WO2023185156A9 - 一类多肽化合物及其制备方法和应用 - Google Patents

一类多肽化合物及其制备方法和应用 Download PDF

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WO2023185156A9
WO2023185156A9 PCT/CN2022/142732 CN2022142732W WO2023185156A9 WO 2023185156 A9 WO2023185156 A9 WO 2023185156A9 CN 2022142732 W CN2022142732 W CN 2022142732W WO 2023185156 A9 WO2023185156 A9 WO 2023185156A9
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WO2023185156A1 (zh
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李卓荣
刘睿
白炜琪
刘蜜敏
崔阿龙
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中国医学科学院医药生物技术研究所
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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/04—Peptides having up to 20 amino acids in a fully defined sequence; Derivatives thereof
    • A61K38/08—Peptides having 5 to 11 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P25/00—Drugs for disorders of the nervous system
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P29/00—Non-central analgesic, antipyretic or antiinflammatory agents, e.g. antirheumatic agents; Non-steroidal antiinflammatory drugs [NSAID]
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P37/00—Drugs for immunological or allergic disorders
    • A61P37/02—Immunomodulators
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention relates to a type of polypeptide molecule composed of 5-20 amino acids, its preparation method and its application in immune regulation, treatment of inflammation-related diseases and neurological diseases.
  • the invention belongs to the field of medical technology.
  • NK Natural killer cells
  • NK are important immune cells in the body. They are not only related to anti-tumor, anti-viral infection and immune regulation, but also participate in the occurrence of hypersensitivity reactions and autoimmune diseases in some cases.
  • Neuroinflammation inflammation in the brain (neuroinflammation) is directly related to a variety of diseases, such as depression, psychosis, and multiple sclerosis, and recently researchers have found that neuroinflammation is directly related to the onset of Alzheimer's disease. Related.
  • AD Alzheimer's disease
  • Senile Plaques senile Plaques
  • the FDA approved the first drug to treat AD Tacrine, which is an acetylcholinesterase inhibitor, and then approved the second-generation acetylcholinesterase inhibitors Donepzil, galantamine, and kabala. Stin and rivastigmine are now widely used in the treatment of AD patients.
  • Memantine is the first drug approved by the FDA for the treatment of patients with moderate to severe AD and can alleviate memory impairment in AD patients.
  • Huperzine A is an alkaloid extracted from plants of the Huperzaceae family. It is widely used in the treatment of AD patients in China. However, because the FDA has not approved this drug, its development abroad is limited.
  • Neuropeptides are endogenous active peptides in the central system and peripheral nervous system. They play an important role in the nervous system and are involved in regulating many brain functions such as metabolism, food intake, learning, memory and sleep. Many studies have found that neuropeptides such as ghrelin, neurotensin, pituitary adenylate cyclase-activating peptide, neuropeptide Y, substance P (Substance P, SP), and orexin are closely related to the physiology and pathology of AD. In AD patients, the content levels of neuropeptides and their receptors are significantly changed.
  • neuropeptides mainly exert neuroprotective effects by inhibiting A ⁇ accumulation, increasing neuronal glucose transport, increasing the production of neurotrophic factors, inhibiting endoplasmic reticulum stress and autophagy, and regulating potassium channels.
  • the discovery of neuroprotective effects of neuropeptides on AD provides a new direction for AD drug research and development.
  • the present invention relates to a type of polypeptide molecule composed of 5-20 amino acids, its preparation method and its application in immune regulation, treatment of inflammation-related diseases and neurological diseases.
  • the present invention proposes polypeptide compounds having the structure shown in Formula I, pharmaceutically acceptable salts, solvates, isomers or polymorphs thereof:
  • numbers 1-20 represent the specific positions of amino acids in Formula I.
  • the minimum number of amino acids in Formula I is 5 and the maximum number of amino acids is 20; the configuration of the amino acid is L-type or D-type. ; The presence or absence of amino acids at positions 1-15 and 20;
  • R 3 is selected from the following group: -(CH 2 ) 2 -COOH, -CH 2 -COOH;
  • R 6 is selected from the following group: -(CH 2 ) 2 -COOH, -CH 2 -CH(CH 3 ) 2 , -CH-(CH 3 ) 2 ;
  • R 7 is selected from the following group: -(CH 2 ) 2 -COOH, -CH-(CH 3 ) 2 , -CH 2 -CONH 2 , -CH 3 , -CH 2 -CH(CH 3 ) 2 , -C 3 H 6 ;
  • R 8 is selected from the following group: -CH 2 -CONH 2 , -CH 2 -CH(CH 3 ) 2 , -C 3 H 6 , -CH 3 , -(CH 2 ) 2 -COOH, -CH 2 -COOH , -CH-(CH 3 ) 2 ;
  • R 9 is selected from the following group: -CH 3 , -(CH 2 ) 2 -COOH, -CH 2 -COOH, -(CH 2 ) 3 -NHC(NH)NH 2 , -CH 2 -C 6 H 4 - OH, -CH(CH 3 )-OH;
  • R 10 is selected from the following group: -(CH 2 ) 2 -COOH, -(CH 2 ) 3 -NHC(NH)NH 2 , -CH 2 -C 6 H 4 -OH, -CH 2 -CH(CH 3 ) 2 , -CH(CH 3 )-CH 2 -CH 3 , -(CH 2 ) 2 -CONH 2 , -C 8 NH 6 , -CH(CH 3 )-OH, -CH 2 -COOH;
  • R 11 is selected from the following group: -CH 2 -C 6 H 4 -OH, -CH 2 -CH(CH 3 ) 2 , -CH(CH 3 )-CH 2 -CH 3 , -(CH 2 ) 2 - CONH 2 , -CH 2 -CONH 2 , -(CH 2 ) 2 -CONH 2 , -CH 2 -C 6 H 5 , -C 3 H 6
  • R 12 is selected from the following group: -CH 2 -CH(CH 3 ) 2 , -CH 2 -CONH 2 , -(CH 2 ) 2 -CONH 2 , -(CH 2 ) 3 -NHC(NH)NH 2 , -CH(CH 3 )-OH, -CH 2 -COOH, -(CH 2 ) 2 -NH 2 , -(CH 2 ) 4 -NH 2 ;
  • R 13 is selected from the following group: -(CH 2 ) 2 -COOH, -(CH 2 ) 3 -NHC(NH)NH 2 , -CH(CH 3 )-OH, -(CH 2 ) 2 -CONH 2 , -CH 2 -COOH, -(CH 2 ) 2 -NH 2 , -C 3 H 6
  • R 14 is selected from the group consisting of: -(CH 2 ) 2 -COOH, -(CH 2 ) 2 -CONH 2
  • R 15 is selected from the following group: -(CH 2 ) 2 -CONH 2 , -(CH 2 ) 2 -COOH;
  • R 20 is selected from the following group: -NH 2 , -OH.
  • polypeptide compound is selected from the following group:
  • (18)A3-M1 (L-Leu)-(L-Ala)-(L-Arg)-(L-Leu)-(L-Leu)-(L-Thr)-(L-Glu)-( L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-NH 2
  • (20)A3-M3 (L-Leu)-(L-Ala)-(L-Arg)-(L-Leu)-(L-Leu)-(L-Thr)-(L-Glu)-( L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(D-Met)-NH 2
  • (21)B1-M1 (L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-( L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-NH 2
  • (22)B1-M2 (L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-( L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)
  • (23)B1-M3 (L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-( L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(D-Met)-NH 2
  • (24)B3-M1 (L-Leu)-(L-Ala)-(L-Arg)-(L-Leu)-(L-Leu)-(L-Thr)-(L-Glu)-( L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-NH 2
  • (25)B3-M2 (L-Leu)-(L-Ala)-(L-Arg)-(L-Leu)-(L-Leu)-(L-Thr)-(L-Glu)-( L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)
  • (28)B5-M2 (L-Asp)-(L-Tyr)-(L-Gln)-(L-Gln)-(L-Asp)-(L-Glu)-(L-Glu)-( L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)
  • (31)C1 (L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Gln)-(L-Glu)-(L-Glu)-(L- Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
  • (32)C2 (L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Asn)-(L-Glu)-(L-Glu)-(L- Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
  • (33)C3 (L-Ala)-(L-Glu)-(L-Tyr)-(L-Phe)-(L-Arg)-(L-Glu)-(L-Glu)-(L- Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
  • (34)C4 (L-Ala)-(L-Glu)-(L-Tyr)-(L-Pro)-(L-Arg)-(L-Glu)-(L-Glu)-(L- Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
  • (36)C6 (L-Ala)-(L-Glu)-(L-Trp)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L- Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
  • (37)C7 (L-Ala)-(L-Thr)-(L-Thr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L- Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
  • (38)C8 (L-Ala)-(L-Asp)-(L-Asp)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L- Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
  • (40)C10 (L-Val)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L- Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
  • (42)D1 (L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Thr)-(L-Gln)-(L-Glu)-(L- Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
  • (43)D2 (L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Asp)-(L-Gln)-(L-Glu)-(L- Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
  • (44)D3 (L-Ala)-(L-Tyr)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Gln)-(L-Glu)-(L- Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
  • (46)D5 (L-Ala)-(L-Thr)-(L-Thr)-(L-Leu)-(L-Arg)-(L-Gln)-(L-Glu)-(L- Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
  • (47)D6 (L-Ala)-(L-Asp)-(L-Asp)-(L-Leu)-(L-Arg)-(L-Gln)-(L-Glu)-(L- Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
  • (49)D8 (L-Leu)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Gln)-(L-Glu)-(L- Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
  • (61)E6 (D-Asp)-(L-Ser)-(L-Leu)-(L-Ala)-(L-Val)-(L-Glu)-(L-Tyr)-(L- Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu) -(L-Met)-NH 2
  • (67)E12 (L-Glu)-(L-Glu)-(L-Asp)-(L-Asp)-(L-Ser)-(L-Leu)-(L-Ala)-(L- Val)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe) -(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2 .
  • the present invention is a method for preparing the polypeptide compound having the structure shown in Formula I, its pharmaceutically acceptable salt, solvate, isomer or polymorph, which method adopts Fmoc solid phase synthesis method , the polypeptide is synthesized according to the amino acid sequence from the C-terminal to the N-terminal of the peptide chain, and the pure polypeptide is obtained after purification and freeze-drying.
  • the resin used in the Fmoc solid phase synthesis method is selected from any of the following: Amide-MBHA Resin 0.52mmol/g, Amide-MBHA Resin 0.646mmol/g, 2-Chlorotrityl Chloride Resin 0.52mmol/g.
  • the Fmoc solid-phase synthesis method uses Fmoc to protect the amino part of the amino acid;
  • the side chain protecting group is selected from any one of the following groups: benzyl (Bzl), tert-butyl (tBu), tert-butyl Oxycarbonyl (Boc), 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf), triphenyl (Trt).
  • the preferred method is to remove the Fmoc protecting group and condense to form a peptide chain;
  • the reagent for removing Fmoc is 20% piperidine/DMF;
  • the condensing agent is benzotriazole-N,N,N',N'-tetramethyl
  • HBTU urea hexafluorophosphate
  • DIEA N,N'-diisopropylcarbodiimide
  • the amino acid at position 20 when it does not exist, it is prepared according to conventional methods.
  • R 20 is -OH
  • the amino acid at position 20 is L-form Met
  • route 1 method is used:
  • reaction conditions of each step are as follows:
  • the Fmoc removal reagent is 20% piperidine/DMF, react for 10-30 minutes, repeat 2 times; DMF/DCM cross-washing for more than 3 times; ninhydrin color reaction detection;
  • peptide cutting solution Prepares the peptide cutting solution, the ratio of the peptide cutting solution is 95% v/v trifluoroacetic acid (TFA), 2.5% v/v triisopropylsilane (TIPS), 2.5% v/v water, 15-20ml/g Resin, react for 1-3 hours; press out the liquid; precipitate with ether, wash and dry and then produce crude peptide;
  • TFA trifluoroacetic acid
  • TIPS triisopropylsilane
  • reaction conditions of each step are as follows:
  • the Fmoc removal reagent is 20% piperidine/DMF, react for 10-30 minutes, repeat 2 times; DMF/DCM cross-washing for more than 3 times; ninhydrin color reaction detection;
  • c' Configure the amino acid solvent according to the molar ratio of amino acid: HBTU: DIEA to 1:0.9:2, react for 0.5-2h, cross-wash with DMF/DCM more than 3 times; ninhydrin color reaction detection;
  • f' Prepare the peptide cutting solution.
  • the ratio of the peptide cutting solution is 95% v/v trifluoroacetic acid (TFA), 2.5% v/v triisopropylsilane (TIPS), 2.5% v/v water, 15-20ml/ g resin, react for 1-3 hours; press out the liquid; precipitate with ether, wash and dry, and then obtain the crude polypeptide.
  • TFA trifluoroacetic acid
  • TIPS triisopropylsilane
  • the present invention also proposes a pharmaceutical composition, comprising the polypeptide compound having the structure shown in Formula I, its pharmaceutically acceptable salt, solvate, isomer or polymorph, and a pharmaceutical composition. acceptable carrier or excipient.
  • the present invention also proposes the polypeptide compound having the structure shown in Formula I, its pharmaceutically acceptable salt, solvate, isomer or polymorph or the pharmaceutical composition prepared with Application of NK cell-related immunomodulatory effects, treatment of inflammation-related diseases, or drugs for diagnosis and treatment of neurological diseases.
  • salts refer to salts that retain the desired biological activity of the target compound and exhibit minimal undesirable toxicological effects.
  • base addition salts can be obtained by contacting the neutral form of the compounds of the invention with a sufficient amount of base in pure solution or in a suitable inert solvent.
  • Pharmaceutically acceptable bases include salts prepared from inorganic bases and organic bases.
  • the salts of the inorganic bases include aluminum salts, ammonium salts, calcium salts, copper salts, iron salts, ferrous salts, lithium salts, magnesium salts, and manganese salts. Salt, manganous salt, potassium salt, sodium salt, zinc salt, etc.
  • Examples of pharmaceutically acceptable acid addition salts include inorganic acids such as hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, phosphoric acid or nitric acid; organic acids such as formic acid, acetic acid, acetoacetic acid, acetone Acid, trifluoroacetic acid, propionic acid, butyric acid, caproic acid, enanthic acid, undecanoic acid, lauric acid, benzoic acid, salicylic acid, 2-(4-hydroxybenzoyl)-benzoic acid, camphoric acid, cinnamon Acid, cyclopentanepropionic acid, digluconic acid, 3-hydroxy-2-naphthoic acid, niacin, pamoic acid, pectic acid, 3-phenylpropionic acid, picric acid, pivalic acid, 2-hydroxy Ethanesulfonic acid, itaconic acid, amidosulfonic acid, trifluoromethanesulfonic acid, dodecyl s
  • N,N'-dibenzylethylene glycol Salts formed from amines, chloroprocaine, choline, diethanolamine, ethylenediamine, N-methylglucamine and procaine.
  • solvate means that a compound of formula I may exist in the form of a solvate (such as a hydrate), wherein the compound of the invention contains as a structural element of the crystal lattice of the compound a polar solvent, in particular such as water , methanol or ethanol.
  • polar solvent in particular such as water , methanol or ethanol.
  • the amount of polar solvent, especially water, may be present in stoichiometric or non-stoichiometric ratios.
  • “isomer” means that in the case where the compound of formula I contains one or more asymmetric centers and/or double bonds, the compound of the present invention can be a racemate, a racemic mixture, a single enantiomer They exist in the form of isomers, diastereoisomer mixtures, single diastereomers, geometric isomers, etc. These compounds may be represented by the symbol “R” or “S”, depending on the configuration of the substituents around the steric carbon atoms.
  • polymorph means that the compounds of Formula I may also exist in various crystalline forms, and their different single crystalline forms as well as polymorphic forms are obtained by recrystallizing the compound or a pharmaceutically acceptable salt thereof in a solvent. Crystal mixture.
  • the present invention provides a pharmaceutical composition, which contains a polypeptide compound represented by Formula I, its pharmaceutically acceptable salt or ester, solvate, isomer, polymorph, metabolite or prodrug, and a pharmaceutically acceptable carrier or excipient.
  • a "pharmaceutical composition” contains a therapeutically effective amount of a polypeptide compound of Formula I thereof, a pharmaceutically acceptable salt or ester, solvate, isomer, polymorph, isotopically labeled compound, metabolite or precursor thereof. medicine, and one or more pharmaceutically acceptable carriers, prepared into tablets, capsules, granules, powders, suspensions, emulsions, powders, solutions, gels, syrups, pills, tinctures, and wines , decoction, lozenge, mixture, suppository, injection, inhalant or spray, etc.
  • the pharmaceutical composition preferably contains the multi-substituted benzoheterocyclic compound of the present invention or a pharmaceutically acceptable salt thereof as an active ingredient in a weight ratio of 0.1% to 99.5%, and more preferably contains an active ingredient in a weight ratio of 0.5% to 99.5%.
  • the agent when used orally, it can be made into oral preparations, such as tablets, capsules, granules and pills, etc., containing fillers (such as sugar derivatives such as lactose, sucrose, glucose, mannitol and sorbitol; Starch derivatives such as corn starch, potato starch, dextrin and carboxymethyl starch; cellulose derivatives such as crystalline cellulose, hydroxypropyl cellulose, carboxymethyl cellulose, carboxymethyl cellulose calcium, carboxymethyl cellulose sodium phosphate; gum arabic; dextran; silicate derivatives such as magnesium aluminum metasilicate; phosphate derivatives such as calcium phosphate; carbonate derivatives such as calcium carbonate; sulfate derivatives such as calcium sulfate, etc.), adhesives (e.g.
  • fillers such as sugar derivatives such as lactose, sucrose, glucose, mannitol and sorbitol; Starch derivatives such as corn starch, potato starch, dextri
  • gelatin polyvinylpyrrolidone and polyethylene glycol
  • disintegrating agents e.g. cellulose derivatives such as sodium carboxymethylcellulose, polyvinylpyrrolidone
  • lubricants e.g. talc, calcium stearate , magnesium stearate, spermaceti, boric acid, sodium benzoate, leucine
  • stabilizers methyl parahydroxybenzoate, propyl parahydroxybenzoate, etc.
  • flavoring agents such as commonly used sweeteners, sour tastes agents and spices, etc.
  • the invention may be administered by any suitable method known in the art, for example, oral, intravenous, intraperitoneal, intramuscular, topical, transdermal, ocular, nasal, inhalation, subcutaneous, intramuscular, buccal,
  • the above-mentioned compound can be administered in any amount ranging from ⁇ g to mg/kg body weight of the subject by sublingual, rectal administration, etc., for example, the above-mentioned compound can be administered in an amount of 0.1 ⁇ g to 1000 mg/kg body weight/day.
  • administration as described above may be performed 4 times daily, 3 times daily, 2 times daily, once daily, once every other day, once weekly, or at other intervals.
  • the dosing regimen as described above is optionally repeated weekly or monthly, as appropriate.
  • the dosage of the compound can be adjusted according to factors such as the severity of the disease, age, weight, gender, administration method, and course of treatment of the patient or subject.
  • the compounds of the present invention can be used alone or in combination with one or more other active ingredients to treat, prevent, inhibit or improve diseases or conditions.
  • the combined use of drugs is safer or better than the single use of any one drug. More effective.
  • Such other drugs may be administered simultaneously or sequentially with the compounds of the invention in routes and amounts customarily used for this purpose.
  • pharmaceutical compositions containing the other drugs and the compounds of the invention in a unit dosage form are preferred, especially in combination with a pharmaceutically acceptable carrier.
  • combination therapy may also include treatments in which a compound of the invention and one or more other drugs are administered on different overlapping schedules.
  • compositions of the present invention also include those compositions containing one or more other active ingredients.
  • the present invention discovers a type of polypeptide compound through structural derivatization research on substance P.
  • This type of polypeptide compound has a neuroprotective effect on AD in vitro cell models and can improve cell viability.
  • the AD in vitro cell model used is APPswe cells, which are human neuroblastoma cells stably transfected with the human-mouse chimeric APP gene. Because the cells overexpress the APP gene, the production of APP increases and promotes ⁇ -secretase. Breakdown of APP generates excess A ⁇ .
  • copper ions When using copper ions to treat cells, copper ions form chelates with APP and A ⁇ , aggravating the production and deposition of A ⁇ , thereby simulating AD pathological damage such as oxidative stress, inflammatory response, and apoptosis degeneration induced by A ⁇ .
  • the present invention uses this in vitro model to screen and evaluate the anti-AD effects of drugs.
  • the polypeptide compound of the present invention can significantly improve the spatial learning and memory abilities of mice with A ⁇ -mediated cognitive impairment.
  • Amyloid- ⁇ (A ⁇ ) is the main pathological mediator of familial and sporadic AD.
  • a ⁇ Amyloid- ⁇
  • Clinical and experimental verification show that this abnormal deposition of A ⁇ blocks long-term synaptic potentiation, impairs synaptic function and induces cognitive deficits. This model is based on the above-mentioned neurotoxic effects of A ⁇ .
  • Injecting oligomerized A ⁇ 1-42 into the brains of mice using a stereotaxic injection method can cause neurotoxic reactions and induce AD-like memory impairment and behavioral disorders. , can better simulate the pathological conditions in the brain of clinical patients, and is therefore a commonly used animal model of AD.
  • polypeptide compounds involved in the present invention have low toxicity and show low toxicity in kidney cell (Vero) toxicity test and hemolysis (rabbit whole blood) test.
  • Figure 1 shows that C10 improves the learning and memory abilities of A ⁇ 1-42 model mice.
  • (A) Platform escape latency in the MWM positioning navigation test; (B) swimming speed in the positioning navigation test; (C) In the space exploration test, the original position of the C10-intervention A ⁇ 1-42 model mouse crossing the platform The number of times increased; (D) In the space exploration test, the duration of C10-intervention A ⁇ 1-42 model mice in the target quadrant was prolonged. The results are expressed as mean ⁇ standard error, n 8, ## P ⁇ 0.01vs.Sham, *P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001vs.A ⁇ 1-42 .
  • FIG. 1 shows that C7 improves the learning and memory abilities of A ⁇ 1-42 model mice.
  • (A) Platform escape latency in the MWM positioning navigation test; (B) swimming speed in the positioning navigation test; (C) In the space exploration test, the original position of the C7-intervention A ⁇ 1-42 model mouse crossing the platform The number of times increased; (D) In the space exploration test, the duration of C7-intervention A ⁇ 1-42 model mice in the target quadrant was prolonged. The results are expressed as mean ⁇ standard error, n 8, ## P ⁇ 0.01, ### P ⁇ 0.001vs.Sham, **P ⁇ 0.01, **P ⁇ 0.001vs.A ⁇ 1-42 .
  • the method for preparing the polypeptide compound having the structure shown in Formula I, its pharmaceutically acceptable salt, solvate, isomer or polymorph adopts the Fmoc solid phase synthesis method, and starts from the C-terminus of the peptide chain according to the method.
  • the polypeptide is synthesized from the amino acid sequence to the N-terminus, purified, and freeze-dried to obtain a pure polypeptide.
  • numbers 1-20 represent the specific positions of amino acids in Formula I.
  • the minimum number of amino acids in Formula I is 5 and the maximum number of amino acids is 20; the configuration of the amino acid is L-type or D-type. ; The presence or absence of amino acids at positions 1-15 and 20;
  • R 3 is selected from the following group: -(CH 2 ) 2 -COOH, -CH 2 -COOH;
  • R 4 is selected from the group consisting of: -CH 2 -COOH;
  • R 5 is selected from the following group: -CH 2 -OH;
  • R 7 is selected from the following group: -(CH 2 ) 2 -COOH, -CH-(CH 3 ) 2 , -CH 2 -CONH 2 , -CH 3 , -CH 2 -CH (CH 3 ) 2 , -C 3 H 6 ;
  • R 8 is selected from the following group: -CH 2 -CONH 2 , -CH 2 -CH(CH 3 ) 2 , -C 3 H 6 , -CH 3 , -(CH 2 ) 2 -COOH, -CH 2 -COOH , -CH-(CH 3 ) 2 ;
  • R 9 is selected from the following group: -CH 3 , -(CH 2 ) 2 -COOH, -CH 2 -COOH, -(CH 2 ) 3 -NHC(NH)NH 2 , -CH 2 -C 6 H 4 - OH, -CH(CH 3 )-OH;
  • R 10 is selected from the following group: -(CH 2 ) 2 -COOH, -(CH 2 ) 3 -NHC(NH)NH 2 , -CH 2 -C 6 H 4 -OH, -CH 2 -CH(CH 3 ) 2 , -CH(CH 3 )-CH 2 -CH 3 , -(CH 2 ) 2 -CONH 2 , -C 8 NH 6 , -CH(CH 3 )-OH, -CH 2 -COOH;
  • R 11 is selected from the following group: -CH 2 -C 6 H 4 -OH, -CH 2 -CH(CH 3 ) 2 , -CH(CH 3 )-CH 2 -CH 3 , -(CH 2 ) 2 - CONH 2 , -CH 2 -CONH 2 , -(CH 2 ) 2 -CONH 2 , -CH 2 -C 6 H 5 , -C 3 H 6 ;
  • R 12 is selected from the following group: -CH 2 -CH(CH 3 ) 2 , -CH 2 -CONH 2 , -(CH 2 ) 2 -CONH 2 , -(CH 2 ) 3 -NHC(NH)NH 2 , -CH(CH 3 )-OH, -CH 2 -COOH, -(CH 2 ) 2 -NH 2 , -(CH 2 ) 4 -NH 2 ;
  • R 13 is selected from the following group: -(CH 2 ) 2 -COOH, -(CH 2 ) 3 -NHC(NH)NH 2 , -CH(CH 3 )-OH, -(CH 2 ) 2 -CONH 2 , -CH 2 -COOH, -(CH 2 ) 2 -NH 2 , -C 3 H 6 ;
  • R 14 is selected from the following group: -(CH 2 ) 2 -COOH, -(CH 2 ) 2 -CONH 2 ;
  • R 15 is selected from the following group: -(CH 2 ) 2 -CONH 2 , -(CH 2 ) 2 -COOH;
  • R 20 is selected from the following group: -NH 2 , -OH;
  • R 21 is selected from the group: -(CH 2 ) 2 -S-CH 3.
  • the resin used in the Fmoc solid-phase synthesis method is selected from any of the following: Amide-MBHA Resin 0.52mmol/g, Amide-MBHA Resin 0.646mmol/g, 2-Chlorotrityl Chloride Resin 0.52mmol/g.
  • the Fmoc solid-phase synthesis method uses Fmoc to protect the amino part of the amino acid;
  • the side chain protecting group is selected from any one of the following groups: benzyl (Bzl), tert-butyl (tBu), tert-butoxycarbonyl ( Boc), 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Pbf), triphenyl (Trt).
  • the Fmoc protecting group is removed and condensed to form a peptide chain;
  • the Fmoc removal reagent is 20% piperidine/DMF;
  • the condensing agent is benzotriazole-N,N,N',N'-tetramethylurea Fluorophosphate (HBTU) and N,N'-diisopropylcarbodiimide (DIEA), the molar ratio is amino acid: HBTU: DIEA is 1:0.9:2.
  • the peptide is removed from the resin and amino acid protecting groups by acid.
  • the ratio of the peptide cutting solution is 95% v/v trifluoroacetic acid (TFA), 2.5% v/v triisopropylsilane (TIPS), and 2.5% v/v water. , 15-20ml/g resin.
  • reaction conditions of each step are as follows:
  • the Fmoc removal reagent is 20% piperidine/DMF, react for 10-30 minutes, repeat 2 times; DMF/DCM cross-washing for more than 3 times; ninhydrin color reaction detection;
  • peptide cutting solution Prepares the peptide cutting solution, the ratio of the peptide cutting solution is 95% v/v trifluoroacetic acid (TFA), 2.5% v/v triisopropylsilane (TIPS), 2.5% v/v water, 15-20ml/g Resin, react for 1-3 hours; press out the liquid; precipitate with ether, wash and dry and then produce crude peptide;
  • TFA trifluoroacetic acid
  • TIPS triisopropylsilane
  • reaction conditions of each step are as follows:
  • the Fmoc removal reagent is 20% piperidine/DMF, react for 10-30 minutes, repeat 2 times; DMF/DCM cross-washing for more than 3 times; ninhydrin color reaction detection;
  • c' Configure the amino acid solvent according to the molar ratio of amino acid: HBTU: DIEA to 1:0.9:2, react for 0.5-2h, cross-wash with DMF/DCM more than 3 times; ninhydrin color reaction detection;
  • d' Repeat steps c' and d', and synthesize the polypeptide according to the amino acid sequence of the remaining polypeptides except for the amino acid numbered (16), (19), (22), (25), (28) and the 20th amino acid. ;
  • f' Prepare the peptide cutting solution.
  • the ratio of the peptide cutting solution is 95% v/v trifluoroacetic acid (TFA), 2.5% v/v triisopropylsilane (TIPS), 2.5% v/v water, 15-20ml/ g resin, react for 1-3 hours; press out the liquid; precipitate with ether, wash and dry, and then obtain the crude polypeptide.
  • TFA trifluoroacetic acid
  • TIPS triisopropylsilane
  • Example 1 A1 (L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L- Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
  • the peptide cutting solution cuts the resin to obtain crude polypeptide A1, which is dissolved in an aqueous solution with a pH of 10 to 11 added with ammonia water.
  • the crude product was filtered through a 0.22 ⁇ m filter membrane for later use.
  • Pure polypeptide A1 was obtained through liquid phase purification, with a yield of 20.94%; HPLC purity of 99%; molecular formula C70H103N16O19S; HRMS: [M+H] + 1503.72925, calcd 1503.73006.
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 0-8min 25% B phase, 8-38min 25%-90% B phase, maintain the B phase gradient for 1 minute and then return to the initial gradient; collect the product peak; the acetonitrile was evaporated under reduced pressure; and the pure peptide was obtained after freeze-drying.
  • Example 2 A2 (L-Glu)-(L-Asn)-(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L- Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
  • the peptide cutting solution cuts the resin to obtain crude polypeptide A2, which is dissolved in an aqueous solution with a pH of 10 to 11 added with ammonia water.
  • the crude product was filtered through a 0.22 ⁇ m filter membrane for later use.
  • Pure polypeptide A2 was obtained through liquid phase purification, with a yield of 10.51%; HPLC purity of 96%; molecular formula C79H116N19O24S; HRMS: [M+H] + 1746.81311, calcd 1746.81558.
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 20% B phase in 0-6min period, 20%-90% B phase in 6-31min period, maintain the B phase gradient for 1min and then return to the initial gradient; collect the product peak; the acetonitrile was evaporated under reduced pressure; and the pure peptide was obtained after freeze-drying.
  • the peptide cutting solution cuts the resin to obtain crude polypeptide A3, which is dissolved in an aqueous solution with a pH of 10 to 11 added with ammonia water.
  • the crude product was filtered through a 0.22 ⁇ m filter membrane for later use.
  • Pure polypeptide A3 was obtained through liquid phase purification, with a yield of 25.08%; HPLC purity of 98%; molecular formula C72H116N17O18S; HRMS: [M+H] + 1538.84229, calcd 1538.84995.
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 0-8min 25% B phase, 8-38min 25%-90% B phase, maintain the B phase gradient for 1 minute and then return to the initial gradient; collect the product peak; the acetonitrile was evaporated under reduced pressure; and the pure peptide was obtained after freeze-drying.
  • Example 4 A4 (L-Val)-(L-Pro)-(L-Glu)-(L-Tyr)-(L-Ile)-(L-Asn)-(L-Gln)-(L- Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
  • the peptide cutting liquid cuts the resin to obtain crude polypeptide A4, which is dissolved in an aqueous solution with a pH of 10 to 11 added with ammonia water.
  • the crude product was filtered through a 0.22 ⁇ m filter membrane for later use.
  • Pure polypeptide A4 was obtained through liquid phase purification, with a yield of 22.69%; HPLC purity ⁇ 95%; molecular formula C80H116N17O23S; HRMS: [M+H] + 1714.81494, calcd1714.81452.
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 0-8min 25% B phase, 8-38min 25%-90% B phase, maintain the B phase gradient for 1 minute and then return to the initial gradient; collect the product peak; the acetonitrile was evaporated under reduced pressure; and the pure peptide was obtained after freeze-drying.
  • Example 5 A5 (L-Asp)-(L-Tyr)-(L-Gln)-(L-Gln)-(L-Asp)-(L-Glu)-(L-Glu)-(L- Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
  • the peptide cutting solution cuts the resin to obtain crude polypeptide A5, which is dissolved in an aqueous solution with a pH of 10 to 11 added with ammonia water.
  • the crude product was filtered through a 0.22 ⁇ m filter membrane for later use.
  • Pure polypeptide A5 was obtained through liquid phase purification, with a yield of 14.57%; HPLC purity ⁇ 95%; molecular formula C68H93N14O24S; HRMS: [M+H] + 1521.63904, calcd1521.63924.
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 20% B phase in 0-6min period, 20%-90% B phase in 6-31min period, maintain the B phase gradient for 1min and then return to the initial gradient; collect the product peak; the acetonitrile was evaporated under reduced pressure; and the pure peptide was obtained after freeze-drying.
  • Example 6 B1 (L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L- Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
  • the peptide cutting solution cuts the resin to obtain crude polypeptide B1, which is dissolved in an aqueous solution with a pH of 10 to 11 and ammonia added.
  • the crude product was filtered through a 0.22 ⁇ m filter membrane for later use.
  • Pure polypeptide B1 was obtained through liquid phase purification, with a yield of 9.125%; HPLC purity of 95.7%; molecular formula C75H111N18O21S; HRMS: [M+H] + 1631.78504, calcd 1631.78864.
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 0-5min 20% B phase, 5-35min 20%-90% B phase, maintain the B phase gradient for 1 minute and then return to the initial gradient; collect the product peak; the acetonitrile was evaporated under reduced pressure; and the pure peptide was obtained after freeze-drying.
  • Example 7 B2 (L-Glu)-(L-Asn)-(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L- Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
  • Synthesis sequence B2 The peptide cutting solution cuts the resin to obtain crude polypeptide B2, which is dissolved in an aqueous solution with a pH of 10 to 11 added with ammonia water. The crude product was filtered through a 0.22 ⁇ m filter membrane for later use. Pure polypeptide B2 was obtained through liquid phase purification, with a yield of 11.29%; HPLC purity of 98%; molecular formula C84H124N18O26S; HRMS: [M+H] + 1874.87142, calcd 1874.87416.
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 23% phase B from 0 to 8 minutes, 23% to 90% phase B from 8 to 38 minutes, maintain the gradient of phase B for 1 minute and then return to the initial gradient; collect the product peak; the acetonitrile was evaporated under reduced pressure; and the pure peptide was obtained after freeze-drying.
  • Example 8 B3 (L-Leu)-(L-Ala)-(L-Arg)-(L-Leu)-(L-Leu)-(L-Thr)-(L-Glu)-(L- Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
  • the peptide cutting solution cuts the resin to obtain crude polypeptide B3, which is dissolved in an aqueous solution with a pH of 10 to 11 added with ammonia.
  • the crude product was filtered through a 0.22 ⁇ m filter membrane for later use.
  • Pure polypeptide B3 was obtained through liquid phase purification, with a yield of 21.72%; HPLC purity of 97%; molecular formula C77H124N19O25S; HRMS: [M+H] + 1666.89551, calcd 1666.89852.
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 0-6min 20% B phase, 6-31min 20%-90% B phase, maintain the B phase gradient for 1 minute and then return to the initial gradient; collect the product peak; the acetonitrile was evaporated under reduced pressure; and the pure peptide was obtained after freeze-drying.
  • Example 9 B4 (L-Val)-(L-Pro)-(L-Glu)-(L-Tyr)-(L-Ile)-(L-Asn)-(L-Gln)-(L- Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
  • the peptide cutting solution cuts the resin to obtain crude polypeptide B4, which is dissolved in an aqueous solution with a pH of 10 to 11 added with ammonia.
  • the crude product was filtered through a 0.22 ⁇ m filter membrane for later use.
  • Pure polypeptide B4 was obtained through liquid phase purification, with a yield of 27.42%; HPLC purity of 99%; molecular formula C85H124N19O25S; HRMS: [M+H] + 1842.87878, calcd 1842.87310.
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 0-6min 20% B phase, 6-31min 20%-90% B phase, maintain the B phase gradient for 1 minute and then return to the initial gradient; collect the product peak; the acetonitrile was evaporated under reduced pressure; and the pure peptide was obtained after freeze-drying.
  • Example 10 B5 (L-Asp)-(L-Tyr)-(L-Gln)-(L-Gln)-(L-Asp)-(L-Glu)-(L-Glu)-(L- Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
  • the peptide cutting solution cuts the resin to obtain crude polypeptide B5, which is dissolved in an aqueous solution with a pH of 10 to 11 added with ammonia.
  • the crude product was filtered through a 0.22 ⁇ m filter membrane for later use.
  • Pure polypeptide B5 was obtained through liquid phase purification, with a yield of 44.215%; HPLC purity of 99%; molecular formula C73H101N16O26S; HRMS: [M+H] + 1648.69727, calcd16488.67099.
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 20% B phase in 0-5min period, 20%-90% B phase in 5-35min period, maintain the B phase gradient for 1min and then return to the initial gradient; collect the product peak; the acetonitrile was evaporated under reduced pressure; and the pure peptide was obtained after freeze-drying.
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 20% B phase in 0-8min period, 20%-90% B phase in 8-38min period, maintain the B phase gradient for 1min and then return to the initial gradient; collect Product peak; acetonitrile was evaporated under reduced pressure; pure polypeptide was obtained after freeze-drying.
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 20% B phase in 0-6min period, 20%-90% B phase in 6-31min period, maintain the B phase gradient for 1min and then return to the initial gradient; collect the product peak; the acetonitrile was evaporated under reduced pressure; and the pure peptide was obtained after freeze-drying.
  • Example 13 EE-6-11 (L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu) -(L-Met)-NH 2
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 0-6min 20% B phase, 6-31min 20%-90% B phase, maintain the B phase gradient for 1 minute and then return to the initial gradient; collect the product peak; the acetonitrile was evaporated under reduced pressure; and the pure peptide was obtained after freeze-drying.
  • Example 14 EE-7-11 (L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met) -NH 2
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 0-6min 20% B phase, 6-31min 20%-90% B phase, maintain the B phase gradient for 1 minute and then return to the initial gradient; collect the product peak; the acetonitrile was evaporated under reduced pressure; and the pure peptide was obtained after freeze-drying.
  • the peptide cutting solution cuts the resin to obtain crude polypeptide A2-M1, which is dissolved in an aqueous solution with a pH of 10 to 11 and ammonia added.
  • the crude product was filtered through a 0.22 ⁇ m filter membrane for later use.
  • Pure polypeptide A2-M1 was obtained through liquid phase purification, with a yield of 36.16%; HPLC purity of 99%; molecular formula C74H107N18O23; HRMS: [M+H] + 1615.77209, calcd 1615.77510.
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 20% B phase in 0-6min period, 20%-90% B phase in 6-31min period, maintain the B phase gradient for 1min and then return to the initial gradient; collect the product peak; the acetonitrile was evaporated under reduced pressure; and the pure peptide was obtained after freeze-drying.
  • the peptide cutting solution cuts the resin to obtain crude polypeptide A2-M2, which is dissolved in an aqueous solution with a pH of 10 to 11 added with ammonia.
  • the crude product was filtered through a 0.22 ⁇ m filter membrane for later use.
  • Pure polypeptide A2-M2 was obtained through liquid phase purification, with a yield of 8.89%; HPLC purity ⁇ 95%; molecular formula C79H115N18O25S; HRMS: [M+H] + 1747.79260, calcd 1747.79960.
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 20% phase B from 0 to 8 minutes, 20% to 90% phase B from 8 to 33 minutes, maintain the gradient of phase B for 1 minute and then return to the initial gradient; collect the product peak; the acetonitrile was evaporated under reduced pressure; and the pure peptide was obtained after freeze-drying.
  • Example 17 A2-M3: (L-Glu)-(L-Asn)-(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-( L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(D-Met)-NH 2
  • the peptide cutting solution cuts the resin to obtain crude polypeptide A2-M3, which is dissolved in an aqueous solution with a pH of 10 to 11 added with ammonia.
  • the crude product was filtered through a 0.22 ⁇ m filter membrane for later use.
  • Pure polypeptide A2-M3 was obtained through liquid phase purification, with a yield of 19.6%; HPLC purity ⁇ 95%; molecular formula C79H115N19O24S; HRMS: [M+H] + 1746.81787, calcd1746.81558.
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 20% phase B from 0 to 8 minutes, 20% to 90% phase B from 8 to 33 minutes, maintain the gradient of phase B for 1 minute and then return to the initial gradient; collect the product peak; the acetonitrile was evaporated under reduced pressure; and the pure peptide was obtained after freeze-drying.
  • Example 18 A3-M1: (L-Leu)-(L-Ala)-(L-Arg)-(L-Leu)-(L-Leu)-(L-Thr)-(L-Glu)-( L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-NH 2
  • the peptide cutting solution cuts the resin to obtain crude polypeptide A3-M1, which is dissolved in an aqueous solution with a pH of 10 to 11 added with ammonia.
  • the crude product was filtered through a 0.22 ⁇ m filter membrane for later use.
  • Pure polypeptide A3-M1 was obtained through liquid phase purification, with a yield of 43.72%; HPLC purity ⁇ 95%; molecular formula C67H107N16O17; HRMS: [M+H] + 1407.80029, calcd 1407.79946.
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 0-7min 20% B phase, 7-32min 20%-90% B phase, maintain the B phase gradient for 1 minute and then return to the initial gradient; collect the product peak; the acetonitrile was evaporated under reduced pressure; and the pure peptide was obtained after freeze-drying.
  • the peptide cutting solution cuts the resin to obtain crude polypeptide A3-M2, which is dissolved in an aqueous solution with a pH of 10 to 11 added with ammonia.
  • the crude product was filtered through a 0.22 ⁇ m filter membrane for later use.
  • Pure polypeptide A3-M2 was obtained through liquid phase purification, with a yield of 22.65%; HPLC purity ⁇ 95%; molecular formula C72H115N16O19S; HRMS: [M+H] + 1539.82805, calcd 1539.82896.
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 0-8min 20% B phase, 8-38min 20%-90% B phase, maintain the B phase gradient for 1 minute and then return to the initial gradient; collect the product peak; the acetonitrile was evaporated under reduced pressure; and the pure peptide was obtained after freeze-drying.
  • Example 20 A3-M3: (L-Leu)-(L-Ala)-(L-Arg)-(L-Leu)-(L-Leu)-(L-Thr)-(L-Glu)-( L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(D-Met)-NH 2
  • the peptide cutting solution cuts the resin to obtain crude polypeptide A3-M3, which is dissolved in an aqueous solution with a pH of 10 to 11 and ammonia added.
  • the crude product was filtered through a 0.22 ⁇ m filter membrane for later use.
  • Pure polypeptide A3-M3 was obtained through liquid phase purification, with a yield of 23.82%; HPLC purity ⁇ 95%; molecular formula C72H116N17O18S; HRMS: [M+H] + 1538.84573, calcd 1538.83995.
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 0-6min 20% B phase, 6-31min 20%-90% B phase, maintain the B phase gradient for 1 minute and then return to the initial gradient; collect the product peak; the acetonitrile was evaporated under reduced pressure; and the pure peptide was obtained after freeze-drying.
  • Example 21 B1-M1 (L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-( L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-NH 2
  • the peptide cutting solution cuts the resin to obtain crude polypeptide B1-M1, which is dissolved in an aqueous solution with a pH of 10 to 11 and ammonia added.
  • the crude product was filtered through a 0.22 ⁇ m filter membrane for later use.
  • Pure polypeptide B1-M1 was obtained through liquid phase purification, with a yield of 22.99%; HPLC purity ⁇ 95%; molecular formula C70H102N17O20; HRMS: [M+H] + 1500.75049, calcd 1500.74851.
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 0-5min 20% B phase, 5-25min 20%-90% B phase, maintain the B phase gradient for 1 minute and then return to the initial gradient; collect the product peak; the acetonitrile was evaporated under reduced pressure; and the pure peptide was obtained after freeze-drying.
  • Example 22 B1-M2 (L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-( L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)
  • the peptide cutting solution cuts the resin to obtain crude polypeptide B1-M2, which is dissolved in an aqueous solution with a pH of 10 to 11 and ammonia added.
  • the crude product was filtered through a 0.22 ⁇ m filter membrane for later use.
  • Pure polypeptide B1-M2 was obtained through liquid phase purification, with a yield of 12.58%; HPLC purity ⁇ 95%; molecular formula C75H110N19O22S; HRMS: [M+H] + 1632.77576, calcd 1632.78864.
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 0-8min 20% B phase, 8-33min 20%-90% B phase, maintain the B phase gradient for 1 minute and then return to the initial gradient; collect the product peak; the acetonitrile was evaporated under reduced pressure; and the pure peptide was obtained after freeze-drying.
  • the peptide cutting solution cuts the resin to obtain crude polypeptide B1-M3, which is dissolved in an aqueous solution with a pH of 10 to 11 added with ammonia.
  • the crude product was filtered through a 0.22 ⁇ m filter membrane for later use.
  • Pure polypeptide B1-M3 was obtained through liquid phase purification, with a yield of 18.5%; HPLC purity ⁇ 95%; molecular formula C75H111N18O21S; HRMS: [M+H] + 1631.79163, calcd 1631.78864.
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 0-8min 20% B phase, 8-33min 20%-90% B phase, maintain the B phase gradient for 1 minute and then return to the initial gradient; collect the product peak; the acetonitrile was evaporated under reduced pressure; and the pure peptide was obtained after freeze-drying.
  • Example 24 B3-M1 (L-Leu)-(L-Ala)-(L-Arg)-(L-Leu)-(L-Leu)-(L-Thr)-(L-Glu)-( L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-NH 2
  • the peptide cutting solution cuts the resin to obtain crude polypeptide B3-M1, which is dissolved in an aqueous solution with a pH of 10 to 11 and ammonia added.
  • the crude product was filtered through a 0.22 ⁇ m filter membrane for later use.
  • Pure polypeptide B3-M1 was obtained through liquid phase purification, with a yield of 9.42%; HPLC purity ⁇ 95%; molecular formula C72H115N18O19; HRMS: [M+H] + 1535.86096, calcd 1535.85804.
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 0-6min 20% B phase, 6-29min 20%-90% B phase, maintain the B phase gradient for 1 minute and then return to the initial gradient; collect the product peak; the acetonitrile was evaporated under reduced pressure; and the pure peptide was obtained after freeze-drying.
  • the peptide cutting solution cuts the resin to obtain crude polypeptide B3-M2, which is dissolved in an aqueous solution with a pH of 10 to 11 added with ammonia.
  • the crude product was filtered through a 0.22 ⁇ m filter membrane for later use.
  • Pure polypeptide B3-M2 was obtained through liquid phase purification, with a yield of 12.58%; HPLC purity ⁇ 95%; molecular formula C75H110N19O22S; HRMS: [M+H] + 1667.88489, calcd1667.88254.
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 0-8min 20% B phase, 8-33min 20%-90% B phase, maintain the B phase gradient for 1 minute and then return to the initial gradient; collect the product peak; the acetonitrile was evaporated under reduced pressure; and the pure peptide was obtained after freeze-drying.
  • the peptide cutting solution cuts the resin to obtain crude polypeptide B3-M3, which is dissolved in an aqueous solution with a pH of 10 to 11 added with ammonia.
  • the crude product was filtered through a 0.22 ⁇ m filter membrane for later use.
  • Pure polypeptide B3-M3 was obtained through liquid phase purification, with a yield of 7.47%; HPLC purity ⁇ 95%; molecular formula C77H124N19O20S; HRMS: [M+H] + 1666.90051, calcd 1666.89852.
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 0-8min 20% B phase, 8-33min 20%-90% B phase, maintain the B phase gradient for 1 minute and then return to the initial gradient; collect the product peak; the acetonitrile was evaporated under reduced pressure; and the pure peptide was obtained after freeze-drying.
  • Example 27 B5-M1 (L-Asp)-(L-Tyr)-(L-Gln)-(L-Gln)-(L-Asp)-(L-Glu)-(L-Glu)-( L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-NH 2
  • the peptide cutting solution cuts the resin to obtain crude polypeptide B5-M1, which is dissolved in an aqueous solution with a pH of 10 to 11 and ammonia added.
  • the crude product was filtered through a 0.22 ⁇ m filter membrane for later use.
  • Pure polypeptide B5-M1 was obtained through liquid phase purification, with a yield of 36.35%; HPLC purity ⁇ 95%; molecular formula C68H92N15O25; HRMS: [M+H] + 1518.65979, calcd 1518.63833.
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 20% phase B from 0 to 8 minutes, 20% to 90% phase B from 8 to 33 minutes, maintain the gradient of phase B for 1 minute and then return to the initial gradient; collect the product peak; the acetonitrile was evaporated under reduced pressure; and the pure peptide was obtained after freeze-drying.
  • the synthesis sequence is B5-M2.
  • the peptide cutting solution cuts the resin to obtain crude polypeptide B5-M2, which is dissolved in an aqueous solution with a pH of 10 to 11 and ammonia added.
  • the crude product was filtered through a 0.22 ⁇ m filter membrane for later use.
  • Pure polypeptide B5-M2 was obtained through liquid phase purification, with a yield of 31.84%; HPLC purity ⁇ 95%; molecular formula C73H100N15O27S; HRMS: [M+H] + 1650.68237, calcd 1650.66283.
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 20% phase B from 0 to 8 minutes, 20% to 90% phase B from 8 to 33 minutes, maintain the gradient of phase B for 1 minute and then return to the initial gradient; collect the product peak; the acetonitrile was evaporated under reduced pressure; and the pure peptide was obtained after freeze-drying.
  • Example 29 B5-M3 (L-Asp)-(L-Tyr)-(L-Gln)-(L-Gln)-(L-Asp)-(L-Glu)-(L-Glu)-( L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(D-Met)-NH 2
  • the peptide cutting solution cuts the resin to obtain crude polypeptide B5-M3, which is dissolved in an aqueous solution with a pH of 10 to 11 and ammonia added.
  • the crude product was filtered through a 0.22 ⁇ m filter membrane for later use.
  • Pure polypeptide B5-M3 was obtained through liquid phase purification, with a yield of 21.4%; HPLC purity ⁇ 95%; molecular formula C73H101N16O26S; HRMS: [M+H] + 1649.69958, calcd 1649.67881.
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 20% phase B from 0 to 8 minutes, 20% to 90% phase B from 8 to 33 minutes, maintain the gradient of phase B for 1 minute and then return to the initial gradient; collect the product peak; the acetonitrile was evaporated under reduced pressure; and the pure peptide was obtained after freeze-drying.
  • Example 30 CO: (L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Gln)-(L-Glu)-(L- Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
  • the peptide cleaving solution cuts the resin to obtain crude polypeptide C0, which is dissolved in an aqueous solution with a pH of 10 to 11 and ammonia added.
  • the crude product was filtered through a 0.22 ⁇ m filter membrane for later use.
  • the pure polypeptide C0 was obtained through liquid phase purification, with a yield of 27.01%; HPLC purity of 98%; molecular formula C75H111N18O21S; HRMS: [M+H] + 1631.79089, calcd 1631.78864.
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 20% B phase for 0-6min, 20%-90% B phase for 6-36min, maintain the B-phase gradient for 1 minute and then return to the initial gradient; collect the product peak; the acetonitrile was evaporated under reduced pressure; and the pure peptide was obtained after freeze-drying.
  • the crude product was filtered through a 0.22 ⁇ m filter membrane for later use.
  • Pure polypeptide C1 was obtained through liquid phase purification, with a yield of 38.85%; HPLC purity of 99%; molecular formula C74H107N16O22S; HRMS: [M+H] + 1603.74829, calcd 1603.74611.
  • Example 32 C2 (L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Asn)-(L-Glu)-(L-Glu)-(L- Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
  • the peptide cutting solution cuts the resin to obtain crude polypeptide C2, which is dissolved in an aqueous solution with a pH of 10 to 11 and ammonia added.
  • the crude product was filtered through a 0.22 ⁇ m filter membrane for later use.
  • Pure polypeptide C2 was obtained through liquid phase purification, with a yield of 21.4%; HPLC purity of 99%; molecular formula C73H105N16O22S; HRMS: [M+H] + 1589.73291, calcd 1589.73046.
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 20% B phase for 0-6min, 20%-90% B phase for 6-36min, maintain the B-phase gradient for 1 minute and then return to the initial gradient; collect the product peak; the acetonitrile was evaporated under reduced pressure; and the pure peptide was obtained after freeze-drying.
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 20% B phase in 0-6min period, 20%-90% B phase in 6-31min period, maintain the B phase gradient for 1min and then return to the initial gradient; collect the product peak; the acetonitrile was evaporated under reduced pressure; and the pure peptide was obtained after freeze-drying.
  • Example 34 C4 (L-Ala)-(L-Glu)-(L-Tyr)-(L-Pro)-(L-Arg)-(L-Glu)-(L-Glu)-(L- Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
  • the peptide cutting liquid cuts the resin to obtain crude polypeptide C4, which is dissolved in an aqueous solution with a pH of 10 to 11 and ammonia added.
  • the crude product was filtered through a 0.22 ⁇ m filter membrane for later use.
  • Pure polypeptide C4 was obtained through liquid phase purification, with a yield of 12.92%; HPLC purity of 97%; molecular formula C74H107N18O21S; HRMS: [M+H] + 1615.76025, calcd 1615.75734.
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 20% B phase in 0-6min period, 20%-90% B phase in 6-31min period, maintain the B phase gradient for 1min and then return to the initial gradient; collect the product peak; the acetonitrile was evaporated under reduced pressure; and the pure peptide was obtained after freeze-drying.
  • the peptide cutting solution cuts the resin to obtain crude polypeptide C5, which is dissolved in an aqueous solution with a pH of 10 to 11 added with ammonia.
  • the crude product was filtered through a 0.22 ⁇ m filter membrane for later use.
  • Pure polypeptide C5 was obtained through liquid phase purification, with a yield of 21.4%; HPLC purity ⁇ 95%; molecular formula C79H113N18O20S; HRMS: [M+H] + 1665.81262, calcd 1665.80937.
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 25% phase B from 0 to 8 minutes, 25% to 90% phase B from 8 to 38 minutes, maintain the gradient of phase B for 1 minute and then return to the initial gradient; collect the product peak; the acetonitrile was evaporated under reduced pressure; and the pure peptide was obtained after freeze-drying.
  • Example 36 C6 (L-Ala)-(L-Glu)-(L-Trp)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L- Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
  • the peptide cutting solution cuts the resin to obtain crude polypeptide C6, which is dissolved in an aqueous solution with a pH of 10 to 11 and ammonia added.
  • the crude product was filtered through a 0.22 ⁇ m filter membrane for later use.
  • Pure polypeptide C6 was obtained through liquid phase purification, with a yield of 10.125%; HPLC purity of 96%; molecular formula C77H112N19O20S; HRMS: [M+H] + 1654.80908, calcd 1654.80462.
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 20% phase B from 0 to 8 minutes, 20% to 90% phase B from 8 to 38 minutes, maintain the phase B gradient for 1 minute and then return to the initial gradient; collect the product peak; the acetonitrile was evaporated under reduced pressure; and the pure peptide was obtained after freeze-drying.
  • the peptide cutting solution cuts the resin to obtain crude polypeptide C7, which is dissolved in an aqueous solution with a pH of 10 to 11 and ammonia added.
  • the crude product was filtered through a 0.22 ⁇ m filter membrane for later use.
  • Pure polypeptide C7 was obtained through liquid phase purification, with a yield of 8.89%; HPLC purity of 99%; molecular formula C69H109N18O22S; HRMS: [M+H] + 1541.78052, calcd 1541.77807.
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 20% B phase in 0-6min period, 20%-90% B phase in 6-31min period, maintain the B phase gradient for 1min and then return to the initial gradient; collect the product peak; the acetonitrile was evaporated under reduced pressure; and the pure peptide was obtained after freeze-drying.
  • Example 38 C8 (L-Ala)-(L-Asp)-(L-Asp)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L- Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
  • the peptide cutting solution cuts the resin to obtain crude polypeptide C8, which is dissolved in an aqueous solution with a pH of 10 to 11 and ammonia added.
  • the crude product was filtered through a 0.22 ⁇ m filter membrane for later use.
  • Pure polypeptide C8 was obtained through liquid phase purification, with a yield of 10.81%; HPLC purity of 98%; molecular formula C69H105N18O22S; HRMS: [M+H] + 1569.74121, calcd 1569.73660.
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 20% B phase in 0-6min period, 20%-90% B phase in 6-31min period, maintain the B phase gradient for 1min and then return to the initial gradient; collect the product peak; the acetonitrile was evaporated under reduced pressure; and the pure peptide was obtained after freeze-drying.
  • the peptide cutting solution cuts the resin to obtain crude polypeptide C9, which is dissolved in an aqueous solution with a pH of 10 to 11 added with ammonia.
  • the crude product was filtered through a 0.22 ⁇ m filter membrane for later use.
  • Pure polypeptide C9 was obtained through liquid phase purification, with a yield of 5.18%; HPLC purity of 99%; molecular formula C78H117N18O21S; HRMS: [M+H] + 1673.83765, calcd 1673.83559.
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 20% B phase in 0-6min period, 20%-90% B phase in 6-31min period, maintain the B phase gradient for 1min and then return to the initial gradient; collect the product peak; the acetonitrile was evaporated under reduced pressure; and the pure peptide was obtained after freeze-drying.
  • Example 40 C10 (L-Val)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L- Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
  • the peptide cutting solution cuts the resin to obtain crude polypeptide C10, which is dissolved in an aqueous solution with a pH of 10 to 11 added with ammonia.
  • the crude product was filtered through a 0.22 ⁇ m filter membrane for later use.
  • Pure polypeptide C10 was obtained through liquid phase purification, with a yield of 21.36%; HPLC purity of 99%; molecular formula C77H115N18O21S; HRMS: [M+H] + 1659.82336, calcd 1659.83994.
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 20% phase B from 0 to 8 minutes, 20% to 90% phase B from 8 to 38 minutes, maintain the phase B gradient for 1 minute and then return to the initial gradient; collect the product peak; the acetonitrile was evaporated under reduced pressure; and the pure peptide was obtained after freeze-drying.
  • the peptide cutting solution cuts the resin to obtain crude polypeptide C11, which is dissolved in an aqueous solution with a pH of 10 to 11 added with ammonia.
  • the crude product was filtered through a 0.22 ⁇ m filter membrane for later use.
  • Pure polypeptide C11 was obtained through liquid phase purification, with a yield of 12.9%; HPLC purity of 97%; molecular formula C73H107N16O21S; HRMS: [M+H] + 1575.75317, calcd 1575.75119.
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 20% B phase in 0-6min period, 20%-90% B phase in 6-31min period, maintain the B phase gradient for 1min and then return to the initial gradient; collect the product peak; the acetonitrile was evaporated under reduced pressure; and the pure peptide was obtained after freeze-drying.
  • Example 42 D1 (L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Thr)-(L-Gln)-(L-Glu)-(L- Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
  • the crude product was filtered through a 0.22 ⁇ m filter membrane for later use.
  • Pure polypeptide D1 was obtained through liquid phase purification, with a yield of 5.81%; HPLC purity ⁇ 95%; molecular formula C73H106N15O22S; HRMS: [M+H] + 1576.73535, calcd 1576.73521.
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 20% phase B from 0 to 8 minutes, 20% to 90% phase B from 8 to 38 minutes, maintain the phase B gradient for 1 minute and then return to the initial gradient; collect the product peak; the acetonitrile was evaporated under reduced pressure; and the pure peptide was obtained after freeze-drying.
  • Example 43 D2 (L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Asp)-(L-Gln)-(L-Glu)-(L- Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
  • the peptide cutting solution cuts the resin to obtain crude polypeptide D2, which is dissolved in an aqueous solution with a pH of 10 to 11 and ammonia added.
  • the crude product was filtered through a 0.22 ⁇ m filter membrane for later use.
  • Pure polypeptide D2 was obtained through liquid phase purification, with a yield of 23.98%; HPLC purity of 96%; molecular formula C73H104N15O23S; HRMS: [M+H] + 1590.71655, calcd 1590.71447.
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 20% B phase in 0-6min period, 20%-90% B phase in 6-31min period, maintain the B phase gradient for 1min and then return to the initial gradient; collect the product peak; the acetonitrile was evaporated under reduced pressure; and the pure peptide was obtained after freeze-drying.
  • Example 44 D3 (L-Ala)-(L-Tyr)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Gln)-(L-Glu)-(L- Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
  • the peptide cutting solution cuts the resin to obtain crude polypeptide D3, which is dissolved in an aqueous solution with a pH of 10 to 11 added with ammonia.
  • the crude product was filtered through a 0.22 ⁇ m filter membrane for later use.
  • Pure polypeptide D3 was obtained through liquid phase purification, with a yield of 11.06%; HPLC purity ⁇ 95%; molecular formula C79H113N18O20S; HRMS: [M+H] + 1665.81335, calcd 1665.80937.
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 20% phase B from 0 to 8 minutes, 20% to 90% phase B from 8 to 35 minutes, maintain the gradient of phase B for 1 minute and then return to the initial gradient; collect the product peak; the acetonitrile was evaporated under reduced pressure; and the pure peptide was obtained after freeze-drying.
  • Example 45 D4 (L-Ala)-(L-Glu)-Trp-(L-Leu)-(L-Arg)-(L-Gln)-(L-Glu)-(L-Glu)-( L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
  • the crude product was filtered through a 0.22 ⁇ m filter membrane for later use.
  • Pure polypeptide D4 was obtained through liquid phase purification, with a yield of 10.63%; HPLC purity of 97%; molecular formula C77H112N19O20S; HRMS: [M+H] + 1654.80676, calcd 1654.80462.
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 20% phase B from 0 to 8 minutes, 20% to 90% phase B from 8 to 38 minutes, maintain the phase B gradient for 1 minute and then return to the initial gradient; collect the product peak; the acetonitrile was evaporated under reduced pressure; and the pure peptide was obtained after freeze-drying.
  • Example 46 D5 (L-Ala)-(L-Thr)-(L-Thr)-(L-Leu)-(L-Arg)-(L-Gln)-(L-Glu)-(L- Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
  • the peptide cutting solution cuts the resin to obtain crude polypeptide D5, which is dissolved in an aqueous solution with a pH of 10 to 11 and ammonia added.
  • the crude product was filtered through a 0.22 ⁇ m filter membrane for later use.
  • Pure polypeptide D5 was obtained through liquid phase purification, with a yield of 18.06%; HPLC purity of 96%; molecular formula C69H109N18O20S; HRMS: [M+H] + 1541.78076, calcd 1541.77807.
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 20% phase B from 0 to 6 minutes, 20% to 90% phase B from 6 to 31 minutes, maintain the gradient of phase B for 1 minute and then return to the initial gradient; collect the product peak; the acetonitrile was evaporated under reduced pressure; and the pure peptide was obtained after freeze-drying.
  • Example 47 D6 (L-Ala)-(L-Asp)-(L-Asp)-(L-Leu)-(L-Arg)-(L-Gln)-(L-Glu)-(L- Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
  • the peptide cutting solution cuts the resin to obtain crude polypeptide D6, which is dissolved in an aqueous solution with a pH of 10 to 11 added with ammonia.
  • the crude product was filtered through a 0.22 ⁇ m filter membrane for later use.
  • Pure polypeptide D6 was obtained through liquid phase purification, with a yield of 8.28%; HPLC purity of 98%; molecular formula C69H105N18O22S; HRMS: [M+H] + 1569.74072, calcd 1569.73660.
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 20% B phase in 0-6min period, 20%-90% B phase in 6-31min period, maintain the B phase gradient for 1min and then return to the initial gradient; collect the product peak; the acetonitrile was evaporated under reduced pressure; and the pure peptide was obtained after freeze-drying.
  • Example 48 D7 (L-Val)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Gln)-(L-Glu)-(L- Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
  • the crude product was filtered through a 0.22 ⁇ m filter membrane for later use.
  • Pure polypeptide D7 was obtained through liquid phase purification, with a yield of 10.51%; HPLC purity of 98%; molecular formula C77H115N18O21S; HRMS: [M+H] + 1659.82361, calcd 1659.81994.
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 20% B phase in 0-8min period, 20%-90% B phase in 8-35min period, maintain the B phase gradient for 1min and then return to the initial gradient; collect Product peak; acetonitrile was evaporated under reduced pressure; pure polypeptide was obtained after freeze-drying.
  • Example 49 D8 (L-Leu)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Gln)-(L-Glu)-(L- Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
  • the crude product was filtered through a 0.22 ⁇ m filter membrane for later use.
  • Pure polypeptide D8 was obtained through liquid phase purification, with a yield of 10.85%; HPLC purity ⁇ 95%; molecular formula C78H117N18O21S; HRMS: [M+H] + 1673.83862, calcd 1673.83559.
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 20% phase B from 0 to 8 minutes, 20% to 90% phase B from 8 to 38 minutes, maintain the phase B gradient for 1 minute and then return to the initial gradient; collect the product peak; the acetonitrile was evaporated under reduced pressure; and the pure peptide was obtained after freeze-drying.
  • Example 50 D9 (L-Ala)-(L-Glu)-(L-Tyr)-Phe-(L-Arg)-(L-Gln)-(L-Glu)-(L-Glu)-( L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
  • the crude product was filtered through a 0.22 ⁇ m filter membrane for later use.
  • Pure polypeptide D9 was obtained through liquid phase purification, with a yield of 21.28%; HPLC purity of 99%; molecular formula C78H109N18O21S; HRMS: [M+H] + 1665.77515, calcd 1665.77299.
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 25% phase B from 0 to 8 minutes, 25% to 90% phase B from 8 to 38 minutes, maintain the gradient of phase B for 1 minute and then return to the initial gradient; collect the product peak; the acetonitrile was evaporated under reduced pressure; and the pure peptide was obtained after freeze-drying.
  • Example 51 D10 (L-Ala)-(L-Glu)-(L-Tyr)-(L-Pro)-(L-Arg)-(L-Gln)-(L-Glu)-(L- Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
  • the peptide cutting solution cuts the resin to obtain crude polypeptide D10, which is dissolved in an aqueous solution with a pH of 10 to 11 added with ammonia.
  • the crude product was filtered through a 0.22 ⁇ m filter membrane for later use.
  • Pure polypeptide D10 was obtained through liquid phase purification, with a yield of 25.17%; HPLC purity of 98%; molecular formula C74H107N18O21S; HRMS: [M+H] + 1615.76147, calcd 1615.75734.
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 20% B phase in 0-6min period, 20%-90% B phase in 6-31min period, maintain the B phase gradient for 1min and then return to the initial gradient; collect the product peak; the acetonitrile was evaporated under reduced pressure; and the pure peptide was obtained after freeze-drying.
  • Example 52 D11: (L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Dab)-(L-Gln)-(L-Glu)-(L- Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
  • the peptide cutting solution cuts the resin to obtain crude polypeptide D11, which is dissolved in an aqueous solution with a pH of 10 to 11 added with ammonia water.
  • the crude product was filtered through a 0.22 ⁇ m filter membrane for later use.
  • Pure polypeptide D11 was obtained through liquid phase purification, with a yield of 14.96%; HPLC purity of 97%; molecular formula C73H107N16O21S; HRMS: [M+H] + 1575.75427, calcd 1575.75119.
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 20% B phase in 0-6min period, 20%-90% B phase in 6-31min period, maintain the B phase gradient for 1min and then return to the initial gradient; collect Product peak; acetonitrile was evaporated under reduced pressure; pure polypeptide was obtained after freeze-drying.
  • Example 53 D12 (L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Dab)-(L-Glu)-(L- Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
  • the peptide cutting solution cuts the resin to obtain crude polypeptide D12, which is dissolved in an aqueous solution with a pH of 10 to 11 and ammonia added.
  • the crude product was filtered through a 0.22 ⁇ m filter membrane for later use.
  • Pure polypeptide D12 was obtained through liquid phase purification, with a yield of 14.43%; HPLC purity of 99%; molecular formula C74H111N18O20S; HRMS: [M+H] + 1603.79761, calcd 1603.79372.
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 20% B phase in 0-6min period, 20%-90% B phase in 6-31min period, maintain the B phase gradient for 1min and then return to the initial gradient; collect the product peak; the acetonitrile was evaporated under reduced pressure; and the pure peptide was obtained after freeze-drying.
  • Liquid phase purification conditions Shimadzu LC-20AT chromatography system, Shim-pack PREP-ODS (20mm ⁇ 250mm i.d., 10 ⁇ m), C18 packing, reversed-phase column; mobile phase A phase 0.08% TFA/water, phase B 0.08% TFA /acetonitrile; 40°C; flow rate 6ml/min; detection wavelength 210nm; gradient conditions are 0-2min 10% B phase, 2-33min 10%-30% B phase, 3-33min 30% B phase, maintain B phase gradient for 1 minute Then return to the initial gradient; collect the product peak; evaporate the acetonitrile under reduced pressure; and obtain pure polypeptide after freeze-drying.
  • stably transfected APPswe cells can promote A ⁇ deposition and cellular oxidative stress response under the stimulation of copper ions, simulating the occurrence of Alzheimer's disease (AD).
  • AD Alzheimer's disease
  • 350 ⁇ M Cu 2+ was used to damage APPswe cells for 24 h, and 0.032 ⁇ M, 0.16 ⁇ M, 0.8 ⁇ M, 4 ⁇ M, 20 ⁇ M, and 100 ⁇ M neurokinin derivatives were added for co-incubation. After 24 hours, CCK-8 reagent was used to determine cell viability.
  • Cell survival rate (%) [(OD drug group -OD blank group )/(OD control group -OD blank group )] ⁇ 100%; (*P ⁇ 0.05, **P ⁇ 0.01, ***P ⁇ 0.001).
  • Effective rate (%) (OD medication group -OD model group )/OD model group *100%;
  • the present invention relates to the protective effect of some polypeptides on APPswe cells
  • Amyloid- ⁇ is the main pathological mediator of familial and sporadic AD.
  • a ⁇ Amyloid- ⁇
  • a large amount of A ⁇ oligomers and plaques are accumulated, causing neuronal cell degeneration and apoptosis.
  • Clinical and experimental verification show that this abnormal deposition of A ⁇ blocks long-term synaptic potentiation, impairs synaptic function and induces cognitive deficits.
  • This model is based on the above-mentioned neurotoxic effects of A ⁇ .
  • the Morris water maze (MWM) experiment was used to detect the spatial learning ability and memory ability of mice in each group after medication.
  • the diameter of the water maze pool is 1.2m.
  • the pool is divided into four quadrants on average. Markers are hung corresponding to the four quadrants to facilitate the animals to remember the location.
  • the water temperature was maintained at 22 ⁇ 2°C during the experiment.
  • the experiment is divided into two parts: positioning navigation test and space exploration test.
  • the positioning navigation test was conducted for five consecutive days, twice a day. During the experiment, the platform was placed in a fixed position, and the mice were put into the water from two quadrants every day. The time for the mice to successfully find the platform, that is, the latency period and swimming speed, were recorded.
  • mice were allowed 60 seconds to complete the task during the experiment, otherwise they were guided to the platform and stayed for 10 seconds to observe learning.
  • the space exploration test was conducted 24 hours after the positioning navigation test to evaluate memory consolidation. During the test, the platform was removed, the mouse was put into the water from the farthest quadrant from the original platform, and allowed to swim for 60 seconds. Record the time the mouse spends swimming in the quadrant of the original platform and the number of times it crosses the original platform location.
  • Results are expressed as mean ⁇ standard error. Platform latency and swimming speed in the MWM experiment were analyzed by one-way analysis of variance for repeated measures data using IBM SPSS Statistics 26.0.
  • the latency of mice to find the platform is an indicator of spatial learning ability.
  • the latency of mice to find the platform shortened as the number of training sessions increased, and there were significant differences in the latency of mice in different treatment groups. Comparing the trend of latency changes over time between groups, compared with mice in the Sham group, the latency of A ⁇ 1-42 model mice was significantly longer ( Figure 1A and Figure 2A, P ⁇ 0.01), suggesting that the learning ability of the mice was significantly improved. decline.
  • African green monkey kidney cells - Vero cells were selected. The cells were cultured using MEM culture medium and 10% fetal calf serum at 37°C and 5% CO2 .
  • the blank control well contains cells plus solvent and serves as a negative control well; the well without cells and only culture medium serves as the background well.
  • the inhibition rate is calculated according to the following calculation formula:
  • polypeptides involved in the present invention show low inhibitory effects at 100 ⁇ g/ml, and the inhibition rates do not exceed 24%, indicating that the polypeptides involved in the present invention have low toxicity to renal cells.
  • This experiment uses the whole blood of Japanese big-eared white rabbits to test the hemolytic properties of polypeptides.
  • the negative control is PBS (0% hemolysis), and the positive control is 1% Triton X-100 (100% hemolysis).
  • the test peptide drug concentrations were set to 2 ⁇ g/mL, 1 ⁇ g/mL, 0.5 ⁇ g/mL, 0.25 ⁇ g/mL, 0.125 ⁇ g/mL, and 0.0625 ⁇ g/mL.
  • Cell survival rate (%) [(OD drug group-OD negative control group)/(OD positive control group-OD blank group)] ⁇ 100%;
  • polypeptides involved in the present invention have good neuroprotective effects, have low nephrotoxicity and hemolysis, and have good development potential.

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Abstract

一种多肽化合物及其制备方法和应用。所述的多肽化合物具有式I所示的结构。所述多肽化合物能够显著提高Aβ介导认知损伤小鼠的空间学习和记忆能力。此外，所述部分多肽化合物的毒性较低，在肾细胞(Vero)毒性检测和溶血性(兔子全血)检测中表现出较低的毒性，可用于制备具有NK细胞相关免疫调节作用、治疗炎症相关疾病或诊断与治疗神经系统疾病药物。所述多肽化合物可以单独使用，也可以和另一种或多种其它活性成分联合用于治疗、预防、抑制或者改善疾病或者病状，其中药物的联合使用比任何一种药物的单独使用更为安全或者更为有效。

Description

一类多肽化合物及其制备方法和应用 技术领域
本发明涉及一类由5-20个氨基酸组成的多肽分子，及其制备方法和在免疫调节、治疗炎症相关疾病以及神经系统疾病中的应用。本发明属于医药技术领域。
背景技术
多肽类化合物具有广泛生物活性。其中，神经激肽P物质及其受体NK-1R介导的神经源性炎症越来越受到重视。P物质与其受体结合后，激发免疫细胞亲润和细胞因子释放的瀑布式级联反应，在多种病理生理过程中发挥重要作用。另一方面，P物质还可以激活NK92-M1细胞活性，发挥重要免疫作用。自然杀伤细胞(natural killer cell，NK)是机体重要的免疫细胞，不仅与抗肿瘤、抗病毒感染和免疫调节有关，而且在某些情况下参与超敏反应和自身免疫性疾病的发生。近期研究结果显示，大脑中的炎症(神经性炎症)与多种疾病直接相关，比如抑郁症、精神病和多发性硬化症等，而且最近研究人员发现神经性炎症竟与阿尔茨海默病发病直接相关。
阿尔茨海默病(Alzheimer's disease,AD)是一种神经退行性慢性疾病，病程5-10年。研究显示，65岁以上老人为AD的高发人群。随着人口老龄化日渐加剧，AD伴随着的治疗与看护成本会持续增加。AD的临床表现以进行性认知功能障碍和记忆损害为主要特征，并伴有行为障碍和社交障碍，晚期AD患者甚至会失去进食和控制身体的能力。AD的病理特征为β-淀粉样蛋白聚集形成老年斑(Senile Plaques)和Tau蛋白过度磷酸化形成神经内神经纤维神经元缠结(neurofibrillary tangles)。
AD为原发性神经系统疾病，发病机制复杂多样，其中β-淀粉样蛋白(Aβ)假说、Tau蛋白异常磷酸化假说、胆碱能神经元假说、氧化应激学说、基因突变学说、胰岛素传导通路障碍学说研究较为深入。另外随着科学技术的进步，研究者们发现线粒体功能障碍、突触及神经递质衰竭、炎症反应、兴奋性氨基酸毒性学说、脂质代谢异常等潜在机制也在影响着AD的疾病进程。环境因素与遗传因素也是诱导AD患者发病的重要因素。
1993年FDA批准了第一个治疗AD的药物—他可林(Tacrine)，为乙酰胆碱酯酶抑制剂，接着又批准了二代乙酰胆碱酯酶抑制剂多奈哌齐(Donepzil)、加兰他敏、卡巴拉汀、利伐斯的明，现已被广泛应用于AD患者的治疗。美金刚为FDA批准的第一个用于中重度AD患者的治疗的药物，可以缓解AD患者的记忆损伤。石杉碱甲 是从石杉科植物中提取出的生物碱，在国内广泛用于对AD患者的治疗，但由于FDA未批准该药物，使得其在国外发展受限。但是当前经FDA批准的用于治疗AD的药物仅可以辅助改善AD患者的生存质量，未能阻止AD的发病进程，同时又存在诸多不良反应。据报道，1998年至2017年期间，在美国就有150项针对AD的治疗方法与药物研究，但是，仅仅只有4个新药被批准用于AD的治疗。因此，开发新的治疗策略与发现新的药物研发靶点也许会为AD的药物研发提供新的希望。
神经肽是中枢系统和外周神经系统内的内源性活性多肽，在神经系统中发挥重要作用，参与调节新陈代谢、食物摄入、学习、记忆与睡眠等诸多大脑功能。许多研究发现，胃饥饿素、神经紧张素、垂体腺苷酸环化酶激活肽、神经肽Y、P物质(Substance P,SP)、食欲素等神经肽与AD的生理病理密切相关。在AD患者中，神经肽及其受体含量水平发生了明显变化。这些神经肽主要通过抑制Aβ积累、增加神经元葡萄糖运输、增加神经营养因子的产生、抑制内质网应激与自噬和调节钾离子通道等机制发挥神经保护作用。神经肽对AD中神经保护作用的发现，为AD药物研发提供了新的方向。
其中，在晚发型AD患者的大脑皮层和海马体中发现P物质含量降低，而脊髓液中P物质含量增加。研究表明，P物质通过抑制Aβ诱导的钾离子通道过表达和钾离子电流增加，发挥神经保护作用，从而减轻AD的认知缺陷症状和细胞凋亡情况。另外，P物质也通过抑制caspase-3诱导的PARP1切割和增加α-分泌酶的活性来保护神经细胞。
基于神经激肽P物质的结构、与炎症的相关性、与NK细胞的相关性以及炎症与AD的相关性等研究成果，设计并合成了一系列新型多肽化合物，发现了该类多肽化合物对NK细胞的调节作用，相关作用的发现为研发相关机制新药提供了可能。并在体内、外研究发现了其作为认知障碍性疾病治疗药物的应用前景与价值。
发明内容
本发明涉及一类由5-20个氨基酸组成的多肽分子，及其制备方法和在免疫调节、治疗炎症相关疾病以及神经系统疾病中的应用。
为达到上述目的，本发明采用了如下技术方案：
首先，本发明提出了具有如式Ⅰ所示结构的多肽化合物、其药用盐、溶剂合物、异构体或多晶型物：
Figure PCTCN2022142732-appb-000001
式Ⅰ中，数字1-20表示氨基酸在式Ⅰ中的具体位置，式I中的最少氨基酸个数为5个、最大氨基酸个数为20个；所述氨基酸的构型为L型或D型；数字1-15以及20所在位置的氨基酸存在或不存在；
式Ⅰ中：
R 3选自以下的组：-(CH 2) 2-COOH、-CH 2-COOH；
R 4选自以下的组：-CH 2-COOH；
R 5选自以下的组：-CH 2-OH；
R 6选自以下的组：-(CH 2) 2-COOH、-CH 2-CH(CH 3) 2、-CH-(CH 3) 2；
R 7选自以下的组：-(CH 2) 2-COOH、-CH-(CH 3) 2、-CH 2-CONH 2、-CH 3、-CH 2-CH(CH 3) 2、-C 3H 6；
R 8选自以下的组：-CH 2-CONH 2、-CH 2-CH(CH 3) 2、-C 3H 6、-CH 3、-(CH 2) 2-COOH、-CH 2-COOH、-CH-(CH 3) 2；
[援引加入（细则20.6）08.12.2023]
R9选自以下的组：-CH3、-(CH2)2-COOH、-CH2-COOH、-(CH2)3-NHC(NH)NH2、-CH2-C6H4-OH、-CH(CH3)-OH；
[援引加入（细则20.6）08.12.2023]

R10选自以下的组：-(CH2)2-COOH、-(CH2)3-NHC(NH)NH2、-CH2-C6H4-OH、-CH2-CH(CH3)2、-CH(CH3)-CH2-CH3、-(CH2)2-CONH2、-C8NH6、-CH(CH3)-OH、-CH2-COOH；
[援引加入（细则20.6）08.12.2023]
R 11选自以下的组：-CH2-C6H4-OH、-CH2-CH(CH3)2、-CH(CH3)-CH2-CH3、-(CH2)2-CONH2、-CH2-CONH2、-(CH2)2-CONH2、-CH2-C6H5、-C3H6
[援引加入（细则20.6）08.12.2023]
R12选自以下的组：-CH2-CH(CH3)2、-CH2-CONH2、-(CH2)2-CONH2、-(CH2)3-NHC(NH)NH2、-CH(CH3)-OH、-CH2-COOH、-(CH2)2-NH2、-(CH2)4-NH2；
[援引加入（细则20.6）08.12.2023]
R13选自以下的组：-(CH2)2-COOH、-(CH2)3-NHC(NH)NH2、-CH(CH3)-OH、-(CH2)2-CONH2、-CH2-COOH、-(CH2)2-NH2、-C3H6
[援引加入（细则20.6）08.12.2023]
R14选自以下的组：-(CH2)2-COOH、-(CH2)2-CONH2
[援引加入（细则20.6）08.12.2023]
R15选自以下的组：-(CH2)2-CONH2、-(CH2)2-COOH；
R 20选自以下的组：-NH 2、-OH。
其中，优选的，所述的多肽化合物选自以下的组：
(1)A1:(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
(2)A2:(L-Glu)-(L-Asn)-(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
(3)A3:(L-Leu)-(L-Ala)-(L-Arg)-(L-Leu)-(L-Leu)-(L-Thr)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
(4)A4:(L-Val)-(L-Pro)-(L-Glu)-(L-Tyr)-(L-Ile)-(L-Asn)-(L-Gln)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
(5)A5:(L-Asp)-(L-Tyr)-(L-Gln)-(L-Gln)-(L-Asp)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
(6)B1:(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
(7)B2:(L-Glu)-(L-Asn)-(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
(8)B3:(L-Leu)-(L-Ala)-(L-Arg)-(L-Leu)-(L-Leu)-(L-Thr)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
(9)B4:(L-Val)-(L-Pro)-(L-Glu)-(L-Tyr)-(L-Ile)-(L-Asn)-(L-Gln)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
(10)B5:(L-Asp)-(L-Tyr)-(L-Gln)-(L-Gln)-(L-Asp)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
(11)SP-6-11:(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
(12)SP-7-11:(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
(13)EE-6-11:(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
(14)EE-7-11:(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
(15)A2-M1:(L-Glu)-(L-Asn)-(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-NH 2
(16)A2-M2:(L-Glu)-(L-Asn)-(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)
(17)A2-M3:(L-Glu)-(L-Asn)-(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)- (L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(D-Met)-NH 2
(18)A3-M1:(L-Leu)-(L-Ala)-(L-Arg)-(L-Leu)-(L-Leu)-(L-Thr)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-NH 2
(19)A3-M2:(L-Leu)-(L-Ala)-(L-Arg)-(L-Leu)-(L-Leu)-(L-Thr)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)
(20)A3-M3:(L-Leu)-(L-Ala)-(L-Arg)-(L-Leu)-(L-Leu)-(L-Thr)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(D-Met)-NH 2
(21)B1-M1:(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-NH 2
(22)B1-M2:(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)
(23)B1-M3:(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(D-Met)-NH 2
(24)B3-M1:(L-Leu)-(L-Ala)-(L-Arg)-(L-Leu)-(L-Leu)-(L-Thr)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-NH 2
(25)B3-M2:(L-Leu)-(L-Ala)-(L-Arg)-(L-Leu)-(L-Leu)-(L-Thr)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)
(26)B3-M3:(L-Leu)-(L-Ala)-(L-Arg)-(L-Leu)-(L-Leu)-(L-Thr)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(D-Met)-NH 2
(27)B5-M1:(L-Asp)-(L-Tyr)-(L-Gln)-(L-Gln)-(L-Asp)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-NH 2
(28)B5-M2:(L-Asp)-(L-Tyr)-(L-Gln)-(L-Gln)-(L-Asp)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)
(29)B5-M3:(L-Asp)-(L-Tyr)-(L-Gln)-(L-Gln)-(L-Asp)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(D-Met)-NH 2
(30)C0:(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Gln)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
(31)C1:(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Gln)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
(32)C2:(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Asn)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
(33)C3:(L-Ala)-(L-Glu)-(L-Tyr)-(L-Phe)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
(34)C4:(L-Ala)-(L-Glu)-(L-Tyr)-(L-Pro)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
(35)C5:(L-Ala)-(L-Tyr)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
(36)C6:(L-Ala)-(L-Glu)-(L-Trp)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
(37)C7:(L-Ala)-(L-Thr)-(L-Thr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
(38)C8:(L-Ala)-(L-Asp)-(L-Asp)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
(39)C9:(L-Leu)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
(40)C10:(L-Val)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
(41)C11:(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Dab)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
(42)D1:(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Thr)-(L-Gln)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
(43)D2:(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Asp)-(L-Gln)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
(44)D3:(L-Ala)-(L-Tyr)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Gln)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
(45)D4:(L-Ala)-(L-Glu)-Trp-(L-Leu)-(L-Arg)-(L-Gln)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
(46)D5:(L-Ala)-(L-Thr)-(L-Thr)-(L-Leu)-(L-Arg)-(L-Gln)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
(47)D6:(L-Ala)-(L-Asp)-(L-Asp)-(L-Leu)-(L-Arg)-(L-Gln)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
(48)D7:(L-Val)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Gln)-(L-Glu)-(L-Glu)- (L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
(49)D8:(L-Leu)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Gln)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
(50)D9:(L-Ala)-(L-Glu)-(L-Tyr)-Phe-(L-Arg)-(L-Gln)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
(51)D10:(L-Ala)-(L-Glu)-(L-Tyr)-(L-Pro)-(L-Arg)-(L-Gln)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
(52)D11:(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Dab)-(L-Gln)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
(53)D12:(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Dab)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
(54)SP:(L-Arg)-(L-Pro)-(L-Lys)-(L-Pro)-(L-Gln)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
(56)E1:(L-Leu)-(L-Ala)-(L-Val)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-Gly-(L-Leu)-(L-Met)-NH 2
(57)E2:(D-Leu)-(L-Ala)-(L-Val)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
(58)E3:(L-Ser)-(L-Leu)-(L-Ala)-(L-Val)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
(59)E4:(D-Ser)-(L-Leu)-(L-Ala)-(L-Val)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
(60)E5:(L-Asp)-(L-Ser)-(L-Leu)-(L-Ala)-(L-Val)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
(61)E6:(D-Asp)-(L-Ser)-(L-Leu)-(L-Ala)-(L-Val)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
(62)E7:(L-Glu)-(L-Asp)-(L-Ser)-(L-Leu)-(L-Ala)-(L-Val)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
(63)E8:(L-Asp)-(L-Asp)-(L-Ser)-(L-Leu)-(L-Ala)-(L-Val)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
(64)E9:(L-Glu)-(L-Glu)-(L-Asp)-(L-Ser)-(L-Leu)-(L-Ala)-(L-Val)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
(65)E10:(L-Glu)-(L-Asp)-(L-Asp)-(L-Ser)-(L-Leu)-(L-Ala)-(L-Val)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
(66)E11:(L-Glu)-(L-Glu)-(L-Glu)-(L-Asp)-(L-Ser)-(L-Leu)-(L-Ala)-(L-Val)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
(67)E12:(L-Glu)-(L-Glu)-(L-Asp)-(L-Asp)-(L-Ser)-(L-Leu)-(L-Ala)-(L-Val)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2。
进一步的，本发明一种制备所述的具有如式Ⅰ所示结构的多肽化合物、其药用盐、溶剂合物、异构体或多晶型物的方法，该方法采用Fmoc固相合成方法，按照肽链从C端到N端的氨基酸顺序合成多肽，经纯化，冷冻干燥后得到多肽纯品。
其中，优选的，所述的Fmoc固相合成方法所用树脂选自以下任意一种：Amide-MBHA Resin 0.52mmol/g、Amide-MBHA Resin 0.646mmol/g、2-Chlorotrityl Chloride Resin 0.52mmol/g。
其中，优选的，所述的Fmoc固相合成方法选用Fmoc保护氨基酸氨基部分；侧链保护基选自以下基团中的任意一种：苄基(Bzl)、叔丁基(tBu)、叔丁氧羰基(Boc)、2,2,4,6,7-五甲基二氢苯并呋喃-5-磺酰基(Pbf)、三苯基(Trt)。
其中，优选的，经脱除Fmoc保护基、缩合形成肽链；脱除Fmoc试剂为20％哌啶/DMF；缩合剂为苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸酯(HBTU)、N,N'-二异丙基碳二亚胺(DIEA)，摩尔比为氨基酸：HBTU：DIEA为1:0.9:2。
其中，优选的，多肽通过酸脱除树脂与氨基酸保护基，切肽液比例为95％v/v三氟乙酸(TFA)、2.5％v/v三异丙基硅烷(TIPS)、2.5％v/v水，15-20ml/g树脂。
其中，优选的，当20位氨基酸不存在时，按照常规方法制备。
其中，优选的，当R 20为-OH时，20位氨基酸为L型Met，采用路线1方法：
路线1：
Figure PCTCN2022142732-appb-000002
Figure PCTCN2022142732-appb-000003
其中，各步骤的反应条件如下：
a：DMF，20-50℃，溶胀10-50min；DMF/DCM交叉洗涤3次以上；茚三酮显色反应检测；
b：Fmoc-Met-OH，DIEA，反应3h或过夜；甲醇0.5ml，30min；DMF/DCM交叉洗涤3次以上；茚三酮显色反应检测；
c：配置脱保护试剂，脱除Fmoc试剂为20％哌啶/DMF，反应10-30min，重复2次；DMF/DCM交叉洗涤3次以上；茚三酮显色反应检测；
d：按照摩尔比为氨基酸：HBTU：DIEA为1:0.9:2配置氨基酸溶剂，反应1-3h，DMF/DCM交叉洗涤3次以上；茚三酮显色反应检测；
e：重复步骤c、d，按照顺序合成多肽；
f：配置切肽液，切肽液比例为95％v/v三氟乙酸(TFA)、2.5％v/v三异丙基硅烷(TIPS)、2.5％v/v水，15-20ml/g树脂，反应1-3h；压出液体；乙醚沉淀洗涤干燥后得多肽粗品；
路线2：
当R 20为-NH 2时，采用路线2方法：
Figure PCTCN2022142732-appb-000004
Figure PCTCN2022142732-appb-000005
其中，各步骤的反应条件如下：
a':DMF，20-50℃，溶胀10-50min；DMF/DCM交叉洗涤3次以上；茚三酮显色反应检测；
b':配置脱保护试剂，脱除Fmoc试剂为20％哌啶/DMF，反应10-30min，重复2次；DMF/DCM交叉洗涤3次以上；茚三酮显色反应检测；
c':按照摩尔比为氨基酸：HBTU：DIEA为1:0.9:2配置氨基酸溶剂，反应0.5-2h，DMF/DCM交叉洗涤3次以上；茚三酮显色反应检测；
d':重复步骤c'、d'，按照顺序合成多肽；
f':配置切肽液，切肽液比例为95％v/v三氟乙酸(TFA)、2.5％v/v三异丙基硅烷(TIPS)、2.5％v/v水，15-20ml/g树脂，反应1-3h；压出液体；乙醚沉淀洗涤干燥后得多肽粗品。
更进一步的，本发明还提出了一种药物组合物，包含所述的具有如式Ⅰ所示结构的多肽化合物、其药用盐、溶剂合物、异构体或多晶型物，以及药学上可以接受的载体或赋形剂。
更进一步的，本发明还提出了所述的具有如式Ⅰ所示结构的多肽化合物、其药用盐、溶剂合物、异构体或多晶型物或所述的药物组合物在制备具有NK细胞相关免疫调节作用、治疗炎症相关疾病或诊断与治疗神经系统疾病药物中的应用。
如本文所用，“药用盐”是指保留目标化合物的所需生物活性并表现出最小的不希望的毒理学效应的盐。当本发明的化合物中含有相对酸性的功能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的碱与本发明化合物的中性形式接触的方式获得碱加成盐。药学上可接受的碱包括无机碱及有机碱制备的盐，所述的无机碱的盐包括铝盐、铵盐、钙盐、铜盐、铁盐、亚铁盐、锂盐、镁盐、锰盐、亚锰盐、钾盐、钠 盐、锌盐等。所述的有机无毒碱的盐，包括伯胺、仲胺和叔胺的盐，包括取取代胺和环状胺。当本发明的化合物中含有相对碱性的官能团时，可以通过在纯的溶液或合适的惰性溶剂中用足够量的酸与这类化合物的中性形式接触的方式获得酸加成盐。药学上可接受的酸加成盐的实例包括无机酸盐，所述无机酸包括例盐酸、氢溴酸、氢碘酸、硫酸、磷酸或硝酸等；有机酸例如甲酸、乙酸、乙酰乙酸、丙酮酸、三氟乙酸、丙酸、丁酸、己酸、庚酸、十一酸、月桂酸、苯甲酸、水杨酸、2-(4-羟基苯甲酰基)-苯甲酸、樟脑酸、肉桂酸、环戊烷丙酸、二葡萄糖酸、3-羟基-2-萘甲酸、烟酸、巴莫酸、果胶酯酸、3-苯基丙酸、苦味酸、特戊酸、2-羟基乙磺酸、衣康酸、胺基磺酸、三氟甲磺酸、十二烷基硫酸、乙磺酸、苯磺酸、对-甲苯磺酸、甲磺酸、2-萘磺酸、萘二磺酸、樟脑磺酸、柠檬酸、酒石酸、硬脂酸、乳酸、草酸、丙二酸、琥珀酸、苹果酸、己二酸、海藻酸、马来酸、富马酸、D-葡萄糖酸、扁桃酸、抗坏血酸、葡庚糖酸、甘油磷酸、天冬胺酸、磺基水杨酸等包括钠、钾、镁、锂、铝、钙、锌、N,N’-二苄基乙二胺、氯代普鲁卡因、胆碱、二乙醇胺、乙二胺、N-甲基葡糖胺和普鲁卡因等形成的盐。
如本文所用，“溶剂合物”是指式I化合物可以溶剂化物(如水合物)的形式存在，其中本发明的化合物包含作为所述化合物晶格的结构要素的极性溶剂，特别是例如水、甲醇或乙醇。极性溶剂特别是水的量可以化学计量比或非化学计量比存在。
如本文所用，“异构体”是指式I化合物含有一个或多个不对称中心和/或双键的情况下，本发明的化合物能够以外消旋物、外消旋混合物、单一对映异构体、非对映异构体混合物、单一非对映异构体、几何异构体等的形式存在。这些化合物可以由符号“R”或“S”表示，这取决于立体碳原子周围的取代基的构型。
如本文所用，“多晶型”是指式I化合物也可以各类晶型的形式存在，通过将化合物或其药学上可接受的盐在溶剂中重结晶，得到它们的不同单一晶型以及多晶型混合物。
本发明提供一种药物组合物，其包含式I所示的多肽化合物、其药用盐或酯、溶剂合物、异构体、多晶型物、代谢物或前药，以及药学上可以接受的载体或赋形剂。
如本文所用，“药物组合物”其中含有治疗有效量的所述式I多肽化合物其药用盐或酯、溶剂合物、异构体、多晶型物、同位素标记的化合物、代谢物或前药，以及一种或多种药学上可接受的载体，制备成片剂、胶囊、颗粒剂、散剂、混悬剂、乳剂、粉剂、溶液、凝胶剂、糖浆剂、丸剂、酊剂、酒剂、煎膏剂、锭剂、合剂、栓剂、注射剂、吸入剂或喷雾剂等形式。该药物组合物优选含有重量比为0.1％-99.5％的本发明的多取代苯并杂环类化合物或其药用盐作为活性成分，更优选含有重量比为0.5％-99.5％的活性成分。
如本文所用，“药学上可接受的载体或赋形剂”包括：稀释剂、填充剂、粘合剂、崩解剂、润滑剂、助流剂、粒化剂、包衣剂、润湿剂、溶剂、共溶剂、助悬剂、乳化剂、增甜剂、调味剂、掩味剂、着色剂、防结块剂、保湿剂、螯合剂、增塑剂、增粘剂、抗氧化剂、防腐剂、稳定剂、表面活性剂和缓冲剂，本领域技术人员将理解，某些药学上可接受的赋形剂可以以多于一种功能和以替代性功能来使用，取决于所述赋形剂在制剂中存在多少和在制剂中存在何种其它成分。例如：当用于口服时，可以制成口服制剂，如片剂、胶囊剂、颗粒剂和丸剂等，包含填充剂(例如糖类衍生物如乳糖、蔗糖、葡萄糖、甘露糖醇和山梨糖醇；淀粉衍生物如玉米淀粉、土豆淀粉、糊精和羧甲基淀粉；纤维素衍生物如结晶纤维素、羟丙基纤维素、羧甲基纤维素、羧甲基纤维素钙、羧甲基纤维素钠；阿拉伯胶；右旋糖酐；硅酸盐衍生物如偏硅酸镁铝；磷酸盐衍生物如磷酸钙；碳酸盐衍生物如碳酸钙；硫酸盐衍生物如硫酸钙等)、粘合剂(例如明胶、聚乙烯吡咯烃酮和聚乙二醇)、崩解剂(例如纤维素衍生物如羧甲基纤维素钠、聚乙烯吡咯烃酮)、润滑剂(例如滑石、硬脂酸钙、硬脂酸镁、鲸蜡、硼酸、苯甲酸钠、亮氨酸)、稳定剂(对羟基苯甲酸甲酯、对羟基苯甲酸丙酯等)、矫味剂(例如常用的甜味剂、酸味剂和香料等)。当用于肠胃外时，可以制成注射剂，包括注射用无菌粉末与注射用溶剂，所用载体或赋形剂包含无菌水、林格氏液和等渗氯化钠溶液，也可根据药物的性质加入适宜的附加剂例如抗氧化剂、缓冲剂和抑菌剂。当用于直肠给药时，所述药物可以制成栓剂等。用于经肺给药时，所述药物可以制成吸入剂或喷雾剂等。有许多本领域技术人员可用的资源，这些资源描述了药学上可接受的赋形剂且其可用于选择合适的药学上可接受的赋形剂，例如《雷明登药学大全》、《中国药学年鉴》、《药剂学》等书籍。
本发明可以通过本领域已知的任何合适的方法来施用，例如，口服、静脉内、腹膜内、肌肉内、局部、透皮、经眼、经鼻、吸入、皮下、肌内、口含、舌下、直肠给予等方式，可以以μg～mg/kg级受试者体重的任何量施用如上所述的化合物，例如以0.1μg～1000mg/kg体重/天的量施用如上所述的化合物。在本发明的一些实施方案中，可以以每日4次、每日3次、每日2次、每日1次、每两日1次、每周1次或其他间隔的方式施用如上所述的化合物，任选地酌情每周或每月重复如上所述的给药方案。在本发明中，所述化合物的给药剂量可根据患者或受试者的病情轻重、年龄、体重、性别、给药方式及疗程等因素进行调整。
本发明化合物可以单独使用，也可以和另一种或多种其它活性成分联合用于治疗、预防、抑制或者改善疾病或者病状，其中药物的联合使用比任何一种药物的单独使用 更为安全或者更为有效。这种其它药物可以以对此通常使用的途径和量与本发明的化合物同时或者依次给药。当本发明的化合物与一种或者多种其它药物同时使用时，在单位剂型中含有其它药物和本发明的化合物的药物组合物是优选的，特别是与药学可接受的载体联合。然而，联合治疗还可以包括在不同重叠日程中给予本发明的化合物和一种或者多种其它药物的治疗。还可以预期，当与一种或者多种其它活性成分联合使用时，本发明化合物和其它活性成分可以以比各自单独使用时更低的剂量使用。因此，除了本发明的化合物外，本发明药物组合物还包括含有一种或者多种其它活性成分的那些组合物。
相较于现有技术，本发明有益效果如下：
本发明通过对P物质进行结构衍生化研究发现一类多肽化合物，该类多肽化合物对AD体外细胞模型有神经保护作用，能够提高细胞活力。所使用的AD体外细胞模型为APPswe细胞，是一种稳定转染人鼠嵌合型APP基因的人神经母细胞瘤细胞，由于细胞过表达APP基因，导致APP的生成增多，促使β-分泌酶分解APP生成过量的Aβ。在使用铜离子处理细胞时，铜离子与APP、Aβ形成螯合物，加重Aβ的产生与沉积，进而模拟Aβ诱导的氧化应激、炎症反应和凋亡退变等AD病理损伤。本发明采用该体外模型用来筛选和评价药物的抗AD作用。
本发明的多肽化合物能够显著提高Aβ介导认知损伤小鼠的空间学习和记忆能力。淀粉样蛋白-β(Aβ)是家族性和散发性AD的主要病理介质，在AD患者的大脑中，观察到大量的Aβ寡聚体和斑块积累，引起神经元细胞变性、凋亡。临床和实验验证表明这种Aβ异常沉积会阻断长时程突触增强、损害突触功能并诱发认知缺陷。该模型是基于Aβ上述的神经毒性作用，采用立体定位注射的方法将寡聚化的Aβ1-42注射入小鼠脑部，可引起神经毒性反应，并诱导产生AD样记忆受损和行为学障碍，能较好地模拟临床上患者脑内的病理情况，因而是常用的AD动物模型。
本发明涉及的部分多肽化合物的毒性较低，在肾细胞(Vero)毒性检测和溶血性(兔子全血)检测中表现出较低的毒性。
附图说明
为了清楚地说明本发明具体实施方式或技术方案，下面将对附表图做简单介绍。
图1为C10改善Aβ 1-42模型小鼠的学习和记忆能力。
其中，(A)MWM定位航行试验中的平台逃避潜伏期；(B)定位航行试验中的游泳速度；(C)在空间探索试验中，C10干预的Aβ 1-42模型小鼠的穿越平台原始位置 的次数增加；(D)在空间探索试验中，C10干预的Aβ 1-42模型小鼠在目标象限内的持续时间延长。结果以均数±标准误表示，n＝8, ##P<0.01vs.Sham,*P<0.05,**P<0.01,***P<0.001vs.Aβ 1-42。
图2为C7改善Aβ 1-42模型小鼠的学习和记忆能力。
其中，(A)MWM定位航行试验中的平台逃避潜伏期；(B)定位航行试验中的游泳速度；(C)在空间探索试验中，C7干预的Aβ 1-42模型小鼠的穿越平台原始位置的次数增加；(D)在空间探索试验中，C7干预的Aβ 1-42模型小鼠在目标象限内的持续时间延长。结果以均数±标准误表示，n＝8, ##P<0.01, ###P<0.001vs.Sham,**P<0.01,**P<0.001vs.Aβ 1-42。
具体实施方式
下面结合具体实施例来进一步描述本发明，本发明的优点和特点将会随具体实施例的描述而更为清楚。但实施例仅是范例性的，并不对本发明的范围构成任何限制。本领域技术人员应该理解的是，在不偏离本发明的精神和范围下可以对本发明技术方案的细节和形式进行修改或替换，但这些修改和替换均落入本发明的保护范围内。
制备所述的具有如式Ⅰ所示结构的多肽化合物、其药用盐、溶剂合物、异构体或多晶型物的方法，该方法采用Fmoc固相合成方法，按照肽链从C端到N端的氨基酸顺序合成多肽，经纯化，冷冻干燥后得到多肽纯品。
Figure PCTCN2022142732-appb-000006
式Ⅰ中，数字1-20表示氨基酸在式Ⅰ中的具体位置，式I中的最少氨基酸个数为5个、最大氨基酸个数为20个；所述氨基酸的构型为L型或D型；数字1-15以及20所在位置的氨基酸存在或不存在；
式Ⅰ中：
R 3选自以下的组：-(CH 2) 2-COOH、-CH 2-COOH；
R 4选自以下的组：-CH 2-COOH；
R 5选自以下的组：-CH 2-OH；
R 6选自以下的组：-(CH 2) 2-COOH、-CH 2-CH(CH 3) 2、-CH-(CH 3) 2；
R 7选自以下的组：-(CH 2) 2-COOH、-CH-(CH 3) 2、-CH 2-CONH 2、-CH 3、-CH 2-CH (CH 3) 2、-C 3H 6；
R 8选自以下的组：-CH 2-CONH 2、-CH 2-CH(CH 3) 2、-C 3H 6、-CH 3、-(CH 2) 2-COOH、-CH 2-COOH、-CH-(CH 3) 2；
[援引加入（细则20.6）08.12.2023]
R9选自以下的组：-CH3、-(CH2)2-COOH、-CH2-COOH、-(CH2)3-NHC(NH)NH2、-CH2-C6H4-OH、-CH(CH3)-OH；
[援引加入（细则20.6）08.12.2023]
R10选自以下的组：-(CH2)2-COOH、-(CH2)3-NHC(NH)NH2、-CH2-C6H4-OH、-CH2-CH(CH3)2、-CH(CH3)-CH2-CH3、-(CH2)2-CONH2、-C8NH6、-CH(CH3)-OH、-CH2-COOH；
[援引加入（细则20.6）08.12.2023]
R11选自以下的组：-CH2-C6H4-OH、-CH2-CH(CH3)2、-CH(CH3)-CH2-CH3、-(CH2)2-CONH2、-CH2-CONH2、-(CH2)2-CONH2、-CH2-C6H5、-C3H6;
[援引加入（细则20.6）08.12.2023]
R12选自以下的组：-CH2-CH(CH3)2、-CH2-CONH2、-(CH2)2-CONH2、-(CH2)3-NHC(NH)NH2、-CH(CH3)-OH、-CH2-COOH、-(CH2)2-NH2、-(CH2)4-NH2；
[援引加入（细则20.6）08.12.2023]
R13选自以下的组：-(CH2)2-COOH、-(CH2)3-NHC(NH)NH2、-CH(CH3)-OH、-(CH2)2-CONH2、-CH2-COOH、-(CH2)2-NH2、-C3H6；
[援引加入（细则20.6）08.12.2023]
R14选自以下的组：-(CH2)2-COOH、-(CH2)2-CONH2；
[援引加入（细则20.6）08.12.2023]
R15选自以下的组：-(CH2)2-CONH2、-(CH2)2-COOH；
R 20选自以下的组：-NH 2、-OH；
[援引加入（细则20.6）08.12.2023]
R21选自以下的组：-(CH2)2-S-CH3。
其中，所述的Fmoc固相合成方法所用树脂选自以下任意一种：Amide-MBHA Resin 0.52mmol/g、Amide-MBHA Resin 0.646mmol/g、2-Chlorotrityl Chloride Resin 0.52mmol/g。
其中，所述的Fmoc固相合成方法选用Fmoc保护氨基酸氨基部分；侧链保护基选自以下基团中的任意一种：苄基(Bzl)、叔丁基(tBu)、叔丁氧羰基(Boc)、2,2,4,6,7-五甲基二氢苯并呋喃-5-磺酰基(Pbf)、三苯基(Trt)。
其中，经脱除Fmoc保护基、缩合形成肽链；脱除Fmoc试剂为20％哌啶/DMF；缩合剂为苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸酯(HBTU)、N,N'-二异丙基碳二亚胺(DIEA)，摩尔比为氨基酸：HBTU：DIEA为1:0.9:2。
其中，多肽通过酸脱除树脂与氨基酸保护基，切肽液比例为95％v/v三氟乙酸(TFA)、2.5％v/v三异丙基硅烷(TIPS)、2.5％v/v水，15-20ml/g树脂。
其中，当20位氨基酸不存在时，按照常规方法制备。
其中，当R 20为-OH时，20位氨基酸为L型Met，采用路线1方法：
路线1：
Figure PCTCN2022142732-appb-000007
其中，各步骤的反应条件如下：
a：DMF，20-50℃，溶胀10-50min；DMF/DCM交叉洗涤3次以上；茚三酮显色反应检测；
b：Fmoc-Met-OH，DIEA，反应3h或过夜；甲醇0.5ml，30min；DMF/DCM交叉洗涤3次以上；茚三酮显色反应检测；
c：配置脱保护试剂，脱除Fmoc试剂为20％哌啶/DMF，反应10-30min，重复2次；DMF/DCM交叉洗涤3次以上；茚三酮显色反应检测；
d：按照摩尔比为氨基酸：HBTU：DIEA为1:0.9:2配置氨基酸溶剂，反应1-3h，DMF/DCM交叉洗涤3次以上；茚三酮显色反应检测；
e：重复步骤c、d，按照编号为(16)、(19)、(22)、(25)、(28)的多肽的氨基酸的顺序合成多肽；
f：配置切肽液，切肽液比例为95％v/v三氟乙酸(TFA)、2.5％v/v三异丙基硅烷(TIPS)、2.5％v/v水，15-20ml/g树脂，反应1-3h；压出液体；乙醚沉淀洗涤干燥后得多肽粗品；
路线2：
当R 20为-NH 2时，采用路线2方法：
Figure PCTCN2022142732-appb-000008
Figure PCTCN2022142732-appb-000009
其中，各步骤的反应条件如下：
a':DMF，20-50℃，溶胀10-50min；DMF/DCM交叉洗涤3次以上；茚三酮显色反应检测；
b':配置脱保护试剂，脱除Fmoc试剂为20％哌啶/DMF，反应10-30min，重复2次；DMF/DCM交叉洗涤3次以上；茚三酮显色反应检测；
c':按照摩尔比为氨基酸：HBTU：DIEA为1:0.9:2配置氨基酸溶剂，反应0.5-2h，DMF/DCM交叉洗涤3次以上；茚三酮显色反应检测；
d':重复步骤c'、d'，按照除编号为(16)、(19)、(22)、(25)、(28)以及20位氨基酸不存在之外的其余多肽的氨基酸顺序合成多肽；
f':配置切肽液，切肽液比例为95％v/v三氟乙酸(TFA)、2.5％v/v三异丙基硅烷(TIPS)、2.5％v/v水，15-20ml/g树脂，反应1-3h；压出液体；乙醚沉淀洗涤干燥后得多肽粗品。
实施例1 A1:(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
选用Amide-MBHA Resin 0.52mmol/g树脂，按照合成路线2，以Fmoc-L-Met-OH、Fmoc-L-Leu-OH、Fmoc-L-Gly-OH、Fmoc-L-Phe-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Arg(pbf)-OH、Fmoc-L-Leu-OH、Fmoc-L-Tyr(tBu)-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Ala-OH为合成原料及合成顺序合成序列A1。切肽液切割树脂，得多肽A1粗品，溶于添加氨水的pH＝10～11的水溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽A1纯品，收率20.94％；HPLC纯度99％；分子式 C70H103N16O19S；HRMS：[M+H] +1503.72925，calcd 1503.73006。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-8min 25％B相、8-38min 25％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例2 A2:(L-Glu)-(L-Asn)-(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
选用Amide-MBHA Resin 0.52mmol/g树脂，按照合成路线2，以Fmoc-L-Met-OH、Fmoc-L-Leu-OH、Fmoc-L-Gly-OH、Fmoc-L-Phe-OH、Fmoc-L-Arg(pbf)-OH、Fmoc-L-Leu-OH、Fmoc-L-Tyr(tBu)-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Ala-OH、Fmoc-L-Asn(Trt)-OH、Fmoc-L-Glu(OtBu)-OH为合成原料及合成顺序合成序列A2。切肽液切割树脂，得多肽A2粗品，溶于添加氨水的pH＝10～11的水溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽A2纯品，收率10.51％；HPLC纯度96％；分子式C79H116N19O24S；HRMS：[M+H] +1746.81311，calcd 1746.81558。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-6min期20％B相、6-31min20％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例3(L-Leu)-(L-Ala)-(L-Arg)-(L-Leu)-(L-Leu)-(L-Thr)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
选用Amide-MBHA Resin 0.52mmol/g树脂，按照合成路线2，以Fmoc-L-Met-OH、Fmoc-L-Leu-OH、Fmoc-L-Gly-OH、Fmoc-L-Phe-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Thr(tBu)-OH、Fmoc-L-Leu-OH、Fmoc-L-Arg(pbf)-OH、Fmoc-L-Ala-OH、Fmoc-L-Leu-OH为合成原料及合成顺序合成序列A3。切肽液切割树脂，得多肽A3粗品，溶于添加氨水的pH＝10～11的水溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽A3纯品，收率25.08％；HPLC纯度98％；分子式C72H116N17O18S；HRMS：[M+H] +1538.84229，calcd 1538.84995。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-8min 25％B相、8-38min 25％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例4 A4:(L-Val)-(L-Pro)-(L-Glu)-(L-Tyr)-(L-Ile)-(L-Asn)-(L-Gln)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
选用Amide-MBHA Resin 0.52mmol/g树脂，按照合成路线2，以Fmoc-L-Met-OH、Fmoc-L-Leu-OH、Fmoc-L-Gly-OH、Fmoc-L-Phe-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Gln(Trt)-OH、Fmoc-L-Asn(Trt)-OH、Fmoc-L-Ile-OH、Fmoc-L-Tyr(tBu)-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Pro-OH、Fmoc-L-Val-OH为合成原料及合成顺序合成序列A4。切肽液切割树脂，得多肽A4粗品，溶于添加氨水的pH＝10～11的水溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽A4纯品，收率22.69％；HPLC纯度≥95％；分子式C80H116N17O23S；HRMS：[M+H] +1714.81494，calcd1714.81452。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-8min 25％B相、8-38min 25％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例5 A5:(L-Asp)-(L-Tyr)-(L-Gln)-(L-Gln)-(L-Asp)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
选用Amide-MBHA Resin 0.52mmol/g树脂，按照合成路线2，以Fmoc-L-Met-OH、Fmoc-L-Leu-OH、Fmoc-L-Gly-OH、Fmoc-L-Phe-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Asp(Obzl)-OH、Fmoc-L-Gln(Trt)-OH、Fmoc-L-Tyr(tBu)-OH、Fmoc-L-Asp(Obzl)-OH为合成原料及合成顺序合成序列A5。切肽液切割树脂，得多肽A5粗品，溶于添加氨水的pH＝10～11的水溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽A5纯品，收率14.57％；HPLC纯度≥95％；分子式C68H93N14O24S；HRMS：[M+H] +1521.63904，calcd1521.63924。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-6min期20％B相、6-31min20％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例6 B1:(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
选用Amide-MBHA Resin 0.52mmol/g树脂，按照合成路线2，以Fmoc-L-Met-OH、Fmoc-L-Leu-OH、Fmoc-L-Gly-OH、Fmoc-L-Phe-OH、Fmoc-L-Gln(Trt)-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Arg(pbf)-OH、Fmoc-L-Leu-OH、Fmoc-L-Tyr(tBu)-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Ala-OH为合成原料及合成顺序合成序列B1。切肽液切割树脂，得多肽B1粗品，溶于添加氨水的pH＝10～11的水溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽B1纯品，收率9.125％；HPLC纯度95.7％；分子式C75H111N18O21S；HRMS：[M+H] +1631.78504，calcd 1631.78864。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-5min 20％B相、5-35min 20％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例7 B2:(L-Glu)-(L-Asn)-(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
选用Amide-MBHA Resin 0.52mmol/g树脂，按照合成路线2，以Fmoc-L-Met-OH、Fmoc-L-Leu-OH、Fmoc-L-Gly-OH、Fmoc-L-Phe-OH、Fmoc-L-Gln(Trt)-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Arg(pbf)-OH、Fmoc-L-Leu-OH、Fmoc-L-Tyr(tBu)-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Ala-OH、Fmoc-L-Asn(Trt)-OH、Fmoc-L-Glu(OtBu)-OH为合成原料及合成顺序合成序列B2。切肽液切割树脂，得多肽B2粗品，溶于添加氨水的pH＝10～11的水溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽B2纯品，收率11.29％；HPLC纯度98％；分子式C84H124N18O26S；HRMS：[M+H] +1874.87142，calcd 1874.87416。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-8min期23％B相、8-38min23％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例8 B3:(L-Leu)-(L-Ala)-(L-Arg)-(L-Leu)-(L-Leu)-(L-Thr)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
选用Amide-MBHA Resin 0.52mmol/g树脂，按照合成路线2，以Fmoc-L-Met-OH、Fmoc-L-Leu-OH、Fmoc-L-Gly-OH、Fmoc-L-Phe-OH、Fmoc-L-Gln(Trt)-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Thr(tBu)-OH、Fmoc-L-Leu-OH、Fmoc-L-Arg(pbf)-OH、Fmoc-L-Ala-OH、Fmoc-L-Leu-OH为合成原料及合成顺序合成序列B3。切肽液切割树脂，得多肽B3粗品，溶于添加氨水的pH＝10～11的水溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽B3纯品，收率21.72％；HPLC纯度97％；分子式C77H124N19O25S；HRMS：[M+H] +1666.89551，calcd 1666.89852。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-6min 20％B相、6-31min 20％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例9 B4:(L-Val)-(L-Pro)-(L-Glu)-(L-Tyr)-(L-Ile)-(L-Asn)-(L-Gln)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
选用Amide-MBHA Resin 0.52mmol/g树脂，按照合成路线2，以Fmoc-L-Met-OH、Fmoc-L-Leu-OH、Fmoc-L-Gly-OH、Fmoc-L-Phe-OH、Fmoc-L-Gln(Trt)-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Gln(Trt)-OH、Fmoc-L-Asn(Trt)-OH、Fmoc-L-Ile-OH、Fmoc-L-Tyr(tBu)-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Pro-OH、Fmoc-L-Val-OH为合成原料及合成顺序合成序列B4。切肽液切割树脂，得多肽B4粗品，溶于添加氨水的pH＝10～11的水溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽B4纯品，收率27.42％；HPLC纯度99％；分子式C85H124N19O25S；HRMS：[M+H] +1842.87878，calcd 1842.87310。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-6min 20％B相、6-31min 20％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例10 B5:(L-Asp)-(L-Tyr)-(L-Gln)-(L-Gln)-(L-Asp)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
选用Amide-MBHA Resin 0.52mmol/g树脂，按照合成路线2，以Fmoc-L-Met-OH、Fmoc-L-Leu-OH、Fmoc-L-Gly-OH、Fmoc-L-Phe-OH、Fmoc-L-Gln(Trt)-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Asp(Obzl)-OH、Fmoc-L-Gln(Trt)-OH、Fmoc-L-Tyr(tBu)-OH、Fmoc-L-Asp(Obzl)-OH为合成原料及合成顺序合成序列B5。切肽液切割树脂，得多肽B5粗品，溶于添加氨水的pH＝10～11的水溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽B5纯品，收率44.215％；HPLC纯度99％；分子式C73H101N16O26S；HRMS：[M+H] +1648.69727，calcd16488.67099。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-5min期20％B相、5-35min20％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例11 SP-6-11:(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
选用Amide-MBHA Resin 0.52mmol/g树脂，按照合成路线2，以Fmoc-L-Met-OH、Fmoc-L-Leu-OH、Fmoc-L-Gly-OH、Fmoc-L-Phe-OH、Fmoc-L-Gln(Trt)-OH为合成原料及合成顺序合成序列SP-6-11。切肽液切割树脂，得多肽SP-6-11粗品，溶于添加氨水的pH＝10～11的水溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽SP-6-11纯品，收率1.48％；HPLC纯度95.716％；分子式C36H53N8O7S；HRMS：[M+H] +741.37439，calcd 741.37524。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-8min期20％B相、8-38min 20％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例12 SP-7-11:(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
选用Amide-MBHA Resin 0.52mmol/g树脂，按照合成路线2，以Fmoc-L-Met-OH、Fmoc-L-Leu-OH、Fmoc-L-Gly-OH、Fmoc-L-Phe-OH为合成原料及合成顺序合成序列SP-7-11。切肽液切割树脂，得多肽SP-7-11粗品，溶于甲醇溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽SP-7-11纯品，收率11.225％；HPLC纯度99％；分子式C31H45N6O5S；HRMS：[M+H] +613.31622，calcd 613.31667。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-6min期20％B相、6-31min20％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例13 EE-6-11:(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
选用Amide-MBHA Resin 0.52mmol/g树脂，按照合成路线2，以Fmoc-L-Met-OH、Fmoc-L-Leu-OH、Fmoc-L-Gly-OH、Fmoc-L-Phe-OH、Fmoc-L-Gln(Trt)-OH、Fmoc-L-Glu(OtBu)-OH为合成原料及合成顺序合成序列EE-6-11。切肽液切割树脂，得多肽EE-6-11粗品，溶于添加氨水的pH＝10～11的水溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽EE-6-11纯品，收率47.64％；HPLC纯度99％；分子式C46H67N10O13S；HRMS：[M+H] +999.45825，calcd 999.46043。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-6min 20％B相、6-31min 20％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例14 EE-7-11:(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
选用Amide-MBHA Resin 0.52mmol/g树脂，按照合成路线2，以Fmoc-L-Met-OH、Fmoc-L-Leu-OH、Fmoc-L-Gly-OH、Fmoc-L-Phe-OH、Fmoc-L-Glu(OtBu)-OH为合成原料及合成顺序合成序列EE-7-11。切肽液切割树脂，得多肽EE-7-11粗品，溶于添加氨水的pH＝10～11的水溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽EE-7-11纯品，收率53.96％；HPLC纯度99％；分子式C41H59N8O11S；HRMS：[M+H] +871.40014，calcd871.40185。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-6min 20％B相、6-31min 20％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例15 A2-M1:(L-Glu)-(L-Asn)-(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-NH 2
选用Amide-MBHA Resin 0.52mmol/g树脂，按照合成路线2，以Fmoc-L-Leu-OH、Fmoc-L-Gly-OH、Fmoc-L-Phe-OH、Fmoc-L-Arg(pbf)-OH、Fmoc-L-Leu-OH、Fmoc-L-Tyr(tBu)-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Ala-OH、Fmoc-L-Asn(Trt)-OH、Fmoc-L-Glu(OtBu)-OH为合成原料及合成顺序合成序列A2-M1。切肽液切割树脂，得多肽A2-M1粗品，溶于添加氨水的pH＝10～11的水溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽A2-M1纯品，收率36.16％；HPLC纯度99％；分子式C74H107N18O23；HRMS：[M+H] +1615.77209，calcd 1615.77510。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-6min期20％B相、6-31min20％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例16 A2-M2:(L-Glu)-(L-Asn)-(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)
选用2-Chlorotrityl Chloride Resin 0.52mmol/g树脂，按照合成路线1，以Fmoc-L-Met-OH、Fmoc-L-Leu-OH、Fmoc-L-Gly-OH、Fmoc-L-Phe-OH、Fmoc-L-Arg(pbf) -OH、Fmoc-L-Leu-OH、Fmoc-L-Tyr(tBu)-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Ala-OH、Fmoc-L-Asn(Trt)-OH、Fmoc-L-Glu(OtBu)-OH为合成原料及合成顺序合成序列A2-M2。切肽液切割树脂，得多肽A2-M2粗品，溶于添加氨水的pH＝10～11的水溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽A2-M2纯品，收率8.89％；HPLC纯度≥95％；分子式C79H115N18O25S；HRMS：[M+H] +1747.79260，calcd 1747.79960。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-8min期20％B相、8-33min20％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例17 A2-M3:(L-Glu)-(L-Asn)-(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(D-Met)-NH 2
选用Amide-MBHA Resin 0.52mmol/g树脂，按照合成路线2，以Fmoc-D-Met-OH、Fmoc-L-Leu-OH、Fmoc-L-Gly-OH、Fmoc-L-Phe-OH、Fmoc-L-Arg(pbf)-OH、Fmoc-L-Leu-OH、Fmoc-L-Tyr(tBu)-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Ala-OH、Fmoc-L-Asn(Trt)-OH、Fmoc-L-Glu(OtBu)-OH为合成原料及合成顺序合成序列A2-M3。切肽液切割树脂，得多肽A2-M3粗品，溶于添加氨水的pH＝10～11的水溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽A2-M3纯品，收率19.6％；HPLC纯度≥95％；分子式C79H115N19O24S；HRMS：[M+H] +1746.81787，calcd1746.81558。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-8min期20％B相、8-33min20％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例18 A3-M1:(L-Leu)-(L-Ala)-(L-Arg)-(L-Leu)-(L-Leu)-(L-Thr)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-NH 2
选用Amide-MBHA Resin 0.52mmol/g树脂，按照合成路线2，以Fmoc-L-Leu-OH、 Fmoc-L-Gly-OH、Fmoc-L-Phe-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Thr(tBu)-OH、Fmoc-L-Leu-OH、Fmoc-L-Arg(pbf)-OH、Fmoc-L-Ala-OH、Fmoc-L-Leu-OH为合成原料及合成顺序合成序列A3-M1。切肽液切割树脂，得多肽A3-M1粗品，溶于添加氨水的pH＝10～11的水溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽A3-M1纯品，收率43.72％；HPLC纯度≥95％；分子式C67H107N16O17；HRMS：[M+H] +1407.80029，calcd 1407.79946。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-7min 20％B相、7-32min 20％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例19 A3-M2:(L-Leu)-(L-Ala)-(L-Arg)-(L-Leu)-(L-Leu)-(L-Thr)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)
选用2-Chlorotrityl Chloride Resin 0.52mmol/g树脂，按照合成路线1，以Fmoc-L-Met-OH、Fmoc-L-Leu-OH、Fmoc-L-Gly-OH、Fmoc-L-Phe-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Thr(tBu)-OH、Fmoc-L-Leu-OH、Fmoc-L-Arg(pbf)-OH、Fmoc-L-Ala-OH、Fmoc-L-Leu-OH为合成原料及合成顺序合成序列A3-M2。切肽液切割树脂，得多肽A3-M2粗品，溶于添加氨水的pH＝10～11的水溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽A3-M2纯品，收率22.65％；HPLC纯度≥95％；分子式C72H115N16O19S；HRMS：[M+H] +1539.82805，calcd 1539.82896。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-8min 20％B相、8-38min 20％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例20 A3-M3:(L-Leu)-(L-Ala)-(L-Arg)-(L-Leu)-(L-Leu)-(L-Thr)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(D-Met)-NH 2
选用Amide-MBHA Resin 0.52mmol/g树脂，按照合成路线2，以Fmoc-D-Met-OH、Fmoc-L-Leu-OH、Fmoc-L-Gly-OH、Fmoc-L-Phe-OH、Fmoc-L-Glu(OtBu)-OH、 Fmoc-L-Thr(tBu)-OH、Fmoc-L-Leu-OH、Fmoc-L-Arg(pbf)-OH、Fmoc-L-Ala-OH、Fmoc-L-Leu-OH为合成原料及合成顺序合成序列A3-M3。切肽液切割树脂，得多肽A3-M3粗品，溶于添加氨水的pH＝10～11的水溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽A3-M3纯品，收率23.82％；HPLC纯度≥95％；分子式C72H116N17O18S；HRMS：[M+H] +1538.84573，calcd 1538.83995。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-6min 20％B相、6-31min 20％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例21 B1-M1:(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-NH 2
选用Amide-MBHA Resin 0.52mmol/g树脂，按照合成路线2，以Fmoc-L-Leu-OH、Fmoc-L-Gly-OH、Fmoc-L-Phe-OH、Fmoc-L-Gln(Trt)-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Arg(pbf)-OH、Fmoc-L-Leu-OH、Fmoc-L-Tyr(tBu)-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Ala-OH为合成原料及合成顺序合成序列B1-M1。切肽液切割树脂，得多肽B1-M1粗品，溶于添加氨水的pH＝10～11的水溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽B1-M1纯品，收率22.99％；HPLC纯度≥95％；分子式C70H102N17O20；HRMS：[M+H] +1500.75049，calcd 1500.74851。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-5min 20％B相、5-25min 20％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例22 B1-M2:(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)
选用2-Chlorotrityl Chloride Resin 0.52mmol/g树脂，按照合成路线1，以Fmoc-L-Met-OH、Fmoc-L-Leu-OH、Fmoc-L-Gly-OH、Fmoc-L-Phe-OH、Fmoc-L-Gln(Trt)-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Arg(pbf)-OH、Fmoc-L-Leu-OH、Fmoc- L-Tyr(tBu)-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Ala-OH为合成原料及合成顺序合成序列B1-M2。切肽液切割树脂，得多肽B1-M2粗品，溶于添加氨水的pH＝10～11的水溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽B1-M2纯品，收率12.58％；HPLC纯度≥95％；分子式C75H110N19O22S；HRMS：[M+H] +1632.77576，calcd 1632.78864。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-8min 20％B相、8-33min 20％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例23 B1-M3:(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(D-Met)-NH 2
选用Amide-MBHA Resin 0.52mmol/g树脂，按照合成路线2，以Fmoc-D-Met-OH、Fmoc-L-Leu-OH、Fmoc-L-Gly-OH、Fmoc-L-Phe-OH、Fmoc-L-Gln(Trt)-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Arg(pbf)-OH、Fmoc-L-Leu-OH、Fmoc-L-Tyr(tBu)-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Ala-OH为合成原料及合成顺序合成序列B1-M3。切肽液切割树脂，得多肽B1-M3粗品，溶于添加氨水的pH＝10～11的水溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽B1-M3纯品，收率18.5％；HPLC纯度≥95％；分子式C75H111N18O21S；HRMS：[M+H] +1631。79163，calcd 1631.78864。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-8min 20％B相、8-33min 20％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例24 B3-M1:(L-Leu)-(L-Ala)-(L-Arg)-(L-Leu)-(L-Leu)-(L-Thr)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-NH 2
选用Amide-MBHA Resin 0.52mmol/g树脂，按照合成路线2，以Fmoc-L-Leu-OH、Fmoc-L-Gly-OH、Fmoc-L-Phe-OH、Fmoc-L-Gln(Trt)-OH、Fmoc-L-Glu(OtBu) -OH、Fmoc-L-Thr(tBu)-OH、Fmoc-L-Leu-OH、Fmoc-L-Arg(pbf)-OH、Fmoc-L-Ala-OH、Fmoc-L-Leu-OH为合成原料及合成顺序合成序列B3-M1。切肽液切割树脂，得多肽B3-M1粗品，溶于添加氨水的pH＝10～11的水溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽B3-M1纯品，收率9.42％；HPLC纯度≥95％；分子式C72H115N18O19；HRMS：[M+H] +1535.86096，calcd 1535.85804。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-6min 20％B相、6-29min 20％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例25 B3-M2:(L-Leu)-(L-Ala)-(L-Arg)-(L-Leu)-(L-Leu)-(L-Thr)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)
选用2-Chlorotrityl Chloride Resin 0.52mmol/g树脂，按照合成路线1，以Fmoc-L-Met-OH、Fmoc-L-Leu-OH、Fmoc-L-Gly-OH、Fmoc-L-Phe-OH、Fmoc-L-Gln(Trt)-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Thr(tBu)-OH、Fmoc-L-Leu-OH、Fmoc-L-Arg(pbf)-OH、Fmoc-L-Ala-OH、Fmoc-L-Leu-OH为合成原料及合成顺序合成序列B3-M2。切肽液切割树脂，得多肽B3-M2粗品，溶于添加氨水的pH＝10～11的水溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽B3-M2纯品，收率12.58％；HPLC纯度≥95％；分子式C75H110N19O22S；HRMS：[M+H] +1667.88489，calcd1667.88254。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-8min 20％B相、8-33min 20％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例26 B3-M3:(L-Leu)-(L-Ala)-(L-Arg)-(L-Leu)-(L-Leu)-(L-Thr)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(D-Met)-NH 2
选用Amide-MBHA Resin 0.52mmol/g树脂，按照合成路线2，以Fmoc-D-Met-OH、Fmoc-L-Leu-OH、Fmoc-L-Gly-OH、Fmoc-L-Phe-OH、Fmoc-L-Gln(Trt)-OH、Fmoc- L-Glu(OtBu)-OH、Fmoc-L-Thr(tBu)-OH、Fmoc-L-Leu-OH、Fmoc-L-Arg(pbf)-OH、Fmoc-L-Ala-OH、Fmoc-L-Leu-OH为合成原料及合成顺序合成序列B3-M3。切肽液切割树脂，得多肽B3-M3粗品，溶于添加氨水的pH＝10～11的水溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽B3-M3纯品，收率7.47％；HPLC纯度≥95％；分子式C77H124N19O20S；HRMS：[M+H] +1666.90051，calcd 1666.89852。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-8min 20％B相、8-33min 20％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例27 B5-M1:(L-Asp)-(L-Tyr)-(L-Gln)-(L-Gln)-(L-Asp)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-NH 2
选用Amide-MBHA Resin 0.52mmol/g树脂，按照合成路线2，以Fmoc-L-Leu-OH、Fmoc-L-Gly-OH、Fmoc-L-Phe-OH、Fmoc-L-Gln(Trt)-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Asp(Obzl)-OH、Fmoc-L-Gln(Trt)-OH、Fmoc-L-Tyr(tBu)-OH、Fmoc-L-Asp(Obzl)-OH为合成原料及合成顺序合成序列B5-M1。切肽液切割树脂，得多肽B5-M1粗品，溶于添加氨水的pH＝10～11的水溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽B5-M1纯品，收率36.35％；HPLC纯度≥95％；分子式C68H92N15O25；HRMS：[M+H] +1518.65979，calcd 1518.63833。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-8min期20％B相、8-33min20％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例28 B5-M2:(L-Asp)-(L-Tyr)-(L-Gln)-(L-Gln)-(L-Asp)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)
选用2-Chlorotrityl Chloride Resin 0.52mmol/g树脂，按照合成路线1，以Fmoc-L-Met-OH、Fmoc-L-Leu-OH、Fmoc-L-Gly-OH、Fmoc-L-Phe-OH、Fmoc-L-Gln(Trt)-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Asp(Obzl)-OH、Fmoc-L-Gln(Trt)-OH、 Fmoc-L-Tyr(tBu)-OH、Fmoc-L-Asp(Obzl)-OH为合成原料及合成顺序合成序列B5-M2。切肽液切割树脂，得多肽B5-M2粗品，溶于添加氨水的pH＝10～11的水溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽B5-M2纯品，收率31.84％；HPLC纯度≥95％；分子式C73H100N15O27S；HRMS：[M+H] +1650.68237，calcd 1650.66283。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-8min期20％B相、8-33min20％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例29 B5-M3:(L-Asp)-(L-Tyr)-(L-Gln)-(L-Gln)-(L-Asp)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(D-Met)-NH 2
选用Amide-MBHA Resin 0.52mmol/g树脂，按照合成路线2，以Fmoc-D-Met-OH、Fmoc-L-Leu-OH、Fmoc-L-Gly-OH、Fmoc-L-Phe-OH、Fmoc-L-Gln(Trt)-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Asp(Obzl)-OH、Fmoc-L-Gln(Trt)-OH、Fmoc-L-Tyr(tBu)-OH、Fmoc-L-Asp(Obzl)-OH为合成原料及合成顺序合成序列B5-M3。切肽液切割树脂，得多肽B5-M3粗品，溶于添加氨水的pH＝10～11的水溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽B5-M3纯品，收率21.4％；HPLC纯度≥95％；分子式C73H101N16O26S；HRMS：[M+H] +1649.69958，calcd 1649.67881。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-8min期20％B相、8-33min20％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例30 C0:(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Gln)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
选用Amide-MBHA Resin 0.52mmol/g树脂，按照合成路线2，以Fmoc-L-Met-OH、Fmoc-L-Leu-OH、Fmoc-L-Gly-OH、Fmoc-L-Phe-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Gln(Trt)-OH、Fmoc-L-Arg(pbf)-OH、Fmoc-L-Leu-OH、Fmoc-L-Tyr (tBu)-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Ala-OH为合成原料及合成顺序合成序列C0。切肽液切割树脂，得多肽C0粗品，溶于添加氨水的pH＝10～11的水溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽C0纯品，收率27.01％；HPLC纯度98％；分子式C75H111N18O21S；HRMS：[M+H] +1631.79089，calcd 1631.78864。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-6min期20％B相、6-36min20％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例31 C1:(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Gln)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
选用Amide-MBHA Resin 0.52mmol/g树脂，按照合成路线2，以Fmoc-L-Met-OH、Fmoc-L-Leu-OH、Fmoc-L-Gly-OH、Fmoc-L-Phe-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Gln(Trt)-OH、Fmoc-L-Leu-OH、Fmoc-L-Tyr(tBu)-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Ala-OH为合成原料及合成顺序合成序列C1。切肽液切割树脂，得多肽C1粗品，溶于添加氨水的pH＝10～11的水溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽C1纯品，收率38.85％；HPLC纯度99％；分子式C74H107N16O22S；HRMS：[M+H] +1603.74829，calcd 1603.74611。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-6min期20％B相、6-36min20％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例32 C2:(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Asn)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
选用Amide-MBHA Resin 0.52mmol/g树脂，按照合成路线2，以Fmoc-L-Met-OH、Fmoc-L-Leu-OH、Fmoc-L-Gly-OH、Fmoc-L-Phe-OH、Fmoc-L-Gln(Trt)-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Asn(Trt)-OH、Fmoc-L-Leu-OH、Fmoc-L-Tyr(tBu)-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Ala-OH为合成原料及合成顺序合成序列 C2。切肽液切割树脂，得多肽C2粗品，溶于添加氨水的pH＝10～11的水溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽C2纯品，收率21.4％；HPLC纯度99％；分子式C73H105N16O22S；HRMS：[M+H] +1589.73291，calcd 1589.73046。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-6min期20％B相、6-36min20％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例33 C3:(L-Ala)-(L-Glu)-(L-Tyr)-(L-Phe)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
选用Amide-MBHA Resin 0.52mmol/g树脂，按照合成路线2，以Fmoc-L-Met-OH、Fmoc-L-Leu-OH、Fmoc-L-Gly-OH、Fmoc-L-Phe-OH、Fmoc-L-Gln(Trt)-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Arg(pbf)-OH、Fmoc-L-Phe-OH、Fmoc-L-Tyr(tBu)-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Ala-OH为合成原料及合成顺序合成序列C3。切肽液切割树脂，得多肽C3粗品，溶于添加氨水的pH＝10～11的水溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽C3纯品，收率11.82％；HPLC纯度97％；分子式C78H109N18O21S；HRMS：[M+H] +1665.77722，calcd 1665.77299。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-6min期20％B相、6-31min20％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例34 C4:(L-Ala)-(L-Glu)-(L-Tyr)-(L-Pro)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
选用Amide-MBHA Resin 0.52mmol/g树脂，按照合成路线2，以Fmoc-L-Met-OH、Fmoc-L-Leu-OH、Fmoc-L-Gly-OH、Fmoc-L-Phe-OH、Fmoc-L-Gln(Trt)-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Arg(pbf)-OH、Fmoc-L-Pro-OH、Fmoc-L-Tyr(tBu)-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Ala-OH为合成原料及合成顺序合成序列C4。切肽液切割树脂，得多肽C4粗品，溶于添加氨水的pH＝10～11的水溶液中。粗 品经0.22μm滤膜过滤后备用。经液相纯化得多肽C4纯品，收率12.92％；HPLC纯度97％；分子式C74H107N18O21S；HRMS：[M+H] +1615.76025，calcd 1615.75734。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-6min期20％B相、6-31min20％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例35 C5:(L-Ala)-(L-Tyr)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
选用Amide-MBHA Resin 0.52mmol/g树脂，按照合成路线2，以Fmoc-L-Met-OH、Fmoc-L-Leu-OH、Fmoc-L-Gly-OH、Fmoc-L-Phe-OH、Fmoc-L-Gln(Trt)-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Arg(pbf)-OH、Fmoc-L-Leu-OH、Fmoc-L-Tyr(tBu)-OH、Fmoc-L-Ala-OH为合成原料及合成顺序合成序列C5。切肽液切割树脂，得多肽C5粗品，溶于添加氨水的pH＝10～11的水溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽C5纯品，收率21.4％；HPLC纯度≥95％；分子式C79H113N18O20S；HRMS：[M+H] +1665.81262，calcd 1665.80937。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-8min期25％B相、8-38min25％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例36 C6:(L-Ala)-(L-Glu)-(L-Trp)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
选用Amide-MBHA Resin 0.52mmol/g树脂，按照合成路线2，以Fmoc-L-Met-OH、Fmoc-L-Leu-OH、Fmoc-L-Gly-OH、Fmoc-L-Phe-OH、Fmoc-L-Gln(Trt)-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Arg(pbf)-OH、Fmoc-L-Leu-OH、Fmoc-L-Trp-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Ala-OH为合成原料及合成顺序合成序列C6。切肽液切割树脂，得多肽C6粗品，溶于添加氨水的pH＝10～11的水溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽C6纯品，收率10.125％；HPLC纯度96％； 分子式C77H112N19O20S；HRMS：[M+H] +1654.80908，calcd 1654.80462。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-8min期20％B相、8-38min20％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例37 C7:(L-Ala)-(L-Thr)-(L-Thr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
选用Amide-MBHA Resin 0.52mmol/g树脂，按照合成路线2，以Fmoc-L-Met-OH、Fmoc-L-Leu-OH、Fmoc-L-Gly-OH、Fmoc-L-Phe-OH、Fmoc-L-Gln(Trt)-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Arg(pbf)-OH、Fmoc-L-Leu-OH、Fmoc-L-Thr(tBu)-OH、Fmoc-L-Ala-OH为合成原料及合成顺序合成序列C7。切肽液切割树脂，得多肽C7粗品，溶于添加氨水的pH＝10～11的水溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽C7纯品，收率8.89％；HPLC纯度99％；分子式C69H109N18O22S；HRMS：[M+H] +1541.78052，calcd 1541.77807。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-6min期20％B相、6-31min20％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例38 C8:(L-Ala)-(L-Asp)-(L-Asp)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
选用Amide-MBHA Resin 0.52mmol/g树脂，按照合成路线2，以Fmoc-L-Met-OH、Fmoc-L-Leu-OH、Fmoc-L-Gly-OH、Fmoc-L-Phe-OH、Fmoc-L-Gln(Trt)-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Arg(pbf)-OH、Fmoc-L-Leu-OH、Fmoc-L-Asp(Trt)-OH、Fmoc-L-Ala-OH为合成原料及合成顺序合成序列C8。切肽液切割树脂，得多肽C8粗品，溶于添加氨水的pH＝10～11的水溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽C8纯品，收率10.81％；HPLC纯度98％；分子式C69H105N18O22S；HRMS：[M+H] +1569.74121，calcd 1569.73660。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-6min期20％B相、6-31min20％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例39 C9:(L-Leu)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
选用Amide-MBHA Resin 0.52mmol/g树脂，按照合成路线2，以Fmoc-L-Met-OH、Fmoc-L-Leu-OH、Fmoc-L-Gly-OH、Fmoc-L-Phe-OH、Fmoc-L-Gln(Trt)-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Arg(pbf)-OH、Fmoc-L-Leu-OH、Fmoc-L-Tyr(tBu)-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Ala-OH为合成原料及合成顺序合成序列C9。切肽液切割树脂，得多肽C9粗品，溶于添加氨水的pH＝10～11的水溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽C9纯品，收率5.18％；HPLC纯度99％；分子式C78H117N18O21S；HRMS：[M+H] +1673.83765，calcd 1673.83559。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-6min期20％B相、6-31min20％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例40 C10:(L-Val)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
选用Amide-MBHA Resin 0.52mmol/g树脂，按照合成路线2，以Fmoc-Met-OH、Fmoc-Leu-OH、Fmoc-Gly-OH、Fmoc-Phe-OH、Fmoc-Gln(Trt)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Arg(pbf)-OH、Fmoc-Leu-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Val-OH为合成原料及合成顺序合成序列C10。切肽液切割树脂，得多肽C10粗品，溶于添加氨水的pH＝10～11的水溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽C10纯品，收率21.36％；HPLC纯度99％；分子式C77H115N18O21S；HRMS：[M+H] +1659.82336，calcd 1659.83994。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm  i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-8min期20％B相、8-38min20％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例41 C11:(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Dab)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
选用Amide-MBHA Resin 0.52mmol/g树脂，按照合成路线2，以Fmoc-L-Met-OH、Fmoc-L-Leu-OH、Fmoc-L-Gly-OH、Fmoc-L-Phe-OH、Fmoc-L-Gln(Trt)-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Dab(tBu)-OH、Fmoc-L-Leu-OH、Fmoc-L-Tyr(tBu)-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Ala-OH为合成原料及合成顺序合成序列C11。切肽液切割树脂，得多肽C11粗品，溶于添加氨水的pH＝10～11的水溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽C11纯品，收率12.9％；HPLC纯度97％；分子式C73H107N16O21S；HRMS：[M+H] +1575.75317，calcd 1575.75119。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-6min期20％B相、6-31min20％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例42 D1:(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Thr)-(L-Gln)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
选用Amide-MBHA Resin 0.52mmol/g树脂，按照合成路线2，以Fmoc-L-Met-OH、Fmoc-L-Leu-OH、Fmoc-L-Gly-OH、Fmoc-L-Phe-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Gln(Trt)-OH、Fmoc-L-Thr(tBu)-OH、Fmoc-L-Leu-OH、Fmoc-L-Tyr(tBu)-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Ala-OH为合成原料及合成顺序合成序列D1。切肽液切割树脂，得多肽D1粗品，溶于添加氨水的pH＝10～11的水溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽D1纯品，收率5.81％；HPLC纯度≥95％；分子式C73H106N15O22S；HRMS：[M+H] +1576.73535，calcd 1576.73521。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm  i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-8min期20％B相、8-38min20％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例43 D2:(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Asp)-(L-Gln)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
选用Amide-MBHA Resin 0.52mmol/g树脂，按照合成路线2，以Fmoc-L-Met-OH、Fmoc-L-Leu-OH、Fmoc-L-Gly-OH、Fmoc-L-Phe-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Gln(Trt)-OH、Fmoc-L-Asp(Obzl)-OH、Fmoc-L-Leu-OH、Fmoc-L-Tyr(tBu)-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Ala-OH为合成原料及合成顺序合成序列D2。切肽液切割树脂，得多肽D2粗品，溶于添加氨水的pH＝10～11的水溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽D2纯品，收率23.98％；HPLC纯度96％；分子式C73H104N15O23S；HRMS：[M+H] +1590.71655，calcd 1590.71447。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-6min期20％B相、6-31min20％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例44 D3:(L-Ala)-(L-Tyr)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Gln)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
选用Amide-MBHA Resin 0.52mmol/g树脂，按照合成路线2，以Fmoc-L-Met-OH、Fmoc-Leu-OH、Fmoc-Gly-OH、Fmoc-Phe-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Gln(Trt)-OH、Fmoc-L-Arg(pbf)-OH、Fmoc-L-Leu-OH、Fmoc-L-Tyr(tBu)-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Ala-OH为合成原料及合成顺序合成序列D3。切肽液切割树脂，得多肽D3粗品，溶于添加氨水的pH＝10～11的水溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽D3纯品，收率11.06％；HPLC纯度≥95％；分子式C79H113N18O20S；HRMS：[M+H] +1665.81335，calcd 1665.80937。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm  i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-8min期20％B相、8-35min20％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例45 D4:(L-Ala)-(L-Glu)-Trp-(L-Leu)-(L-Arg)-(L-Gln)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
选用Amide-MBHA Resin 0.52mmol/g树脂，按照合成路线2，以Fmoc-L-Met-OH、Fmoc-L-Leu-OH、Fmoc-L-Gly-OH、Fmoc-L-Phe-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Gln(Trt)-OH、Fmoc-L-Arg(pbf)-OH、Fmoc-L-Leu-OH、Fmoc-L-Trp-OH、Fmoc-L-Tyr(tBu)-OH、Fmoc-L-Ala-OH为合成原料及合成顺序合成序列D4。切肽液切割树脂，得多肽D4粗品，溶于添加氨水的pH＝10～11的水溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽D4纯品，收率10.63％；HPLC纯度97％；分子式C77H112N19O20S；HRMS：[M+H] +1654.80676，calcd 1654.80462。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-8min期20％B相、8-38min20％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例46 D5:(L-Ala)-(L-Thr)-(L-Thr)-(L-Leu)-(L-Arg)-(L-Gln)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
选用Amide-MBHA Resin 0.52mmol/g树脂，按照合成路线2，以Fmoc-L-Met-OH、Fmoc-L-Leu-OH、Fmoc-L-Gly-OH、Fmoc-L-Phe-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Gln(Trt)-OH、Fmoc-L-Arg(pbf)-OH、Fmoc-L-Leu-OH、Fmoc-L-Thr(tBu)-OH、Fmoc-L-Ala-OH为合成原料及合成顺序合成序列D5。切肽液切割树脂，得多肽D5粗品，溶于添加氨水的pH＝10～11的水溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽D5纯品，收率18.06％；HPLC纯度96％；分子式C69H109N18O20S；HRMS：[M+H] +1541.78076，calcd 1541.77807。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈； 40℃；流速6ml/min；检测波长210nm；梯度条件为0-6min期20％B相、6-31min20％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例47 D6:(L-Ala)-(L-Asp)-(L-Asp)-(L-Leu)-(L-Arg)-(L-Gln)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
选用Amide-MBHA Resin 0.52mmol/g树脂，按照合成路线2，以Fmoc-L-Met-OH、Fmoc-L-Leu-OH、Fmoc-L-Gly-OH、Fmoc-L-Phe-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Gln(Trt)-OH、Fmoc-L-Arg(pbf)-OH、Fmoc-L-Asp(Obzl)-OH、Fmoc-L-Ala-OH为合成原料及合成顺序合成序列D6。切肽液切割树脂，得多肽D6粗品，溶于添加氨水的pH＝10～11的水溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽D6纯品，收率8.28％；HPLC纯度98％；分子式C69H105N18O22S；HRMS：[M+H] +1569.74072，calcd 1569.73660。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-6min期20％B相、6-31min20％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例48 D7:(L-Val)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Gln)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
选用Amide-MBHA Resin 0.52mmol/g树脂，按照合成路线2，以Fmoc-L-Met-OH、Fmoc-L-Leu-OH、Fmoc-L-Gly-OH、Fmoc-L-Phe-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Gln(Trt)-OH、Fmoc-L-Arg(pbf)-OH、Fmoc-L-Leu-OH、Fmoc-L-Tyr(tBu)-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Ala-OH为合成原料及合成顺序合成序列D7。切肽液切割树脂，得多肽D7粗品，溶于添加氨水的pH＝10～11的水溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽D7纯品，收率10.51％；HPLC纯度98％；分子式C77H115N18O21S；HRMS：[M+H] +1659.82361，calcd 1659.81994。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-8min期20％B相、8-35min 20％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例49 D8:(L-Leu)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Gln)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
选用Amide-MBHA Resin 0.52mmol/g树脂，按照合成路线2，以Fmoc-Met-OH、Fmoc-Leu-OH、Fmoc-Gly-OH、Fmoc-Phe-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Gln(Trt)-OH、Fmoc-Arg(pbf)-OH、Fmoc-Leu-OH、Fmoc-Tyr(tBu)-OH、Fmoc-Glu(OtBu)-OH、Fmoc-Leu-OH为合成原料及合成顺序合成序列D8。切肽液切割树脂，得多肽D8粗品，溶于添加氨水的pH＝10～11的水溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽D8纯品，收率10.85％；HPLC纯度≥95％；分子式C78H117N18O21S；HRMS：[M+H] +1673.83862，calcd 1673.83559。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-8min期20％B相、8-38min20％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例50 D9:(L-Ala)-(L-Glu)-(L-Tyr)-Phe-(L-Arg)-(L-Gln)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
选用Amide-MBHA Resin 0.52mmol/g树脂，按照合成路线2，以Fmoc-L-Met-OH、Fmoc-L-Leu-OH、Fmoc-L-Gly-OH、Fmoc-L-Phe-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Gln(Trt)-OH、Fmoc-L-Arg(pbf)-OH、Fmoc-L-Phe-OH、Fmoc-L-Tyr(tBu)-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Ala-OH为合成原料及合成顺序合成序列D9。切肽液切割树脂，得多肽D9粗品，溶于添加氨水的pH＝10～11的水溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽D9纯品，收率21.28％；HPLC纯度99％；分子式C78H109N18O21S；HRMS：[M+H] +1665.77515，calcd 1665.77299。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-8min期25％B相、8-38min25％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈； 冷冻干燥后得纯品多肽。
实施例51 D10:(L-Ala)-(L-Glu)-(L-Tyr)-(L-Pro)-(L-Arg)-(L-Gln)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
选用Amide-MBHA Resin 0.52mmol/g树脂，按照合成路线2，以Fmoc-L-Met-OH、Fmoc-L-Leu-OH、Fmoc-L-Gly-OH、Fmoc-L-Phe-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Gln(Trt)-OH、Fmoc-L-Arg(pbf)-OH、Fmoc-L-Pro-OH、Fmoc-L-Tyr(tBu)-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Ala-OH为合成原料及合成顺序合成序列D10。切肽液切割树脂，得多肽D10粗品，溶于添加氨水的pH＝10～11的水溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽D10纯品，收率25.17％；HPLC纯度98％；分子式C74H107N18O21S；HRMS：[M+H] +1615.76147，calcd 1615.75734。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-6min期20％B相、6-31min20％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例52 D11:(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Dab)-(L-Gln)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
选用Amide-MBHA Resin 0.52mmol/g树脂，按照合成路线2，以Fmoc-L-Met-OH、Fmoc-L-Leu-OH、Fmoc-L-Gly-OH、Fmoc-L-Phe-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Gln(Trt)-OH、Fmoc-L-Dab(tBu)-OH、Fmoc-L-Leu-OH、Fmoc-L-Tyr(tBu)-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Ala-OH为合成原料及合成顺序合成序列D11。切肽液切割树脂，得多肽D11粗品，溶于添加氨水的pH＝10～11的水溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽D11纯品，收率14.96％；HPLC纯度97％；分子式C73H107N16O21S；HRMS：[M+H] +1575.75427，calcd 1575.75119。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-6min期20％B相、6-31min 20％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例53 D12:(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Dab)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
选用Amide-MBHA Resin 0.52mmol/g树脂，按照合成路线2，以Fmoc-L-Met-OH、Fmoc-L-Leu-OH、Fmoc-L-Gly-OH、Fmoc-L-Phe-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Dab(tBu)-OH、Fmoc-L-Arg(pbf)-OH、Fmoc-L-Leu-OH、Fmoc-L-Tyr(tBu)-OH、Fmoc-L-Glu(OtBu)-OH、Fmoc-L-Ala-OH为合成原料及合成顺序合成序列D12。切肽液切割树脂，得多肽D12粗品，溶于添加氨水的pH＝10～11的水溶液中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽D12纯品，收率14.43％；HPLC纯度99％；分子式C74H111N18O20S；HRMS：[M+H] +1603.79761，calcd 1603.79372。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-6min期20％B相、6-31min20％-90％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压蒸去乙腈；冷冻干燥后得纯品多肽。
实施例54 SP:(L-Arg)-(L-Pro)-(L-Lys)-(L-Pro)-(L-Gln)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
选用Amide-MBHA Resin 0.52mmol/g树脂，按照合成路线2，以Fmoc-L-Met-OH、Fmoc-L-Leu-OH、Fmoc-L-Gly-OH、Fmoc-L-Phe-OH、Fmoc-L-Gln(Trt)-OH、Fmoc-L-Pro-OH、Fmoc-L-Lys(Trt)-OH、Fmoc-L-Pro-OH、Fmoc-L-Arg(pbf)-OH为合成原料及合成顺序合成序列SP。切肽液切割树脂，得多肽SP粗品，溶于水中。粗品经0.22μm滤膜过滤后备用。经液相纯化得多肽SP纯品，收率12.74％；HPLC纯度96.079％；分子式C63H99N15O13S；HRMS：[M+H] +1347.73401，calcd 1347.73302。
液相纯化条件：岛津LC-20AT色谱系统，Shim-pack PREP-ODS(20mm×250mm i.d.，10μm)，C18填料，反相柱；流动相A相0.08％TFA/水，B相0.08％TFA/乙腈；40℃；流速6ml/min；检测波长210nm；梯度条件为0-2min 10％B相、2-33min 10％-30％B相、3-33min 30％B相、维持B相梯度1min后回到初始梯度；收集产物峰；减压 蒸去乙腈；冷冻干燥后得纯品多肽。
实验例1：部分多肽对体外AD细胞的保护作用
使用稳转株APPswe细胞，在铜离子的刺激下可以促进Aβ的沉积和细胞氧化应激反应，模拟阿尔茨海默病(Alzheimer’s disease，AD)的发生。实验中应用350μM Cu 2+损伤APPswe细胞24h，同时加入0.032μM、0.16μM、0.8μM、4μM、20μM、100μM神经激肽衍生物共同孵育。24h后应用CCK-8试剂测定细胞活力。
细胞存活率计算公式：
细胞存活率(％)＝[(OD 加药组-OD 空白组)/(OD 对照组-OD 空白组)]×100％；(*P<0.05，**P<0.01，***P<0.001)。
有效率(％)计算公式：
有效率(％)＝(OD 加药组-OD 模型组)/OD 模型组*100％；
结果如表1所示：
表1.本发明涉及部分多肽对APPswe细胞的保护作用
Figure PCTCN2022142732-appb-000010
Figure PCTCN2022142732-appb-000011
Figure PCTCN2022142732-appb-000012
实验例2：多肽化合物提高寡聚化Aβ 1-42诱导认知损伤小鼠的空间学习记忆能力
在整体动物水平，应用Morris水迷宫实验确证多肽衍生物对寡聚化Aβ 1-42诱导认知损伤模型小鼠的学习记忆能力的改善作用。淀粉样蛋白-β(Aβ)是家族性和散发性AD的主要病理介质，在AD患者的大脑中，观察到大量的Aβ寡聚体和斑块积累，引起神经元细胞变性、凋亡。临床和实验验证表明这种Aβ异常沉积会阻断长时程突触增强、损害突触功能并诱发认知缺陷。该模型是基于Aβ上述的神经毒性作用，采用立体定位注射的方法将寡聚化的Aβ 1-42注射入小鼠侧脑室，引起神经毒性反应，并诱导产生学习记忆和行为学障碍，能较好地模拟临床上AD患者脑内的病理和认知损伤情况，是常用的AD动物模型。
Morris水迷宫(Morris water maze，MWM)实验用于检测用药后各组小鼠的空间学习能力以及记忆能力。水迷宫水池直径为1.2m，水池平均分为四个象限，对应四个象限悬挂标志物以方便动物记忆位置，实验时水温保持在22±2℃。实验分为定位航行试验和空间探索试验2个部分。定位航行试验连续进行五天，每天进行两次。试验期间将平台置于固定位置，每天将小鼠分别从两个象限放入水中，记录小鼠成功找到平台的时间即潜伏期以及游泳速度。试验中允许小鼠在60秒内完成任务，否则将它们引导到平台上并停留10秒以观察学习。空间探索试验在定位航行试验结束后24h进行，评估记忆的巩固。试验期间将平台撤走，将小鼠从距离原平台最远象限处放入水中，让其游泳60秒。记录小鼠在原平台所在象限游泳的时间和穿越原平台位置的次数。
结果以均数±标准误差表示。使用IBM SPSS Statistics 26.0通过重复测量数据的单因素方差分析，对MWM实验中的平台潜伏期和游泳速度进行分析。
在定位航行试验中，小鼠寻找平台的潜伏期长短是空间学习能力的指标。试验中小鼠寻找平台的潜伏期随着训练次数的增加而缩短，不同处理组小鼠的潜伏期出现了显著性差异。对潜伏期随时间变化的趋势进行组间比较，与Sham组小鼠相比，Aβ 1-42 模型小鼠的潜伏期显著延长(图1A和图2A，P<0.01)，提示小鼠的学习能力显著下降。与Aβ 1-42模型小鼠相比，给予多肽C10的0.4μg/kg和2μg/kg显著降低了Aβ 1-42小鼠的潜伏期(图1A，P<0.05)，给予多肽C7的0.4μg/kg和2μg/kg也显著降低了Aβ 1-42小鼠的潜伏期(图2A，P<0.01)，并且多肽C10和C7的药理作用均呈现剂量依赖性。此外，与Aβ 1-42模型小鼠相比，多肽SP的50μg/kg及临床一线抗AD药物多奈哌齐(Donepezil)的1mg/kg也缩短了Aβ 1-42小鼠的潜伏期(图1A、图2A，P<0.001、P<0.01)。鉴于各组小鼠之间的游泳速度没有显著性差异(图2A、图2B)，提示实验小鼠的运动能力没有受到不同处理因素的影响，进一步说明多肽C10和C7能够提高Aβ 1-42模型小鼠的空间学习能力。
在空间探索试验中，小鼠在目标象限的持续时间和穿过平台原始位置的次数是空间记忆能力的两个指标。与Sham组小鼠相比，Aβ 1-42模型小鼠穿越平台原始位置的次数减少(图1C、图2C，P<0.01)，在目标象限内游泳的时间缩短(图1D、图2D，P<0.01、P<0.001)，提示小鼠的记忆能力明显降低。与Aβ 1-42模型小鼠相比，给予0.4μg/kg和2μg/kg的多肽C10与C7，显著增加了Aβ 1-42小鼠穿越平台原始位置的次数(图1C、图2C，P<0.05、P<0.01、P<0.001)和在目标象限内持续的时间(图1D、图2D，P<0.05、P<0.01、P<0.001)，并且药物作用具有剂量依赖性。此外，多肽SP的50μg/kg及多奈哌齐(Donepezil)的1mg/kg也增加了Aβ 1-42小鼠穿越平台位置的次数(图1C、图2C，P<0.01)和在目标象限内持续的时间(图1D、图2D，P<0.05、P<0.01、P<0.001)。上述结果表明，多肽C10和C7能够提高Aβ 1-42模型小鼠的空间记忆能力，改善记忆障碍。
实验例3：本发明涉及部分多肽的肾细胞毒性
本实验选择非洲绿猴肾细胞——Vero细胞，使用MEM培养液、10％胎牛血清于37℃、5％CO 2条件下培养细胞。
多肽预处理：
(1)将待配制的多肽化合物以及溶剂(水、DMSO)放置至室温，称取一定质量化合物，加入溶剂配制成指定浓度，上下颠倒混匀配合涡旋振荡器振荡，直至全部溶解，配制成20mg/mL储液。
(2)取1.5mL离心管，标记为管I，II，III-VI，进行药物稀释：
管I中加入40μL储液；
管II加入36μL溶剂和4μL管I中溶液，充分混匀；
管III加入36μL溶剂和4μL管II中溶液，充分混匀；
依次稀释各管；
管VI加入40μL溶剂。
(3)空白对照孔为细胞加溶剂，作为阴性对照孔；无细胞只有培养基的孔作为背景孔。
CCK-8方法检测：
(1)细胞在25cm 2培养瓶培生长至对数期，用0.25％Trypsin-EDTA消化收集细胞并计数。
(2)将细胞悬液加入96孔板，每孔200μL，细胞密度为4000/孔。
(3)培养24h后，如表3所示，依次加入1μL各管中的化合物，CO 2培养箱孵育70h。
(4)弃上清，加入CCK-8检测试剂，孵育2h后，振荡混匀，酶标仪450nm检测并记录各孔吸光值。
抑制率按照以下计算公式计算：
Figure PCTCN2022142732-appb-000013
其中，
Figure PCTCN2022142732-appb-000014
为化合物组OD平均值；
Figure PCTCN2022142732-appb-000015
为无细胞培养基组OD平均值，
Figure PCTCN2022142732-appb-000016
Figure PCTCN2022142732-appb-000017
为DMSO阴性对照组OD平均值。
使用IBM SPSS Statistics 26.0进行回归分析计算。
结果如表2所示。
表2.本发明涉及部分多肽的肾细胞毒性
Figure PCTCN2022142732-appb-000018
Figure PCTCN2022142732-appb-000019
Figure PCTCN2022142732-appb-000020
本发明涉及部分多肽在100μg/ml下表现出较低的抑制作用，抑制率均不超过24％，表明本发明涉及多肽对肾细胞毒性较低。
实验例4：本发明涉及部分多肽的溶血性
本实验采用日本大耳白兔全血测试多肽溶血性。
(1)取1kg日本大耳白兔1只，耳缘静脉取血3mL，置于含有抗凝剂的离心管内，1500rpm离心15min，去除上层的白细胞和纤维蛋白。
(2)用冷的PBS将细胞洗3次。
(3)然后将洗涤好的红细胞用PBS配成2％的红细胞悬液备用。
(4)阴性对照为PBS(0％溶血)，阳性对照为1％Triton X-100(100％溶血)。测试多肽药物浓度设置为2μg/mL，1μg/mL，0.5μg/mL，0.25μg/mL，0.125μg/mL，0.0625μg/mL。
(5)将200μL 2％的红细胞悬液与200μL 2×多肽药物浓度混合，37℃静置培养60min，1500rpm离心15min，取100μL上清于96孔板中。用酶标仪检测OD570吸光值，梯度稀释1％Triton X-100制作标准曲线，计算测试药物引起红细胞溶血的百分比。结果如表3所示。
(6)细胞存活率计算公式：
细胞存活率(％)＝[(OD加药组-OD阴性对照组)/(OD阳性对照组-OD空白组)]× 100％；
使用Prism7.0软件计算EC 50；
表3.本发明涉及部分多肽的溶血性
Figure PCTCN2022142732-appb-000021
Figure PCTCN2022142732-appb-000022
综上所述，本发明所涉及的部分多肽具有较好的神经保护作用，并且肾毒性与溶血性较低，具有良好的发展潜力。

Claims (10)

  1. [援引加入（细则20.6）08.12.2023]
    具有如式Ⅰ所示结构的多肽化合物、其药用盐、溶剂合物、异构体或多晶型物：
    Figure PCTCN2022142732-appb-100001

    式Ⅰ中，数字1-20表示氨基酸在式Ⅰ中的具体位置，式I中的最少氨基酸个数为5个、最大氨基酸个数为20个；所述氨基酸的构型为L型或D型；数字1-15以及20所在位置的氨基酸存在或不存在；
    式Ⅰ中：
    R 3选自以下的组：-(CH 2) 2-COOH、-CH 2-COOH；
    R 4选自以下的组：-CH 2-COOH；
    R 5选自以下的组：-CH 2-OH；
    R 6选自以下的组：-(CH 2) 2-COOH、-CH 2-CH(CH 3) 2、-CH-(CH 3) 2；
    R 7选自以下的组：-(CH 2) 2-COOH、-CH-(CH 3) 2、-CH 2-CONH 2、-CH 3、-CH 2-CH(CH 3) 2、-C 3H 6；
    R 8选自以下的组：-CH 2-CONH 2、-CH 2-CH(CH 3) 2、-C 3H 6、-CH 3、-(CH 2) 2-COOH、-CH 2-COOH、-CH-(CH 3) 2；
    R 9选自以下的组：-CH3、-(CH2)2-COOH、-CH2-COOH、-(CH2)3-NHC(NH)NH2、-CH2-C6H4-OH、-CH(CH3)-OH；
    R 10选自以下的组：-(CH2)2-COOH、-(CH2)3-NHC(NH)NH2、-CH2-C6H4-OH、-CH2-CH(CH3)2、-CH(CH3)-CH2-CH3、-(CH2)2-CONH2、-C8NH6、-CH(CH3)-OH、-CH2-COOH；
    R 11选自以下的组：-CH2-C6H4-OH、-CH2-CH(CH3)2、-CH(CH3)-CH2-CH3、-(CH2)2-CONH2、-CH2-CONH2、-(CH2)2-CONH2、-CH2-C6H5、-C3H6；
    R 12选自以下的组：-CH2-CH(CH3)2、-CH2-CONH2、-(CH2)2-CONH2、-(CH2)3-NHC(NH)NH2、-CH(CH3)-OH、-CH2-COOH、-(CH2)2-NH2、-(CH2)4-NH2；
    R 13选自以下的组：-(CH2)2-COOH、-(CH2)3-NHC(NH)NH2、-CH(CH3)-OH、-(CH2)2-CONH2、-CH2-COOH、-(CH2)2-NH2、-C3H6；
    R 14选自以下的组：-(CH2)2-COOH、-(CH2)2-CONH2；
    R 15选自以下的组：-(CH2)2-CONH2、-(CH2)2-COOH；
    R 20选自以下的组：-NH2、-OH；
    R 21选自以下的组：-(CH2)2-S-CH3。
  2. 如权利要求1所述的具有如式Ⅰ所示结构的多肽化合物、其药用盐、溶剂合物、异构体或多晶型物，其特征在于，所述的多肽化合物选自以下的组：
    (1)A1:(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
    (2)A2:(L-Glu)-(L-Asn)-(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
    (3)A3:(L-Leu)-(L-Ala)-(L-Arg)-(L-Leu)-(L-Leu)-(L-Thr)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
    (4)A4:(L-Val)-(L-Pro)-(L-Glu)-(L-Tyr)-(L-Ile)-(L-Asn)-(L-Gln)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
    (5)A5:(L-Asp)-(L-Tyr)-(L-Gln)-(L-Gln)-(L-Asp)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
    (6)B1:(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
    (7)B2:(L-Glu)-(L-Asn)-(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
    (8)B3:(L-Leu)-(L-Ala)-(L-Arg)-(L-Leu)-(L-Leu)-(L-Thr)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
    (9)B4:(L-Val)-(L-Pro)-(L-Glu)-(L-Tyr)-(L-Ile)-(L-Asn)-(L-Gln)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
    (10)B5:(L-Asp)-(L-Tyr)-(L-Gln)-(L-Gln)-(L-Asp)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
    (11)SP-6-11:(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
    (12)SP-7-11:(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
    (13)EE-6-11:(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
    (14)EE-7-11:(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
    (15)A2-M1:(L-Glu)-(L-Asn)-(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-NH 2
    (16)A2-M2:(L-Glu)-(L-Asn)-(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)
    (17)A2-M3:(L-Glu)-(L-Asn)-(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(D-Met)-NH 2
    (18)A3-M1:(L-Leu)-(L-Ala)-(L-Arg)-(L-Leu)-(L-Leu)-(L-Thr)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-NH 2
    (19)A3-M2:(L-Leu)-(L-Ala)-(L-Arg)-(L-Leu)-(L-Leu)-(L-Thr)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)
    (20)A3-M3:(L-Leu)-(L-Ala)-(L-Arg)-(L-Leu)-(L-Leu)-(L-Thr)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(D-Met)-NH 2
    (21)B1-M1:(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-NH 2
    (22)B1-M2:(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)
    (23)B1-M3:(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(D-Met)-NH 2
    (24)B3-M1:(L-Leu)-(L-Ala)-(L-Arg)-(L-Leu)-(L-Leu)-(L-Thr)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-NH 2
    (25)B3-M2:(L-Leu)-(L-Ala)-(L-Arg)-(L-Leu)-(L-Leu)-(L-Thr)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)
    (26)B3-M3:(L-Leu)-(L-Ala)-(L-Arg)-(L-Leu)-(L-Leu)-(L-Thr)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(D-Met)-NH 2
    (27)B5-M1:(L-Asp)-(L-Tyr)-(L-Gln)-(L-Gln)-(L-Asp)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-NH 2
    (28)B5-M2:(L-Asp)-(L-Tyr)-(L-Gln)-(L-Gln)-(L-Asp)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)
    (29)B5-M3:(L-Asp)-(L-Tyr)-(L-Gln)-(L-Gln)-(L-Asp)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(D-Met)-NH 2
    (30)C0:(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Gln)-(L-Glu)-(L-Glu)- (L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
    (31)C1:(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Gln)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
    (32)C2:(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Asn)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
    (33)C3:(L-Ala)-(L-Glu)-(L-Tyr)-(L-Phe)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
    (34)C4:(L-Ala)-(L-Glu)-(L-Tyr)-(L-Pro)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
    (35)C5:(L-Ala)-(L-Tyr)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
    (36)C6:(L-Ala)-(L-Glu)-(L-Trp)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
    (37)C7:(L-Ala)-(L-Thr)-(L-Thr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
    (38)C8:(L-Ala)-(L-Asp)-(L-Asp)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
    (39)C9:(L-Leu)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
    (40)C10:(L-Val)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
    (41)C11:(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Dab)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
    (42)D1:(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Thr)-(L-Gln)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
    (43)D2:(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Asp)-(L-Gln)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
    (44)D3:(L-Ala)-(L-Tyr)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Gln)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
    (45)D4:(L-Ala)-(L-Glu)-Trp-(L-Leu)-(L-Arg)-(L-Gln)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
    (46)D5:(L-Ala)-(L-Thr)-(L-Thr)-(L-Leu)-(L-Arg)-(L-Gln)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
    (47)D6:(L-Ala)-(L-Asp)-(L-Asp)-(L-Leu)-(L-Arg)-(L-Gln)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
    (48)D7:(L-Val)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Gln)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
    (49)D8:(L-Leu)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Gln)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
    (50)D9:(L-Ala)-(L-Glu)-(L-Tyr)-Phe-(L-Arg)-(L-Gln)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
    (51)D10:(L-Ala)-(L-Glu)-(L-Tyr)-(L-Pro)-(L-Arg)-(L-Gln)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
    (52)D11:(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Dab)-(L-Gln)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
    (53)D12:(L-Ala)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Dab)-(L-Glu)-(L-Glu)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
    (54)SP:(L-Arg)-(L-Pro)-(L-Lys)-(L-Pro)-(L-Gln)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
    (56)E1:(L-Leu)-(L-Ala)-(L-Val)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-Gly-(L-Leu)-(L-Met)-NH 2
    (57)E2:(D-Leu)-(L-Ala)-(L-Val)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
    (58)E3:(L-Ser)-(L-Leu)-(L-Ala)-(L-Val)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
    (59)E4:(D-Ser)-(L-Leu)-(L-Ala)-(L-Val)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
    (60)E5:(L-Asp)-(L-Ser)-(L-Leu)-(L-Ala)-(L-Val)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
    (61)E6:(D-Asp)-(L-Ser)-(L-Leu)-(L-Ala)-(L-Val)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
    (62)E7:(L-Glu)-(L-Asp)-(L-Ser)-(L-Leu)-(L-Ala)-(L-Val)-(L-Glu)-(L-Tyr)- (L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
    (63)E8:(L-Asp)-(L-Asp)-(L-Ser)-(L-Leu)-(L-Ala)-(L-Val)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
    (64)E9:(L-Glu)-(L-Glu)-(L-Asp)-(L-Ser)-(L-Leu)-(L-Ala)-(L-Val)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
    (65)E10:(L-Glu)-(L-Asp)-(L-Asp)-(L-Ser)-(L-Leu)-(L-Ala)-(L-Val)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
    (66)E11:(L-Glu)-(L-Glu)-(L-Glu)-(L-Asp)-(L-Ser)-(L-Leu)-(L-Ala)-(L-Val)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2
    (67)E12:(L-Glu)-(L-Glu)-(L-Asp)-(L-Asp)-(L-Ser)-(L-Leu)-(L-Ala)-(L-Val)-(L-Glu)-(L-Tyr)-(L-Leu)-(L-Arg)-(L-Glu)-(L-Glu)-(L-Gln)-(L-Phe)-(L-Phe)-(L-Gly)-(L-Leu)-(L-Met)-NH 2。
  3. 一种制备权利要求1或2所述的具有如式Ⅰ所示结构的多肽化合物、其药用盐、溶剂合物、异构体或多晶型物的方法，其特征在于，采用Fmoc固相合成方法，按照肽链从C端到N端的氨基酸顺序合成多肽，经纯化，冷冻干燥后得到多肽纯品。
  4. 如权利要求3所述的方法，其特征在于，所述的Fmoc固相合成方法所用树脂选自以下任意一种：Amide-MBHA Resin 0.52mmol/g、Amide-MBHA Resin 0.646mmol/g、2-Chlorotrityl Chloride Resin 0.52mmol/g。
  5. 如权利要求3所述的方法，其特征在于，所述的Fmoc固相合成方法选用Fmoc保护氨基酸氨基部分；侧链保护基选自以下基团中的任意一种：苄基(Bzl)、叔丁基(tBu)、叔丁氧羰基(Boc)、2,2,4,6,7-五甲基二氢苯并呋喃-5-磺酰基(Pbf)、三苯基(Trt)。
  6. 如权利要求3所述的方法，其特征在于，经脱除Fmoc保护基、缩合形成肽链；脱除Fmoc试剂为20％哌啶/DMF；缩合剂为苯并三氮唑-N,N,N',N'-四甲基脲六氟磷酸酯(HBTU)、N,N'-二异丙基碳二亚胺(DIEA)，摩尔比为氨基酸：HBTU：DIEA为1:0.9:2。
  7. 如权利要求3所述的方法，其特征在于，多肽通过酸脱除树脂与氨基酸保护基，切肽液比例为95％v/v三氟乙酸(TFA)、2.5％v/v三异丙基硅烷(TIPS)、2.5％v/v水，15-20ml/g树脂。
  8. 如权利要求3所述的方法，其特征在于，当R 20为-OH时，20位氨基酸为L型Met，采用路线1方法：
    路线1：
    Figure PCTCN2022142732-appb-100002
    其中，各步骤的反应条件如下：
    a：DMF，20-50℃，溶胀10-50min；DMF/DCM交叉洗涤3次以上；茚三酮显色反应检测；
    b：Fmoc-Met-OH，DIEA，反应3h或过夜；甲醇0.5ml，30min；DMF/DCM交叉洗涤3次以上；茚三酮显色反应检测；
    c：配置脱保护试剂，脱除Fmoc试剂为20％哌啶/DMF，反应10-30min，重复2次；DMF/DCM交叉洗涤3次以上；茚三酮显色反应检测；
    d：按照摩尔比为氨基酸：HBTU：DIEA为1:0.9:2配置氨基酸溶剂，反应1-3h，DMF/DCM交叉洗涤3次以上；茚三酮显色反应检测；
    e：重复步骤c、d，按照顺序合成多肽；
    f：配置切肽液，切肽液比例为95％v/v三氟乙酸(TFA)、2.5％v/v三异丙基硅烷(TIPS)、2.5％v/v水，15-20ml/g树脂，反应1-3h；压出液体；乙醚沉淀洗涤干燥后得多肽粗品；
    路线2：
    当R 20为-NH 2时，采用路线2方法：
    Figure PCTCN2022142732-appb-100003
    其中，各步骤的反应条件如下：
    a':DMF，20-50℃，溶胀10-50min；DMF/DCM交叉洗涤3次以上；茚三酮显色反应检测；
    b':配置脱保护试剂，脱除Fmoc试剂为20％哌啶/DMF，反应10-30min，重复2次；DMF/DCM交叉洗涤3次以上；茚三酮显色反应检测；
    c':按照摩尔比为氨基酸：HBTU：DIEA为1:0.9:2配置氨基酸溶剂，反应0.5-2h，DMF/DCM交叉洗涤3次以上；茚三酮显色反应检测；
    d':重复步骤c'、d'，按照顺序合成多肽；
    f':配置切肽液，切肽液比例为95％v/v三氟乙酸(TFA)、2.5％v/v三异丙基硅烷(TIPS)、2.5％v/v水，15-20ml/g树脂，反应1-3h；压出液体；乙醚沉淀洗涤干燥后得多肽粗品。
  9. 一种药物组合物，其特征在于，包含权利要求1或2所述的具有如式Ⅰ所示结构的多肽化合物、其药用盐、溶剂合物、异构体或多晶型物，以及药学上可以接受的载体或赋形剂。
  10. 权利要求1或2所述的具有如式Ⅰ所示结构的多肽化合物、其药用盐、溶剂合物、异构体或多晶型物或权利要求9所述的药物组合物在制备具有NK细胞相关免疫调节作用、治疗炎症相关疾病或诊断与治疗神经系统疾病药物中的应用。
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