutamine, N-methyl aspartic acid, N-methyl glutamic acid, N-methyl aspartate Amide or N-methyl glutamine;

T (threonine) is substituted with L or D type serine, N-methylthreonine or N-methylserine;

E (glutamic acid) is substituted with L or D type aspartic acid, asparagine, glutamine, N-methyl aspartic acid, N-methyl glutamic acid, N-methyl aspartate Amide or N-methyl glutamine;

I (isoleucine) is substituted with L or D leucine, β-leucine, β-leucine, norleucine, tert-leucine, iso- leucine, valine, --cyclopropylalanine, β-cyclopentylalanine, β-cyclohexylalanine, 2-amino-5-methylhexanoic acid, iso-proline, N-methyl-leucine , N-methyl-isoleucine, N-methyl-β-leucine, N-methyl-β-leucine, N-methyl-norleucine, N-methyl-tert Leucine, N-methyl-isoisoleucine or N-methyl-proline.
The compound of claim 1 or a pharmaceutically acceptable salt thereof, wherein the functional variant of LDTEI is a variant in which one or more amino acids in LDTEI are substituted with the corresponding D-form amino acid.
The compound of claim 8 or a pharmaceutically acceptable salt thereof, wherein the functional variant of LDTEI is selected from the group consisting of: (D)-Leu-DTEI, L-(D)-Asp-TEI, LD-(D )-Thr-EI, LDT-(D)-Glu-I and LDTE-(D)-Ile.
The compound of claim 1 or a pharmaceutically acceptable salt thereof, which has a structure selected from the group consisting of:

Ac-YGRKKRRQRRRYEKLLDTEI (SEQ ID NO: 10),

YGRKKRRQRRRYEKLLDTEI-NH 2 (SEQ ID NO: 11),

YGRKKRRQRRRYEKL-(D)-Leu-DTEI (SEQ ID NO: 12),

YGRKKRRQRRRYEKLL-(D)-Asp-TEI (SEQ ID NO: 13),

YGRKKRRQRRRYEKLLD-(D)-Thr-EI (SEQ ID NO: 14),

YGRKKRRQRRRYEKLLDT-(D)-Glu-I (SEQ ID NO: 15), or

YGRKKRRQRRRYEKLLDTE-(D)-Ile (SEQ ID NO: 16).
The compound of any one of the preceding claims, or a pharmaceutically acceptable salt thereof, wherein the salt is selected from the group consisting of trifluoroacetate, acetate, hydrochloride or phosphate.
A pharmaceutical composition comprising a compound of any one of the preceding claims, or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier, excipient and/or diluent.
A pharmaceutical composition according to claim 12 which is a pre-lyophilized preparation, preferably comprising histidine and trehalose.
A pharmaceutical composition according to claim 12 which is a lyophilized preparation, preferably prepared by lyophilizing the pre-lyophilized preparation of claim 13.
A pharmaceutical composition according to claim 12 which is a reconstituted preparation, preferably prepared by combining the lyophilized preparation of claim 14 with an aqueous solution.
The pharmaceutical composition according to any one of claims 12 to 15 for use in treating, ameliorating or preventing a nervous system injury, a disease or pain associated with nervous system damage, a neurodegenerative disease, anxiety or epilepsy in an individual, or Used as a neuroprotective agent.
A method of treating, ameliorating or preventing a nervous system injury, a disease or pain associated with a nervous system injury, a neurodegenerative disease, anxiety or epilepsy in an individual, the method comprising administering to the individual in need thereof any one of claims 1-11 The compound or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition according to any one of claims 12-15.
A compound according to any one of claims 1 to 11, or a pharmaceutically acceptable salt thereof, or a pharmaceutical composition according to any one of claims 12 to 15 for use in the preparation, treatment or amelioration or prevention of the nervous system in an individual A drug for injury, a disease or pain associated with nervous system damage, a neurodegenerative disease, anxiety or epilepsy or use in the preparation of a neuroprotective agent.
The pharmaceutical composition according to claim 16, the method of claim 17, or the use of claim 18, wherein the nervous system damage is a nervous system damage caused by excitotoxicity.
The pharmaceutical composition, method or use according to claim 19, wherein the neurological damage caused by the excitatory neurotoxicity comprises an ischemic or traumatic injury selected from the group consisting of stroke, spinal cord injury, brain or spinal cord, central nervous system Systemic (CNS) neuronal damage, including acute CNS injury, ischemic stroke, or spinal cord injury, as well as hypoxia, ischemia, mechanical injury, and neurodegenerative diseases, anxiety, epilepsy, and stroke-induced damage.
The pharmaceutical composition according to claim 16, the method of claim 17, or the use of claim 18, wherein the neurodegenerative disease is selected from the group consisting of Alzheimer's disease, amyotrophic lateral sclerosis (ALS), Parkinson's disease or Huntington's disease.
The pharmaceutical composition according to claim 16, the method of claim 17, or the use of claim 18, wherein the nervous system injury or pain is located in the peripheral nervous system or the central nervous system.
The pharmaceutical composition according to claim 16, the method of claim 17, or the use of claim 18, wherein the neurological damage-related disease is stroke.
The pharmaceutical composition, method or use of claim 23, wherein the stroke is selected from the group consisting of ischemic stroke, hemorrhagic stroke, and hemorrhagic stroke converted from ischemic stroke.
The pharmaceutical composition, method or use of claim 24, wherein the stroke is an ischemic stroke.