Chemically modified polypeptide

Abstract

A polypeptide compound or a variant thereof, or a pharmaceutically acceptable salt. The polypeptide compound or the variant thereof, or the pharmaceutically acceptable salt thereof can be used for preparing a drug for treating traumatic brain injury.

Description

A chemically modified peptide

[0001] This disclosure claims priority to Chinese patent application CN202311639052.9 filed on December 1, 2023, and the entire contents of the aforementioned patent application are incorporated into this disclosure by reference. Technical Field

[0002] The present application belongs to the field of biomedicine technology and relates to a chemically modified polypeptide, and more specifically to a modified peptide that can be used to treat traumatic brain injury and / or inhibit neural damage after traumatic brain injury and its use. Background Art

[0003] Traumatic brain injury (TBI) is a direct or indirect injury to the head caused by external violence and is one of the leading causes of death in children and young people. TBI is a serious public health problem, and there are currently few measures available to reverse the initial brain damage caused by trauma. Due to the severe damage and limited available treatments, survivors often suffer from sequelae such as motor disabilities, cognitive impairment, and epilepsy. The neurological, economic, and social consequences of TBI are devastating for patients, their families, and society as a whole.

[0004] According to incomplete statistics, there are 1.5-2 million people in China disabled by head injuries, with 80,000-100,000 new cases each year. 200,000 people die annually from these injuries. Approximately 60%-80% of these deaths are attributed to head injuries, while 17% are due to disability. Spinal cord injuries have a cumulative total of 500,000-600,000 people disabled, with 230,000 new cases each year. The resulting medical costs amount to 200-300 billion yuan annually. These statistics demonstrate the urgent need for effective treatments to improve functional recovery after trauma.

[0005] CN103230581A provides a method for inhibiting nerve cell damage after traumatic brain injury by using a short peptide sequence (KKNRNKLRRQHSY (SEQ ID NO: 1)).

[0006] CN111363014A provides a short peptide sequence (YGRKKRRQRRRKKNRNKLRRQHSY (SEQ ID NO: 2)), which can inhibit the death of necrotic nerve cells after traumatic brain injury.

[0007] Despite these discoveries, there remains a need for short peptide sequences having one or more of the following properties: further enhanced polypeptide biological activity, improved polypeptide stability, ability to cross cell membranes, and reduced side effects. Summary of the Invention

[0008] In a first aspect, the present application provides a polypeptide compound comprising a structure represented by general formula (I):

[0009] KX 2 X 3 RX 5 KX 7 X 8 RX 10 HSY (SEQ ID NO: 3)-R 1 (I),

[0010] wherein X2 is selected from Lys, HoLys, Dap, DAB, Cit, Orn, D-Lys, D-HoLys, D-Dap, D-DAB, D-Cit, D-Orn, or Lys whose side chain amino group is modified by Z1; X3 and X5 are independently selected from Asn, Asp, D-Asn, or D-Asp; X7 is selected from Ile, Leu, Val, Tel, D-Ile, D-Leu, D-Val, or D-Tel; X8 is selected from Arg, HomoArg, D-Arg, or D-HomoArg; and X10 is selected from Glu, Gln, D-Glu, or D-Gln;

[0011] R 1 Selected from -OH, -ol, -NR 2 R 3 AND-OR 4 , where R 2 and R 3 are independently selected from hydrogen, C 1-16 Alkyl, C 3-12 Cycloalkyl and 3 to 12 membered heterocycloalkyl, or R 2 、R 3 Together with the nitrogen atom to which it is connected, it forms a 3- to 12-membered heterocycloalkyl group; R 4 Selected from C 1-16 Alkyl, C 3-12 Cycloalkyl and 3- to 12-membered heterocycloalkyl;

[0012] The alkyl, cycloalkyl, heterocycloalkyl are optionally replaced by one or more identical or different R 5 Replacement, R 5 Selected from halogen, nitro, cyano, hydroxy, carbonyl, carboxyl, -NR a R b , thiol, C 1-8 Alkyl, C 1-8 Alkoxy, halogenated C 1-8 Alkyl, halogenated C 1-8 Alkoxy, C 3-8 Cycloalkyl, C 3-8Cycloalkyloxy, 3 to 8 membered heterocycloalkyl, -SC 1-3 Alkyl, -SO2C 1-3 Alkyl, -SO2NR a R b 、-S(O)C 1-3 Alkyl, -C(O)NR a R b 、-C(O)C 1-3 Alkyl, -C(O)OC 1-3 Alkyl, -OC(O)C 1-3 Alkyl, C 6-10 aryl, 5- to 10-membered heteroaryl;

[0013] R a 、R b are each independently hydrogen or C 1-8 Alkyl; or R a 、R b Together with the nitrogen atom to which it is attached, it forms a 3- to 6-membered heterocycloalkyl group; and

[0014] Z 1 Selected from-AEEA-COR 6 , where R 6 C 1-15 of alkyl.

[0015] In some embodiments, the polypeptide compound further comprises a cell-penetrating peptide at its N-terminus or C-terminus.

[0016] In some embodiments, the N-terminus of the polypeptide compound contains a cell-penetrating peptide, and the N-terminus of the cell-penetrating peptide is R 7 The structure of the polypeptide compound is shown in the general formula (II):

[0017] R 7 -Cell-penetrating peptide-KX 2 X 3 RX 5 KX 7 X 8 RX 10 HSY (SEQ ID NO: 3)-R 1 (II),

[0018] Among them, R 7 Selected from hydrogen, C 1-16 Alkyl, C 3-12 Cycloalkyl, 3 to 12 membered heterocycloalkyl, R 8 CO-, and phosphate groups, where R 8 Selected from C 1-16 Alkyl, C 3-12 Cycloalkyl and 3 to 12 membered heterocycloalkyl, C 6-10aryl, 5- to 10-membered heteroaryl.

[0019] In some embodiments, the polypeptide compound comprises a cell-penetrating peptide at its C-terminus, i.e., 2 X 3 RX 5 KX 7 X 8 RX 10 HSY (SEQ ID NO: 1) and R 1 The groups contain a membrane-penetrating peptide.

[0020] In some specific embodiments, the cell-penetrating peptide in the general formula (II) is selected from TAT sequence, MAP sequence, MTS sequence or R9 sequence or variants thereof.

[0021] In some specific embodiments, the cell-penetrating peptide in the general formula (II) is a TAT sequence or a variant thereof, wherein the TAT sequence at least includes the amino acid fragment at positions 49 to 57 in the transcriptional transactivator protein.

[0022] In some specific embodiments, the cell-penetrating peptide in the polypeptide compound represented by general formula (II) is a TAT sequence, wherein X 2 Selected from Lys;X 3 and X 5 Selected from Asn; X 7 Selected from Leu;X 8 Selected from Arg; X 10 Selected from Gln; the structure of the polypeptide compound is shown in the general formula (III):

[0023] R 7 -TAT-KKNRNKLRRQHSY(SEQ ID NO:1)-R 1 (III),

[0024] Wherein, TAT is connected to KKNRNKLRRQHSY (SEQ ID NO: 1) through a main chain amide bond, wherein the nitrogen atom in the main chain amide bond is optionally replaced by C 1-3 Alkylation, preferably methylation;

[0025] In the general formula (III), R 1 Selected from -OH, -ol, -NR 2 R 3 AND-OR 4 , where R 2 and R 3 are independently selected from hydrogen, C 1-16 Alkyl, C 3-12 Cycloalkyl and 3 to 12 membered heterocycloalkyl, or R 2 、R 3Together with the nitrogen atom to which it is connected, it forms a 3- to 12-membered heterocycloalkyl group; R 4 Selected from C 1-16 Alkyl, C 3-12 Cycloalkyl and 3- to 12-membered heterocycloalkyl;

[0026] The alkyl, cycloalkyl, heterocycloalkyl are optionally replaced by one or more identical or different R 5 replace;

[0027] R 5 Selected from halogen, nitro, cyano, hydroxy, carbonyl, carboxyl, -NR a R b , thiol, C 1-8 Alkyl, C 1-8 Alkoxy, halogenated C 1-8 Alkyl, halogenated C 1-8 Alkoxy, C 3-8 Cycloalkyl, C 3-8 Cycloalkyloxy, 3 to 8 membered heterocycloalkyl, -SC 1-3 Alkyl, -SO2C 1-3 Alkyl, -SO2NR a R b 、-S(O)C 1-3 Alkyl, -C(O)NR a R b 、-C(O)C 1-3 Alkyl, -C(O)OC 1-3 Alkyl, -OC(O)C 1-3 Alkyl, C 6-10 aryl, 5- to 10-membered heteroaryl;

[0028] R a 、R b are each independently hydrogen or C 1-8 Alkyl; or R a 、R b Together with the nitrogen atom to which it is attached, it forms a 3- to 6-membered heterocycloalkyl group; and R 7 When it is hydrogen, R 1 is not -OH; the TAT sequence includes at least the 49th to 57th amino acid fragment of the transcriptional transactivator protein;

[0029] R 7 Selected from hydrogen, C 1-16 Alkyl, C 3-12 Cycloalkyl, 3 to 12 membered heterocycloalkyl, R 8 CO-, and phosphate groups, where R 8 Selected from C 1-16 Alkyl, C 3-12 Cycloalkyl and 3 to 12 membered heterocycloalkyl, C 6-10 aryl, 5- to 10-membered heteroaryl.

[0030] In some specific embodiments, the compound represented by the general formula (III) of the present application can also be selected from its variant forms or pharmaceutically acceptable salts.

[0031] In some specific embodiments, the present application provides a polypeptide compound or a variant thereof, or a pharmaceutically acceptable salt thereof, wherein the structure of the polypeptide compound is shown in the general formula (III):

[0032] R 7 -TAT-KKNRNKLRRQHSY(SEQ ID NO:1)-R 1 (III),

[0033] wherein TAT is linked to KKNRNKLRRQHSY (SEQ ID NO: 1) via a main chain amide bond, wherein the nitrogen atom in the main chain amide bond is optionally C1-3 alkylated, preferably methylated;

[0034] R 7 Selected from hydrogen, C 1-16 Alkyl, C 3-12 Cycloalkyl, 3 to 12 membered heterocycloalkyl, R 8 CO-, and phosphate groups, where R 8 Selected from C 1-16 Alkyl, C 3-12 Cycloalkyl and 3 to 12 membered heterocycloalkyl, C 6-10 aryl, 5- to 10-membered heteroaryl;

[0035] R 1 Selected from -OH, -ol, -NR2R3 and -OR4, wherein R 2 and R 3 are independently selected from hydrogen, C 1-16 Alkyl, C 3-12 Cycloalkyl and 3 to 12 membered heterocycloalkyl, or R 2 、R 3 Together with the nitrogen atom to which it is connected, it forms a 3- to 12-membered heterocycloalkyl group; R 4 Selected from C 1-16 Alkyl, C 3-12 Cycloalkyl and 3- to 12-membered heterocycloalkyl;

[0036] The alkyl, cycloalkyl, heterocycloalkyl are optionally replaced by one or more identical or different R 5 replace;

[0037] R 5 Selected from halogen, nitro, cyano, hydroxy, carbonyl, carboxyl, -NR a R b , thiol, C 1-8 Alkyl, C 1-8Alkoxy, halogenated C 1-8 Alkyl, halogenated C 1-8 Alkoxy, C 3-8 Cycloalkyl, C 3-8 Cycloalkyloxy, 3 to 8 membered heterocycloalkyl, -SC 1-3 Alkyl, -SO2C 1-3 Alkyl, -SO2NR a R b 、-S(O)C 1-3 Alkyl, -C(O)NR a R b 、-C(O)C 1-3 Alkyl, -C(O)OC 1-3 Alkyl, -OC(O)C 1-3 Alkyl, C 6-10 aryl, 5- to 10-membered heteroaryl;

[0038] R a 、R b are each independently hydrogen or C 1-8 Alkyl; or R a 、R b Together with the nitrogen atom to which it is attached, it forms a 3- to 6-membered heterocycloalkyl group; and R 7 When it is hydrogen, R 1 It is not -OH; the TAT sequence at least includes the 49th to 57th amino acid fragment in the transcriptional transactivator protein.

[0039] In some specific embodiments, the TAT sequence is a short peptide selected from YGRKKRRQRRR (SEQ ID NO: 4), GRKKRRQRRR (SEQ ID NO: 33), YGRKKRRQRRRPPQ (SEQ ID NO: 34) or GRKKRRQRRRQ (SEQ ID NO: 35).

[0040] In some specific embodiments, the variant of the TAT sequence has 1, 2 or 3 amino acid substitutions, additions or deletions in the TAT sequence.

[0041] In some specific embodiments, the variant of the TAT sequence has a sequence selected from at least 80%, at least 85%, at least 90%, at least 95%, at least 99%, or 100% identical to YGRKKRRQRRR (SEQ ID NO: 4).

[0042] In some specific embodiments, the variant of the TAT sequence is an amino acid sequence in which at least one Gln in the TAT sequence is replaced by Glu, and / or at least one L-Arg is replaced by D-Arg, and / or at least one L-Lys is replaced by D-Lys.

[0043] In some specific embodiments, R 1 Selected from -OH, -NR 2 R 3 , where R 2 and R 3 are independently selected from hydrogen, C 1-3 alkyl.

[0044] In some specific embodiments, R 1 Selected from -OH or -NH2.

[0045] In some specific embodiments, R 1 It is -OH.

[0046] In some specific embodiments, R 1 is -NH2.

[0047] In other specific embodiments, R 7 is selected from hydrogen, methyl, acetyl or phosphate.

[0048] In some specific embodiments, R 7 Selected from hydrogen, C 1-3 Alkyl or acetyl.

[0049] In some specific embodiments, R 7 For hydrogen.

[0050] In some specific embodiments, R 7 It is a methyl group.

[0051] In some specific embodiments, R 7 It is a phosphate group.

[0052] In some specific embodiments, the polypeptide compound is selected from the following polypeptide compounds shown in SEQ ID NO: 5 to SEQ ID NO: 29:

[0053] Me-YGRKKRRQRRRKKNRNKLRRQHSY(SEQ ID NO:5)、

[0054] pYGRKKRRQRRRKKNRNKLRRQHSY(SEQ ID NO:6),

[0055] Ac-YGRKKRRQRRRKKNRNKLRRQHSY(SEQ ID NO:7),

[0056] C 15 H 31 CO-YGRKKRRQRRRKKNRNKLRRQHSY(SEQ ID NO:8),

[0057] YGRKKRRQRRRRKNRNKLRRQHSY-NH2(SEQ ID NO:9)、

[0058] Ac-YGRKKRRQRRRKKNRNKLRRQHSY-NH2(SEQ ID NO:10)、

[0059] pYGRKKRRQRRRRKNRNKLRRQHSY-NH2(SEQ ID NO:11)、

[0060] Me-YGRKKRRQRRRKNRNKLRRQHSY-NH2(SEQ ID NO:12)、

[0061] C 15 H 31 CO-YGRKKRRQRRRKNRNKLRRQHSY-NH2(SEQ ID NO:13)、

[0062] YGRKKRRQRRRKKNRNKIRRQHSY(SEQ ID NO:14)、

[0063] YGRKKRRQRRRKKNRNKIRRQHSY-NH2(SEQ ID NO:15)、

[0064] YGRKKRRQRRRKKDRDKLRRQHSY(SEQ ID NO:16)、

[0065] YGRKKRRQRRR-K(N-AEEA-Palmitic acid)-KNRNKLRRQHSY(SEQ ID NO:17)、

[0066] YGRKKRRQRRRKKNRNKLRREHSY(SEQ ID NO:18)、

[0067] YGRKKRRQRRRKKNRDKLRREHSY(SEQ ID NO:19)、

[0068] YGRKKRRERRRKKDRNKLRRQHSY(SEQ ID NO:20)、

[0069] Ac-YGRKKRRERRRKKDRDKLRREHSY-NH2(SEQ ID NO:21)、

[0070] Me-YGRKKRRERRRKKDRDKLRREHSY-NH2 (SEQ ID NO: 22),

[0071] Ac-YGRKKRRERRRKKDRDK(DL)RREHSY-NH2(SEQ ID NO:23),

[0072] Ac-YGRKKRRERRRKKDRDKLRRQHSY-NH2 (SEQ ID NO: 24),

[0073] Me-YGRKKRRERRRKKDRDKLRRQHSY-NH2 (SEQ ID NO:25),

[0074] Ac-YGRKKRRERRRKKDRDKVRREHSY-NH2 (SEQ ID NO: 26),

[0075] Me-YAibRKKRRERRRKKDRDKTleRREHSY-NH2 (SEQ ID NO: 27),

[0076] Ac-YGRKKRRERRRKKDRDKTleRREHSY-NH2 (SEQ ID NO:28) and

[0077] Ac-YGRK(DK)(DR)RER(DR)RKKDRDK(DL)(DR)REHSY-NH2 (SEQ ID NO: 29).

[0078] In a second aspect, the present application provides an isolated polynucleotide encoding the polypeptide compound of the present application.

[0079] In a third aspect, the present application provides a plasmid comprising the polynucleotide as described above.

[0080] In a fourth aspect, the present application provides an expression vector comprising the polynucleotide as described above.

[0081] In a fifth aspect, the present application provides a host cell, wherein the cell contains the above-mentioned expression vector or plasmid or the genome of the cell is integrated with the exogenous isolated polynucleotide as described above.

[0082] In a sixth aspect, the present application provides a pharmaceutical composition comprising the aforementioned polypeptide compound or a variant thereof or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable excipient.

[0083] In a seventh aspect, the present application provides the use of the aforementioned polypeptide compound or variant thereof, or pharmaceutically acceptable salt, or pharmaceutical composition in the preparation of a medicament. In some embodiments, the medicament is a medicament for treating and / or preventing traumatic brain injury and / or inhibiting neurological damage after traumatic brain injury.

[0084] In an eighth aspect, the present application provides a method for treating and / or preventing traumatic brain injury and / or inhibiting neural damage after traumatic brain injury, the method comprising administering the above-mentioned polypeptide compound or its variant, or a pharmaceutically acceptable salt, or the pharmaceutical composition described in the sixth aspect to a subject.

[0085] In a ninth aspect, the present application provides the use of the above-mentioned pharmaceutical composition for treating trauma and / or preventing traumatic brain injury and / or inhibiting neural damage after traumatic brain injury.

[0086] Without being bound by theory, the inventors of the present application have discovered that certain specific modifications in the polypeptide compounds provided herein can significantly improve the stability of the modified polypeptide, and / or can reduce glutamate-induced neuronal excitotoxicity and improve the survival rate of neurons, thereby significantly improving the efficacy of the polypeptides of the present application in treating traumatic brain injury and inhibiting neural damage after traumatic brain injury. DETAILED DESCRIPTION

[0087] The technical solution of the present application is further described below through specific implementation methods. Those skilled in the art should understand that the embodiments are only used to help understand the present application and should not be regarded as specific limitations of the present application.

[0088] In order to more clearly understand the technical content of this application, the terms of this application are further explained below.

[0089] "Variant" refers to an amino acid sequence in which one or more amino acids are altered. A variant can have a "conservative" change, wherein the substituted amino acids have similar structural or chemical properties, such as replacing leucine (L) with isoleucine (I), replacing asparagine (N) with aspartic acid (D), replacing glutamine (Q) with glutamic acid (E). A variant can have a "non-conservative" change, such as replacing leucine (L) with valine (V), replacing glycine (G) with tryptophan (W), or replacing natural amino acids with non-natural amino acids. Alternatively, a variant can replace leucine (L) with L-tert-leucine (Tle), or replace glycine (G) with 2-aminoisobutyric acid (Aib). Similar lesser changes can also include amino acid deletions and / or insertions.

[0090] "Y-ol" refers to tyrosinol.

[0091] The amino acids are linked together by amide bonds to form covalent peptide bonds linking the backbone carboxylic acid group of one amino acid to the backbone amino group of another amino acid, forming a peptide bond (-C(=O)-NH-) or a backbone amide bond.

[0092] "Alkyl" refers to a fully saturated aliphatic hydrocarbon group. 1-16 Alkyl refers to a straight or branched chain alkyl group having 1 to 16 carbon atoms in the chain. 1-6 Alkyl refers to a straight or branched chain alkyl group having 1 to 6 carbon atoms in the chain. Examples of alkyl groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl, isopentyl, tert-pentyl, hexyl, and isohexyl.

[0093] "Cycloalkyl" refers to a saturated or partially saturated monocyclic, fused, bridged, or spirocyclic ring having at least 3 ring atoms per carbon ring. 3-18 Cycloalkyl refers to a carbon ring having 3 to 18 ring atoms. 3-6 Cycloalkyl refers to a carbon ring having 3 to 6 ring atoms. Illustrative examples of cycloalkyl include, but are not limited to, the following entities:

[0094] "Heterocycloalkyl" refers to a saturated or partially saturated monocyclic, fused polycyclic, bridged, or spirocyclic ring structure having at least one heteroatom selected from nitrogen, oxygen, and sulfur in the ring structure. Illustrative examples of heterocyclic groups include, but are not limited to, the following entities:

[0095] "Aryl" refers to an all-carbon monocyclic or fused polycyclic (ie, rings that share adjacent pairs of carbon atoms) group having a conjugated π electron system. 6-10 The term "aryl" refers to a monocyclic or bicyclic aromatic group having 6 to 10 carbon atoms. Illustrative examples of aryl include, but are not limited to, the following entities: phenyl, naphthyl, and the like.

[0096] "Heteroaryl" refers to a monocyclic, bicyclic, or polycyclic 4n+2 aromatic ring system (e.g., having 6 or 10 π electrons shared in a cyclic arrangement) having ring carbon atoms and ring heteroatoms, wherein each heteroatom is independently selected from nitrogen, oxygen, and sulfur. Illustrative examples of heteroaryl include, but are not limited to, the following entities: thienyl, furanyl, thiazolyl, isothiazolyl, imidazolyl, pyrrolyl, pyrazolyl, pyridinyl, pyridazinyl, pyrimidinyl, pyrazinyl, and the like.

[0097] "Halogen" refers to fluorine, chlorine, bromine or iodine.

[0098] "Halo" refers to a group in which one or more (eg, 1, 2, 3, 4, or 5) hydrogen atoms are replaced by a halogen.

[0099] The comparison between amino acid abbreviations and Chinese names is shown in Table 1.

[0100] Table 1: Comparison table of amino acid abbreviations and Chinese names

[0101] HoLys stands for hydroxylysine, the full name of which is 5-hydroxylysine, 5-hydroxylysine.

[0102] Dap stands for 2,6-Diaminoheptanedioic acid.

[0103] DAB stands for 2,4-diaminobutyric acid.

[0104] Cit stands for Citrulline.

[0105] Orn stands for ornithine.

[0106] HomoArg stands for homoarginine.

[0107] The amino acid residues in the polypeptide sequences of the present application are represented by amino acid abbreviations.

[0108] As used herein, "Ac" and "NH2" refer to acetyl and amidated termini, respectively.

[0109] Unless otherwise noted, amino acids described herein are "L-amino acids," represented by a capital single letter or a three-letter abbreviation of the amino acid. "D-amino acids" described herein are represented by a lowercase single letter of the amino acid abbreviation, or by a three-letter abbreviation preceded by the letter "D," or by a capital single letter of the amino acid abbreviation preceded by "D." For example, "L-leucine" can be represented by "Leu" or "L," and "D-leucine" can be represented by "D-Leu," "l," or "DL."

[0110] Aib stands for 2-aminoisobutyric acid.

[0111] Tle stands for L-tert-leucine.

[0112] In one example, the sequence of the amino acid fragment at positions 49 to 57 in the transcriptional transactivator protein is RKKRRQRRR (SEQ ID NO: 36).

[0113] As used herein, "treatment" refers to administering an effective amount of the polypeptide compound of the present application to an individual to alleviate symptoms or complications, delay the disease, hinder the progression of the disease, alleviate or relieve symptoms and complications, and / or cure or eliminate the disease or disease and prevent the disease, wherein prevention should be understood as administering the polypeptide compound of the present application to an individual for the purpose of combating the disease or disease to prevent the onset of symptoms or complications. In one embodiment, "treatment" refers to administering an effective amount of the polypeptide compound of the present application for the purpose of alleviating, improving, preventing or eradicating (curing) symptoms or disease states.

[0114] As used herein, "effective amount" or "therapeutically effective amount" refers to a sufficient amount of a drug or pharmaceutical agent to achieve the desired effect.

[0115] Peptides of this application:

[0116] The peptide of the present application can further include the peptide that can guide the application across blood-brain barrier and nerve cell membrane at its N-terminal or C-terminal end.Suitable membrane-penetrating peptides as known in the art can be applied to the present application, as long as they can guide the peptide of the present application across blood-brain barrier and nerve cell membrane.Exemplary membrane-penetrating peptides can be selected from TAT sequence (selected from " YGRKKRRQRRR (SEQ ID NO:4)", "GRKKRRQRRR (SEQ ID NO:33)", "YGRKKRRQRRRPPQ (SEQ ID NO:34)" or "GRKKRRQRRRQ (SEQ ID NO:35)"), model amphipathic peptide (modelamphipathic peptide, MAP) sequence (" KLALKLALKALKAALKLA (SEQ ID NO:30)"), membrane translocation peptide (membranetranslocating peptide, MTS) sequence (" AAVALLPAVLLALLAP (SEQ ID NO:31)") or R9 (" RRRRRRRRR (SEQ ID NO:32)") sequence. In a particularly preferred embodiment, the membrane-penetrating peptide of the present application is a TAT sequence. Optionally, the peptide of the present application can be chemically modified to facilitate its translocation across the BBB and nerve cell membranes, including but not limited to amidation, acetylation, phosphorylation, side chain modification, palmitate, variants, etc.

[0117] "Pharmaceutically acceptable salt" refers to a salt obtained by reacting a compound with a pharmaceutically acceptable (relatively non-toxic, safe, and suitable for use by patients) acid or base. When the compound contains a relatively acidic functional group, a base addition salt can be obtained by contacting the free form of the compound with a sufficient amount of a pharmaceutically acceptable base in a suitable inert solvent. Pharmaceutically acceptable base addition salts include, but are not limited to, sodium salts, potassium salts, calcium salts, aluminum salts, magnesium salts, bismuth salts, ammonium salts, and the like. When the compound contains a relatively basic functional group, an acid addition salt can be obtained by contacting the free form of the compound with a sufficient amount of a pharmaceutically acceptable acid in a suitable inert solvent. Pharmaceutically acceptable acid addition salts include, but are not limited to, hydrochlorides, sulfates, methanesulfonates, and the like.

[0118] "Pharmaceutical composition" refers to a mixture of one or more compounds of the present invention or their pharmaceutically acceptable salts with other chemical components, such as pharmaceutically acceptable carriers and excipients. The purpose of a pharmaceutical composition is to facilitate the administration of the compound to an organism.

[0119] Examples of types of "excipients" include, but are not limited to, binders, disintegrants, lubricants, glidants, stabilizers, fillers, diluents, and the like.

[0120] EC 50 The half-maximal effect concentration (HEC) is the concentration of drug required to produce a 50% effect.

[0121] mmol means millimole.

[0122] eq stands for equivalent, which refers to the molar ratio of two reagents or materials.

[0123] The compound represented by DIC is N,N'-diisopropylcarbodiimide.

[0124] The compound represented by TBTU is O-benzotriazole-N,N,N',N'-tetramethyluronium tetrafluoroborate.

[0125] DIPEA stands for N,N-diisopropylethylamine.

[0126] The Fmoc group refers to fluorenylmethoxycarbonyl.

[0127] 2-Cl-CTC-ResinChloro(o-chlorophenyl)diphenylmethane.

[0128] Fmoc-Tyr(tBu)-OH: N-Fluorenylmethoxycarbonyl-O-tert-butyl-L-tyrosine.

[0129] Fmoc-Ser(tBu)-OH: N-Fluorenylmethoxycarbonyl-O-tert-butyl-L-serine.

[0130] Fmoc-His(Trt)-OH: N-Fmoc-N'-trityl-L-histidine.

[0131] Fmoc-Gln(Trt)-OH: N-fluorenylmethoxycarbonyl-N'-trityl-L-glutamine.

[0132] Fmoc-Leu-OH: N-Fmoc-L-leucine.

[0133] Fmoc-Lys(Boc)-OH: N-fluorenylmethoxycarbonyl-N-tert-butyloxycarbonyl-L-lysine.

[0134] Fmoc-Asn(Trt)-OH: fluorenylmethoxycarbonyl-N-trityl-L-asparagine.

[0135] Fmoc-Arg(Pbf)-OH: N-Fluorenylmethoxycarbonyl-2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl-L-arginine.

[0136] Fmoc-Gln(Trt)-OH: N-fluorenylmethoxycarbonyl-N'-trityl-L-glutamine.

[0137] Fmoc-Gly-OH: N-Fluorenylmethoxycarbonyl-glycine.

[0138] Fmoc-N-Me-Tyr(tBu)-OH: O-tert-butyl-N-[(9H-fluoren-9-ylmethoxy)carbonyl]-N-methyl-L-tyrosine.

[0139] Fmoc-Tyr(H2PO3)-OH: N-Fluorenylmethoxycarbonyl-O-phospho-L-tyrosine.

[0140] TFA stands for trifluoroacetic acid.

[0141] MPA stands for 2-mercaptopropionic acid.

[0142] TIS stands for triisopropylsilane.

[0143] HOBt stands for 1-hydroxybenzotriazole.

[0144] The Chinese name of TBTU is: O-Benzotriazol-N,N,N',N'-tetramethyluronium tetrafluoroborate DIPEA, and the English name is: O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate.

[0145] MK-801: Dizocilpine maleate (maleate) is a potent, selective, non-competitive NMDA receptor antagonist.

[0146] Material Source:

[0147] The amino acid materials were purchased from Jier Biochemical (Shanghai) Co., Ltd.; the condensation agents DIC, HOBT and TBTU were purchased from Suzhou Haofan Biotechnology Co., Ltd.

[0148] DMEM / F12 culture medium was purchased from White Shark Biotechnology.

[0149] Fetal bovine serum was purchased from Suzhou Yikesai Biotechnology Co., Ltd.

[0150] Penicillin / streptomycin mixed solution was purchased from Hyclone.

[0151] Glutamic acid was purchased from Sigma.

[0152] Propidium iodide / Hoechst staining solution was purchased from Sigma.

[0153] 4% paraformaldehyde was purchased from MacLean's reagent.

[0154] Phosphate buffered saline was purchased from White Shark Biotechnology.

[0155] The corresponding example numbers and amino acid sequence numbers are shown in Table 2:

[0156] Table 2: Correspondence between Example Numbers and Amino Acid Sequence Numbers

[0157] Preparation Example:

[0158] Example 1: Preparation of YGRKKRRQRRRKKNRNKLRRQHSY (SEQ ID NO: 2) polypeptide

[0159] YGRKKRRQRRRKKNRNKLRRQHSY (SEQ ID NO: 2) was prepared according to the method in patent CN111363014A.

[0160] Example 2: Synthesis of Me-YGRKKRRQRRRKKNRNKLRRQHSY (SEQ ID NO: 5)

[0161] Taking 1mmol synthesis scale as an example

[0162] 2.1 Synthesis of Fmoc-Tyr(tBu)-CTC-Resin

[0163] 3.0 g of 2-Cl-CTC-Resin with a substitution density of 1.08 mmol / g was added to the peptide reaction column, washed once with 10 mL of N,N-dimethylformamide, swelled with 20 mL of dichloromethane for 20 minutes, and the solution was removed by aspiration.

[0164] Weigh 1.4 eq of Fmoc-Tyr(tBu)-OH and 4.2 eq of N,N-diisopropylethylamine and add them to the peptide reaction column. Add 20 mL of N,N-dimethylformamide and react for three hours, maintaining a constant temperature of 30°C. Aspirate the solution, wash three times with N,N-dimethylformamide, and block twice with 20 mL of a blocking solution consisting of dichloromethane / methanol / N,N-diisopropylethylamine in a ratio of 17 / 2 / 1, each for 20 minutes. Wash four times with N,N-dimethylformamide, shrink three times with methanol, and vacuum dry.

[0165] Weigh 1 mmol of the corresponding resin and swell it in N,N-dimethylformamide for 30 minutes. Deprotect the resin twice with 20% piperidine / N,N-dimethylformamide solution (20 mL each time, 5 and 10 minutes respectively). Wash the column with N,N-dimethylformamide six times (20 mL each time), maintaining a constant temperature of 30°C. A small amount of the resin was tested for ninhydrin, which indicated a blue-black color.

[0166] 2.2 Coupling of Fmoc-Ser(tBu)-OH

[0167] Weigh Fmoc-Ser(tBu)-OH (4 eq), TBTU (4 eq), and N,N-diisopropylethylamine (6 eq) and add them to the peptide reaction column. Add 20 mL of N,N-dimethylformamide and react for two hours, maintaining a constant temperature of 30°C. Aspirate the solution and wash with N,N-dimethylformamide three times, 20 mL each time. Take a small amount of resin for inspection. The resin is transparent and colorless, indicating that the reaction is complete.

[0168] Maintain the peptide reaction column at 30°C and deprotect the peptide using a 20% piperidine / N,N-dimethylformamide solution (20 mL each) for 5 and 10 minutes, respectively. Wash the column six times with N,N-dimethylformamide (20 mL each). A small amount of resin was tested for ninhydrin. A blue-black color indicates complete deprotection.

[0169] 2.3 Coupling of Fmoc-His(Trt)-OH

[0170] Weigh Fmoc-His(Trt)-OH (4 eq), HOBt (4 eq), and DIC (6 eq) and add them to the peptide reaction column. Add 20 mL of N,N-dimethylformamide and react for two hours, maintaining a constant temperature of 30°C. Aspirate the solution and wash with N,N-dimethylformamide three times, 20 mL each time. Take a small amount of resin for examination. If the resin is transparent and colorless, the reaction is complete.

[0171] Maintain the peptide reaction column at 30°C and deprotect the peptide using a 20% piperidine / N,N-dimethylformamide solution (20 mL each) for 5 and 10 minutes, respectively. Wash the column six times with N,N-dimethylformamide (20 mL each). A small amount of resin was tested for ninhydrin. A blue-black color indicates complete deprotection.

[0172] 2.4 Coupling of Fmoc-Gln(Trt)-OH

[0173] Weigh Fmoc-Gln(Trt)-OH (4 eq), HOBt (4 eq), and DIC (6 eq) and add them to the peptide reaction column. Add 20 mL of N,N-dimethylformamide and react for two hours, maintaining a constant temperature of 30°C. Aspirate the solution and wash with N,N-dimethylformamide three times, 20 mL each time. Take a small amount of resin for examination. If the resin is transparent and colorless, it indicates that the reaction is complete.

[0174] Maintain the peptide reaction column at 30°C and deprotect the peptide using a 20% piperidine / N,N-dimethylformamide solution (20 mL each) for 5 and 10 minutes, respectively. Wash the column six times with N,N-dimethylformamide (20 mL each). A small amount of resin was tested for ninhydrin. A blue-black color indicates complete deprotection.

[0175] 2.5 Coupling of Fmoc-Arg(Pbf)-OH

[0176] Weigh Fmoc-Arg(Pbf)-OH (4 eq), HOBt (4 eq), and DIC (6 eq) and add them to the peptide reaction column. Add 20 mL of N,N-dimethylformamide and react for two hours, maintaining a constant temperature of 30°C. Aspirate the solution and wash with N,N-dimethylformamide three times, 20 mL each time. Take a small amount of resin for examination. If the resin is transparent and colorless, the reaction is complete.

[0177] Maintain the peptide reaction column at 30°C and deprotect the peptide using a 20% piperidine / N,N-dimethylformamide solution (20 mL each) for 5 and 10 minutes, respectively. Wash the column six times with N,N-dimethylformamide (20 mL each). A small amount of resin was tested for ninhydrin. A blue-black color indicates complete deprotection.

[0178] 2.6 Coupling of Fmoc-Arg(Pbf)-OH

[0179] Weigh Fmoc-Arg(Pbf)-OH (4 eq), HOBt (4 eq), and DIC (6 eq) and add them to the peptide reaction column. Add 20 mL of N,N-dimethylformamide and react for two hours, maintaining a constant temperature of 30°C. Aspirate the solution and wash with N,N-dimethylformamide three times, 20 mL each time. Take a small amount of resin for examination. If the resin is transparent and colorless, the reaction is complete.

[0180] Maintain the peptide reaction column at 30°C and deprotect the peptide using a 20% piperidine / N,N-dimethylformamide solution (20 mL each) for 5 and 10 minutes, respectively. Wash the column six times with N,N-dimethylformamide (20 mL each). A small amount of resin was tested for ninhydrin. A blue-black color indicates complete deprotection.

[0181] 2.7 Coupling of Fmoc-Leu-OH

[0182] Weigh Fmoc-Leu-OH (4 eq), HOBt (4 eq), and DIC (6 eq) and add them to the peptide reaction column. Add 20 mL of N,N-dimethylformamide and react for two hours, maintaining a constant temperature of 30°C. Aspirate the solution and wash with N,N-dimethylformamide three times, 20 mL each time. Take a small amount of resin for examination. If the resin is transparent and colorless, it indicates that the reaction is complete.

[0183] Maintain the peptide reaction column at 30°C and deprotect the peptide using a 20% piperidine / N,N-dimethylformamide solution (20 mL each) for 5 and 10 minutes, respectively. Wash the column six times with N,N-dimethylformamide (20 mL each). A small amount of resin was tested for ninhydrin. A blue-black color indicates complete deprotection.

[0184] 2.8 Coupling of Fmoc-Lys(Boc)-OH

[0185] Weigh Fmoc-Lys(Boc)-OH (4 eq), HOBt (4 eq), and DIC (6 eq) and add them to the peptide reaction column. Add 20 mL of N,N-dimethylformamide and react for two hours, maintaining a constant temperature of 30°C. Aspirate the solution and wash with N,N-dimethylformamide three times, 20 mL each time. Take a small amount of resin for examination. If the resin is transparent and colorless, it indicates that the reaction is complete.

[0186] Maintain the peptide reaction column at 30°C and deprotect the peptide using a 20% piperidine / N,N-dimethylformamide solution (20 mL each) for 5 and 10 minutes, respectively. Wash the column six times with N,N-dimethylformamide (20 mL each). A small amount of resin was tested for ninhydrin. A blue-black color indicates complete deprotection.

[0187] 2.9 Coupling of Fmoc-Asn(Trt)-OH

[0188] Weigh Fmoc-Asn(Trt)-OH (4 eq), HOBt (4 eq), and DIC (6 eq) and add them to the peptide reaction column. Add 20 mL of N,N-dimethylformamide and react for two hours, maintaining a constant temperature of 30°C. Aspirate the solution and wash with N,N-dimethylformamide three times, 20 mL each time. Take a small amount of resin for examination. If the resin is transparent and colorless, it indicates that the reaction is complete.

[0189] Maintain the peptide reaction column at 30°C and deprotect the peptide using a 20% piperidine / N,N-dimethylformamide solution (20 mL each) for 5 and 10 minutes, respectively. Wash the column six times with N,N-dimethylformamide (20 mL each). A small amount of resin was tested for ninhydrin. A blue-black color indicates complete deprotection.

[0190] 2.10 Coupling of Fmoc-Arg(Pbf)-OH

[0191] Weigh Fmoc-Arg(Pbf)-OH (4 eq), HOBt (4 eq), and DIC (6 eq) and add them to the peptide reaction column. Add 20 mL of N,N-dimethylformamide and react for two hours, maintaining a constant temperature of 30°C. Aspirate the solution and wash with N,N-dimethylformamide three times, 20 mL each time. Take a small amount of resin for examination. If the resin is transparent and colorless, the reaction is complete.

[0192] Maintain the peptide reaction column at 30°C and deprotect the peptide using a 20% piperidine / N,N-dimethylformamide solution (20 mL each) for 5 and 10 minutes, respectively. Wash the column six times with N,N-dimethylformamide (20 mL each). A small amount of resin was tested for ninhydrin. A blue-black color indicates complete deprotection.

[0193] 2.11 Coupling of Fmoc-Asn(Trt)-OH

[0194] Weigh Fmoc-Asn(Trt)-OH (4 eq), HOBt (4 eq), and DIC (6 eq) and add them to the peptide reaction column. Add 20 mL of N,N-dimethylformamide and react for two hours, maintaining a constant temperature of 30°C. Aspirate the solution and wash with N,N-dimethylformamide three times, 20 mL each time. Take a small amount of resin for examination. If the resin is transparent and colorless, it indicates that the reaction is complete.

[0195] Maintain the peptide reaction column at 30°C and deprotect the peptide using a 20% piperidine / N,N-dimethylformamide solution (20 mL each) for 5 and 10 minutes, respectively. Wash the column six times with N,N-dimethylformamide (20 mL each). A small amount of resin was tested for ninhydrin. A blue-black color indicates complete deprotection.

[0196] 2.12 Coupling of Fmoc-Lys(Boc)-OH

[0197] Weigh Fmoc-Lys(Boc)-OH (4 eq), HOBt (4 eq), and DIC (6 eq) and add them to the peptide reaction column. Add 20 mL of N,N-dimethylformamide and react for two hours, maintaining a constant temperature of 30°C. Aspirate the solution and wash with N,N-dimethylformamide three times, 20 mL each time. Take a small amount of resin for examination. If the resin is transparent and colorless, it indicates that the reaction is complete.

[0198] Maintain the peptide reaction column at 30°C and deprotect the peptide using a 20% piperidine / N,N-dimethylformamide solution (20 mL each) for 5 and 10 minutes, respectively. Wash the column six times with N,N-dimethylformamide (20 mL each). A small amount of resin was tested for ninhydrin. A blue-black color indicates complete deprotection.

[0199] 2.13 Coupling of Fmoc-Lys(Boc)-OH

[0200] Weigh Fmoc-Lys(Boc)-OH (4 eq), HOBt (4 eq), and DIC (6 eq) and add them to the peptide reaction column. Add 20 mL of N,N-dimethylformamide and react for two hours, maintaining a constant temperature of 30°C. Aspirate the solution and wash with N,N-dimethylformamide three times, 20 mL each time. Take a small amount of resin for examination. If the resin is transparent and colorless, it indicates that the reaction is complete.

[0201] Maintain the peptide reaction column at 30°C and deprotect the peptide using a 20% piperidine / N,N-dimethylformamide solution (20 mL each) for 5 and 10 minutes, respectively. Wash the column six times with N,N-dimethylformamide (20 mL each). A small amount of resin was tested for ninhydrin. A blue-black color indicates complete deprotection.

[0202] 2.14 Coupling of Fmoc-Arg(Pbf)-OH

[0203] Weigh Fmoc-Arg(Pbf)-OH (4 eq), HOBt (4 eq), and DIC (6 eq) and add them to the peptide reaction column. Add 20 mL of N,N-dimethylformamide and react for two hours, maintaining a constant temperature of 30°C. Aspirate the solution and wash with N,N-dimethylformamide three times, 20 mL each time. Take a small amount of resin for examination. If the resin is transparent and colorless, the reaction is complete.

[0204] Maintain the peptide reaction column at 30°C and deprotect the peptide using a 20% piperidine / N,N-dimethylformamide solution (20 mL each) for 5 and 10 minutes, respectively. Wash the column six times with N,N-dimethylformamide (20 mL each). A small amount of resin was tested for ninhydrin. A blue-black color indicates complete deprotection.

[0205] 2.15 Coupling of Fmoc-Arg(Pbf)-OH

[0206] Weigh Fmoc-Arg(Pbf)-OH (4 eq), HOBt (4 eq), and DIC (6 eq) and add them to the peptide reaction column. Add 20 mL of N,N-dimethylformamide and react for two hours, maintaining a constant temperature of 30°C. Aspirate the solution and wash with N,N-dimethylformamide three times, 20 mL each time. Take a small amount of resin for examination. If the resin is transparent and colorless, the reaction is complete.

[0207] Maintain the peptide reaction column at 30°C and deprotect the peptide using a 20% piperidine / N,N-dimethylformamide solution (20 mL each) for 5 and 10 minutes, respectively. Wash the column six times with N,N-dimethylformamide (20 mL each). A small amount of resin was tested for ninhydrin. A blue-black color indicates complete deprotection.

[0208] 2.16 Coupling of Fmoc-Arg(Pbf)-OH

[0209] Weigh Fmoc-Arg(Pbf)-OH (4 eq), HOBt (4 eq), and DIC (6 eq) and add them to the peptide reaction column. Add 20 mL of N,N-dimethylformamide and react for two hours, maintaining a constant temperature of 30°C. Aspirate the solution and wash with N,N-dimethylformamide three times, 20 mL each time. Take a small amount of resin for examination. If the resin is transparent and colorless, the reaction is complete.

[0210] Maintain the peptide reaction column at 30°C and deprotect the peptide using a 20% piperidine / N,N-dimethylformamide solution (20 mL each) for 5 and 10 minutes, respectively. Wash the column six times with N,N-dimethylformamide (20 mL each). A small amount of resin was tested for ninhydrin. A blue-black color indicates complete deprotection.

[0211] 2.17 Coupling of Fmoc-Gln(Trt)-OH

[0212] Weigh Fmoc-Gln(Trt)-OH (4 eq), HOBt (4 eq), and DIC (6 eq) and add them to the peptide reaction column. Add 20 mL of N,N-dimethylformamide and react for two hours, maintaining a constant temperature of 30°C. Aspirate the solution and wash with N,N-dimethylformamide three times, 20 mL each time. Take a small amount of resin for examination. If the resin is transparent and colorless, it indicates that the reaction is complete.

[0213] Maintain the peptide reaction column at 30°C and deprotect the peptide using a 20% piperidine / N,N-dimethylformamide solution (20 mL each) for 5 and 10 minutes, respectively. Wash the column six times with N,N-dimethylformamide (20 mL each). A small amount of resin was tested for ninhydrin. A blue-black color indicates complete deprotection.

[0214] 2.18 Coupling of Fmoc-Arg(Pbf)-OH

[0215] Weigh Fmoc-Arg(Pbf)-OH (4 eq), HOBt (4 eq), and DIC (6 eq) and add them to the peptide reaction column. Add 20 mL of N,N-dimethylformamide and react for two hours, maintaining a constant temperature of 30°C. Aspirate the solution and wash with N,N-dimethylformamide three times, 20 mL each time. Take a small amount of resin for examination. If the resin is transparent and colorless, the reaction is complete.

[0216] Maintain the peptide reaction column at 30°C and deprotect the peptide using a 20% piperidine / N,N-dimethylformamide solution (20 mL each) for 5 and 10 minutes, respectively. Wash the column six times with N,N-dimethylformamide (20 mL each). A small amount of resin was tested for ninhydrin. A blue-black color indicates complete deprotection.

[0217] 2.19 Coupling of Fmoc-Arg(Pbf)-OH

[0218] Weigh Fmoc-Arg(Pbf)-OH (4 eq), HOBt (4 eq), and DIC (6 eq) and add them to the peptide reaction column. Add 20 mL of N,N-dimethylformamide and react for two hours, maintaining a constant temperature of 30°C. Aspirate the solution and wash with N,N-dimethylformamide three times, 20 mL each time. Take a small amount of resin for examination. If the resin is transparent and colorless, the reaction is complete.

[0219] Maintain the peptide reaction column at 30°C and deprotect the peptide using a 20% piperidine / N,N-dimethylformamide solution (20 mL each) for 5 and 10 minutes, respectively. Wash the column six times with N,N-dimethylformamide (20 mL each). A small amount of resin was tested for ninhydrin. A blue-black color indicates complete deprotection.

[0220] 2.20 Coupling of Fmoc-Lys(Boc)-OH

[0221] Weigh Fmoc-Lys(Boc)-OH (4 eq), HOBt (4 eq), and DIC (6 eq) and add them to the peptide reaction column. Add 20 mL of N,N-dimethylformamide and react for two hours, maintaining a constant temperature of 30°C. Aspirate the solution and wash with N,N-dimethylformamide three times, 20 mL each time. Take a small amount of resin for examination. If the resin is transparent and colorless, it indicates that the reaction is complete.

[0222] Maintain the peptide reaction column at 30°C and deprotect the peptide using a 20% piperidine / N,N-dimethylformamide solution (20 mL each) for 5 and 10 minutes, respectively. Wash the column six times with N,N-dimethylformamide (20 mL each). A small amount of resin was tested for ninhydrin. A blue-black color indicates complete deprotection.

[0223] 2.21 Coupling of Fmoc-Lys(Boc)-OH

[0224] Weigh Fmoc-Lys(Boc)-OH (4 eq), HOBt (4 eq), and DIC (6 eq) and add them to the peptide reaction column. Add 20 mL of N,N-dimethylformamide and react for two hours, maintaining a constant temperature of 30°C. Aspirate the solution and wash with N,N-dimethylformamide three times, 20 mL each time. Take a small amount of resin for examination. If the resin is transparent and colorless, it indicates that the reaction is complete.

[0225] Maintain the peptide reaction column at 30°C and deprotect the peptide using a 20% piperidine / N,N-dimethylformamide solution (20 mL each) for 5 and 10 minutes, respectively. Wash the column six times with N,N-dimethylformamide (20 mL each). A small amount of resin was tested for ninhydrin. A blue-black color indicates complete deprotection.

[0226] 2.22 Coupling of Fmoc-Arg(Pbf)-OH

[0227] Weigh Fmoc-Arg(Pbf)-OH (4 eq), HOBt (4 eq), and DIC (6 eq) and add them to the peptide reaction column. Add 20 mL of N,N-dimethylformamide and react for two hours, maintaining a constant temperature of 30°C. Aspirate the solution and wash with N,N-dimethylformamide three times, 20 mL each time. Take a small amount of resin for examination. If the resin is transparent and colorless, the reaction is complete.

[0228] Maintain the peptide reaction column at 30°C and deprotect the peptide using a 20% piperidine / N,N-dimethylformamide solution (20 mL each) for 5 and 10 minutes, respectively. Wash the column six times with N,N-dimethylformamide (20 mL each). A small amount of resin was tested for ninhydrin. A blue-black color indicates complete deprotection.

[0229] 2.23 Coupling of Fmoc-Gly-OH

[0230] Weigh Fmoc-Gly-OH (4 eq), HOBt (4 eq), and DIC (6 eq) and add them to the peptide reaction column. Add 20 mL of N,N-dimethylformamide and react for two hours, maintaining a constant temperature of 30°C. Aspirate the solution and wash with N,N-dimethylformamide three times, 20 mL each time. Take a small amount of resin for examination. The resin is transparent and colorless, indicating that the reaction is complete.

[0231] Maintain the peptide reaction column at 30°C and deprotect the peptide using a 20% piperidine / N,N-dimethylformamide solution (20 mL each) for 5 and 10 minutes, respectively. Wash the column six times with N,N-dimethylformamide (20 mL each). A small amount of resin was tested for ninhydrin. A blue-black color indicates complete deprotection.

[0232] 2.24 Coupling of Fmoc-N-Me-Tyr(tBu)-OH

[0233] Weigh Fmoc-N-Me-Tyr(tBu)-OH (4 eq), HOBt (4 eq), and DIC (6 eq) and add them to the peptide reaction column. Add 20 mL of N,N-dimethylformamide and react for two hours, maintaining a constant temperature of 30°C. Aspirate the solution and wash with N,N-dimethylformamide three times, 20 mL each time. Take a small amount of resin for examination. The resin is transparent and colorless, indicating that the reaction is complete.

[0234] Maintain the peptide reaction column at 30°C and deprotect the column twice with 20% piperidine / N,N-dimethylformamide solution (20 mL each) for 5 and 10 minutes, respectively. Wash the column six times with N,N-dimethylformamide (20 mL each). A small amount of resin was tested with ninhydrin. If the resin turned blue-black, deprotection was complete. The column was then condensed with methanol three times for 3 minutes each (20 mL each), and the column was vacuum-dried to obtain the peptide resin.

[0235] 2.25 Cleavage of Peptide Resin

[0236] A volume of 10 eq of frozen lysate (lysate ratio: TFA / MPA / TIS / water = 90 / 5 / 2.5 / 2.5) was added to the peptide resin in a flask. The mixture was maintained at 25°C for 2 hours, filtered, and the filtrate was added to 10 eq of lysate in frozen methyl tert-butyl ether, allowed to settle for 10 minutes, and centrifuged. The crude peptide was washed four times with room temperature methyl tert-butyl ether and dried under vacuum (<50°C) for 3 hours.

[0237] 2.26 Purification

[0238] Mobile phase: 0.1% trifluoroacetic acid in water / 0.1% trifluoroacetic acid in acetonitrile; preparative column: ODS-PK C18, 30 mm×250 mm×8 mm; flow rate: 25 mL / min; column temperature: 25°C; wavelength: 220 nm.

[0239] Gradient: water / acetonitrile: 5:95 to 95:5, 40 min.

[0240] The collected samples were concentrated under reduced pressure and lyophilized to obtain pure peptides. ESI-MS m / z[M+1] + :3282.94.

[0241] Example 3: Synthesis of pYGRKKRRQRRRKKNRNKLRRQHSY (SEQ ID NO: 6)

[0242] The preparation method of reference example 2 was carried out, except that Fmoc-Tyr(H2PO3BZL)-OH was used to replace Fmoc-N-Me-Tyr(tBu)-OH in step 2.24.

[0243] 3.1 Purification

[0244] Mobile phase: 0.1% trifluoroacetic acid in water / 0.1% trifluoroacetic acid in acetonitrile; preparative column: ODS-PK C18, 30 mm×250 mm×8 mm; flow rate: 25 mL / min; column temperature: 25°C; wavelength: 220 nm.

[0245] Gradient: water / acetonitrile: 5:95 to 95:5, 40 min.

[0246] The collected samples were concentrated under reduced pressure and lyophilized to obtain pure peptides. ESI-MS m / z[M+1] + :3348.89.

[0247] Example 4: Synthesis of Ac-YGRKKRRQRRRKKNRNKLRRQHSY (SEQ ID NO: 7)

[0248] The preparation method of Reference Example 2 was used, except that Fmoc-Tyr(tBu)-OH was substituted for Fmoc-N-Me-Tyr(tBu)-OH in step 2.24. After Fmoc-Tyr(tBu)-OH coupling, Fmoc deprotection, and elution, 30 eq of acetic anhydride, 15 eq of pyridine, and 20 mL of N,N-dimethylformamide were added. The reaction was allowed to proceed for one hour, and the mixture was washed six times with N,N-dimethylformamide. The mixture was condensed with methanol three times for 3 minutes each, with a 20 mL reaction mixture, and vacuum-dried to obtain the peptide resin.

[0249] 4.1 Cleavage of peptide resin

[0250] A volume of 10 eq of frozen lysate (lysate ratio TFA / MPA / TIS / water = 90 / 5 / 2.5 / 2.5) was added to frozen methyl tert-butyl ether and allowed to settle for 10 minutes, followed by centrifugation. The crude peptide was washed four times with room temperature methyl tert-butyl ether and dried under vacuum (<50°C) for 3 hours.

[0251] 4.2 Purification

[0252] Mobile phase: 0.1% trifluoroacetic acid in water / 0.1% trifluoroacetic acid in acetonitrile; preparative column: ODS-PK C18, 30 mm×250 mm×8 mm; flow rate: 25 mL / min; column temperature: 25°C; wavelength: 220 nm.

[0253] Gradient: water / acetonitrile: 5:95 to 95:5, 40 min.

[0254] The collected samples were concentrated under reduced pressure and lyophilized to obtain pure peptides. ESI-MS m / z[M+1] + :3310.93.

[0255] Example 5: C 15 H 31 Synthesis of CO-YGRKKRRQRRRKKNRNKLRRQHSY (SEQ ID NO:8)

[0256] The preparation method of Reference Example 2 was used, except that Fmoc-Tyr(tBu)-OH was used instead of Fmoc-N-Me-Tyr(tBu)-OH in step 2.24. After coupling with Fmoc-Tyr(tBu)-OH, deprotection with Fmoc, and elution, palmitic acid (4 eq), HOBt (4 eq), and DIC (6 eq) were added to the peptide reaction column. 20 mL of N,N-dimethylformamide was added and the reaction was continued for two hours, maintaining a constant temperature of 30°C. The solution was removed and the column was washed with N,N-dimethylformamide three times, 20 mL each time. A small amount of resin was taken for inspection. The resin was transparent and colorless, indicating that the reaction was complete. The column was washed with 20 mL of N,N-dimethylformamide six times and condensed with methanol three times, 3 minutes each time, 20 mL each time. The column was then vacuum-dried to obtain the peptide resin.

[0257] 5.1 Cleavage of peptide resin

[0258] A volume of 10 eq of frozen lysate (lysate ratio: TFA / MPA / TIS / water = 90 / 5 / 2.5 / 2.5) was added to the peptide resin in a flask. The mixture was maintained at 25°C for 2 hours, filtered, and the filtrate was added to 10 eq of lysate in frozen methyl tert-butyl ether, allowed to settle for 10 minutes, and centrifuged. The mixture was washed four times with room temperature methyl tert-butyl ether and vacuum dried (<50°C) for 3 hours to obtain the crude peptide.

[0259] 5.2 Purification

[0260] Mobile phase: 0.1% trifluoroacetic acid in water / 0.1% trifluoroacetic acid in acetonitrile; preparative column: ODS-PK C18, 30 mm×250 mm×8 mm; flow rate: 25 mL / min; column temperature: 25°C; wavelength: 220 nm.

[0261] Gradient: water / acetonitrile: 5:95 to 95:5, 40 min.

[0262] The collected samples were concentrated under reduced pressure and lyophilized to obtain pure peptides. ESI-MS m / z[M+1] + :3507.15.

[0263] Example 6: Synthesis of YGRKKRRQRRRKKNRNKLRRQHSY-NH2 (SEQ ID NO: 9)

[0264] Taking 1mmol synthesis scale as an example

[0265] 6.1 Coupling of Fmoc-Tyr(tBu)-OH

[0266] A peptide reaction column was maintained at 30°C. 3.0 g of Rink-Amide-AM-Resin (0.338 mmol / g) was added to the column. The column was washed once with 10 mL of N,N-dimethylformamide and swelled with 20 mL of dichloromethane for 20 minutes. The solution was then removed. The protective groups were deprotected twice with 20% piperidine / N,N-dimethylformamide solution (20 mL each) for 5 minutes and 10 minutes respectively. After deprotection, the column was washed six times with 20 mL of N,N-dimethylformamide and vacuum-dried to obtain the peptide resin. A small amount of the resin was tested with ninhydrin. The resin was blue-black, indicating complete deprotection.

[0267] Weigh Fmoc-Tyr(tBu)-OH (4 eq), HOBt (4 eq), and DIC (6 eq) and add them to the peptide reaction column. Add 20 mL of N,N-dimethylformamide and react for two hours, maintaining a constant temperature of 30°C. Aspirate the solution and wash with N,N-dimethylformamide three times, 20 mL each time. Take a small amount of resin for examination. If the resin is transparent and colorless, it indicates that the reaction is complete.

[0268] Maintain the peptide reaction column at 30°C and deprotect the peptide using a 20% piperidine / N,N-dimethylformamide solution (20 mL each) for 5 and 10 minutes, respectively. Wash the column six times with N,N-dimethylformamide (20 mL each). A small amount of resin was tested for ninhydrin. A blue-black color indicates complete deprotection.

[0269] Steps 6.2-6.24. Refer to steps 2.2-2.24 of Example 2, except that HOBt is used to replace TBTU in step 2.2, DIC is used to replace DIPEA in step 2.2; and Fmoc-Tyr(tBu)-OH is used to replace Fmoc-N-Me-Tyr(tBu)-OH in step 2.24.

[0270] 6.25 Cleavage of Peptide Resin

[0271] A volume of 10 eq of frozen lysate (lysate ratio: TFA / MPA / TIS / water = 90 / 5 / 2.5 / 2.5) was added to the peptide resin in a flask. The mixture was maintained at 25°C for 2 hours, filtered, and the filtrate was added to 10 eq of lysate in frozen methyl tert-butyl ether, allowed to settle for 10 minutes, and centrifuged. The mixture was washed four times with room temperature methyl tert-butyl ether and vacuum dried (<50°C) for 3 hours to obtain the crude peptide.

[0272] 6.26 Purification

[0273] Mobile phase: 0.1% trifluoroacetic acid in water / 0.1% trifluoroacetic acid in acetonitrile; preparative column: ODS-PK C18, 30 mm×250 mm×8 mm; flow rate: 25 mL / min; column temperature: 25°C; wavelength: 220 nm.

[0274] Gradient: water / acetonitrile: 5:95 to 95:5, 40 min.

[0275] The collected samples were concentrated under reduced pressure and lyophilized to obtain pure peptides. ESI-MS m / z[M+1] + :3267.94.

[0276] Example 7: Synthesis of Ac-YGRKKRRQRRRKKNRNKLRRQHSY-NH2 (SEQ ID NO: 10)

[0277] 7.1 Acetylation

[0278] The product from step 6.24 of Example 6 was used as the starting material. 30 eq of acetic anhydride, 15 eq of pyridine, and 20 mL of N,N-dimethylformamide were added. The mixture was reacted for one hour and washed six times with N,N-dimethylformamide. The mixture was then condensed with methanol three times for 3 minutes each, using 20 mL of solution. The mixture was then vacuum dried to obtain the peptide resin.

[0279] 7.2 Cleavage of Peptide Resin

[0280] A volume of 10 eq of frozen lysate (lysate ratio: TFA / MPA / TIS / water = 90 / 5 / 2.5 / 2.5) was added to the peptide resin in a flask. The mixture was maintained at 25°C for 2 hours, filtered, and the filtrate was added to 10 eq of lysate in frozen methyl tert-butyl ether, allowed to settle for 10 minutes, and centrifuged. The mixture was washed four times with room temperature methyl tert-butyl ether and vacuum dried (<50°C) for 3 hours to obtain the crude peptide.

[0281] 7.3 Purification

[0282] Mobile phase: 0.1% trifluoroacetic acid in water / 0.1% trifluoroacetic acid in acetonitrile; preparative column: ODS-PK C18, 30 mm×250 mm×8 mm; flow rate: 25 mL / min; column temperature: 25°C; wavelength: 220 nm.

[0283] Gradient: water / acetonitrile: 5:95 to 95:5, 40 min.

[0284] The collected samples were concentrated under reduced pressure and lyophilized to obtain pure peptides. ESI-MS m / z[M+1] + :3309.95.

[0285] Example 8: Synthesis of pYGRKKRRQRRRKKNRNKLRRQHSY-NH2 (SEQ ID NO: 11)

[0286] 8.1 Coupling of Fmoc-Tyr(H2PO3BZL)-OH

[0287] Using the product from step 6.23 of Example 6 as the starting material, Fmoc-Tyr(H2PO3BZL)-OH (4 eq), HOBt (4 eq), and DIC (6 eq) were weighed and added to a peptide reaction column. 20 mL of N,N-dimethylformamide was added and the reaction was allowed to proceed for two hours, maintaining a constant temperature of 30°C. The solution was removed and washed three times with 20 mL of N,N-dimethylformamide. A small amount of the resin was sampled for examination; the resin was transparent and colorless, indicating a complete reaction.

[0288] Maintain the peptide reaction column at 30°C and deprotect the column twice with 20% piperidine / N,N-dimethylformamide solution (20 mL each) for 5 and 10 minutes, respectively. Wash the column six times with N,N-dimethylformamide (20 mL each). A small amount of resin was tested with ninhydrin. If the resin turned blue-black, deprotection was complete. The column was then condensed with methanol three times for 3 minutes each (20 mL each). The crude peptide resin was then vacuum-dried to dryness.

[0289] 8.2 Cleavage of Peptide Resin

[0290] A volume of 10 eq of frozen lysate (lysate ratio: TFA / MPA / TIS / water = 90 / 5 / 2.5 / 2.5) was added to the peptide resin in a flask. The mixture was maintained at 25°C for 2 hours, filtered, and the filtrate was added to 10 eq of lysate in frozen methyl tert-butyl ether, allowed to settle for 10 minutes, and centrifuged. The mixture was washed four times with room temperature methyl tert-butyl ether and vacuum dried (<50°C) for 3 hours to obtain the crude peptide.

[0291] 8.3 Purification

[0292] Mobile phase: 0.1% trifluoroacetic acid in water / 0.1% trifluoroacetic acid in acetonitrile; preparative column: ODS-PK C18, 30 mm×250 mm×8 mm; flow rate: 25 mL / min; column temperature: 25°C; wavelength: 220 nm.

[0293] Gradient: water / acetonitrile: 5:95 to 95:5, 40 min.

[0294] The collected samples were concentrated under reduced pressure and lyophilized to obtain pure peptides. ESI-MS m / z[M+1] + :3347.90.

[0295] Example 9: Synthesis of Me-YGRKKRRQRRRKKNRNKLRRQHSY-NH2 (SEQ ID NO: 12)

[0296] 9.1 Coupling of Fmoc-N-Me-Tyr(tBu)-OH

[0297] Using the product from step 6.23 of Example 6 as the starting material, Fmoc-N-Me-Tyr(tBu)-OH (4 eq), HOBt (4 eq), and DIC (6 eq) were weighed and added to a peptide reaction column. 20 mL of N,N-dimethylformamide was added and the reaction was allowed to proceed for two hours, maintaining a constant temperature of 30°C. The solution was removed and washed three times with 20 mL of N,N-dimethylformamide. A small amount of the resin was sampled for examination; the resin was transparent and colorless, indicating that the reaction was complete.

[0298] Maintain the peptide reaction column at 30°C and deprotect the column twice with 20% piperidine / N,N-dimethylformamide solution (20 mL each) for 5 and 10 minutes, respectively. Wash the column six times with N,N-dimethylformamide (20 mL each). A small amount of resin was tested with ninhydrin. If the resin turned blue-black, deprotection was complete. The column was then condensed with methanol three times for 3 minutes each (20 mL each), and the column was vacuum-dried to obtain the peptide resin.

[0299] 9.2 Cleavage of Peptide Resin

[0300] A volume of 10 eq of frozen lysate (lysate ratio: TFA / MPA / TIS / water = 90 / 5 / 2.5 / 2.5) was added to the peptide resin in a flask. The mixture was maintained at 25°C for 2 hours, filtered, and the filtrate was added to 10 eq of lysate in frozen methyl tert-butyl ether, allowed to settle for 10 minutes, and centrifuged. The mixture was washed four times with room temperature methyl tert-butyl ether and vacuum dried (<50°C) for 3 hours to obtain the crude peptide.

[0301] 9.3 Purification

[0302] Mobile phase: 0.1% trifluoroacetic acid in water / 0.1% trifluoroacetic acid in acetonitrile; preparative column: ODS-PK C18, 30 mm×250 mm×8 mm; flow rate: 25 mL / min; column temperature: 25°C; wavelength: 220 nm.

[0303] Gradient: water / acetonitrile: 5:95 to 95:5, 40 min.

[0304] The collected samples were concentrated under reduced pressure and lyophilized to obtain pure peptides. ESI-MS m / z[M+1] + :3281.95.

[0305] Example 10: C 15 H 31Synthesis of CO-YGRKKRRQRRRKKNRNKLRRQHSY-NH2 (SEQ ID NO:13)

[0306] 10.1 Palmitylation

[0307] The product from step 6.24 of Example 6 was used as the starting material. Palmitic acid (4 eq), HOBt (4 eq), and DIC (6 eq) were added to a peptide reaction column. 20 mL of N,N-dimethylformamide was added and the reaction was continued for two hours, maintaining a constant temperature of 30°C. The solution was removed and the column was washed three times with 20 mL of N,N-dimethylformamide. A small amount of resin was taken for inspection. The resin was transparent and colorless, indicating that the reaction was complete. The column was washed six times with 20 mL of N,N-dimethylformamide and contracted three times with methanol for 3 minutes each, 20 mL each time. The column was then vacuum-dried to obtain the peptide resin.

[0308] 10.2 Cleavage of Peptide Resin

[0309] A volume of 10 eq of frozen lysate (lysate ratio: TFA / MPA / TIS / water = 90 / 5 / 2.5 / 2.5) was added to the peptide resin in a flask. The mixture was maintained at 25°C for 2 hours, filtered, and the filtrate was added to 10 eq of lysate in frozen methyl tert-butyl ether, allowed to settle for 10 minutes, and centrifuged. The mixture was washed four times with room temperature methyl tert-butyl ether and vacuum dried (<50°C) for 3 hours to obtain the crude peptide.

[0310] 10.3 Purification

[0311] Mobile phase: 0.1% trifluoroacetic acid in water / 0.1% trifluoroacetic acid in acetonitrile; preparative column: ODS-PK C18, 30 mm×250 mm×8 mm; flow rate: 25 mL / min; column temperature: 25°C; wavelength: 220 nm.

[0312] Gradient: water / acetonitrile: 5:95 to 95:5, 40 min.

[0313] The collected samples were concentrated under reduced pressure and lyophilized to obtain pure peptides. ESI-MS m / z[M+1] + :3506.17.

[0314] Example 11: Synthesis of YGRKKRRQRRRKKNRNKIRRQHSY (SEQ ID NO: 14)

[0315] The preparation method of reference example 1 was used, except that Fmoc-Leu-OH in preparation step 7 was replaced with Fmoc-Ile-OH. The pure peptide was prepared, and ESI-MS m / z [M+1] + :3268.92.

[0316] Example 12: Synthesis of YGRKKRRQRRRKKNRNKIRRQHSY-NH2 (SEQ ID NO: 15)

[0317] The preparation method of reference example 6 was used, except that Fmoc-Leu-OH in preparation step 6.7 was replaced with Fmoc-Ile-OH to prepare a pure peptide. ESI-MS m / z [M+1] + :3267.94.

[0318] Example 13: Synthesis of YGRKKRRQRRRKKDRDKLRRQHSY (SEQ ID NO: 16)

[0319] The preparation method of Example 1 was used, except that Fmoc-Asn(Trt)-OH in Steps 9 and 11 of Example 1 was replaced with Fmoc-Asp(otBu)-OH. The pure peptide was prepared, and ESI-MS m / z[M+1] + :3270.89.

[0320] Example 14: Synthesis of YGRKKRRQRRRK(N-AEEA-Palmitic acid)KNRNKLRRQHSY (SEQ ID NO: 17)

[0321] Taking the synthesis scale of 1 mmol as an example, the synthesis steps are the same as those in Example 1, except that Fmoc-Lys(Boc)-OH in Step 13 of Example 1 is replaced with Fmoc-Lys(Dde)-OH.

[0322] 14.1 Coupling of Fmoc-Lys(Dde)-OH

[0323] Weigh Fmoc-Lys(Dde)-OH (4 eq), HOBt (4 eq), and DIC (6 eq) and add them to the peptide reaction column. Add 20 mL of N,N-dimethylformamide and react for two hours, maintaining a constant temperature of 30°C. Aspirate the solution and wash with N,N-dimethylformamide three times, 20 mL each time. Take a small amount of resin for examination. If the resin is transparent and colorless, the reaction is complete.

[0324] Deprotection of the Dde group was performed using 20 mL of a 5% hydrazine hydrate / DMF solution over 60 minutes. The column was then washed six times with 20 mL of DMF, maintaining a constant temperature of 30°C. A small amount of the resin was tested for ninhydrin. The resin was blue-black, indicating complete deprotection.

[0325] 14.2 Coupling of Fmoc-AEEA-OH

[0326] Weigh 4 eq of Fmoc-AEEA-OH, 4 eq of HOBT, and 6 eq of DIC and add them to the peptide reaction column. Add 20 mL of DMF and react for two hours, maintaining a constant temperature of 30°C. Drain the solution and wash with DMF three times, 20 mL each time. Take a small amount of resin for examination. If the resin is transparent and colorless, the reaction is complete.

[0327] The peptide reaction column was maintained at 30°C and the Fmoc protecting group was removed using a 20% piperidine / DMF solution (20 mL each) for 5 and 10 minutes, respectively. The column was then washed with DMF six times (20 mL each) and vacuum-dried to obtain the peptide resin. A small amount of the resin was tested for ninhydrin. The resin was blue-black, indicating complete deprotection.

[0328] 14.3 Palmitylation

[0329] Palmitic acid (4 eq), HOBt (4 eq), and DIC (6 eq) were added to the peptide reaction column. 20 mL of N,N-dimethylformamide was added and the reaction was allowed to proceed for two hours, maintaining a constant temperature of 30°C. The solution was removed and the column was washed three times with 20 mL of N,N-dimethylformamide each time. A small amount of resin was taken for inspection. The resin was transparent and colorless, indicating that the reaction was complete. The column was washed six times with 20 mL of N,N-dimethylformamide and contracted three times with methanol for 3 minutes each time, 20 mL each time. The column was then vacuum-dried to obtain the peptide resin.

[0330] 14.4 Cleavage of Peptide Resin

[0331] A volume of 10 eq of frozen lysate (lysate ratio: TFA / MPA / TIS / water = 90 / 5 / 2.5 / 2.5) was added to the peptide resin in a flask. The mixture was maintained at 25°C for 2 hours, filtered, and the filtrate was added to 10 eq of lysate in frozen methyl tert-butyl ether, allowed to settle for 10 minutes, and centrifuged. The mixture was washed four times with room temperature methyl tert-butyl ether and vacuum dried (<50°C) for 3 hours to obtain the crude peptide.

[0332] 14.5 Purification

[0333] Mobile phase: 0.1% trifluoroacetic acid in water / 0.1% trifluoroacetic acid in acetonitrile; preparative column: ODS-PK C18, 30 mm×250 mm×8 mm; flow rate: 25 mL / min; column temperature: 25°C; wavelength: 220 nm.

[0334] Gradient: water / acetonitrile: 5:95 to 95:5, 40 min.

[0335] The collected samples were concentrated under reduced pressure and lyophilized to obtain pure peptides. ESI-MS m / z[M+1] + :3652.2251.

[0336] Example 15: Synthesis of YGRKKRRQRRRKKNRNKLRREHSY (SEQ ID NO: 18)

[0337] The preparation method of Example 1 was used with the exception that Fmoc-Gln(Trt)-OH in step 4 of Example 1 was replaced with Fmoc-Glu(otBu)-OH. The pure peptide was obtained, and ESI-MS m / z[M+1] + :3269.91.

[0338] Example 16: Synthesis of YGRKKRRQRRRKKNRDKLRREHSY (SEQ ID NO: 19)

[0339] The preparation method of Example 1 was used with the exception that Fmoc-Gln(Trt)-OH in step 4 and Fmoc-Asn(Trt)-OH in step 9 were replaced with Fmoc-Glu(otBu)-OH and Fmoc-Asp(otBu)-OH, respectively. A pure peptide was obtained, with ESI-MS m / z [M+1]: + :3270.89.

[0340] Example 17: Synthesis of YGRKKRRERRRKKDRNKLRRQHSY (SEQ ID NO: 20)

[0341] The preparation method of Example 1 was used with the exception that Fmoc-Asn(Trt)-OH in step 11 and Fmoc-Gln(Trt)-OH in step 17 of Example 1 were replaced with Fmoc-Asp(otBu)-OH and Fmoc-Glu(otBu)-OH, respectively. A pure peptide was obtained, with ESI-MS m / z [M+1]: + :3270.89.

[0342] Example 18: Synthesis of Ac-YGRKKRRERRRKKDRDKLRREHSY-NH2 (SEQ ID NO: 21)

[0343] The preparation method of Reference Example 7 was used, except that Fmoc-Gln(Trt)-OH in step 7.4, Fmoc-Asn(Trt)-OH in step 7.9, Fmoc-Asn(Trt)-OH in step 11, and Fmoc-Gln(Trt)-OH in step 17 were replaced with Fmoc-Glu(otBu)-OH, Fmoc-Asp(otBu)-OH, Fmoc-Asp(otBu)-OH, and Fmoc-Glu(otBu)-OH, respectively. A pure peptide was prepared, and ESI-MS m / z [M+1] +:3314.89.

[0344] Example 19: Synthesis of Me-YGRKKRRERRRKKDRDKLRREHSY-NH2 (SEQ ID NO: 22)

[0345] The preparation method of Reference Example 9 was used, except that Fmoc-Gln(Trt)-OH in step 9.4, Fmoc-Asn(Trt)-OH in step 9.9, Fmoc-Asn(Trt)-OH in step 9.11, and Fmoc-Gln(Trt)-OH in step 9.17 were replaced with Fmoc-Glu(otBu)-OH, Fmoc-Asp(otBu)-OH, Fmoc-Asp(otBu)-OH, and Fmoc-Glu(otBu)-OH, respectively. A pure peptide was prepared, with ESI-MS m / z [M+1] + :3286.89.

[0346] Example 20: Synthesis of Ac-YGRKKRRERRRKKDRDK1RREHSY-NH2 (SEQ ID NO: 23)

[0347] The preparation method of reference example 18 was used, except that Fmoc-Leu-OH in step 18.7 was replaced with Fmoc-Leu-OH (D configuration). The pure peptide was prepared, ESI-MS m / z [M+1] + :3314.89.

[0348] Example 21: Synthesis of Ac-YGRKKRRERRRKKDRDKLRRQHSY-NH2 (SEQ ID NO: 24)

[0349] The preparation method of Example 7 was used, except that Fmoc-Asn(Trt)-OH in step 7.9, Fmoc-Asn(Trt)-OH in step 7.11, and Fmoc-Gln(Trt)-OH in step 7.17 were replaced with Fmoc-Asp(otBu)-OH, Fmoc-Asp(otBu)-OH, and Fmoc-Glu(otBu)-OH, respectively. A pure peptide was prepared, with ESI-MS m / z [M+1] + :3313.90.

[0350] Example 22: Synthesis of Me-YGRKKRRERRRKKDRDKLRRQHSY-NH2 (SEQ ID NO: 25)

[0351] The preparation method of Reference Example 9 was used, except that Fmoc-Asn(Trt)-OH in step 9.9, Fmoc-Asn(Trt)-OH in step 9.11, and Fmoc-Gln(Trt)-OH in step 9.17 were replaced with Fmoc-Asp(otBu)-OH, Fmoc-Asp(otBu)-OH, and Fmoc-Glu(otBu)-OH, respectively. A pure peptide was obtained, with ESI-MS m / z [M+1]: + :3285.91.

[0352] Example 23: Synthesis of Ac-YGRKKRRERRRKKDRDKVRREHSY-NH2 (SEQ ID NO: 26)

[0353] The preparation method of reference example 18 was used, except that Fmoc-Leu-OH in step 18.7 was replaced with Fmoc-Val-OH. The pure peptide was prepared, ESI-MS m / z [M+1] + :3300.87.

[0354] Example 24: Synthesis of Me-YAibRKKRRERRRKKDRDKTleRREHSY-NH2 (SEQ ID NO: 27)

[0355] The preparation method of Example 19 was used, except that Fmoc-Leu-OH in step 19.7 and Fmoc-Gly-OH in step 19.23 were replaced with Fmoc-Tle-OH and Fmoc-Aib-OH, respectively. The pure peptide was prepared, and ESI-MS m / z [M+1] + :3313.92.

[0356] Example 25: Synthesis of Ac-YGRKKRRERRRKKDRDKTleRREHSY-NH2 (SEQ ID NO: 28)

[0357] The preparation method of reference example 18 was used, except that Fmoc-Leu-OH in step 18.7 was replaced with Fmoc-Tle-OH. The pure peptide was prepared, ESI-MS m / z [M+1] + :3313.88.

[0358] Example 26: Synthesis of Ac-YGRK(DK)(DR)RER(DR)RKKDRDK(DL)(DR)REHSY-NH2 (SEQ ID NO: 29)

[0359] The preparation method of Reference Example 18 was used, except that Fmoc-Arg(Pbf)-OH in step 18.6, Fmoc-Leu-OH in step 18.7, Fmoc-Arg(Pbf)-OH in step 18.15, Fmoc-Arg(Pbf)-OH in step 18.19, and Fmoc-Lys(Boc)-OH in step 18.20 were replaced with Fmoc-Arg(Pbf)-OH (D configuration), Fmoc-Leu-OH (D configuration), Fmoc-Arg(Pbf)-OH (D configuration), Fmoc-Arg(Pbf)-OH (D configuration), and Fmoc-Lys(Boc)-OH (D configuration), respectively. A pure peptide was prepared, and ESI-MS m / z [M+1] + :3312.89.

[0360] Effect Example 1: When PC-12 nerve cells are stimulated by glutamate, the polypeptide compound of the present application increases the cell survival rate

[0361] 1.1 PC-12 cell line culture and excitotoxic cell death model

[0362] PC-12 cells (rat adrenal medulla neuroblastoma cell line) were cultured in DMEM / F12 complete medium until the confluence was 80-90%. The cells were digested, collected, resuspended, and plated at 5×10 4 Cells were seeded / well in a 24-well plate covered with a slide. After 26 hours of cell culture, glutamate was used to induce excitotoxicity. The model was established by weighing an appropriate amount of glutamate, preparing a stock solution in DMEM / F12 medium, filtering it, and diluting it with culture medium. The cells were then administered as a medium exchange. Incubation was continued for 24 hours after administration.

[0363] 1.2. Administration of small molecules

[0364] Before glutamate modeling, the cells incubated in the previous step were pretreated with 10 nM of the polypeptide compound of the present application for 30 minutes, and then stimulated with glutamate.

[0365] 1.3. Propidium iodide staining

[0366] The cells treated with the polypeptide compound of the present application were stained with propidium iodide to assess cell death. After discarding the culture medium, the cells were washed with PBS, propidium iodide / Hoechst was added, and the cells were taken out after incubation for 30 minutes. The staining solution was discarded, and the cells were fixed and sealed after washing with PBS. The images were collected under a fluorescence microscope. The propidium iodide-positive stained cells and Hoechst-positive stained cells in the collected images were counted and the cell survival rate was calculated. With MK-801 as the positive compound, the standardized survival rate calculation formula was: (compound group survival rate-glutamate group survival rate) / (positive control group survival rate-glutamate group survival rate)*100%. Specific data are shown in Table 3.

[0367] Based on the dose-effect relationship study of the above compounds, the inhibitory effects of different small molecule compounds on excitotoxicity of PC-12 neurons at the same dose (10 nM) were further compared. The specific data are shown in Table 3.

[0368] Table 3: Standardized survival rate (%) of each polypeptide compound in promoting the survival of PC-12 neural cells

[0369] The results showed that the polypeptide compound of the present application was significantly superior to Example 1 in effectively inhibiting glutamate-induced excitotoxicity in PC-12 cells, demonstrating a significant improvement in the biological activity of the polypeptide compound of the present application. Based on this, a dose-effect relationship analysis of the neuroprotective effect of the polypeptide compound of the present application on PC-12 cells was performed, with specific data shown in Table 4.

[0370] Table 4: Dose-effect relationship study of various polypeptide compounds on promoting PC-12 neuronal cell survival

[0371] The results showed that the polypeptide compound of the present application can effectively inhibit the excitotoxicity of PC-12 cells induced by glutamate; compared with Example 1, its EC 50 All of them were significantly reduced, indicating that the biological activity of the polypeptide compound of the present application was significantly improved.

[0372] Effect Example 2: Pharmacokinetic characteristics of each compound in rats

[0373] Intravenous administration: 3 SD rats per group were administered Example 1, Example 2, Example 3, Example 6, Example 7, Example 17, and Example 19 via the tail vein at a dose of 10 mg / kg. After administration, all SD rats were free to eat and drink. Plasma was collected 3 minutes, 10 minutes, 30 minutes, 1 hour, 2 hours, 6 hours, 10 hours, and 24 hours after administration, with 3 rats per time point.

[0374] Sample processing: Whole blood was anticoagulated with EDTA-K2. Within 30 minutes after whole blood collection, the blood was centrifuged at 2-8°C and a centrifugal force of 3000g for 10 minutes. After centrifugation, the separated plasma was transferred to a new 1.5mL centrifuge tube and frozen at -80°C.

[0375] Analytical Methods: LC-MS / MS was used on a Shimadzu LC30AD and AB Sciex QTRAP 6500+ instrument to measure the concentrations of each compound in rat plasma and calculate the pharmacokinetic parameters after administration. The results are shown in Table 5.

[0376] Table 5: Pharmacokinetic parameters after administration

[0377] The results show that the polypeptide compound of the present application can effectively improve the stability of the polypeptide itself in the body and its ability to cross cells; compared with Example 1, its plasma half-life (T 1 / 2 ) was significantly prolonged, and the tissue distribution volume (V ss ) increased significantly, and the average residence time in the body was significantly prolonged (MRT inf ), indicating that the polypeptide compound of the present application can be better distributed from the blood to the tissues and can play a role in the body for a longer time.

[0378] Although various embodiments of the compositions and methods for treating nerve damage have been described in great detail herein, such embodiments are provided merely as non-limiting examples of the application described herein. Therefore, it will be understood by those skilled in the art that various changes and modifications may be made to the application without departing from the scope of the application. In fact, the present application is not intended to be exhaustive or to limit the scope of the application.

[0379] Further, in the description of representative embodiments, the present application content has given the method and / or process of the present application in a specific order of steps. However, the method or process should not be limited to the specific order of steps described. Other order of steps are possible. Therefore, the specific order of steps applied for herein should not be interpreted as limiting the present application. In addition, the application content for the method and / or process should not be limited to performing their steps in the order described. Such an order can be varied and still be within the scope of the present application.

[0380] The foregoing is merely a list of specific embodiments of the present application, intended to enable those skilled in the art to understand and implement the present application. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the present application. Therefore, the present application is not limited to the embodiments described herein, but is intended to conform to the broadest scope consistent with the principles and novel features of the present application.

Claims

1. A polypeptide compound having a structure represented by the general formula (I): KX 2 X 3 RX 5 KX 7 X 8 RX 10 HSY (SEQ ID NO: 3)-R 1 (I) in, X 2 is selected from Lys, HoLys, Dap, DAB, Cit, Orn, D-Lys, D-HoLys, D-Dap, D-DAB, D-Cit, D-Orn or a side chain amino group is Z 1 Modified Lys;X 3 and X 5 are independently selected from Asn, Asp, D-Asn or D-Asp; X 7 is selected from Ile, Leu, Val, Tel, D-Ile, D-Leu, D-Val or D-Tel; X 8 is selected from Arg, HomoArg, D-Arg or D-HomoArg; X 10 Selected from Glu, Gln, D-Glu or D-Gln; R 1 Selected from -OH, -ol, -NR 2 R 3 AND-OR 4 , where R 2 and R 3 are independently selected from hydrogen, C 1-16 Alkyl, C 3-12 Cycloalkyl and 3 to 12 membered heterocycloalkyl, or R 2 , R 3 Together with the nitrogen atom to which it is connected, it forms a 3- to 12-membered heterocycloalkyl group; R 4 Selected from C 1-16 Alkyl, C 3-12 Cycloalkyl and 3- to 12-membered heterocycloalkyl; The alkyl, cycloalkyl, heterocycloalkyl group may be optionally replaced by one or more identical or different R 5 replace; R 5 Selected from halogen, nitro, cyano, hydroxyl, carbonyl, carboxyl, -NR a R b , thiol, C 1-8 Alkyl, C 1-8 Alkoxy, halogenated C 1-8 Alkyl, halogenated C 1-8 Alkoxy, C 3-8 Cycloalkyl, C 3-8 Cycloalkyloxy, 3- to 8-membered heterocycloalkyl, -SC 1-3 Alkyl, -SO2C 1-3 Alkyl, -SO2NR a R b 、-S(O)C 1-3 Alkyl, -C(O)NR a R b 、-C(O)C 1-3 Alkyl, -C(O)OC 1-3 Alkyl, -OC(O)C 1-3 Alkyl, C 6-10 Aryl, 5- to 10-membered heteroaryl; R a , R b are independently hydrogen or C 1-8 Alkyl; or R a , R b Together with the nitrogen atom to which it is attached, it forms a 3- to 6-membered heterocycloalkyl group; and Z 1 Selected from-AEEA-COR 6 , where R 6 C 1-15 of alkyl.

2. The polypeptide compound according to claim 1, wherein The polypeptide compound further comprises a cell-penetrating peptide at its N-terminus or C-terminus.

3. The polypeptide compound according to claim 2, wherein: The N-terminus of the polypeptide compound contains a cell-penetrating peptide, and the N-terminus of the cell-penetrating peptide is R 7 The structure of the polypeptide compound is shown in the general formula (II): 7 -Cell-penetrating peptide-KX 2 X 3 RX 5 KX 7 X 8 RX 10 HSY (SEQ ID NO: 3)-R 1 (II), Among them, R 7 Selected from hydrogen, C 1-16 Alkyl, C 3-12 Cycloalkyl, 3- to 12-membered heterocycloalkyl, R 8 CO-, and phosphate groups, where R 8 Selected from C 1-16 Alkyl, C 3-12 Cycloalkyl and 3 to 12 membered heterocycloalkyl, C 6-10 Aryl, 5- to 10-membered heteroaryl.

4. The polypeptide compound according to claim 2 or 3, wherein: The cell-penetrating peptide is selected from TAT sequence, MAP sequence, MTS sequence or R9 sequence or variants thereof.

5. The polypeptide compound according to any one of claims 2 to 4, wherein: The cell-penetrating peptide is a TAT sequence or a variant thereof, and the TAT sequence at least includes the 49th to 57th amino acid fragment in the transcriptional transactivator protein.

6. The polypeptide compound according to any one of claims 2 to 4, wherein: The cell-penetrating peptide is a TAT sequence, wherein X 2 Selected from Lys;X 3 and X 5 Selected from Asn;X 7 Selected from Leu;X 8 Selected from Arg; X 10 is selected from Gln; the structure of the polypeptide compound is shown in the general formula (III): R 7 -TAT-KKNRNKLRRQHSY(SEQ ID NO:1)-R 1 (III), Wherein, TAT is connected to KKNRNKLRRQHSY (SEQ ID NO: 1) via a main chain amide bond, wherein the nitrogen atom in the main chain amide bond is optionally replaced by C 1-3 Alkylation, such as methylation; R 1 Selected from -OH, -ol, -NR 2 R 3 AND-OR 4 , where R 2 and R 3 are independently selected from hydrogen, C 1-16 Alkyl, C 3-12 Cycloalkyl and 3 to 12 membered heterocycloalkyl, or R 2 , R 3 Together with the nitrogen atom to which it is connected, it forms a 3- to 12-membered heterocycloalkyl group; R 4 Selected from C 1-16 Alkyl, C 3-12 Cycloalkyl and 3- to 12-membered heterocycloalkyl; The alkyl, cycloalkyl, heterocycloalkyl group may be optionally replaced by one or more identical or different R 5 replace; R 5 Selected from halogen, nitro, cyano, hydroxyl, carbonyl, carboxyl, -NR a R b , thiol, C 1-8 Alkyl, C 1-8 Alkoxy, halogenated C 1-8 Alkyl, halogenated C 1-8 Alkoxy, C 3-8 Cycloalkyl, C 3-8 Cycloalkyloxy, 3- to 8-membered heterocycloalkyl, -SC 1-3 Alkyl, -SO2C 1-3 Alkyl, -SO2NR a R b 、-S(O)C 1-3 Alkyl, -C(O)NR a R b 、-C(O)C 1-3 Alkyl, -C(O)OC 1-3 Alkyl, -OC(O)C 1-3 Alkyl, C 6-10 Aryl, 5- to 10-membered heteroaryl; R a , R b are independently hydrogen or C 1-8 Alkyl; or R a , R b Together with the nitrogen atom to which it is attached, it forms a 3- to 6-membered heterocycloalkyl group; and R 7 When it is hydrogen, R 1 is not -OH; the TAT sequence at least includes the 49th to 57th amino acid fragment of the transcriptional transactivator protein; R 7 Selected from hydrogen, C 1-16 Alkyl, C 3-12 Cycloalkyl, 3- to 12-membered heterocycloalkyl, R 8 CO-, and phosphate groups, where R 8 Selected from C 1-16 Alkyl, C 3-12 Cycloalkyl and 3 to 12 membered heterocycloalkyl, C 6-10 Aryl, 5- to 10-membered heteroaryl.

7. The polypeptide compound according to any one of claims 4 to 6, wherein: The TAT sequence is a short peptide selected from YGRKKRRQRRR (SEQ ID NO: 4), GRKKRRQRRR (SEQ ID NO: 33), YGRKKRRQRRRPPQ (SEQ ID NO: 34) or GRKKRRQRRRQ (SEQ ID NO: 35).

8. The polypeptide compound according to claim 6 or 7, wherein The variant of the TAT sequence has 1, 2 or 3 amino acid substitutions, additions or deletions in the TAT sequence.

9. The polypeptide compound according to any one of claims 6 to 8, wherein The variant of the TAT sequence has a sequence selected from YGRKKRRQRRR (SEQ ID NO: 4) that is at least 80%, at least 85%, at least 90%, at least 95%, at least 99% or 100% identical.

10. The polypeptide compound according to any one of claims 6 to 9, wherein: The variant of the TAT sequence is an amino acid sequence in which at least one Gln in the TAT sequence is replaced by Glu, and / or at least one L-Arg is replaced by D-Arg, and / or at least one L-Lys is replaced by D-Lys.

11. The polypeptide compound according to any one of claims 1 to 10, wherein R 1 Selected from -OH or -NH2.

12. The polypeptide compound according to any one of claims 3 to 11, wherein: R 7 Selected from hydrogen, C 1-3 Alkyl or acetyl.

13. The polypeptide compound according to any one of claims 1 to 12, wherein: The polypeptide compound is selected from the following polypeptide compounds shown in SEQ ID NO: 5-SEQ ID NO: 29: Me-YGRKKRRQRRRKKNRNKLRRQHSY (SEQ ID NO: 5), pYGRKKRRQRRRKKNRNKLRRQHSY (SEQ ID NO: 6), Ac-YGRKKRRQRRRKKNRNKLRRQHSY (SEQ ID NO: 7), C 15 H 31 CO-YGRKKRRQRRRKKNRNKLRRQHSY(SEQ ID NO:8)、YGRKKRRQRRRKKNRNKLRRQHSY-NH2(SEQ ID NO:9)、Ac-YGRKKRRQRRRKKNRNKLRRQHSY-NH2(SEQ ID NO:10)、 Me-YGRKKRRQRRRKKNRNKLRRQHSY-NH2 (SEQ ID NO: 12), C 15 H 31 CO-YGRKKRRQRRRKKNRNKLRRQHSY-NH2(SEQ ID NO:13)、 YGRKKRRQRRRKKNRNKIRRQHSY(SEQ ID NO:14)、 YGRKKRRQRRRKKNRNKIRRQHSY-NH2(SEQ ID NO:15)、 YGRKKRRQRRRKKDRDKLRRQHSY(SEQ ID NO:16)、 YGRKKRRQRRR-K(N-AEEA-Palmitic acid)-KNRNKLRRQHSY(SEQ ID NO:17)、 YGRKKRRQRRRKNRNKLRREHSY(SEQ ID NO:18)、 YGRKKRRQRRQHSY(SEQ ID NO:18)、 YGRKKRRQRRRKKNRDKLRREHSY(SEQ ID NO:19)、 YGRKKRRERRRKKDRNKLRRQHSY(SEQ ID NO:20)、 Ac-YGRKKRRERRRKKDRDKLRREHSY-NH2(SEQ ID NO:21) NO:22)、 Ac-YGRKKRRERRRKKKDRDK(DL)RREHSY-NH2(SEQ ID NO:23)、 Ac-YGRKKRRERRRKKDRDKLRRQHSY-NH2(SEQ ID NO:24)、 Me-YGRKKRRERRRKKDRKKLRQXQXY-NH2(SEQ ID NO:24)、 NO: Ac-YGRKKRRERRRKKDRDKVRREHSY-NH2(SEQ ID NO:26)、 Me-YAibRKKRRERRRKKDRDKTleRREHSY-NH2(SEQ ID NO:27)、 Ac-YGRKKRRERRRKKDRKTleRREHSY-NH2(SEQ ID NO:28) Ac-YGRK(DK)(DR)RER(DR)RKKDRDK(DL)(DR)REHSY-NH2(SEQ ID NO:29)。 14. A pharmaceutical composition comprising the polypeptide compound or variant thereof, or a pharmaceutically acceptable salt thereof according to any one of claims 1 to 13, and a pharmaceutically acceptable excipient.

15. Use of the polypeptide compound according to any one of claims 1 to 13, or a variant or a pharmaceutically acceptable salt of the polypeptide compound, or the pharmaceutical composition according to claim 14 in the preparation of a drug, wherein: The drug is a drug for treating traumatic brain injury and / or inhibiting nerve damage after traumatic brain injury.

16. A method for treating traumatic brain injury and / or inhibiting neural damage after traumatic brain injury, comprising administering to a subject the polypeptide compound according to any one of claims 1 to 13, or a variant or a pharmaceutically acceptable salt of the polypeptide compound, or the pharmaceutical composition according to claim 14.

17. The polypeptide compound according to any one of claims 1 to 13, or a variant or a pharmaceutically acceptable salt of the polypeptide compound, or the pharmaceutical composition according to claim 14, for use in treating traumatic brain injury and / or inhibiting neural damage after traumatic brain injury.

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