Neuroprotective PSD-95 polypeptide inhibitors and uses thereof

Abstract

Neuroprotective PSD-95 polypeptide inhibitors and uses thereof are provided. Specifically, a series of active peptides capable of binding to the PDZ1 domain or PDZ2 domain of PSD-95, and pharmaceutically acceptable salts thereof, as well as pharmaceutical compositions, are provided. The structures of the active peptides are shown in formula (I), and (I) can be used as inhibitors of PSD-95-mediated protein-protein interactions to treat stroke and nervous system-related diseases.

Description

[Technical Field]

[0001] This application claims priority to Chinese patent application CN202310027484.8 filed on January 9, 2023 and Chinese patent application CN202311125942.8 filed on September 1, 2023.

[0002] The present disclosure belongs to the field of biomedicine, and specifically relates to active peptides that can bind to the PDZ1 domain or PDZ2 domain of PSD-95, and can be used as inhibitors of PSD-95-mediated protein-protein interactions in the treatment of stroke and nervous system-related diseases. [Background technology]

[0003] Postsynaptic density protein-95 (PSD-95) is an important scaffolding protein in postsynaptic membranes in the central nervous system. It consists of three repeating PDZ domains at the N-terminus: PDZ1, PDZ2, and PDZ3; an intermediate SH3 domain; and a C-terminal guanylate kinase domain. PDZ domains are common protein-protein interaction domains, and PSD-95 binds to the tSXV-COOH of NMDARs (N-methyl-D-aspartate receptors) via PDZ1 and PDZ2, and to the PDZ of nNOS (neuronal nitric oxide synthase) via PDZ2 (Acta Pharmacol. Sin., 2018, 39:661-668).

[0004] Impaired local cerebral blood flow after ischemic stroke causes glutamate to accumulate in the extracellular space, and excessively released excitatory glutamate continues to act on glutamate receptors, causing neuronal depolarization and calcium influx, resulting in excessive Ca 2+ activates nNOS via calmodulin, which leads to an abnormal increase in the production of nitric oxide (NO), ultimately causing neuronal damage.

[0005] PSD-95 inhibitors effectively disrupt the intracellular interaction between NMDA receptors and PSD-95, disrupting the GluN2B-PSD95-nNOS complex, inhibiting NMDA-mediated NO production and protecting neurons. Nerinetide (also known as TAT-NR2B9c or NA-1) is a PSD-95 inhibitor developed by NoNO Inc., Canada (Science, 2002, 298:846-50) and belongs to the neuroexcitotoxicity inhibitor family. Nerinetide consists of 20 amino acid residues, of which TAT is derived from the human (HIV-1) transcriptional transactivator and can cross multiple cell membranes. NR2B9c is selected from the C-terminal 9 amino acids of the NR2B subunit.

[0006] In preclinical animal models (rats and cynomolgus monkeys), administration of nerinetide after ischemic stroke significantly reduced infarct size and improved neurobehavioral function (Sci.Transl.Med., 2021, 13, eabb1498). Results from a phase II clinical study in patients undergoing endovascular aneurysm repair therapy (ENACT) and a phase III clinical study in patients with severe acute ischemic stroke (AIS) undergoing endovascular thrombectomy therapy (EVT) demonstrated no drug-related serious adverse events and that nerinetide's safety profile was comparable to that of placebo.

[0007] Currently, research on PSD-95 inhibitors includes WO2015078477A, ​​WO2022150655A, etc., and in order to meet clinical needs, there is still a need to develop PSD-95 inhibitors with high stability and good pharmacokinetic properties that are suitable for the treatment of diseases such as ischemic stroke. Summary of the Invention

[0008] The present disclosure provides a compound of formula (G) or a pharmaceutically acceptable salt thereof: [ka] wherein X1 is selected from Gly or a deletion; X2 is selected from Aib, Ala, Ile, Cha, Phe, Trp, 1-Nal, Ser, Lys, Arg, Nle, Nva, Orn, cyclopropyl Ala, 4-thiazole Ala, homoLeu, or a deletion; X3 is selected from Dap, Dab, Thr, Aib, Ala, Cha, Phe, Trp, 1-NaI, Asn, Glu, Lys, Arg, 1Me-Trp, 2-NaI, Gln, Thr, 4-thiazole Ala or a deletion; X4 is selected from Thr, homoArg, Ser, Phe, or a deletion; X5 is selected from Tle, homoArg, Arg, Lys, 4-thiazoleAla, Chg, Cha, homoPhe, 4F-Phe, 3F-Phe, 2F-Phe, 4-Pal, 3-Pal, 2-Pal, Cit, Orn, or Phe; X6 is selected from Tle, Ile, Phe, Trp, Leu, 1-Nal, Nle, D-Nle, D-Trp or D-Phe; X7 is selected from Thr or Ser, X8 is Asp, Cha, Leu, Trp, 1-Nal, Phe, Val, Ala, Abu, Ser or Thr; The above X9 is Val.

[0009] In another aspect, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof: [ka] wherein X1 is selected from Gly or a deletion; wherein X1 is selected from Gly or a deletion; X2 is selected from Aib, Ala, Ile, Cha, Phe, Trp, 1-Nal, Ser, Lys, Arg, Nle, Nva, Orn, cyclopropyl Ala, 4-thiazole Ala, homoLeu, or a deletion; X3 is selected from Dap, Dab, Thr, Aib, Ala, Cha, Phe, Trp, 1-NaI, Asn, Glu, Lys, Arg, 1Me-Trp, 2-NaI, Gln, Thr, 4-thiazole Ala or a deletion; X4 is selected from Thr, homoArg, Ser, Phe, or a deletion; X5 is selected from Tle, homoArg, Arg, Lys, 4-thiazoleAla, Chg, Cha, homoPhe, 4F-Phe, 3F-Phe, 2F-Phe, 4-Pal, 3-Pal, 2-Pal, Cit, Orn, or Phe; X6 is selected from Tle, Ile, Phe, Trp, Leu, 1-Nal, Nle, D-Nle, D-Trp or D-Phe; X7 is selected from Thr or Ser, X8 is Asp, Cha, Leu, Trp, 1-Nal, Phe, Val, Ala, Abu, Ser or Thr; The above X9 is Val, The L1 is a chemical bond or comprises polyethylene glycol, one or two oxygen atoms of which are optionally replaced with nitrogen atoms, and the CPP is an internalizing peptide.

[0010] In an alternative embodiment, the compound of formula (G), formula (I), or a pharmaceutically acceptable salt thereof provided by the present disclosure is wherein X1 is selected from Gly or a deletion; X2 is selected from Aib, Ala, Ile, Cha, Phe, Trp, 1-Nal, Ser, Lys, Arg, Nle, Nva, Orn, cyclopropyl Ala, 4-thiazole Ala, homoLeu, or a deletion; X3 is selected from Dap, Dab, Thr, Aib, Ala, Cha, Phe, Trp, 1-NaI, Asn, Glu, Lys, Arg, 1Me-Trp, 2-NaI, Gln, Thr, 4-thiazolylalanine or a deletion; X4 is selected from Thr, homoArg, Ser, or a deletion; X5 is selected from Tle, homoArg, and Chg; X6 is selected from Tle or Ile; X7 is selected from Thr or Ser, X8 is Asp, The above X9 is Val.

[0011] In some embodiments, the present disclosure provides a compound of formula (G), formula (I), or a pharmaceutically acceptable salt thereof, wherein X7 is Thr.

[0012] In some embodiments, the present disclosure provides a compound of formula (G), formula (I), or a pharmaceutically acceptable salt thereof, wherein X6 is Tle.

[0013] In some embodiments, the present disclosure provides a compound of Formula (G), Formula (I), or a pharmaceutically acceptable salt thereof, wherein X5 is Tle.

[0014] In some embodiments, the present disclosure provides a compound represented by Formula (G), Formula (I), or a pharmaceutically acceptable salt thereof, wherein X6-X7 are selected from Tle-Thr, Ile-Thr, or Tle-Ser.

[0015] In some embodiments, the present disclosure provides a compound of Formula (G), Formula (I), or a pharmaceutically acceptable salt thereof, wherein X6-X7 is selected from Tle-Thr or Ile-Thr.

[0016] In some embodiments, the present disclosure provides a compound of Formula (G), Formula (I), or a pharmaceutically acceptable salt thereof, wherein X6-X7 is Tle-Thr.

[0017] In some embodiments, the present disclosure provides a compound represented by Formula (G), Formula (I), or a pharmaceutically acceptable salt thereof, wherein X5-X6 is selected from Tle-Tle, homoArg-Ile, or Chg-Tle.

[0018] In some embodiments, the present disclosure provides a compound represented by Formula (G), Formula (I), or a pharmaceutically acceptable salt thereof, wherein X5-X6 is selected from Tle-Tle, Chg-Tle.

[0019] In some embodiments, the present disclosure provides a compound represented by Formula (G), Formula (I), or a pharmaceutically acceptable salt thereof, wherein X5-X6 is Tle-Tle.

[0020] In some embodiments, the present disclosure provides a compound of Formula (G), Formula (I), or a pharmaceutically acceptable salt thereof, wherein X5-X6-X7 is Tle-Tle-Thr.

[0021] In some embodiments, the present disclosure provides a compound represented by formula (G), formula (I), or a pharmaceutically acceptable salt thereof, wherein one or more of X1, X2, X3, and X4 are optionally deleted.

[0022] In some embodiments, the present disclosure provides a compound of formula (G), formula (I), or a pharmaceutically acceptable salt thereof, wherein X 1 is absent.

[0023] In some embodiments, the present disclosure provides a compound represented by formula (G), formula (I), or a pharmaceutically acceptable salt thereof, wherein X2 and / or X3 are absent.

[0024] In some embodiments, the present disclosure provides a compound represented by formula (G), formula (I), or a pharmaceutically acceptable salt thereof, in which all of X1, X2, X3, and X4 are absent.

[0025] In some embodiments, the present disclosure provides a compound of Formula (G), Formula (I), or a pharmaceutically acceptable salt thereof, wherein X4 is selected from Thr, homoArg, or Ser.

[0026] In some embodiments, the present disclosure provides a compound of Formula (G), Formula (I), or a pharmaceutically acceptable salt thereof, wherein X4 is selected from Thr or Ser.

[0027] In some embodiments, the present disclosure provides a compound of formula (G), formula (I), or a pharmaceutically acceptable salt thereof, wherein X4 is Thr.

[0028] In some embodiments, the present disclosure provides a compound of Formula (G), Formula (I), or a pharmaceutically acceptable salt thereof, wherein X3 is selected from Dap or Dab.

[0029] In some embodiments, the present disclosure provides a compound of Formula (G), Formula (I), or a pharmaceutically acceptable salt thereof, wherein X3 is Dab.

[0030] In some embodiments, the present disclosure provides a compound of Formula (G), Formula (I), or a pharmaceutically acceptable salt thereof, wherein X2 is selected from Aib, Ala, or Ile.

[0031] In some embodiments, the present disclosure provides a compound of formula (G), formula (I), or a pharmaceutically acceptable salt thereof, wherein X2 is Aib.

[0032] In some embodiments, the present disclosure provides a compound of Formula (G), Formula (I), or a pharmaceutically acceptable salt thereof, wherein X 1 is selected from Nle, Gly, or a deletion.

[0033] In some embodiments, the present disclosure provides a compound of formula (G), formula (I), or a pharmaceutically acceptable salt thereof, wherein X 1 is Gly.

[0034] In some embodiments, the present disclosure provides a compound of Formula (I) or a pharmaceutically acceptable salt thereof, wherein L 1 is a chemical bond.

[0035] In some embodiments, the present disclosure provides a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, wherein L1 comprises polyethylene glycol, and one or two oxygen atoms of the polyethylene glycol are optionally replaced by nitrogen atoms.

[0036] In alternative embodiments, L1 comprises polyethylene glycol and the number of polyethylene glycols is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28.

[0037] In some embodiments, the present disclosure provides a compound of Formula (I) or a pharmaceutically acceptable salt thereof, wherein L1 is AEEA.

[0038] In some embodiments, the present disclosure provides a compound of formula (G), formula (I), or a pharmaceutically acceptable salt thereof, [ka] is selected from SEQ ID NO: 1 to SEQ ID NO: 8.

[0039] [Table 1]

[0040] In some embodiments, the present disclosure provides a compound of Formula (I) or a pharmaceutically acceptable salt thereof, wherein the internalization peptide is derived from the HIV virus.

[0041] In some embodiments, the present disclosure provides a compound represented by Formula (I) or a pharmaceutically acceptable salt thereof, wherein the internalization peptide has a structure described in WO2022150655A, WO2010072406A, WO2010072405A, or WO2021140485A.

[0042] In some embodiments, the present disclosure provides a compound represented by Formula (I) or a pharmaceutically acceptable salt thereof, wherein the internalization peptide comprises an amino acid sequence set forth in SEQ ID NO:9 to SEQ ID NO:11 or has an amino acid sequence selected from among these, and the amino acid residues in SEQ ID NO:9 to SEQ ID NO:11 are optionally D-amino acids.

[0043] [Table 2] In some embodiments, the present disclosure provides a compound of Formula (I) or a pharmaceutically acceptable salt thereof, wherein the internalization peptide comprises or has an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NO: 11, and the amino acid residues in SEQ ID NO: 11 are optionally D amino acids.

[0044] In some embodiments, the present disclosure provides a compound of Formula (I) or a pharmaceutically acceptable salt thereof, wherein the internalization peptide comprises or has an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NO: 9, and the amino acid residues in SEQ ID NO: 9 are optionally D amino acids.

[0045] In an alternative embodiment, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 of the amino acid residues of the internalization peptide are D-amino acids.

[0046] In an alternative embodiment, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein 4, 5, 6, 7, 8, 9, 10, or 11 of the amino acid residues of the internalization peptide are D-amino acids.

[0047] In an alternative embodiment, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein 9, 10, or 11 of the amino acid residues of the internalization peptide are D-amino acids.

[0048] In an alternative embodiment, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein the amino acid residue R of the internalization peptide is a D-amino acid.

[0049] In an alternative embodiment, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein 1, 2, 3, 4, 5, or 6 of the amino acid residues R are D-amino acids.

[0050] In an alternative embodiment, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein 3, 4, 5, or 6 of the amino acid residues R are D-amino acids.

[0051] In an alternative embodiment, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein five or six of the amino acid residues R are D-amino acids.

[0052] In an alternative embodiment, the present disclosure provides a compound of Formula (I) or a pharmaceutically acceptable salt thereof, wherein the internalization peptide has, from the C-terminus, a first amino acid residue R that is a D-amino acid, and optionally, the amino acid residues spaced therefrom by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids are D-amino acids.

[0053] In an alternative embodiment, the present disclosure provides a compound of Formula (I) or a pharmaceutically acceptable salt thereof, wherein the internalization peptide has, from the C-terminus, a first amino acid residue R which is a D-amino acid, and optionally, the amino acid residues spaced therefrom by 0, 1, 2, 3, 4, 5, 6, 7, or 8 amino acids are D-amino acids.

[0054] In an alternative embodiment, the present disclosure provides a compound of Formula (I) or a pharmaceutically acceptable salt thereof, wherein the internalization peptide has, from the C-terminus, a first amino acid residue R which is a D-amino acid, and the amino acid residues spaced therefrom, optionally by 0, 1, 2, or 3 amino acids, are D-amino acids.

[0055] In an alternative embodiment, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, wherein the internalization peptide comprises or has an amino acid sequence selected from the amino acid sequence set forth in SEQ ID NO: 12 or SEQ ID NO: 13; [Table 3] Note: Single lowercase letters represent D-amino acids.

[0056] In an alternative embodiment, the present disclosure provides a compound of formula (I) or a pharmaceutically acceptable salt thereof, which is selected from SEQ ID NO: 14 to SEQ ID NO: 22, [Table 4] Note: Single lowercase letters represent D-amino acids.

[0057] In another aspect, the present disclosure provides an active peptide or a pharmaceutically acceptable salt thereof, said active peptide comprising or consisting of formula (G): [ka] The above X1, X2, X3, X4, X5, X6, X7, X8, and X9 are each as defined in the compound represented by formula (I).

[0058] In some embodiments, the number of amino acid residues in the active peptide is selected from 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or 15.

[0059] In some embodiments, the active peptide comprises or is selected from the amino acid sequences set forth in SEQ ID NO:1 to SEQ ID NO:8.

[0060] In another aspect, the present disclosure provides a peptide comprising an active peptide and an internalization peptide, wherein the active peptide comprises or is selected from the amino acid sequences set forth in SEQ ID NOs: 1 to 8, and the internalization peptide is as defined by the present disclosure. In some embodiments, the internalization peptide comprises or is selected from the amino acid sequences set forth in SEQ ID NOs: 12 and 13.

[0061] In some embodiments, the peptide comprises or is selected from the amino acid sequences set forth in SEQ ID NO:14 to SEQ ID NO:22.

[0062] In another aspect, the present disclosure provides an active peptide comprising or selected from the active peptides shown in SEQ ID NO: 23 to SEQ ID NO: 39 below, [Table 5]

[0063] In some embodiments, an active peptide selected from the active peptides set forth in SEQ ID NO: 23 to SEQ ID NO: 39 provided by the present disclosure is linked to an internalization peptide.

[0064] In alternative embodiments, the internalization peptide has a structure described in WO2022150655A, WO2010072406A, WO2010072405A, WO2021140485A.

[0065] In an alternative embodiment, the internalization peptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 9 to 11, or has an amino acid sequence selected from these, and the amino acid residues in SEQ ID NOs: 9 to 11 are optionally D amino acids; [Table 6]

[0066] In an alternative embodiment, the active peptides and internalization peptides shown in SEQ ID NO: 23 to SEQ ID NO: 39 are linked via L2, which comprises polyethylene glycol, and one or two oxygen atoms of the polyethylene glycol are optionally substituted with nitrogen atoms.

[0067] In alternative embodiments, L2 comprises polyethylene glycol and the number of polyethylene glycols is selected from 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, or 28.

[0068] In some embodiments, L2 is AEEA.

[0069] In an alternative embodiment, the active peptides and internalization peptides shown in SEQ ID NOs: 23 to 39 form the peptides shown in SEQ ID NOs: 40 to 61 below, [Table 7] Note: Single lowercase letters represent D-amino acids.

[0070] In some embodiments, a peptide according to the present disclosure comprises or is selected from the amino acid sequences set forth in SEQ ID NO:40 to SEQ ID NO:61.

[0071] The active peptides described herein (without the internalization peptide) have a length of 3 to 25 amino acids, 4 to 15 amino acids, 4 to 10 amino acids, for example, 9 amino acids, such as 3, 4, 5, 6, 7, 8, 9 amino acids.

[0072] The compound represented by formula (G) or a pharmaceutically acceptable salt thereof, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, and the active peptide or peptide or a pharmaceutically acceptable salt thereof provided by the present disclosure are active peptides capable of binding to the PDZ1 domain or PDZ2 domain of PSD-95.

[0073] The compound of formula (G) or a pharmaceutically acceptable salt thereof, the compound of formula (I) or a pharmaceutically acceptable salt thereof, and the active peptide or peptide or a pharmaceutically acceptable salt thereof provided by the present disclosure can be used as an inhibitor of PSD-95-mediated protein-protein interaction.

[0074] In another aspect, the present disclosure further provides a pharmaceutical composition comprising a therapeutically effective amount of the above-described compound or a pharmaceutically acceptable salt thereof, and / or the above-described active peptide or peptide or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier and / or excipient.

[0075] In some embodiments, the unit dose of the pharmaceutical composition is 0.001 mg to 1000 mg.

[0076] In one embodiment, the pharmaceutical composition contains 0.01 to 99.99% of the compound or a pharmaceutically acceptable salt thereof, or the active peptide or peptide or a pharmaceutically acceptable salt thereof, based on the total weight of the composition. In one embodiment, the pharmaceutical composition contains 0.1 to 99.9% of the compound or a pharmaceutically acceptable salt thereof, or the active peptide or peptide or a pharmaceutically acceptable salt thereof. In one embodiment, the pharmaceutical composition contains 0.5 to 99.5% of the compound or a pharmaceutically acceptable salt thereof, or the active peptide or peptide or a pharmaceutically acceptable salt thereof. In one embodiment, the pharmaceutical composition contains 1 to 99% of the compound or a pharmaceutically acceptable salt thereof, or the active peptide or peptide or a pharmaceutically acceptable salt thereof. In one embodiment, the pharmaceutical composition contains 2 to 98% of the compound or a pharmaceutically acceptable salt thereof, or the active peptide or peptide or a pharmaceutically acceptable salt thereof.

[0077] In some embodiments, the pharmaceutical composition contains 0.01% to 99.99% pharmaceutically acceptable excipients, based on the total weight of the composition. In some embodiments, the pharmaceutical composition contains 0.1% to 99.9% pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical composition contains 0.5% to 99.5% pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical composition contains 1% to 99% pharmaceutically acceptable excipients. In some embodiments, the pharmaceutical composition contains 2% to 98% pharmaceutically acceptable excipients.

[0078] The present disclosure provides a compound represented by the above formula (G) or a pharmaceutically acceptable salt thereof, a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, or the above active peptide or peptides or a pharmaceutically acceptable salt thereof, as a drug.

[0079] The present disclosure provides a compound represented by the above formula (G) or a pharmaceutically acceptable salt thereof, a compound represented by the above formula (I) or a pharmaceutically acceptable salt thereof, the above active peptide or peptide or a pharmaceutically acceptable salt thereof, or the above pharmaceutical composition, which can improve neuronal function in the brain and reduce the area of ​​cerebral infarction.

[0080] The present disclosure provides a compound represented by the above formula (G) or a pharmaceutically acceptable salt thereof, a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the above active peptide or peptide or a pharmaceutically acceptable salt thereof, or the above pharmaceutical composition, which are capable of reducing histamine-releasing ability.

[0081] The present disclosure provides a compound represented by formula (G) or a pharmaceutically acceptable salt thereof, a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the active peptide or peptide or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition, which can increase plasma exposure.

[0082] This disclosure relates to T 1 / 2 The present invention provides a compound represented by the above formula (G) or a pharmaceutically acceptable salt thereof, a compound represented by the formula (I) or a pharmaceutically acceptable salt thereof, the above active peptide or peptide or a pharmaceutically acceptable salt thereof, or the above pharmaceutical composition, which can extend the

[0083] The present disclosure provides a compound represented by formula (G) or a pharmaceutically acceptable salt thereof, a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the active peptide or peptide or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition, which have high plasma stability.

[0084] In another aspect, the present disclosure provides use of the compound represented by formula (G) or a pharmaceutically acceptable salt thereof, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the active peptide or peptide or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition in the preparation of a medicament, wherein the medicament is used for the treatment and / or prevention of stroke, cerebral ischemia, central nervous system traumatic injury, reperfusion injury, subarachnoid hemorrhage, concussion, pain, anxiety, epilepsy or neurodegenerative disease (Alzheimer's disease or Parkinson's disease), or a disease in which a person is at risk as described above.

[0085] In another aspect, the present disclosure provides a method for treating and / or preventing stroke, cerebral ischemia, central nervous system traumatic injury, reperfusion injury, subarachnoid hemorrhage, concussion, pain, anxiety, epilepsy or neurodegenerative disease (Alzheimer's disease or Parkinson's disease), or a disease at risk of the above, by administering to a patient a therapeutically effective amount or a prophylactically effective amount of the compound represented by formula (G) or a pharmaceutically acceptable salt thereof, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the active peptide or peptide or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition.

[0086] In another aspect, the present disclosure provides use of the compound represented by formula (G) or a pharmaceutically acceptable salt thereof, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the active peptide or peptide or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition in combination with a thrombolytic agent in the preparation of a medicament for the treatment and / or prevention of acute ischemic stroke (AIS). In another aspect, the present disclosure provides a method for the treatment and / or prevention of acute ischemic stroke (AIS), comprising administering to a patient a therapeutically or prophylactically effective amount of the compound represented by formula (G) or a pharmaceutically acceptable salt thereof, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the active peptide or peptide or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition and a thrombolytic agent.

[0087] In another aspect, the present disclosure provides a compound represented by formula (G) or a pharmaceutically acceptable salt thereof, a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the active peptide or peptide or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition, which are used for the treatment and / or prevention of acute ischemic stroke (AIS), wherein the compound represented by formula (G) or a pharmaceutically acceptable salt thereof, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the active peptide or peptide or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition is used in combination with a thrombolytic drug. The two different active ingredients are contained in the same or different containers.

[0088] In another aspect, the present disclosure provides a thrombolytic agent for the treatment and / or prevention of acute ischemic stroke (AIS), wherein the thrombolytic agent is used in combination with the compound represented by formula (G) or a pharmaceutically acceptable salt thereof, the compound represented by formula (I) or a pharmaceutically acceptable salt thereof, the active peptide or peptide or a pharmaceutically acceptable salt thereof, or the pharmaceutical composition. The two different active ingredients are in the same or different containers.

[0089] The thrombolytic agents described in the present disclosure are selected from urokinase, streptokinase, anistreplase, glucokinase, recombinant glucokinase, pro-urokinase, vampire bat saliva plasminogen activator, lanoteplase, pamiteplase, monteplase, alteplase, reteplase, tenecteplase, and the like.

[0090] In an alternative embodiment, the thrombolytic agent described in this disclosure is alteplase (rt-PA).

[0091] In another aspect, the present disclosure provides a method for treating central nervous system damage caused by focal ischemia, comprising administering a therapeutically effective amount of a compound represented by formula (G) or a pharmaceutically acceptable salt thereof, a compound represented by formula (I) or a pharmaceutically acceptable salt thereof, an active peptide or peptide or a pharmaceutically acceptable salt thereof, or the composition to a patient suffering from focal ischemia or at risk of focal ischemia, and performing reperfusion therapy on the patient. For the interpretation of "focal ischemia," "reperfusion," and "reperfusion therapy" in the present disclosure, reference may be made to WO2012176172A.

[0092] In the polypeptide compound sequences provided by the present disclosure, a single lowercase letter represents a D-amino acid. Unless otherwise specified, a single uppercase letter represents an L-amino acid (of which Gly and Aib have no stereochemistry), for example, in RKKRRQRRR (SEQ ID NO: 9), where the amino acid residue in SEQ ID NO: 9 is optionally limited to being a D-amino acid, the amino acid is optionally a D-amino acid.

[0093] The compounds and derivatives thereof provided by the present disclosure are synthesized using a solid-phase synthesis method, in which the synthetic support is Fmoc-Val-Wang resin. The α-amino groups of the amino acid derivatives used in the synthesis process are protected by Fmoc (fluorenylmethoxycarbonyl) groups, and the side chains of the amino acids are selected from the following protecting groups according to their different functional groups: the amino group of glutamine (L / D) side chain is protected by Trt (trityl group), and the guanidino groups of homoarginine (homoArg) and arginine (L / D) side chains are protected by Pb The amino groups on the tryptophan side chain are protected with 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl group (f), the indolyl group on the tryptophan side chain, the amino group on the lysine side chain, and the amino groups on the Dab and Orn side chains are protected with tert-butoxycarbonyl group (Boc), and the hydroxyl group on the threonine side chain, the phenolic group on the tyrosine side chain, the hydroxyl group on the serine side chain, the carboxyl group on the aspartic acid side chain, and the carboxyl group on the glutamic acid side chain are protected with tert-butyl group (t-Bu). The Fmoc-Val-Wang resin is first swollen thoroughly in N,N-dimethylformamide (DMF), and the Fmoc protecting group on the α-amino group is removed using a DMF solution containing 20% ​​4-methylpiperidine. The carboxyl group of the C-terminal amino acid residue is then condensed to the polymer-insoluble resin in the form of an amide bond. Next, the Fmoc protecting group on the α-amino group is removed using a DMF solution containing 20% ​​4-methylpiperidine. Next, if in excess, the solid support is condensed with the next amino acid derivative in the sequence to form an amide bond, thereby extending the peptide chain. The desired polypeptide chain length is achieved by repeating the cycle of washing → deprotection → washing → condensation with the next amino acid → washing. Finally, the polypeptide is cleaved from the solid support by reacting with the resin in a mixture of trifluoroacetic acid:water:triisopropylsilane (90:5:5, v:v:v). The solid is then precipitated with frozen methyl tert-butyl ether. After centrifugation, the supernatant is removed to obtain the crude polypeptide, which is then dried overnight. The solid crude polypeptide is dissolved in deionized water and purified and isolated using a C-18 reverse-phase preparative chromatography column to obtain the pure polypeptide and its derivatives. Detailed Description of the Invention

[0094] "Internalization peptides" are a well-known group of relatively short peptides that enable many cellular or viral proteins to pass through cell membranes. Internalization peptides, also known as intracellular delivery peptides or membrane-penetrating peptides, typically contain 5 to 30 amino acids. These peptides typically have a cationic charge derived from arginine and / or lysine residues (usually for proteins) that are thought to facilitate their membrane penetration. Some such polypeptides contain at least 5, 6, 7, or 8 arginine and / or lysine residues.

[0095] "Naturally occurring amino acids" refers to the 20 common amino acids, namely, alanine (A), cysteine ​​(C), aspartic acid (D), glutamic acid (E), phenylalanine (F), glycine (G), histidine (H), isoleucine (I), lysine (K), leucine (L), methionine (M), asparagine (N), proline (P), glutamine (Q), arginine (R), serine (S), threonine (T), valine (V), tryptophan (W), and tyrosine (Y).

[0096] "Unnatural amino acids" refer to amino acids that are not naturally encoded or found in the genetic code of any living organism. They may also be purely synthetic compounds. Examples of unnatural amino acids include hydroxyproline, γ-carboxyglutamic acid, O-phosphoserine, azetidinecarboxylic acid, 2-aminoadipic acid, 3-aminoadipic acid, β-alanine, aminopropionic acid, 2-aminobutyric acid, 4-aminobutyric acid, 6-aminocaproic acid, 2-aminoheptanoic acid, 2-aminoisobutyric acid (Aib), 3-aminoisobutyric acid, 2-aminopimelic acid, tert-butylglycine, 2,4-diaminoisobutyric acid (Dap), tert-leucine (Tle), desmosine, 2,2'-diaminopimelic acid, 2,3-diaminopropionic acid (Dab), N-ethylglycine, N-methylglycine, and N-ethylasparagine. Non-naturally occurring amino acids include, but are not limited to, homoproline, hydroxylysine, allo-hydroxylysine, 3-hydroxyproline, 4-hydroxyproline, isodesmosine, alloisoleucine, N-methylalanine, N-methylglycine, N-methylisoleucine, N-methylpentylglycine, N-methylvaline, naphthylalanine, norvaline, norleucine, ornithine (Orn), D-ornithine, D-arginine, homoarginine (homoArg), D-tyrosine, D-lysine, D-glutamine, p-aminophenylalanine, pentylglycine, pipecolic acid, and thioproline. Non-naturally occurring amino acids further include natural or non-naturally occurring amino acids whose C-terminal carboxy group, N-terminal amino group, and / or side chain functional groups have been chemically modified.

[0097] The correspondence between some amino acid abbreviations and structural abbreviations in the present disclosure is as follows:

[0098] [ka] [ka] [ka] [ka] .

[0099] The structure of the term "AEEA" is as follows: [ka] .

[0100] "Optionally" or "optionally" means that the subsequently described event or circumstance may or may not occur, and the description includes cases where the event or circumstance occurs and cases where it does not. For example, "a C1-6 alkyl group optionally substituted with a halogen or a cyano group" means that a halogen or cyano group may or may not be present, and the description includes cases where the alkyl group is substituted with a halogen or cyano group and cases where the alkyl group is not substituted with a halogen or cyano group.

[0101] In the chemical structures of the compounds described in this disclosure, unless otherwise specified: [ka] indicates that the configuration is not specified, i.e., chiral isomers exist in the chemical structure, [ka] The bond [ka] or [ka] or [ka] and [ka] For convenience, all of the above structural formulas are depicted in one isomeric form, but the present disclosure also includes all isomers, including tautomers, rotamers, geometric isomers, diastereomers, racemates and enantiomers.

[0102] The term "subject" or "patient" includes humans and veterinary animals, such as mammals, and experimental animal models, such as mice or rats used in preclinical research.

[0103] In the present disclosure, "Ac-y" means that the amino group of the amino acid y is acetylated. [Brief explanation of the drawings]

[0104] [Figure 1A] Rat whole blood hemolysis risk test, [Figure 1B] Human whole blood hemolysis risk test, [Figure 1C] Rabbit whole blood hemolytic risk test, [Figure 2A] Human plasma stability test of polypeptide compounds, [Figure 2B] Rat plasma stability test of polypeptide compounds, [Figure 2C] Human plasma stability test of polypeptide compounds, [Figure 2D] Rat plasma stability test of polypeptide compounds, [Figure 3] Pharmacokinetic results of polypeptide compounds in rats [Figure 4] Pharmacokinetic results of different doses of polypeptide compound 14. [Figure 5A] Nephrotoxicity results of polypeptide compounds in rats - serum urea nitrogen level, ns indicates no statistical difference, * indicates P<0.05, ** indicates P<0.01; [Figure 5B] Nephrotoxicity results of polypeptide compounds in rats - serum creatinine levels, ns indicates no statistical difference, [Figure 6] Pharmacokinetic results of polypeptide compounds in beagle dogs [Figure 7] Ability of polypeptide compounds to induce histamine release in beagle dogs [Figure 8A] Efficacy results of polypeptide compound 14 in rat tMCAO model - cerebral infarct size, ** indicates P<0.01, *** indicates P<0.001. [Figure 8B] Efficacy results of polypeptide compound 14 in rat tMCAO model - nerve function damage score, * indicates P<0.05, ** indicates P<0.01. [Figure 8C] Efficacy results of polypeptide compound 22 in a rat tMCAO model - cerebral infarct size. * indicates P<0.05, ** indicates P<0.01. [Figure 8D] Efficacy results of polypeptide compound 22 in a rat tMCAO model—neural function damage score, * indicates P<0.05, ** indicates P<0.01. DETAILED DESCRIPTION OF THE INVENTION

[0105] To more fully describe the present disclosure, the present specification provides the following detailed description of the invention, but the present disclosure is not limited to these detailed description.

[0106] 1. Experimental Reagents [Table 8] [Table 9]

[0107] 2. Experimental equipment [Table 10]

[0108] 3. Specific Experimental Plan 3.1 Chemical synthesis of polypeptide compound 14 yGrkkrrqrrrG-Aib-Dab-T-Tle-Tle-TDV (SEQ ID NO: 14) [ka] 3.1.1 Resin swelling and removal of Fmoc protecting group The solid-phase synthesis support Fmoc-Val-Wang resin (286 mg, 0.1 mmol, degree of substitution: 0.349 mmol / g) was weighed and placed in a disposable polypropylene solid-phase reaction tube for polypeptide synthesis. DMF (10 mL) was added to swell the resin for 10 minutes, and the DMF was removed under vacuum. The resin was washed twice with 5 mL of DMF and then dried by suction. The resin was then added to 4-methylpiperidine / DMF (20% v / v, 5 mL) and reacted at room temperature for 8 minutes with shaking, then drained to dryness. Next, 4-methylpiperidine / DMF (20% v / v, 5 mL) was added and reacted at room temperature for 8 minutes with shaking, then removed by suction. After deprotection, the resin was washed four times with 5 mL of DMF.

[0109] 3.1.2 Coupling of peptide chain sequences Polypeptide compound 14 was synthesized in the order from the carboxyl end to the amino end according to the peptide chain sequence. First, Fmoc-Asp(OtBu)-OH (1 mmol) was weighed and dissolved in DMF to prepare a 0.34 M solution. 2-(7-azabenzotriazazole)-N,N,N',N'-tetramethyluronium hexafluorophosphate (HATU) (1 mmol) was weighed and dissolved in DMF to prepare a 0.34 M solution. 4-Methylmorpholine (NMM, 2 mmol) was weighed and dissolved in DMF to prepare a 1 M solution. 3 mL of the Fmoc-Asp(OtBu)-OH solution, 3 mL of the HATU solution, and 1.5 mL of the 4-methylmorpholine solution were mixed and added to the resin obtained in step 3.1.1. The mixture was reacted at room temperature for 35 minutes with shaking. After the reaction was complete, the mixture was washed three times with DMF. To remove the Fmoc protecting group at the N-terminus of the amino acid, 4-methylpiperidine / DMF (20% v / v, 5 mL) was added to the resin. After 8 minutes of reaction with shaking at room temperature, the solvent was removed by suction. Next, 4-methylpiperidine / DMF (20% v / v, 5 mL) was added, and the reaction was continued with shaking at room temperature for 8 minutes, after which the solvent was removed by suction. After the reaction was complete, the resin was washed three times with DMF (5 mL).

[0110] The condensation process of the above amino acid derivatives was repeated to obtain Fmoc-Thr(tBu)-OH, Fmoc-Tle-OH, Fmoc-Tle-OH, Fmoc-Thr(tBu)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Aib-OH, Fmoc-Gly-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-Gly ...Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, Fmoc-Gly-OH, F The condensation reaction proceeded in the order of oc-D-Gln(Trt)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-Gly-OH, and Fmoc-D-Tyr(tBu)-OH, finally yielding the intact resin peptide containing polypeptide compound 14.

[0111] 3.1.3 Cleavage of the resin peptide The resin peptide containing polypeptide compound 14 obtained in the above step was washed three times with DMF and DCM, then dried under vacuum. After that, 8 mL of freshly prepared solution (trifluoroacetic acid:triisopropylsilane:water = 90:5:5, v:v:v) was added and the mixture was allowed to react with shaking at room temperature for 6 hours. After the reaction was completed, the resin was filtered, washed twice with trifluoroacetic acid, and the filtrate was combined. A large amount of frozen methyl tert-butyl ether was added to precipitate the solid. After centrifugation, the supernatant was removed to obtain the crude polypeptide product, which was then dried overnight.

[0112] 3.1.4 Purification of crude peptide by reversed-phase liquid chromatography The crude peptide was purified twice, first with a TFA system and then with a 25 mM ammonium bicarbonate (pH 8.0) system. Because the target molecule contains multiple basic amino acids, the TFA system is relatively polar. The crude peptide was dissolved in water and sonicated to completely dissolve it. The solution was then filtered through a 0.22 μm membrane and separated using a WATERS Prep150 HPLC preparative high-performance liquid chromatography system. The mobile phases were A (0.1% trifluoroacetic acid, 10% acetonitrile, aqueous solution, v / v) and B (0.1% trifluoroacetic acid, 90% acetonitrile, aqueous solution, v / v). The column used was an X-SELECT OBD C-18 (WATERS, 10 μm, 19 × 250 mm) reversed-phase column. The chromatographic detection wavelength was set at 220 nm, and the flow rate was 15 mL / min. The product-related distillates were collected and lyophilized, followed by a secondary purification using a 25 mM ammonium bicarbonate (pH 8.0) system. The product obtained in the first purification step was dissolved in 25 mM ammonium bicarbonate buffer and then applied to a column. In the ammonium bicarbonate system, the mobile phases were A (25 mM ammonium bicarbonate aqueous solution) and B (100% acetonitrile). The chromatography column was an X-SELECT OBD C-18 (WATERS, 5 μm, 19 × 250 mm) reverse-phase chromatography column. During the purification process, the chromatographic detection wavelength was set to 220 nm and the flow rate was 10 mL / min. After collecting and lyophilizing the product-related distillates, the pure polypeptide compound 14 was obtained with a yield of approximately 20%. The purity of the polypeptide was determined using a WATERS H-CLASS analytical ultra-high-performance liquid chromatography system (chromatographic column: ReproSil-Pur Basic C-18 (Dr. Maisch, 5 μm, 4.6 × 150 mm)), which was 97.92%. The molecular weight of the compound was confirmed using an Agilent Q-TOF 6530 system (column: ReproSil-Pur Basic C-18 (Dr. Maisch, 5 μm, 4.6 × 150 mm)). The ion current shown in the mass spectrum was 815.4899 [M+3H]. 3+ / 3.

[0113] 3.2 Chemical synthesis of polypeptide compound 15 yGrkkrrqrrr-AEEA-Tle-Tle-TDV (SEQ ID NO: 15) [ka] Polypeptide compound 15 was synthesized by referring to the synthesis procedure of polypeptide compound 14, except that the condensation order of amino acid derivatives was Fmoc-Asp(OtBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tle-OH, Fmoc-Tle-OH, Fmoc-AEEA-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Arg(Pbf)-OH. The polypeptides were purified using a WATERS H-CLASS analytical ultra-high performance liquid chromatography system (chromatography column: ReproSil-Pur Basic C-18 (Dr. Maisch, 5 μm, 4.6 × 150 mm)) and found to be 98.73% pure. The molecular weight of the compound was confirmed using an Agilent Q-TOF 6530 system (column: ReproSil-Pur Basic C-18 (Dr. Maisch, 5 μm, 4.6 × 150 mm)). The ion current shown in the mass spectrum was 749.4533 [M+3H]. 3+ / 3.

[0114] 3.3 Chemical synthesis of polypeptide compound 16 yGrkkrrqrrr-Nle-TT-Tle-Tle-TDV (SEQ ID NO: 16) [ka] Polypeptide compound 16 was synthesized with reference to the synthesis procedure for polypeptide compound 14, except that the condensation order of amino acid derivatives was Fmoc-Asp(OtBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tle-OH, Fmoc-Tle-OH, Fmoc-Thr(tBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Nle-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-Asp(OtBu)-OH, Fmoc-Thr( ...Thr(tBu)-OH, The polypeptides were purified using a WATERS H-CLASS analytical ultra-high performance liquid chromatography system (chromatography column: ReproSil-Pur Basic C-18 (Dr. Maisch, 5 μm, 4.6 × 150 mm)) to determine their purity, which was 97.93%. The molecular weight of the compound was confirmed using an Agilent Q-TOF 6530 system (column: ReproSil-Pur Basic C-18 (Dr. Maisch, 5 μm, 4.6 × 150 mm)). The ion current shown in the mass spectrum was 806.1549 [M+3H]. 3+ / 3.

[0115] 3.4 Chemical synthesis of polypeptide compound 17 yGrkkrrqrrrT-homoArg-homoArg-ITDV (SEQ ID NO: 17) [ka] Polypeptide compound 17 was synthesized with reference to the synthesis procedure for polypeptide compound 14, except that the condensation order of amino acid derivatives was Fmoc-Asp(OtBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Ile-OH, Fmoc-homoArg(Pbf)-OH, Fmoc-homoArg(Pbf)-OH, Fmoc-Thr(tBu)-OH, Fmoc-D-Arg(Pbf)-OH, The polypeptides were Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Gln(Trt)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-Gly-OH, and Fmoc-D-Tyr(tBu)-OH. The purity of the polypeptides was determined using a WATERS H-CLASS analytical ultra-high performance liquid chromatography system (chromatography column: ReproSil-Pur Basic C-18 (Dr. Maisch, 5 μm, 4.6 × 150 mm)), and was found to be 98.77%. The molecular weight of the compound was confirmed using an Agilent Q-TOF 6530 system (column: ReproSil-Pur Basic C-18 (Dr. Maisch, 5 μm, 4.6 × 150 mm)). The ion current shown in the mass spectrum was 810.4980 [M+3H]. 3+ / 3.

[0116] 3.5 Chemical Synthesis of Polypeptide Compound 18 yGrkkrrqrrrG-Aib-Dab-homoArg-Tle-Tle-TDV (SEQ ID NO: 18) [ka] Polypeptide compound 18 was synthesized with reference to the synthesis procedure for polypeptide compound 14, except that the condensation order of amino acid derivatives was Fmoc-Asp(OtBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tle-OH, Fmoc-Tle-OH, Fmoc-homoArg(Pbf)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Aib-OH, Fmoc-Gly-OH, Fmoc-D-Arg(P The polypeptides were purified using a WATERS H-CLASS analytical ultra-high performance liquid chromatography system (chromatography column: ReproSil-Pur Basic C-18 (Dr. Maisch, 5 μm, 4.6 × 150 mm)) to determine their purity, which was 98.68%. The molecular weight of the compound was confirmed using an Agilent Q-TOF 6530 system (column: ReproSil-Pur Basic C-18 (Dr. Maisch, 5 μm, 4.6 × 150 mm)). The ion current shown in the mass spectrum was 838.5120 [M+3H]. 3+ / 3.

[0117] 3.6 Chemical Synthesis of Polypeptide Compound 19 yGrkkrrqrrrG-Aib-Dab-T-Chg-Tle-SDV (SEQ ID NO: 19) [ka] Polypeptide compound 19 was synthesized with reference to the synthesis procedure for polypeptide compound 14, except that the condensation order of the amino acid derivatives was Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tle-OH, Fmoc-Chg-OH, Fmoc-Thr(tBu)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Aib-OH, Fmoc-Gly-OH, and Fmoc-D-Arg(Pbf The polypeptides were: Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Gln(Trt)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Lys(Boc)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-Gly-OH, and Fmoc-D-Tyr(tBu)-OH. The purity of the polypeptides was determined using a WATERS H-CLASS analytical ultra-high performance liquid chromatography system (chromatography column: ReproSil-Pur Basic C-18 (Dr. Maisch, 5 μm, 4.6 × 150 mm)), and was found to be 98.95% pure. The molecular weight of the compound was confirmed using an Agilent Q-TOF 6530 system (column: ReproSil-Pur Basic C-18 (Dr. Maisch, 5 μm, 4.6 × 150 mm)). The ion current shown in the mass spectrum was 819.4897 [M+3H]. 3+ / 3.

[0118] 3.7 Chemical Synthesis of Polypeptide Compound 20 yGrkkrrqrrrGT-Tle-Tle-TDV (SEQ ID NO: 20) [ka] Polypeptide compound 20 was synthesized with reference to the synthesis procedure for polypeptide compound 14, except that the condensation order of amino acid derivatives was Fmoc-Asp(OtBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tle-OH, Fmoc-Tle-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Ar The polypeptides were purified using a WATERS H-CLASS analytical ultra-high performance liquid chromatography system (chromatography column: ReproSil-Pur Basic C-18 (Dr. Maisch, 5 μm, 4.6 × 150 mm)) to determine their purity, which was 97.06%. The molecular weight of the compound was confirmed using an Agilent Q-TOF 6530 system (column: ReproSil-Pur Basic C-18 (Dr. Maisch, 5 μm, 4.6 × 150 mm)). The ion current shown in the mass spectrum was 753.7843 [M+3H]. 3+ / 3.

[0119] 3.8 Chemical Synthesis of Polypeptide Compound 21 yGrkkrrqrrrGT-Chg-Tle-SDV (SEQ ID NO: 21) [ka] Polypeptide compound 21 was synthesized with reference to the synthesis procedure for polypeptide compound 14, except that the condensation order of amino acid derivatives was Fmoc-Asp(OtBu)-OH, Fmoc-Ser(tBu)-OH, Fmoc-Tle-OH, Fmoc-Chg-OH, Fmoc-Thr(tBu)-OH, Fmoc-Gly-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-D-Ar The polypeptides were purified using a WATERS H-CLASS analytical ultra-high performance liquid chromatography system (chromatography column: ReproSil-Pur Basic C-18 (Dr. Maisch, 5 μm, 4.6 × 150 mm)) to determine their purity, which was 98.54%. The molecular weight of the compound was confirmed using an Agilent Q-TOF 6530 system (column: ReproSil-Pur Basic C-18 (Dr. Maisch, 5 μm, 4.6 × 150 mm)). The ion current shown in the mass spectrum was 757.7843 [M+3H]. 3+ / 3.

[0120] 3.9 Chemical Synthesis of Polypeptide Compound 22 rKKRrQRRrG-Aib-Dab-T-Tle-Tle-TDV (SEQ ID NO: 22) [ka] Polypeptide compound 22 was synthesized with reference to the synthesis procedure of polypeptide compound 14, except that the condensation order of amino acid derivatives was Fmoc-Asp(OtBu)-OH, Fmoc-Thr(tBu)-OH, Fmoc-Tle-OH, Fmoc-Tle-OH, Fmoc-Thr(tBu)-OH, Fmoc-Dab(Boc)-OH, Fmoc-Aib-OH, Fmoc The polypeptides were: Fmoc-Gly-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH, Fmoc-D-Arg(Pbf)-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Lys(Boc)-OH, Fmoc-Lys(Boc)-OH, and Fmoc-D-Arg(Pbf)-OH. The purity of the polypeptides was determined using a WATERS H-CLASS analytical ultra-high performance liquid chromatography system (chromatography column: ReproSil-Pur Basic C-18 (Dr. Maisch, 5 μm, 4.6 × 150 mm)), and was found to be 97.82%. The molecular weight of the compound was confirmed using an Agilent Q-TOF 6530 system (column: ReproSil-Pur Basic C-18 (Dr. Maisch, 5 μm, 4.6 × 150 mm)). The ion current shown in the mass spectrum was 742.1292 [M+3H]. 3+ / 3.

[0121] Biological Testing and Evaluation The present disclosure will be further explained below in conjunction with test examples, but these examples are not intended to limit the scope of the present disclosure.

[0122] 1. Experimental Reagents [Table 11]

[0123] 2. Experimental equipment [Table 12]

[0124] 3. Test Example 3.1. Evaluation of the affinity of polypeptide compounds for human PSD-95 3.1.1 Purpose of the experiment The purpose of this test example is to measure the affinity of polypeptide compounds for human PSD95-PDZ2 using a competitive ELISA method.

[0125] 3.1.2 Expression and purification of human PSD95-PDZ2 protein Human PSD95 (Uniprot Entry: P78352) was used as a template for PDZ, and the amino acid sequence of the PDZ2 protein was designed: PDZ2 (155-249, the italicized part is a Flag-His-Avitag-TEVsite tag, used for subsequent purification and biotin labeling): [ka] The PDZ2 gene was engineered into a PET expression vector and induced in BL21-DE3 E. coli cells with 1 mM IPTG for 4 hours at 37°C. The bacterial pellet was then collected by centrifugation at 10,000 g for 10 minutes at 4°C, resuspended in 1x PBS, and homogenized. The supernatant was collected by high-speed centrifugation and filtered at 0.45 μM. A 5-column Ni-Sepharose affinity column was equilibrated with 20 mM phosphate buffer (pH 8.0). The sample was centrifuged at high speed to remove impurities and then loaded onto the column for binding. The column was washed with 20 mM phosphate buffer until the A280 reading was reduced to baseline. Then, a gradient elution was performed with 20 mM phosphate buffer, 0-500 mM imidazole, and the protein was collected and identified. The purified sample was then exchanged with 1x PBS solution, concentrated to a volume of 2 mL, and further purified using a gel chromatograph Superdex 200 (GE) equilibrated with 1x PBS. The peak of interest was collected and dispensed for use.

[0126] 3.1.3 Experimental method Site-specific biotin Biotin-NA-1 (sequence: Biotin-YGRKKRRQRRRKLSSIESDV, SEQ ID NO: 63) was used in a competitive ELISA binding assay. The positive controls in this example were NA-1 (sequence: YGRKKRRQR RRKLSSIESDV, SEQ ID NO: 64) and NoNO42 (sequence: YGrKKRrQrRRkLSSIESDV, SEQ ID NO: 65), and the negative control was NA-1, an Ala mutant at positions 0 and -2 of NA-1. (ADA) (Sequence: YGRKKRRQRRRKLSSIEADA, SEQ ID NO: 66), which was demonstrated to have no PDZ2 binding ability (Science, 2002, 298:846-50).

[0127] PSD95-PDZ2 (tag removed with TEV protease) was diluted to 1 μg / mL in 1x PBS buffer and added to a 96-well microplate (Corning, 9018, 25 / box 96-well clear flat bottom plate) at a volume of 100 μL / well and left overnight at 4°C for 16–20 hours. After discarding the liquid, the plate was washed three times with PBST (pH 7.4, 0.05% Tween-20). 4% BSA blocking solution (300 μL / well) diluted in PBST buffer was added and incubated in a 37°C incubator for 1 hour for blocking. After blocking, the blocking solution was discarded and the plate was washed three times with PBST buffer. Biotin-NA-1 at a constant concentration of 0.3 μM and test compounds initially diluted 100 μM with PBS buffer at seven 10-fold gradients (100, 10, 1, 0.1, 0.01, 0.001, 0.0001, and 0 μM) were added and incubated for 1 hour in a 37°C incubator. After incubation, the reaction solution in the microplate was discarded, the plate was washed three times with PBST, and 100 μL of HRP-SA secondary antibody (1:2000 dilution) was added to each well and incubated for 1 hour at 37°C. After washing the plate three times with PBST, 100 μL of TMB chromogenic substrate was added and incubated at room temperature for 1-3 minutes. The reaction was stopped by adding 100 μL of 1M sulfuric acid.

[0128] 3.1.4 Sample analysis and data processing Absorbance values ​​were read at 450 nm using a SpectraMax M5 plate reader, and nonlinear fitting was performed using GraphPad Prism 9 to calculate the binding IC of test compounds to the PSD95-PDZ2 protein. 50 The values ​​were calculated and the specific data are shown in Table 1.

[0129] [Table 13] [Table 14] *The error of this test is within 3 times, **rIC 50 represents the affinity ratio of the polypeptide compound to NA-1.

[0130] 3.1.5 Experimental conclusions: The results demonstrate that all of the disclosed compounds can efficiently bind to the target protein, human PSD95-PDZ2. Given the high conservation of the PSD95-PDZ domain across different species, this is convenient for subsequent animal evaluation.

[0131] 3.2. Evaluation of the specificity of polypeptide compounds for human PSD95-PDZ2 binding 3.2.1 Purpose of the experiment The three PDZ domains in the PSD95 protein are structurally similar, and binding to the PDZ1 and PDZ2 domains can effectively block PSD95-mediated neuronal excitotoxicity, but the biological function of binding to the PDZ3 domain is unknown. Therefore, to avoid potential safety issues, this test example considers the selectivity of polypeptide compounds for the PDZ2 and PDZ3 domains and selects polypeptide compounds that specifically bind to the PDZ2 domain.

[0132] 3.2.2 Expression and purification of human PSD95-PDZ3 protein Referring to step 3.1.2, PSD95-PDZ3 protein was expressed and purified. The amino acid sequence of the PDZ3 protein is as follows: PDZ3 (309-401, the italicized part is a Flag-His-Avitag-TEVsite tag, used for subsequent purification and biotin labeling): [ka] , sequence number 67.

[0133] 3.2.3 Experimental method Site-specific biotinylated Biotin-PDZ2 protein and Biotin-PDZ3 protein were used in a binding ELISA assay. The positive controls in this test example were NA-1 and NoNO42 (WO2022150655), and the negative control was NA-1. (ADA) Test compounds were diluted to 2 μM in 1x PBS buffer and added to a 96-well microplate (Corning, 9018, 25 / box 96-well clear flat bottom plate) at a volume of 100 μL per well and left overnight at 4°C for 16–20 hours. After discarding the liquid, the plate was washed three times with PBST (pH 7.4, 0.05% Tween-20). Then, 300 μL per well of 4% BSA blocking solution diluted in PBST buffer was added and incubated in a 37°C incubator for 1 hour for blocking. After blocking, the blocking solution was discarded, and the plate was washed three times with PBST buffer. Biotin-PDZ2 or Biotin-PDZ3, initially at 10 μM and diluted 1:10 with 1x PBS buffer to seven gradient concentrations (10, 1, 0.1, 0.01, 0.001, 0.0001, 0.00001, 0 μM), was added and incubated for 1 hour in a 37°C incubator. After incubation was complete, the reaction solution in the microplate was discarded, the plate was washed three times with PBST, and 100 μL of HRP-SA secondary antibody (1:2000 dilution) was added to each well and incubated for 1 hour at 37°C. After washing the plate three times with PBST, 100 μL of TMB chromogenic substrate was added and incubated for 1-3 minutes at room temperature. The reaction was stopped by adding 100 μL of 1M sulfuric acid.

[0134] 3.2.4 Sample analysis and data processing The absorbance at 450 nm was read using a SpectraMax M5 plate reader, and the binding EC50 values ​​of Biotin-PDZ2 or Biotin-PDZ3 for the test compounds were calculated using GraphPad Prism 9. The specific data are shown in Tables 2-1 and 2-2.

[0135] [Table 15]

[0136] [Table 16]

[0137] 3.2.5 Experimental Conclusions The results show that all of the compounds disclosed herein selectively bind to the PSD95-PDZ2 domain, and among them, the selectivity of polypeptide compounds 14, 15, 16, 19, 21, and 22 is comparable to that of the positive drugs NA-1 and NoNO42.

[0138] 3.3 Hemolytic risk assessment of polypeptide compounds in rat, rabbit, and human whole blood Hemolysis refers to the destruction of the cell membrane of red blood cells, which increases their transparency and causes a deep red color. Some drug ingredients and additives contain hemolytic components, which can cause hemolysis in the human body and lead to adverse reactions such as local swelling and circulatory disorders. Based on the principle that hemoglobin released by the destruction of red blood cells has absorption in the visible light wavelength range, a test compound solution was added to a rat red blood cell suspension, and after incubation, the degree of hemolysis was measured using a microplate reader.

[0139] 3.3.1 Purpose of the experiment In this test example, we examine whether polypeptide compounds induce hemolysis in whole blood of rats, rabbits, and humans.

[0140] 3.3.2 Experimental method Preparation of red blood cell suspension: 100 μL of fresh whole blood was collected, and then 900 μL of 1×PBS solution was added. The blood was placed on a plate shaker and shaken at 30 rpm for 5 minutes, followed by centrifugation at 1000 g for 5 minutes. The supernatant was discarded, and the above washing steps were repeated until the supernatant was no longer red, and the blood was then prepared for testing.

[0141] Preparation of test polypeptide solutions: Add an appropriate amount of 1x PBS solution to the polypeptide powder to dissolve it and obtain a standard stock solution. Then, dilute with 1x PBS to prepare test polypeptide solutions at concentrations of 1, 3, 10, 30, 100, and 300 μg / mL. Two parallel wells were prepared for each concentration. Blank 1x PBS was used as a negative control, and 0.1% Triton X-100 in 1x PBS was used as a positive control.

[0142] Incubation process: 500 μL of the test solution was added to the erythrocyte suspension, which was then shaken at 30 rpm for 5 minutes on a plate shaker to ensure thorough and uniform mixing. The suspension was then incubated at 37°C for 1 hour and centrifuged at 1000 g for 5 minutes.

[0143] 3.3.3 Sample analysis and data processing 100 μL of the supernatant was added to one well of a microplate, and the absorbance at 540 nm was measured using a SpectraMax M5 plate reader. Data analysis was performed using GraphPad Prism 9. The hemolysis rate (%) was calculated as follows: (test sample absorbance - negative control absorbance) / (positive control absorbance - negative control absorbance) × 100%. A result of less than 5% was considered non-hemolytic, and a result of more than 5% was considered hemolytic. The experimental results are shown in Table 3 and Figures 1A and 1B.

[0144] [Table 17]

[0145] 3.3.4 Experimental conclusions: The results show that in rat and human whole blood, polypeptide compound 14 and the positive drugs NA-1 and NoNO42 all met the requirement of a hemolysis rate of less than 5% at concentrations ranging from 1 to 50 μM, posing no risk of hemolysis.

[0146] 3.3.5 Assessment of hemolytic risk of polypeptide compounds in rabbit whole blood The method used was similar to that described in Section 3.3.2, except that fresh whole rabbit blood was used in this test. The experimental results are shown in Figure 1C. The results show that in rabbit whole blood, both polypeptide compounds 14 and 22 and the positive drugs NA-1 and NoNO42 met the hemolysis rate requirement of less than 5% at concentrations ranging from 1 to 50 μM, eliminating the risk of hemolysis.

[0147] 3.4 In vitro stability considerations of polypeptide compounds in rat and human plasma There are multiple hydrolytic enzymes in plasma that can decompose and metabolize drug molecules, which causes the concentration of drug molecules in plasma to rapidly decrease and prevent them from reaching an effective concentration, resulting in relatively high clearance and a relatively short half-life, and poor pharmacokinetic and pharmacodynamic properties. Therefore, plasma stability is an important indicator that affects the feasibility of drug discovery.

[0148] 3.4.1 Purpose of the experiment This example examines the in vitro stability of polypeptide compounds in rat and human plasma and is divided into two parts: 1) the stability of the polypeptide compounds alone in rat and human plasma, and 2) the stability of the polypeptide compounds in rat and human plasma when administered simultaneously with alteplase (rt-PA).

[0149] 3.4.2 Experimental method Solution preparation: Weigh out an appropriate amount of test polypeptide compound and add an appropriate amount of 1x PBS to dissolve it to obtain a 1mM test solution. NA-1 and NoNO42 are positive drugs, propantheline bromide is the test control for human plasma, and lovastatin is the test control for rat plasma.

[0150] Plasma stability test: 12.5 μL of 1 mM test solution was added to 495 μL of pre-incubated plasma sample. 50 μL of the prepared plasma sample was divided into centrifuge tubes with different incubation times (0, 10, 20, 30, 60, and 120 minutes) and then incubated in a 37°C water bath with shaking at 60 rpm. Two parallel samples were prepared for each incubation time. 200 μL of stop solution was added to stop the incubation and vortex-mixed for 5 minutes. The samples were then centrifuged at 10,000 rpm for 10 minutes at 4°C to remove proteins. 70 μL of the supernatant was then removed and placed in a new 96-well plate (70 μL of water had been added to each well beforehand) and mixed uniformly.

[0151] Plasma stability test with alteplase: An appropriate amount of solid alteplase was weighed, added with an appropriate volume of water, and dissolved by vortexing to obtain a 1 mg / mL alteplase solution. 250 μL of the 1 mg / mL alteplase solution was added to 4750 μL of plasma to obtain a plasma containing 50 μg / mL alteplase. The remaining procedures were the same as those in the "Plasma Stability Test" above, except that plasma containing 50 μg / mL alteplase was used instead of plasma.

[0152] 3.4.3 Sample analysis and data processing: The content of the remaining compound at each time point was measured by LC-MS / MS, and the relative content of the remaining compound at other time points was calculated based on 0 min (100%).

[0153] Sample pretreatment method: Take 30 μL of plasma sample, add 120 μL of 5% formic acid methanol solution containing 1 ng / mL of internal standard (verapamil), mix by vortex for 5 minutes, centrifuge at 10,000 rpm at low temperature for 10 minutes, take 70 μL of the supernatant, add 70 μL of 0.1% formic acid aqueous solution, mix uniformly, and analyze by LC-MS.

[0154] LC-MS analysis method: (1) Chromatography conditions: Mobile phase A was 0.1% formic acid in water, mobile phase B was 0.1% formic acid in acetonitrile, flow rate was 0.5 mL / min, injection volume was 10 μL, column was nanometric Unisil C18aq (4.6 mm × 150 mm, 5 μm), column temperature was 40 °C. (2) Mass spectrometry conditions: Mass spectrometry was performed using an electrospray ion source (ESI), positive ion analysis mode, and multiple reaction monitoring (MRM) scanning. The experimental results are shown in Table 4-1, Table 4-2, Figure 2A, Figure 2B, Figure 2C, and Figure 2D.

[0155] [Table 18] [Table 19]

[0156] [Table 20]

[0157] 3.4.4 Experimental Conclusions The results showed that NA-1 underwent significant degradation within 2 hours in human and rat plasma, while neither NoNO42 nor polypeptide compound 14 were degraded. After co-incubation with alteplase, the degradation rate of NA-1 accelerated. Polypeptide compound 14 remained stable in both plasma species, whereas NoNO42 exhibited reduced stability in rat plasma but remained stable in human plasma. These results are consistent with the results of a Phase III clinical trial of NA-1, in which the addition of alteplase rapidly disrupted the molecular structure of NA-1, resulting in its loss of biological activity. The resistance of polypeptide compound 14 to alteplase in plasma may make it a promising molecule to address the clinical shortcomings of NA-1.

[0158] Polypeptide Compound 22 is stable in human and rat plasma, and after the addition of alteplase, polypeptide Compound 22 is stable in human plasma and its degradation rate in rat plasma is significantly slower than that of NA-1.

[0159] Polypeptide compounds 14 and 22 can compensate for the clinical shortcomings of NA-1 due to their resistance to alteplase in plasma.

[0160] 3.5 Pharmacokinetic studies of polypeptide compounds in rats 3.5.1 Purpose of the experiment Male SD rats were used as test animals to study the pharmacokinetic behavior of a polypeptide compound in the rat body (plasma) after a single intravenous injection.

[0161] 3.5.2 Experimental method Male SD rats, weighing 170-200 g and aged 4-6 weeks, were purchased from Beijing Weitong Lihua Laboratory Animal Technology Co., Ltd. Polypeptide compound solutions were prepared by dissolving in 1x PBS. Administration was completed by intravenous injection over 10 minutes at a dose of 3 nmol / g, with three animals per group. After the end of the injection, 0.2 mL of blood was collected at 2, 8, 15, 30, 45, 60, and 120 minutes and transferred to centrifuge tubes containing EDTA-K2 anticoagulant. The whole blood samples were centrifuged at 4000 g for 5 minutes at 4°C to obtain plasma, which was then frozen and stored at -80°C.

[0162] A similar method was used to study the pharmacokinetic parameters of intravenous injection of polypeptide compound 22 in rats, except that the enzyme inhibitor was added immediately after blood sampling.

[0163] 3.5.3 Sample analysis and data processing The blood concentration of each test substance in plasma was measured using the method of Test Example 3.4.3, blood concentration-time curves were created, and pharmacokinetic parameters were calculated using PKSolver software. The experimental results are shown in Table 5-1, Table 5-2, and Figure 3.

[0164] 3.5.4 Experimental Conclusions The results in Table 5-1 and Figure 3 show that the positive drugs NA-1 and NoNO42 exhibited similar pharmacokinetic properties in rats, with both having half-lives of less than 3 minutes. The pharmacokinetic properties of the tested polypeptide compounds 14, 19, and 21 were significantly superior to those of the two positive drugs. Compared with NA-1, the half-life was significantly extended by 18-23 times, the area under the curve (AUC) increased by 18-33 times, and the maximum blood concentration (Cmax) increased by 4-5 times. Under the same concentration conditions, polypeptide compounds 14, 19, and 21 had a longer duration of action and greater drug exposure.

[0165] The results in Table 5-2 show that the addition of enzyme inhibitors reduced the degradation of NA-1 after blood collection and improved both the AUC and Cmax values ​​obtained. Compared with NA-1, the half-life of polypeptide compound 22 was significantly extended by 29 times, the area under the curve (AUC) increased by 19.4 times, and the maximum blood concentration (Cmax) increased by 2.3 times.

[0166] [Table 21]

[0167] [Table 22]

[0168] 3.6 Pharmacokinetic studies of different doses of polypeptide compounds in rats 3.6.1 Purpose of the experiment The neuroprotective agent NA-1 has been reported to have dose-related safety issues in both preclinical and clinical studies, such as excessive histamine release at high doses, which can cause symptoms such as tachycardia and hypotension. Therefore, we selected three doses of polypeptide compound 14: a high dose (3 nmol / g), a medium dose (1 nmol / g), and a low dose (0.3 nmol / g), and compared their pharmacokinetic properties with those of the high dose of NA-1 (3 nmol / g) to provide a dose reference for subsequent efficacy studies.

[0169] 3.6.2 Experimental method The test method of Test Example 3.5.2 was used, with three animals in each group, and blood samples were taken at 2, 8, 15, 60, 120 and 180 minutes after the end of intravenous injection.

[0170] 3.6.3 Sample analysis and data processing The blood concentration of each test substance in plasma was measured using the method of Test Example 3.4.3, blood concentration-time curves were plotted, and pharmacokinetic parameters were calculated using WinNonlin software. The experimental results are shown in Table 6 and Figure 4.

[0171] [Table 23] [Table 24]

[0172] 3.6.4 Experimental Conclusions As shown in Table 6, the plasma exposure at low doses of polypeptide compound 14 was equivalent to that at high doses of NA-1, and since plasma exposure is related to efficacy, it is estimated that polypeptide compound 14 may remain effective even if the dose is reduced by 10-fold. The Cmax value is thought to be related to histamine release, and the Cmax of polypeptide compound 14 at both the medium and low doses was lower than that of the high dose of NA-1, suggesting better safety, which will be verified in subsequent studies.

[0173] 3.7 Test for the ability of polypeptide compounds to induce histamine release Histamine is an important endogenous component that mediates allergic and inflammatory responses. Drugs can induce mast cell degranulation, which releases large amounts of histamine, causing inflammatory symptoms such as redness, rash, decreased blood pressure, and decreased heart rate. The histamine-releasing effect of drugs is often evaluated by measuring histamine levels in plasma, and the usual detection method is liquid chromatography-mass spectrometry.

[0174] 3.7.1 Purpose of the experiment Male SD rats were used as test animals to study the ability of a polypeptide compound to induce histamine release in the rat body (plasma) by a single intravenous injection.

[0175] 3.7.2 Experimental Method This test was carried out in the same manner as Test Example 3.6, with three animals in each group, and blood samples were taken before administration and 2, 8 and 15 minutes after the end of intravenous injection.

[0176] 3.7.3 Sample analysis and data processing The histamine and 3-methylhistamine contents were measured at each time point using LC-MS / MS. Compound detection method by LC-MS: (1) Chromatography conditions: Mobile phase A was acetonitrile / water / 100 mM ammonium acetate, 50 / 45 / 5 (v / v / v), 2% FA; mobile phase B was acetonitrile / 100 mM ammonium acetate, 95 / 5 (v / v), 2% FA; flow rate was 0.4 mL / min; column was a Waters BEH HILIC 1.7 μm, 2.1 × 150 mm column; column temperature was 50 °C; injection volume was 20 μL; (2) Mass spectrometry conditions: Mass spectrometry was performed using an electrospray ionization source (ESI), positive ion analysis mode, and multiple reaction monitoring (MRM) scanning.

[0177] Sample preparation method: 30 μL of plasma sample was taken and added to 200 μL of acetonitrile solution containing D4-histamine (100 ng / mL), mixed by vortexing for 1 minute, then centrifuged at 5800 rpm for 10 minutes. 100 μL of the supernatant was transferred to the sample disc and analyzed by the instrument. Table 7 shows the total histamine and 3-methylhistamine detected.

[0178] [Table 25]

[0179] 3.7.4 Experimental Conclusions As can be seen from Table 7, histamine release after administration is a transient response of the organism to external stimuli. At the same dose, high concentrations of NA-1 and polypeptide compound 14 both stimulated histamine release most potently within 2 minutes, showing a significant difference compared to pre-administration. This decreased shortly thereafter, with the peak value of polypeptide compound 14 being nearly twice that of NA-1. Meanwhile, no significant increase in histamine levels was detected within 15 minutes for medium and low doses of polypeptide compound 14. This result corroborates the pharmacokinetic results, indicating that medium and low doses of polypeptide compound 14 have lower blood concentrations than high doses of NA-1, and therefore have a weaker ability to induce histamine release than NA-1, making them safer.

[0180] 3.8 Evaluation of Nephrotoxicity of Polypeptide Compounds in Rats The kidney is an important target organ for drug toxicity, and nephrotoxicity is a significant factor limiting new drug development. Therefore, it is necessary to evaluate potential nephrotoxicity in the process of new drug development to reduce the risk of drug development. Serum urea nitrogen (BUN) and creatinine are important indicators of kidney function, and abnormal increases in these two indicators in the blood may indicate impaired kidney function or damage.

[0181] 3.8.1 Experimental Objectives Male SD rats were used as test animals to study the nephrotoxicity of a polypeptide compound in vivo (serum) following a single intravenous injection.

[0182] 3.8.2 Experimental Method This test was carried out in the same manner as Test Example 3.6, and blood was collected before administration (day 0), and on days 2, 4, and 7 after administration. At each time point, 0.3 mL of blood was collected, transferred to a centrifuge tube, and allowed to stand at room temperature for 1 hour. The whole blood sample was then centrifuged at 4°C and 4000 g for 5 minutes to separate the blood and obtain serum samples. The serum samples were then frozen and stored at -80°C. 3.8.3 Sample analysis and data processing The serum urea nitrogen (BUN) and creatinine contents at each time point were measured using commercially available reagent kits. Blood urea nitrogen was measured using the urease continuous monitoring method, and blood creatinine was measured using the sarcosine oxidase method. After thawing the samples in a refrigerator at 2-8°C, they were mixed uniformly using a vortex mixer. 100 μL was then aspirated and placed in a sample cup, which was then placed in the sample position of the biochemical analyzer. The test was performed according to the instructions in the reagent kit. The experimental results are shown in Tables 8-1 and 8-2 and Figures 5A and 5B.

[0183] 3.8.4 Experimental Conclusions During the 7-day monitoring period, the blood urea nitrogen level of NA-1 increased significantly on days 4 and 7 compared to before administration, but the numerical increase was within 2-fold and not clinically significant. Therefore, the test results indicate that NA-1 and polypeptide compound 14 do not pose any obvious renal safety issues within the test concentration range.

[0184] [Table 26]

[0185] [Table 27]

[0186] 3.9 Pharmacokinetic studies of polypeptide compounds in beagle dogs 3.9.1 Experimental Objectives Conventional beagle dogs were used as test animals to study the pharmacokinetic behavior of a polypeptide compound in the body (plasma) of beagle dogs after a single intravenous injection.

[0187] 3.9.2 Experimental Method Polypeptide compound 22 was administered in two dose groups, at 1.2 nmol / g and 0.4 nmol / g, and NA-1 was administered in one dose group at 1.2 nmol / g. Each group consisted of four animals, half male and half female. Polypeptide compound solutions were prepared by dissolving in 1x PBS and administered intravenously over 10 minutes. Blood was collected at the following time points: before injection (0 hours), 5 minutes after the start of injection, and 0, 2, 5, 10, 15, 30 minutes, 1 hour, 2 hours, 4 hours, 8 hours, and 24 hours after the end of injection. At each time point, 0.5 mL of blood was collected and transferred to a centrifuge tube containing EDTA-K2 anticoagulant. 50 μL of a protease inhibitor was also added, mixed uniformly, and centrifuged at 2000 g for 10 minutes (4°C). Plasma was separated within 1 hour, dispensed into cryopreservation tubes, and stored at -60 to -90°C. All procedures from blood collection to centrifugation were performed under ice bath conditions.

[0188] 3.9.3 Sample analysis and data processing The blood concentration of each test substance in plasma was measured using the method of Test Example 3.4.3, blood concentration-time curves were plotted, and pharmacokinetic parameters were calculated using PKSolver software. The experimental results are shown in Table 9 and Figure 6.

[0189] 3.9.4 Experimental Conclusions The results show that the pharmacokinetic properties of the tested polypeptide compound 22 are clearly superior to those of NA-1. 1 / 2 The mean AUC for polypeptide compound 22 was 21.3 times that of NA-1, and the mean C max The exposure of polypeptide compound 22 was 1.3 times that of NA-1. The drug exposure of polypeptide compound 22 showed a positive correlation with the dose.

[0190] [Table 28]

[0191] 3.10 Test for the ability of polypeptide compounds to induce histamine release 3.10.1 Experimental Objectives Using beagle dogs as test animals, we investigated the ability of a polypeptide compound to induce histamine release in the dog's body (plasma) after a single intravenous injection.

[0192] 3.10.2 Experimental Method This test was carried out in the same manner as Test Example 3.9, with four animals in each group, and blood samples were taken before administration (0 hour), and 2, 5, and 15 minutes after the end of intravenous injection.

[0193] 3.10.3 Sample analysis and data processing Sample analysis and data processing were performed in accordance with the method in 3.7.3. Table 10 and Figure 7 show the total results of detected histamine and 3-methylhistamine.

[0194] [Table 29]

[0195] 3.10.4 Experimental Conclusion Compared with the level before administration, no significant change in histamine level was observed at either of the two doses of polypeptide compound 22, demonstrating its favorable safety.

[0196] 3.11 Pharmacodynamic studies of polypeptide compounds in the rat tMCAO model 3.11.1 Experimental Objectives This study aimed to investigate the pharmacodynamic effects of polypeptide compounds in a rat model of transient middle cerebral artery occlusion (tMCAO). High-dose NA-1 (3 nmol / g) was the active agent, and polypeptide compounds 14 and 22 were administered at three doses: high (3 nmol / g), medium (1 nmol / g), and low (0.3 nmol / g). By comparing the cerebral infarction size and neurological function scores in rats, and in combination with previous studies, the safe and effective doses of polypeptide compounds 14 and 22 were provided as a reference for further evaluation.

[0197] 3.11.2 Experimental method Model Construction and Administration: Male SD rats, 6-8 weeks old and weighing 240-260 g, were purchased from Beijing Weitong Lihua Experimental Animal Technology Co., Ltd. A rat MCAO model was established using the embolization method. Administration was initiated 60 minutes after embolization, followed by removal of the embolization and reperfusion 90 minutes later. Cerebral blood flow baseline was monitored before embolization. The enrollment criteria were a 50% or greater decrease in cerebral blood flow from baseline after embolization and a rapid recovery of cerebral blood flow to 50% or greater after embolization. Animals that did not meet these criteria were excluded. Polypeptide compounds were dissolved in saline to the desired concentration and injected slowly into the tail vein in a single dose over 4-5 minutes 1 hour after MCAO embolization. A control group received an intravenous injection of saline over 4-5 minutes. The study endpoint was 24 hours after administration. Observations of the animals' general condition during the administration period included spontaneous activity, fasting, drinking, death, and other abnormalities.

[0198] Measurement of infarct area: 24 hours after MCAO, surviving rats from each group were dissected, perfused transcardially with pre-chilled PBS, and decapitated to remove the whole brain. Brain sections were stained with 2% triphenyltetrazolium chloride (TTC) solution to calculate the infarct area. The percentage of infarct area was calculated as follows: Infarct area / total brain area × 100%.

[0199] Neurological function damage score: Before model construction (animals with abnormal behavioral scores before model construction were excluded), animals were blindly evaluated for the degree of neurological function damage using the scoring criteria in the attached table below at 24 hours after administration. A total of 16 points were scored. A higher score indicates a more severe degree of damage. The detailed scoring criteria are shown in Table 11.

[0200] [Table 30] [Table 31]

[0201] 3.11.3 Sample analysis and data processing Data analysis was performed using IBM SPSS Statistics 25.0 statistical software.

number

[0202] [Table 32]

[0203] [Table 33]

[0204] 3.11.4 Experimental Conclusion The results show that polypeptide compound 14 and polypeptide compound 22 have a dose-dependent effect on both neuronal function and cerebral infarct size in tMCAO rats after a single administration. A medium dose (1 nmol / g) of polypeptide compound 14 and polypeptide compound 22 showed efficacy equivalent to a high dose (3 nmol / g) of the active drug NA-1.

Claims

1. A compound of formula (I) or a pharmaceutically acceptable salt thereof, 【Chemistry 1】 The X １ is selected from Gly or a deletion, The X ２ is selected from Aib, Ala, Ile, Cha, Phe, Trp, 1-Nal, Ser, Lys, Arg, Nle, Nva, Orn, cyclopropyl Ala, 4-thiazole Ala, homoLeu, or a deletion; The X ３ is selected from Dap, Dab, Thr, AiB, Ala, Cha, Phe, Trp, 1-NaI, Asn, Glu, Lys, Arg, 1Me-Trp, 2-NaI, Gln, Thr, 4-thiazolylalanine or a deletion; The X ４ is selected from Thr, homoArg, Ser or a deletion, The X ５ is selected from Tle, homoArg, and Chg; The X ６ is selected from Tle or Ile, The X ７ is selected from Thr or Ser, The X ８ is Asp, The X ９ is Val, Said L １ is a chemical bond or comprises polyethylene glycol, one or two oxygen atoms of which are optionally replaced by nitrogen atoms; The CPP is an internalization peptide. A compound of formula (I) or a pharmaceutically acceptable salt thereof.

2. The X ７ is Thr, 2. A compound of formula (I) according to claim 1 or a pharmaceutically acceptable salt thereof.

3. The X ６ is Tle, A compound of formula (I) according to any one of claims 1 and 2, or a pharmaceutically acceptable salt thereof.

4. The X ５ is Tle, A compound of formula (I) according to any one of claims 1 to 3 or a pharmaceutically acceptable salt thereof.

5. The X ６ -X ７ is selected from Tle-Thr, Ile-Thr, or Tle-Ser, preferably Tle-Thr or Ile-Thr, and most preferably Tle-Thr; 2. A compound of formula (I) according to claim 1 or a pharmaceutically acceptable salt thereof.

6. The X ５ -X ６ is selected from Tle-Tle, homoArg-Ile, or Chg-Tle, preferably Tle-Tle or Chg-Tle, and most preferably Tle-Tle; 2. A compound of formula (I) according to claim 1 or a pharmaceutically acceptable salt thereof.

7. The X ５ -X ６ -X ７ is Tle-Tle-Thr, A compound of formula (I) according to any one of claims 1 to 6 or a pharmaceutically acceptable salt thereof.

8. The X １ , X ２ , X ３ , X ４ is optionally one or more deleted, and preferably ２ and / or X ３ is deleted or X １ , X ２ , X ３ , X ４ are all missing, A compound of formula (I) according to any one of claims 1 to 7 or a pharmaceutically acceptable salt thereof.

9. The X ４ is selected from Thr, homoArg, or Ser, preferably Thr or Ser, and most preferably Thr; A compound of formula (I) according to any one of claims 1 to 8 or a pharmaceutically acceptable salt thereof.

10. The X ３ is selected from Dap or Dab, preferably Dab; A compound of formula (I) according to any one of claims 1 to 9 or a pharmaceutically acceptable salt thereof.

11. The X ２ is selected from Aib, Ala, or Ile, preferably Aib; A compound of formula (I) according to any one of claims 1 to 10, or a pharmaceutically acceptable salt thereof.

12. The X １ is selected from Nle, Gly, or a deletion, preferably Gly; A compound of formula (I) according to any one of claims 1 to 11 or a pharmaceutically acceptable salt thereof.

13. Said L １ is a chemical bond, A compound of formula (I) according to any one of claims 1 to 12 or a pharmaceutically acceptable salt thereof.

14. Said L １ comprises polyethylene glycol, one or two oxygen atoms of which are optionally replaced by nitrogen atoms, and is preferably AEEA; A compound of formula (I) according to any one of claims 1 to 12 or a pharmaceutically acceptable salt thereof.

15. The aforementioned 【Chemistry 2】 is selected from any one of SEQ ID NO: 1 to SEQ ID NO: 8; Table 1 A compound of formula (I) according to any one of claims 1 to 14, or a pharmaceutically acceptable salt thereof.

16. The internalization peptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 9 to 11, and preferably comprises the amino acid sequence set forth in SEQ ID NO: 11; Optionally, the amino acid residues of SEQ ID NOs:9-11 are D amino acids. Table 2 A compound of formula (I) according to any one of claims 1 to 15, or a pharmaceutically acceptable salt thereof.

17. Among the amino acid residues of the internalization peptide, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 amino acid residues are D-amino acids, preferably 4, 5, 6, 7, 8, 9, 10, or 11 amino acid residues of the internalization peptide are D-amino acids, and most preferably 9, 10, or 11 amino acid residues of the internalization peptide are D-amino acids.

17. A compound of formula (I) according to claim 16 or a pharmaceutically acceptable salt thereof.

18. the amino acid residues R of the internalization peptide are D-amino acids, preferably 1, 2, 3, 4, 5 or 6 of the amino acid residues R are D-amino acids, preferably 3, 4, 5 or 6 of the amino acid residues R are D-amino acids, and most preferably 5 or 6 of the amino acid residues R are D-amino acids; 17. A compound of formula (I) according to claim 16 or a pharmaceutically acceptable salt thereof.

19. The internalization peptide has, from the C-terminus, a first amino acid residue R is a D-amino acid, and an amino acid residue optionally spaced therefrom by 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, or 10 amino acids is a D-amino acid, preferably an amino acid residue optionally spaced therefrom by 0, 1, 2, 3, 4, 5, 6, 7, or 8 amino acids is a D-amino acid, and most preferably an amino acid residue optionally spaced therefrom by 0, 1, 2, or 3 amino acids is a D-amino acid.

17. A compound of formula (I) according to claim 16 or a pharmaceutically acceptable salt thereof.

20. The internalization peptide comprises the amino acid sequence shown in SEQ ID NO: 12 or SEQ ID NO:

13. Table 3 A compound of formula (I) according to any one of claims 16 to 19 or a pharmaceutically acceptable salt thereof.

21. Selected from any one of SEQ ID NOs: 14 to 22; Table 4 A compound of formula (I) according to any one of claims 16 to 20, or a pharmaceutically acceptable salt thereof.

22. An active peptide comprising the formula (G) or a pharmaceutically acceptable salt thereof, 【Transformation 3】 The X １ , X ２ , X ３ , X ４ , X ５ , X ６ , X ７ , X ８ , X ９ is as defined in any one of claims 1 to 12 or 15, respectively. An active peptide or a pharmaceutically acceptable salt thereof.

23. The active peptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 1 to 8.

23. The active peptide of claim 22 or a pharmaceutically acceptable salt thereof.

24. A peptide or a pharmaceutically acceptable salt thereof, An active peptide as defined in claim 22 or 23, and 22. The method of claim 16, comprising administering to a subject an internalization peptide as defined in any one of claims 16 to 21. A peptide or a pharmaceutically acceptable salt thereof.

25. The peptide comprises an amino acid sequence set forth in any one of SEQ ID NOs: 14 to 22.

25. The peptide of claim 24, or a pharmaceutically acceptable salt thereof.

26. 1. A pharmaceutical composition comprising: A compound of formula (I) according to any one of claims 1 to 21 or a pharmaceutically acceptable salt thereof, and / or an active peptide according to any one of claims 22 to 23 or a pharmaceutically acceptable salt thereof, and / or a peptide according to any one of claims 24 to 25 or a pharmaceutically acceptable salt thereof, and a pharmaceutically acceptable carrier and / or excipient, Pharmaceutical compositions.

27. Use of a compound of formula (I) according to any one of claims 1 to 21 or a pharmaceutically acceptable salt thereof, and / or an active peptide according to any one of claims 22 to 23 or a pharmaceutically acceptable salt thereof, and / or a peptide according to any one of claims 24 to 25 or a pharmaceutically acceptable salt thereof, and / or a pharmaceutical composition according to claim 26 in the preparation of a drug, The drug is used in the treatment and / or prevention of stroke, cerebral ischemia, central nervous system traumatic injury, reperfusion injury, subarachnoid hemorrhage, concussion, pain, anxiety, epilepsy, neurodegenerative diseases, and / or diseases at risk of the same; use.

28. 26. Use of a compound of formula (I) according to any one of claims 1 to 21 or a pharmaceutically acceptable salt thereof, and / or an active peptide according to any one of claims 22 to 23 or a pharmaceutically acceptable salt thereof, and / or a peptide according to any one of claims 24 to 25 or a pharmaceutically acceptable salt thereof, and / or a pharmaceutical composition according to claim 26 in combination with a thrombolytic agent in the preparation of a medicament for the treatment and / or prevention of acute ischemic stroke, wherein the thrombolytic agent is selected from urokinase, streptokinase, anistreplase, glucokinase, recombinant glucokinase, prourokinase, vampire bat saliva plasminogen activator, lanoteplase, pamiteplase, monteplase, alteplase, reteplase, tenecteplase, preferably alteplase. use.

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