Neuropeptide FF receptor peptide ligand as well as preparation and application thereof

Abstract

According to the invention, peptide ligands of a neuropeptide FF receptor are designed, namely, endogenous peptide agonists neuropeptide FF (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2) and neuropeptide VF (Val-Pro-Asn-Leu-Pro-Gln-Arg-Phe-NH2) of the neuropeptide FF receptor are used as chemical template molecules, and a series of analogues are derived by virtue of scanning of propargyl glycine (Pra) and scanning of azido alanine [Ala (N3)]. Further, Pra side chain alkynyl and Ala( N3) side chain azide of the analogue are chemically derived from small molecule compounds containing azide and alkynyl structures respectively through click chemistry reaction, and a peptide ligand of a neuropeptide FF receptor with a brand new structure is obtained by combining a substitution modification strategy of a single-site amino acid. In-vitro calcium mobilization experiments show that 44 agonists and 12 antagonists exist in the neuropeptide FF receptor peptide ligands, and the neuropeptide FF receptor peptide ligands with novel structures can be potentially used for relieving or treating side effects of tolerance, constipation, addiction, respiratory depression and the like of opioid drugs.

Description

Technical Field

[0001] The invention belongs to the technical field of biochemistry, and specifically relates to a class of neuropeptide FF receptor peptide ligands and preparation and use thereof. Background Art

[0002] Neuropeptide FF (NPFF) is an octapeptide ending with -Arg-Phe-NH2 structure in vivo. It has the same C-terminal structure as the mollusk cardiac excitatory peptide FMRFa and belongs to the FMRFa-related peptide family. NPFF was first isolated and identified in the bovine brain. Subsequently, two G protein-coupled receptors, namely NPFF1 receptor and NPFF2 receptor, were successfully cloned. NPFF1 receptor is mainly distributed above the spinal cord level, while NPFF2 receptor is distributed in both the brain and spinal cord, and both are only distributed in small amounts in the periphery. NPFF-related peptides are derived from two precursors, pro-NPFFA and pro-NPFFB, of which NPFF is produced from pro-NPFFA and NPVF is produced from pro-NPFFB. NPFF shows a certain selective preference for NPFF2 receptor, while NPVF has a significant selective advantage for NPFF1 receptor. In addition, the NPFF system participates in the regulation of multiple physiological functions in vivo, including pain perception, insulin release, cardiovascular activity, feeding behavior, neuroendocrine, energy balance, inflammatory response and other physiological processes. In addition, the NPFF system has been shown to play a key role in regulating opioid abuse, pain sensitivity, analgesic tolerance, and respiratory depression.

[0003] Several studies have shown that the effects of NPFF on opioid regulation depend on the route of administration and the receptor subtype selectivity of the agonist and antagonist. In terms of the regulation of analgesic activity, intrathecal injection of NPFF and NPFF's metabolically stable analog 1DMe can produce analgesia and enhance the analgesic effect of morphine (Br J Pharmacol. 1996, 117: 493-501), while supraspinal injection of NPSF (1-37) (NPFF1 receptor agonist) and dNPA (NPFF2 receptor agonist) as well as non-selective agonists of NPFF receptors NPFF and 1DMe all produce anti-opioid analgesic effects, while intracerebroventricular injection of NPFF1 receptor selective agonist NPVF produces the opposite effect of enhancing morphine analgesia (Biochem Biophys Res Commun. 2005, 336: 197-203; Eur J Pharmacol. 2005, 508: 107-114; Peptides. 1993, 14: 919-924; Neuropeptides. 1997, 31: 1-7). In terms of the regulation of opioid analgesia tolerance and pain sensitivity, intracerebroventricular injection of IgG of NPFF antiserum restored the analgesic response of morphine-tolerant rats to intravenous morphine (radiant heat tail-flick test); subcutaneous administration of RF9 or oral administration of NPFF1 receptor antagonist RF313 can enhance opioid analgesia and regulate pain sensitivity (Neurosci Lett. 1991, 132: 29-32; Proc Natl Acad Sci USA 2006, 103: 466-471; ACS Chem Neurosci. 2017, 8: 2290-2308; Neuropharmacology. 2017, 118: 188-198).In terms of the regulation of opioid addiction, NPFF or its analogs activate NPFF receptors to induce withdrawal symptoms, while NPFF receptor antagonists (Dansyl-PQRamide, RF9 or AC-262620) can attenuate the withdrawal reaction induced by chronic morphine treatment (Peptides. 1990, 11: 277-280; Peptides. 1999, 20: 1211-1217; Neurosci Lett. 2018, 684: 98-103); and co-injection of NPFF receptor agonists (NPFF and 1DMe) at the lateral ventricular level can block the rewarding effect and motor function induced by chronic morphine or cocaine treatment. The NPFF receptor antagonist RF9 itself has no effect, but it enhances the rewarding effect induced by chronic morphine or cocaine treatment (Peptides. 2006, 27: 964-972; Peptides. 2007, 28: 2235-2242); In addition, our research group previously used a combination of NPFF non-selective antagonist RF9, NPFF1 receptor biased antagonist RF3286 and gene knockout mice to prove that NPFF2 receptors at the spinal cord level are mainly involved in the regulation of analgesia, while NPFF1 receptors above the spinal cord level are mainly involved in the regulation of opioid pain sensitivity (Eur J Pharmacol. 2024, 969: 176457).

[0004] Opioids are still very effective analgesics for many pain conditions, especially chronic pain; however, the adverse effects associated with the use of opioids, such as physical dependence, hyperalgesia, and tolerance, have become obstacles to accurate dosing and effective pain control for many pain patients. Integrating opioids with other drugs that can enhance their efficacy and / or reduce adverse reactions into a combined treatment plan provides a highly promising alternative strategy for pain management. In patents 201210098832.2 and 201610252648.7, a new chimeric peptide BN-9 and DN-9 targeting opioid and NPFF receptors are disclosed. After central and peripheral administration, it can produce stronger and non-tolerable analgesic effects than morphine in different pain models, while its opioid side effects such as constipation and addiction are significantly reduced (Br J Pharmacol. 2016, 173: 1864-80; J Med Chem. 2016, 59: 10198-10208; J Pain. 2020, 21: 477-493; Br J Pharmacol. 2020, 177: 93-109). Patent 201810932993.4 and Patent 202110272260.4 respectively disclose a disulfide bond and amide bond cyclized analogue for opioid and NPFF receptors. After central and peripheral administration, it can produce analgesic effects stronger than morphine and without tolerance in different pain models. It can also produce significant analgesic effects after oral administration in different pain models, and its opioid side effects such as constipation, addiction and respiratory depression are significantly reduced (ACS Chem Neurosci. 2022, 13: 3078-3092; JMed Chem. 2021, 64: 13394-13409.). Given that its biological activity is believed to be regulated by NPFF, our research group is exploring compounds and compositions that can inhibit or activate the functional effects of NPFF. Summary of the invention

[0005] The purpose of the present invention is to provide a more druggable peptide ligand based on NPFF receptor. After a large number of experimental studies, the inventors introduced amino acid residues containing alkyne or azide structures in the side chain into the parent molecule, and directly obtained a series of peptide ligands based on NPFF receptor through copper-catalyzed cycloaddition reaction and classic single-site amino acid substitution modification. Through calcium mobilization experiments, it was found that this type of NPFF receptor ligand contained 44 agonists and 12 antagonists, and the activity of some agonists was much stronger than that of the parent compound, among which the agonist activity of compound 35 was 297 times higher than that of the parent NPVF. In addition, the 12 antagonists included 6 non-selective antagonists, 5 NPFF1 receptor selective antagonists and 1 NPFF2 receptor selective antagonist. Among these antagonists, compound 24 is a non-selective antagonist stronger than RF9, compound 29 is a NPFF1 receptor biased antagonist stronger than RF3286, and it is also the first time that the selective antagonist compound 13 of NPFF2 receptor has been reported.

[0006] Another object of the present invention is to provide a preparation method based on neuropeptide FF receptor peptide ligand or its pharmaceutically acceptable salt and a pharmaceutically acceptable carrier.

[0007] Another object of the present invention is to provide therapeutic uses of the above-mentioned NPFF receptor peptide ligands or pharmaceutically acceptable salts thereof. Such novel ligands can be used as a new generation of potential drugs for treating side effects of opioids such as tolerance, constipation, addiction and respiratory depression.

[0008] The present disclosure relates to the discovery of novel peptide ligands based on NPFF and NPVF. In one aspect, the present disclosure relates to such neuropeptide FF receptor peptide ligands based on Pra scanning, Ala (N3) substitution, copper-catalyzed click chemistry direct derivatization and single-site amino acid substitution modification according to formula (I-V):

[0009] Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2(I)

[0010] In the formula,

[0011] The amino acids at positions 1-8 were scanned by Pra substitution;

[0012] In another preferred embodiment, the neuropeptide FF receptor peptide ligand is formed by a click chemistry reaction of Pra of structural formula (I) with 3-azidopropylamine, 3-azidopropionic acid and benzyl azide to generate a triazole derivative with the general structural formula (II), and the Pra replacement and derivatization at other sites are the same as those at position 1, wherein R1 is a propylamine group, R2 is a propionic acid group, and R3 is a benzyl group.

[0013]

[0014] In another preferred embodiment, the general structural formula of the neuropeptide FF receptor peptide ligand is shown as (III).

[0015] Xaa1-Xaa2-Xaa3-Pra-Pro-Gln-Arg-Phe-NH2(Ⅲ)

[0016] In the formula,

[0017] Xaa1 is selected from the group consisting of Val, Nva, Ala, Gly, AllylGly;

[0018] Xaa2 is selected from Trp, Tpi, HomoPhe, 2-Nal, 4-Bpa;

[0019] Xaa3 is selected from 2-Nal, 4-Bpa, HomoPhe, 2-Trp;

[0020] In another preferred embodiment, the general structural formula of the neuropeptide FF receptor peptide ligand is as shown in (IV), wherein R1 is a propylamine group, R2 is a propionic acid group, and R3 is a benzyl group.

[0021]

[0022] In another preferred embodiment, the general structural formula of the neuropeptide FF receptor peptide ligand is as shown in (V), wherein X1 is ethylamine, X2 is acetic acid, X3 is benzyl, X4 is n-butanol, and X5 is ethylene glycol ethyl ether.

[0023]

[0024] In another aspect, the present disclosure relates to a pharmaceutical composition comprising a neuropeptide FF receptor-based peptide ligand represented by Formula I, Formula II, Formula III, Formula IV and Formula V or a pharmaceutically acceptable salt thereof and a pharmaceutically acceptable carrier.

[0025] More preferred NPFF receptor ligands are compounds 1 to 56, which contain the above-mentioned amino acid sequences.

[0026] Throughout the specification of this application, conventional three-letter codes are used to represent natural amino acids, and recognized three-letter codes are used to represent other amino acids, such as Pra (propargylglycine), Ala(N3) (3-azido-L-alanine), Nva (norvaline), AllylGly (allylglycine), Tpi (L-1,2,3,4-tetrahydro-B-porphyrin-3-carboxylic acid), HomoPhe (homophenylalanine), 2-Nal (3-(2-naphthyl)-L-alanine) and 4-Bpa (4-benzoylphenylalanine).

[0027] In the third aspect of the present invention, a method for preparing the neuropeptide FF linear peptide and the triazole-containing peptide molecule directly derived from the side chain alkynyl or azide group is provided, characterized in that the preparation method comprises the following process steps:

[0028] (a) synthesizing the linear polypeptide targeting the neuropeptide FF receptor according to claim 1 by using the Fmoc solid phase polypeptide synthesis method according to the amino acid sequences of structural formulas I and III of claim 1;

[0029] (b) The linear peptides of structural formulas ⅠⅠ, Ⅳ and Ⅴ are directly derivatized by azidoalkynyl cycloaddition reaction to generate NPFF receptor peptide ligands containing triazole.

[0030] In another preferred embodiment, in step (b), a click chemistry reaction is performed on the side chains of Pra and Ala(N3), thereby forming a triazole bond between Pra and the azide compound or Ala(N3) and the alkynyl compound.

[0031] In another preferred embodiment, the method further comprises: separating the NPFF receptor peptide ligand or a pharmaceutically acceptable salt thereof obtained in steps (a) and (b) to obtain purified NPFF receptor peptide ligand or a pharmaceutically acceptable salt thereof.

[0032] In another preferred embodiment, in step (a), the solid phase synthesis method includes process steps selected from the following groups: pretreatment of the solid phase carrier, amino acid condensation, extension and cyclization of the peptide chain, compression, drying and cutting of the polypeptide, extraction, purification, or a combination thereof.

[0033] In another preferred embodiment, the method comprises the following process steps:

[0034] i. Resin pretreatment: Rink-Amide-MBHA resin was used in the experiment. First, the resin was swollen in dichloromethane (DCM) for 30 minutes, and then the resin was rinsed with N,N-dimethylformamide (DMF) 2 to 4 times, with a stirring rate of 60 to 100 rpm and a stirring time of 2 to 5 minutes, preferably 3 minutes. The volume mass ratio of DCM to resin is 8 to 12 mL / g. Ninhydrin test, under normal circumstances, the solution is light yellow and the resin is colorless.

[0035] ii. Cyclic condensation of amino acids

[0036] ii.i Removal of fluorenylmethoxycarbonyl (Fmoc) protecting group: Use a mixed solvent of 1,8-diazabicycloundec-7-ene (DBU), piperidine and DMF (1:1:98) to remove the Fmoc protecting group, stir the reaction for 3 times, the stirring rate is 60-100 rpm, the first two stirring times are 2-6 min, and the last stirring time is 8-12 min; the time is preferably 5 min for the first two times and 10 min for the last time. Finally, add DMF for washing. Ninhydrin test, under normal circumstances, the solution is blue-purple and the resin is blue-purple.

[0037] ii.ii Amino acid condensation: Weigh N-α-Fmoc protected amino acid, O-benzotriazole-N,N,N',N'-tetramethylurea-hexafluorophosphate (HBTU), N-hydroxybenzotriazole (HOBt) and N,N-diisopropylethylamine (DIEA) in a molar ratio of 1:0.2-1.5:0.2-1.5:1.5-3 and dissolve them in a small amount of DMF. DIEA is added last, and the volume mass ratio of DMF to N-α-Fmoc protected amino acid is 5-10 mL / g; then add it to the resin obtained in step ii.i, the volume mass ratio of the mixed solution to the resin is 4-6 mL / g, and the stirring rate is 60-100 rpm; the condensation reaction is reacted at room temperature for 40-100 min. After the condensation reaction is completed, rinse the resin with DMF. Ninhydrin test, under normal circumstances, the solution is light yellow and the resin is colorless.

[0038] ii.iii Peptide chain extension: According to the peptide sequence, different Fmoc protected amino acids are connected to the obtained peptide resin in accordance with steps ii.i and ii.ii. The last amino acid is usually protected by tert-butyloxycarbonyl (Boc), and the last step of removing Fmoc is omitted. The condensation method of all amino acids is the same as described above. A linear peptide resin is obtained.

[0039] iii. Compression of resin: Wash the linear peptide resin alternately with DCM and MeOH, and drain for 3 to 5 hours.

[0040] iv. Peptide cleavage and precipitation extraction: Add a cleavage agent (trifluoroacetic acid (TFA), triisopropylsilane (Tis) and water (H2O) in a volume ratio of 95:2.5:2.5) to the resin obtained in step iii. React at room temperature for 1.5 to 4 hours, stirring every 15 minutes. The volume mass ratio of the cleavage agent to the linear peptide resin is 10 to 20 mL / g. Use a rotary evaporator to spin dry the cutting liquid at a temperature not higher than 40°C. Then place the remaining liquid in a refrigerator for precooling, add precooled ether, and centrifuge to collect the precipitate. Dissolve the precipitate with acetonitrile and water, and lyophilize to obtain the crude peptide.

[0041] vi. Purification and analysis of peptides: Use reverse phase high performance liquid chromatography (RP-HPLC) semi-preparative columns to separate and purify crude peptides, collect the main peak after separation, and obtain pure peptides after freeze-drying. Use RP-HPLC analytical columns to identify purity, and then use electrospray mass spectrometry (ESI) to identify the molecular weight of the peptide.

[0042] v. Direct derivatization of peptides: Weigh the linear pure peptide synthesized in step vi, azide or alkynyl small molecule compound, CuSO4·H2O, sodium ascorbate and tris(3-hydroxypropyltriazinemethyl)amine (THPTA) in a mixed solvent of dimethyl sulfoxide (DMSO) and water (5:95) in a ratio of 1:2:2:10:2, and shake the reaction for 1 to 3 hours at a shaking rate of 500 to 700 rpm. After the reaction, purity analysis and in vitro activity evaluation were performed directly.

[0043] In a fourth aspect of the present invention, a pharmaceutical composition is provided, comprising:

[0044] (a) the peptide ligand for neuropeptide FF receptor according to claim 1 or a pharmaceutically acceptable salt thereof as an active ingredient;

[0045] (b) pharmaceutically acceptable carriers and / or excipients.

[0046] In another preferred embodiment, the pharmaceutical composition is administered by an administration method selected from the group consisting of oral administration, transdermal administration, intrathecal administration, intravenous administration, intramuscular administration, topical administration, nasal administration, etc.

[0047] In another preferred embodiment, the preparation of the pharmaceutical composition is selected from the following group: tablets, capsules, sugar-coated tablets, granules, oral solutions and syrups, aerosols, nasal sprays, dry powder injections, injections, ointments and patches for skin surfaces.

[0048] In another preferred embodiment, the pharmaceutical composition is in the form of an injection.

[0049] In another preferred embodiment, the pharmaceutical injection can be used for parenteral administration.

[0050] In another preferred embodiment, the pharmaceutical dosage of the polypeptide of the present invention can vary within a wide range, and those skilled in the art can easily determine it based on objective factors, such as the type of disease, severity of the disease, patient weight, dosage form, route of administration, etc.

[0051] Advantages of the present invention:

[0052] (1) Compared with the parent NPVF, most compounds showed significantly enhanced agonist activity, with compound 33 showing a 297-fold increase in agonist activity;

[0053] (2) Like the non-selective antagonist RF9, compound 24 is also a non-selective antagonist of the NPFF receptor, but its antagonistic activity against NPFF1 and NPFF2 receptors is increased by 21 and 73 times, respectively;

[0054] (3) Like the NPFF1 receptor selective antagonist RF3286, compound 29 is also a NPFF1 receptor selective antagonist, but its antagonistic activity against NPFF1 receptor is increased by 1.7 times;

[0055] (4) No peptide or small molecule selective antagonists of NPFF2 receptor have been reported before this patent. This patent discloses a highly selective peptide antagonist compound 13 of NPFF2 receptor;

[0056] (5) The NPFF1 receptor-biased (Compound 29) and NPFF1 receptor non-selective antagonist (Compound 24) disclosed in the present invention can prevent the formation of acute fentanyl-induced hyperalgesia after intracerebroventricular injection; in addition, intrathecal injection of NPFF2 receptor selective antagonist (Compound 13) can prevent the formation of acute fentanyl-induced hyperalgesia.

[0057] May potentiate acute fentanyl-induced analgesic activity.

[0058] In a specific embodiment, the following neuropeptide FF receptor peptide ligand is involved, and its specific sequence is shown in Table 1:

[0059] Table 1 Amino acid sequences of NPFF receptor peptide ligands

[0060]

[0061] Table 1 Amino acid sequences of NPFF receptor peptide ligands

[0062]

[0063] Table 1 Amino acid sequences of NPFF receptor peptide ligands

[0064]

[0065] Table 1 Amino acid sequences of NPFF receptor peptide ligands

[0066] BRIEF DESCRIPTION OF THE DRAWINGS

[0067] Figure 1 is the dose-effect curve of the compound's antagonistic activity on NPFF1 receptor;

[0068] Figure 2 is the dose-effect curve of the compound's antagonistic activity on NPFF2 receptor;

[0069] Figure 3 The dose-dependent regulation of mouse body temperature by neuropeptide FF, neuropeptide VF and compound 35;

[0070] Figure 4 The third ventricle injection of RF9, compound 13, compound 24 and compound 29 regulated the cooling response caused by the third ventricle injection of NPFF;

[0071] Figure 5 Subcutaneous injection of RF9 modulates fentanyl-induced acute analgesia and hyperalgesia in a carrageenan-induced inflammatory pain model;

[0072] Figure 6 The intracerebroventricular injection of compounds 13, 24, and 29 modulates fentanyl-induced acute analgesia and hyperalgesia in the carrageenan-induced inflammatory pain model;

[0073] Figure 7 The intrathecal injection of compound 13, compound 24 and compound 29 modulates fentanyl-induced acute analgesia and hyperalgesia in the carrageenan-induced inflammatory pain model; DETAILED DESCRIPTION

[0074] The embodiments of the present invention will be described in detail below in conjunction with the examples, but it will be appreciated by those skilled in the art that the following examples are only used to illustrate the present invention and should not be considered as limiting the scope of the present invention. If no specific conditions are specified in the examples, the conditions are carried out according to normal conditions or manufacturer recommendations. If the manufacturer is not specified for the reagents or instruments used, they are all conventional products that can be obtained commercially.

[0075] The instruments and main experimental materials used are as follows:

[0076] Experimental reagents: Resin is Rink-Amide-MBHA resin (Tianjin Nankai Hecheng Company), O-benzotriazole-N,N,N',N'-tetramethyluronium-hexafluorophosphate (HBTU, Suzhou Haofan Biotechnology Co., Ltd.), N-hydroxybenzotriazole (HOBt, Suzhou Haofan Biotechnology Co., Ltd.), N,N-diisopropylethylamine (DIEA, Shanghai Anaiji Chemical Co., Ltd.), 1,8-diazabicycloundec-7-ene (DBU, Shanghai Anaiji Chemical Co., Ltd.), N,N-dimethylformamide (DMF, Sichuan Xilong Science Co., Ltd.). Ninhydrin is a product of Shanghai Reagent Factory No. 3. Deuterated dimethyl sulfoxide (Cambridge Isotope Laboratories, USA). Copper sulfate pentahydrate (CuSO4·H2O), sodium ascorbate, and tris(3-hydroxypropyltriazinemethyl)amine (THPTA) were all purchased from Shanghai Bid Pharmaceutical, dichloromethane (DCM), piperidine, methanol (MeOH), and pyridine were all purchased from Tianjin Second Reagent Factory, and trifluoroacetic acid (TFA), phenol, and pyridine were all products of Tianjin Reagent Factory I; all of the above organic reagents were redistilled before use.

[0077] Experimental instruments: solid phase peptide synthesis syringe (Biotech Shanghai Co., Ltd.), sieve plate (Shenzhen Comma Biotechnology Co., Ltd.), rotary evaporator (RE-5298A, Shanghai Yarong), freeze dryer (Ningbo Shuangjia Instrument Co., Ltd.), mass spectrometer (sciex 500QTRAPmass spectrometer, AB, USA), circulating water pump (SHB-Ⅲ, Zhengzhou Great Wall), preparative high performance liquid chromatography (LC-3050N, Shandong Runyang Instrument Co., Ltd.), preparative column (C18 reversed phase chromatography column, 19 mm×250 mm, Suzhou Mike Wangzhi), analytical high performance liquid chromatography (Waters, Delta 600), analytical column (XBridge TM BEH 130Prep C18, 19 mm×250 mm).

[0078] Example 1. Synthesis of Compound 1

[0079] (1) Resin swelling: Weigh 0.5-0.6 g of Rink-Amide-MBHA resin (substitution value 0.48-0.65 mmol / g), add 8 mL of DCM, and swell the resin at an oscillation speed of 750 rpm for 30 min. Then, drain the resin and wash it with DMF three times, each time for 3 min.

[0080] (2) Removal of Fmoc protecting group: Add 8 mL of DBU, piperidine and DMF in a volume ratio of 1:1:98 to the swollen resin; shake at 750 rpm for 5 min, repeat twice. After draining, add the above mixed solution again, shake at 750 rpm for 10 min. Finally, add DMF to wash 4 times, each time for 3 min.

[0081] (3) Ninhydrin test: The ninhydrin test reagent is a 1:2:1 volume ratio of phenol:pyridine:ninhydrin solution. Dissolve 20g of phenol in 5mL of anhydrous ethanol to prepare a phenol solution, and dissolve 0.5g of ninhydrin in 10mL of anhydrous ethanol to prepare a ninhydrin solution, wherein phenol, pyridine and anhydrous ethanol are all redistilled. In the ninhydrin test, the Fmoc is removed to expose the amino group, the solution is blue-purple, and the resin is blue-purple.

[0082] (4) Condensation of amino acids: Weigh Fmoc-Phe-OH, HOBt and HBTU in a molar ratio of 1:1:1, dissolve in 4 mL of DMF, add 2 times the molar amount of DIEA, and immediately add to the resin from which the Fmoc protecting group has been removed in step (2), and stir at 700 rpm at room temperature under argon protection. After 60 minutes, drain the reaction solution and perform an indole assay according to the method of step (3). If the solution is light yellow and the resin is colorless, it indicates that the amino acid has condensed onto the resin. Then, remove the Fmoc protecting group according to step (2), and perform an indole assay again according to step (3). If both the solution and the resin are dark blue, it indicates that the Fmoc protecting group has been completely removed, and a resin peptide without an Fmoc protecting group is obtained.

[0083] (5) Peptide chain extension: The peptide resin obtained in step (4) is condensed with Fmoc-Arg(pbf)-Phe-OH, Fmoc-Gln(Trt)-OH, Fmoc-Pro-OH, Fmoc-Gln(Trt)-OH, Fmoc-Phe-OH, Fmoc-Leu-OH and Fmoc-Pra-OH in sequence onto the peptide resin according to the method of steps (2-4) to obtain the peptide resin Pra-Leu-Phe-Gln(Trt)-Pro-Gln(Trt)-Arg(Pbf)-Phe-Resin.

[0084] (6) Compression and drying of the peptide chain: wash alternately with DCM (2×3 min), MeOH (1×3 min), DCM (1×3 min), and MeOH (2×3 min), and drain for 3 h.

[0085] (7) Peptide chain cleavage: Add 15 mL of cleavage agent (TFA:H2O:Tis=95:2.5:2.5) to the drained peptide resin Pra-Leu-Phe-Gln(Trt)-Pro-Gln(Trt)-Arg(Pbf)-Phe-Resin. React at room temperature at an oscillation speed of 100 r / min for 3 h. The filtrate is fully decompressed and spin-dried at a temperature not higher than 40°C, and ice ether is added and fully oscillated and mixed. The crude peptide is fully precipitated in the form of a white precipitate. Finally, the aqueous solution of the white precipitate is freeze-dried to obtain 390 mg of a white crude peptide solid powder, with a crude peptide yield of 97%.

[0086] (8) Purification of crude peptide: The crude peptide compound was separated and purified by reverse phase high performance liquid chromatography (RP-HPLC) C18 column (19 mm×250 mm), using acetonitrile (containing 0.1% TFA) and water (containing 0.1% TFA) by gradient elution, separating and collecting the main peak sample of the chromatogram to obtain purified compound 1, with a sample load of 100 mg. The white pure peptide solid powder was obtained by freeze drying, and the pure peptide yield was 71 mg. The mass spectrometry and chromatographic results are shown in Table 2.

[0087] Example 2. Synthesis of Compound 2

[0088] According to the same method as in Example 1, a linear peptide Phe-Pra-Phe-Gln-Pro-Gln-Arg-Phe-NH2 was obtained as a white solid powder, 345 mg of crude peptide, 100% crude peptide yield, and 73% pure peptide yield after purification. The mass spectrometry and chromatographic results are shown in Table 2.

[0089] Example 3. Synthesis of Compound 3

[0090] According to the same method as Example 1, a linear peptide Phe-Leu-Pra-Gln-Pro-Gln-Arg-Phe-NH2 was obtained as a white solid powder, 308 mg of crude peptide, 92% crude peptide yield, and 69% pure peptide yield after purification. The mass spectrometry and chromatographic results are shown in Table 2.

[0091] Example 4. Synthesis of Compound 4

[0092] According to the same method as Example 1, a linear peptide Phe-Leu-Phe-Pra-Pro-Gln-Arg-Phe-NH2 was obtained as a white solid powder, 341 mg of crude peptide, 100% crude peptide yield, and 86% pure peptide yield after purification. The mass spectrometry and chromatographic results are shown in Table 2.

[0093] Example 5. Synthesis of Compound 5

[0094] According to the same method as Example 1, a linear peptide Phe-Leu-Phe-Gln-Pra-Gln-Arg-Phe-NH2 was obtained as a white solid powder, 349 mg of crude peptide, 99% crude peptide yield, and 51% pure peptide yield after purification. The mass spectrometry and chromatographic results are shown in Table 2.

[0095] Example 6. Synthesis of Compound 6

[0096] According to the same method as in Example 1, a linear peptide Phe-Leu-Phe-Gln-Pro-Pra-Arg-Phe-NH2 was obtained as a white solid powder, 341 mg of crude peptide, 100% crude peptide yield, and 86% pure peptide yield after purification. The mass spectrometry and chromatographic results are shown in Table 2.

[0097] Example 7. Synthesis of Compound 7

[0098] According to the same method as in Example 1, a linear peptide Phe-Leu-Phe-Gln-Pro-Gln-Pra-Phe-NH2 was obtained as a white solid powder, 304 mg of crude peptide, 92% crude peptide yield, and 42% pure peptide yield after purification. The mass spectrometry and chromatographic results are shown in Table 2.

[0099] Example 8. Synthesis of Compound 8

[0100] According to the same method as Example 1, a linear peptide Phe-Leu-Phe-Gln-Pro-Gln-Arg-Pra-NH2 was obtained as a white solid powder, 318 mg of crude peptide, 95% crude peptide yield, and 79% pure peptide yield after purification. The mass spectrometry and chromatographic results are shown in Table 2.

[0101] Example 9. Synthesis of Compound 9

[0102] Direct derivatization of peptides: The linear pure peptide synthesized in Example 1, 3-azidopropylamine, CuSO4·H2O, sodium ascorbate and THPTA were weighed and dissolved in a mixed solvent of DMSO and water (5:95) in a ratio of 1:2:2:10:2, and the reaction was oscillated for 2 hours at a rate of 500-700 rpm. Purity analysis was performed directly after the reaction, and the yield of pure peptide was 95.9%.

[0103] Example 10. Synthesis of Compound 10

[0104] Direct derivatization of peptides: The linear pure peptide synthesized in Example 2, 3-azidopropylamine, CuSO4·H2O, sodium ascorbate and THPTA were weighed and dissolved in a mixed solvent of DMSO and water (5:95) in a ratio of 1:2:2:10:2, and the reaction was oscillated for 2 hours at a rate of 500-700 rpm. Purity analysis was performed directly after the reaction, and the yield of pure peptide was 98.1%.

[0105] Example 11. Synthesis of Compound 11

[0106] Direct derivatization of peptides: The linear pure peptide synthesized in Example 3, 3-azidopropylamine, CuSO4·H2O, sodium ascorbate and THPTA were weighed and dissolved in a mixed solvent of DMSO and water (5:95) in a ratio of 1:2:2:10:2, and the reaction was oscillated for 2 hours at a rate of 500-700 rpm. Purity analysis was performed directly after the reaction, and the yield of pure peptide was 96.5%.

[0107] Example 12. Synthesis of Compound 12

[0108] Direct derivatization of peptides: The linear pure peptide synthesized in Example 4, 3-azidopropylamine, CuSO4·H2O, sodium ascorbate and THPTA were weighed and dissolved in a mixed solvent of DMSO and water (5:95) in a ratio of 1:2:2:10:2, and the reaction was oscillated for 2 hours at a rate of 500-700 rpm. Purity analysis was performed directly after the reaction, and the yield of pure peptide was 95.0%.

[0109] Example 13. Synthesis of Compound 13

[0110] Direct derivatization of peptides: The linear pure peptide synthesized in Example 5, 3-azidopropylamine, CuSO4·H2O, sodium ascorbate and THPTA were weighed and dissolved in a mixed solvent of DMSO and water (5:95) in a ratio of 1:2:2:10:2, and the reaction was oscillated for 2 hours at a rate of 500-700 rpm. Purity analysis was performed directly after the reaction, and the yield of pure peptide was 96.5%.

[0111] Example 14. Synthesis of Compound 14

[0112] Direct derivatization of peptides: The linear pure peptide synthesized in Example 6, 3-azidopropylamine, CuSO4·H2O, sodium ascorbate and THPTA were weighed and dissolved in a mixed solvent of DMSO and water (5:95) in a ratio of 1:2:2:10:2, and the reaction was oscillated for 2 hours at a rate of 500-700 rpm. Purity analysis was performed directly after the reaction, and the yield of pure peptide was 97.1%.

[0113] Example 15. Synthesis of Compound 15

[0114] Direct derivatization of peptides: The linear pure peptide synthesized in Example 7, 3-azidopropylamine, CuSO4·H2O, sodium ascorbate and THPTA were weighed and dissolved in a mixed solvent of DMSO and water (5:95) in a ratio of 1:2:2:10:2, and the reaction was oscillated for 2 hours at a rate of 500-700 rpm. Purity analysis was performed directly after the reaction, and the yield of pure peptide was 94.1%.

[0115] Example 16. Synthesis of Compound 16

[0116] Direct derivatization of peptides: The linear pure peptide synthesized in Example 8, 3-azidopropylamine, CuSO4·H2O, sodium ascorbate and THPTA were weighed and dissolved in a mixed solvent of DMSO and water (5:95) in a ratio of 1:2:2:10:2, and the reaction was oscillated for 2 hours at a rate of 500-700 rpm. Purity analysis was performed directly after the reaction, and the yield of pure peptide was 98.8%.

[0117] Example 17. Synthesis of Compound 17

[0118] Direct derivatization of peptides: The linear pure peptide synthesized in Example 1, 3-azidopropionic acid, CuSO4·H2O, sodium ascorbate and THPTA were weighed and dissolved in a mixed solvent of DMSO and water (5:95) in a ratio of 1:2:2:10:2, and the reaction was oscillated for 2 hours at a rate of 500-700 rpm. Purity analysis was performed directly after the reaction, and the yield of pure peptide was 100.0%.

[0119] Example 18. Synthesis of Compound 18

[0120] Direct derivatization of peptides: The linear pure peptide synthesized in Example 2, 3-azidopropionic acid, CuSO4·H2O, sodium ascorbate and THPTA were weighed and dissolved in a mixed solvent of DMSO and water (5:95) in a ratio of 1:2:2:10:2, and the reaction was oscillated for 2 hours at a rate of 500-700 rpm. Purity analysis was performed directly after the reaction, and the yield of pure peptide was 98.1%.

[0121] Example 19. Synthesis of Compound 19

[0122] Direct derivatization of peptides: The linear pure peptide synthesized in Example 3, 3-azidopropionic acid, CuSO4·H2O, sodium ascorbate and THPTA were weighed and dissolved in a mixed solvent of DMSO and water (5:95) in a ratio of 1:2:2:10:2, and the reaction was oscillated for 2 hours at a rate of 500-700 rpm. Purity analysis was performed directly after the reaction, and the yield of pure peptide was 98.3%.

[0123] Example 20. Synthesis of Compound 20

[0124] Direct derivatization of peptides: The linear pure peptide synthesized in Example 4, 3-azidopropionic acid, CuSO4·H2O, sodium ascorbate and THPTA were weighed and dissolved in a mixed solvent of DMSO and water (5:95) in a ratio of 1:2:2:10:2, and the reaction was oscillated for 2 hours at a rate of 500-700 rpm. Purity analysis was performed directly after the reaction, and the yield of pure peptide was 98.6%.

[0125] Example 21. Synthesis of Compound 21

[0126] Direct derivatization of peptides: The linear pure peptide synthesized in Example 5, 3-azidopropionic acid, CuSO4·H2O, sodium ascorbate and THPTA were weighed and dissolved in a mixed solvent of DMSO and water (5:95) in a ratio of 1:2:2:10:2, and the reaction was oscillated for 2 hours at a rate of 500-700 rpm. Purity analysis was performed directly after the reaction, and the yield of pure peptide was 97.8%.

[0127] Example 22. Synthesis of Compound 22

[0128] Direct derivatization of peptides: The linear pure peptide synthesized in Example 6, 3-azidopropionic acid, CuSO4·H2O, sodium ascorbate and THPTA were weighed and dissolved in a mixed solvent of DMSO and water (5:95) in a ratio of 1:2:2:10:2, and the reaction was oscillated for 2 hours at a rate of 500-700 rpm. Purity analysis was performed directly after the reaction, and the yield of pure peptide was 100.0%.

[0129] Example 23. Synthesis of Compound 23

[0130] Direct derivatization of peptides: The linear pure peptide synthesized in Example 7, 3-azidopropionic acid, CuSO4·H2O, sodium ascorbate and THPTA were weighed and dissolved in a mixed solvent of DMSO and water (5:95) in a ratio of 1:2:2:10:2, and the reaction was oscillated for 2 hours at a rate of 500-700 rpm. Purity analysis was performed directly after the reaction, and the yield of pure peptide was 99.4%.

[0131] Example 24. Synthesis of Compound 24

[0132] Direct derivatization of peptides: The linear pure peptide synthesized in Example 8, 3-azidopropionic acid, CuSO4·H2O, sodium ascorbate and THPTA were weighed and dissolved in a mixed solvent of DMSO and water (5:95) in a ratio of 1:2:2:10:2, and the reaction was oscillated for 2 hours at a rate of 500-700 rpm. Purity analysis was performed directly after the reaction, and the yield of pure peptide was 100.0%.

[0133] Example 25. Synthesis of Compound 25

[0134] Direct derivatization of peptides: The linear pure peptide synthesized in Example 1, benzyl azide, CuSO4·H2O, sodium ascorbate and THPTA were weighed and dissolved in a mixed solvent of DMSO and water (5:95) in a ratio of 1:2:2:10:2, and the reaction was oscillated for 2 hours at a rate of 500-700 rpm. Purity analysis was performed directly after the reaction, and the yield of pure peptide was 99.4%.

[0135] Example 26. Synthesis of Compound 26

[0136] Direct derivatization of peptides: The linear pure peptide synthesized in Example 2, benzyl azide, CuSO4·H2O, sodium ascorbate and THPTA were weighed and dissolved in a mixed solvent of DMSO and water (5:95) in a ratio of 1:2:2:10:2, and the reaction was oscillated for 2 hours at a rate of 500-700 rpm. Purity analysis was performed directly after the reaction, and the yield of pure peptide was 100.0%.

[0137] Example 27. Synthesis of Compound 27

[0138] Direct derivatization of peptides: The linear pure peptide synthesized in Example 3, benzyl azide, CuSO4·H2O, sodium ascorbate and THPTA were weighed and dissolved in a mixed solvent of DMSO and water (5:95) in a ratio of 1:2:2:10:2, and the reaction was oscillated for 2 hours at a rate of 500-700 rpm. Purity analysis was performed directly after the reaction, and the yield of pure peptide was 97.2%.

[0139] Example 28. Synthesis of Compound 28

[0140] Direct derivatization of peptides: The linear pure peptide synthesized in Example 4, benzyl azide, CuSO4·H2O, sodium ascorbate and THPTA were weighed and dissolved in a mixed solvent of DMSO and water (5:95) in a ratio of 1:2:2:10:2, and the reaction was oscillated for 2 hours at a rate of 500-700 rpm. Purity analysis was performed directly after the reaction, and the yield of pure peptide was 100.0%.

[0141] Example 29. Synthesis of Compound 29

[0142] Direct derivatization of peptides: The linear pure peptide synthesized in Example 5, benzyl azide, CuSO4·H2O, sodium ascorbate and THPTA were weighed and dissolved in a mixed solvent of DMSO and water (5:95) in a ratio of 1:2:2:10:2, and the reaction was oscillated for 2 hours at a rate of 500-700 rpm. Purity analysis was performed directly after the reaction, and the yield of pure peptide was 98.1%.

[0143] Example 30. Synthesis of Compound 30

[0144] Direct derivatization of peptides: The linear pure peptide synthesized in Example 6, benzyl azide, CuSO4·H2O, sodium ascorbate and THPTA were weighed and dissolved in a mixed solvent of DMSO and water (5:95) in a ratio of 1:2:2:10:2, and the reaction was oscillated for 2 hours at a rate of 500-700 rpm. Purity analysis was performed directly after the reaction, and the yield of pure peptide was 100.0%.

[0145] Example 31. Synthesis of Compound 31

[0146] Direct derivatization of peptides: The linear pure peptide synthesized in Example 7, benzyl azide, CuSO4·H2O, sodium ascorbate and THPTA were weighed and dissolved in a mixed solvent of DMSO and water (5:95) in a ratio of 1:2:2:10:2, and the reaction was oscillated for 2 hours at a rate of 500-700 rpm. Purity analysis was performed directly after the reaction, and the yield of pure peptide was 98.8%.

[0147] Example 32. Synthesis of Compound 32

[0148] Direct derivatization of peptides: The linear pure peptide synthesized in Example 8, benzyl azide, CuSO4·H2O, sodium ascorbate and THPTA were weighed and dissolved in a mixed solvent of DMSO and water (5:95) in a ratio of 1:2:2:10:2, and the reaction was oscillated for 2 hours at a rate of 500-700 rpm. Purity analysis was performed directly after the reaction, and the yield of pure peptide was 100.0%.

[0149] Example 33. Synthesis of Compound 33

[0150] According to the same method as in Example 1, a linear peptide Val-His-Phe-Pra-Pro-Gln-Arg-Phe-NH2 was obtained as a white solid powder, 315 mg of crude peptide, 95% crude peptide yield, and 62% pure peptide yield after purification. The mass spectrometry and chromatographic results are shown in Table 2.

[0151] Example 34. Synthesis of Compound 34

[0152] According to the same method as in Example 1, a linear peptide Nva-His-Phe-Pra-Pro-Gln-Arg-Phe-NH2 was obtained as a white solid powder, 283 mg of crude peptide, 85% crude peptide yield, and 35% pure peptide yield after purification. The mass spectrometry and chromatographic results are shown in Table 2.

[0153] Example 35. Synthesis of Compound 35

[0154] According to the same method as in Example 1, a linear peptide Ala-His-Phe-Pra-Pro-Gln-Arg-Phe-NH2 was obtained as a white solid powder, 416 mg of crude peptide, 100% crude peptide yield, and 47% pure peptide yield after purification. The mass spectrometry and chromatographic results are shown in Table 2.

[0155] Example 36. Synthesis of Compound 36

[0156] According to the same method as in Example 1, a linear peptide Gly-His-Phe-Pra-Pro-Gln-Arg-Phe-NH2 was obtained as a white solid powder, 362 mg of crude peptide, 95% crude peptide yield, and 25% pure peptide yield after purification. The mass spectrometry and chromatographic results are shown in Table 2.

[0157] Example 37. Synthesis of Compound 37

[0158] According to the same method as Example 1, a linear peptide AllylGly-His-Phe-Pra-Pro-Gln-Arg-Phe-NH2 was obtained as a white solid powder, 349 mg of crude peptide, 83% crude peptide yield, and 26% pure peptide yield after purification. The mass spectrometry and chromatographic results are shown in Table 2.

[0159] Example 38. Synthesis of Compound 38

[0160] According to the same method as in Example 1, a linear peptide Val-Trp-Phe-Pra-Pro-Gln-Arg-Phe-NH2 was obtained as a white solid powder, 306 mg of crude peptide, 73% crude peptide yield, and 22% pure peptide yield after purification. The mass spectrometry and chromatographic results are shown in Table 2.

[0161] Example 39. Synthesis of Compound 39

[0162] According to the same method as in Example 1, a linear peptide Val-Tpi-Phe-Pra-Pro-Gln-Arg-Phe-NH2 was obtained as a white solid powder, 264 mg of crude peptide, 76% crude peptide yield, and 24% pure peptide yield after purification. The mass spectrometry and chromatographic results are shown in Table 2.

[0163] Example 40. Synthesis of Compound 40

[0164] According to the same method as Example 1, a linear peptide Val-HomoPhe-Phe-Phe-Pra-Pro-Gln-Arg-Phe-NH2 was obtained as a white solid powder, 270 mg of crude peptide, 89% crude peptide yield, and 32% pure peptide yield after purification. The mass spectrometry and chromatographic results are shown in Table 2.

[0165] Example 41. Synthesis of Compound 41

[0166] According to the same method as Example 1, a linear peptide Val-2-Nal-Phe-Pra-Pro-Gln-Arg-Phe-NH2 was obtained as a white solid powder, 287 mg of crude peptide, 92% crude peptide yield, and 15% pure peptide yield after purification. The mass spectrometry and chromatographic results are shown in Table 2.

[0167] Example 42. Synthesis of Compound 42

[0168] According to the same method as in Example 1, a linear peptide Val-4-Bpa-Phe-Pra-Pro-Gln-Arg-Phe-NH2 was obtained as a white solid powder, 201 mg of crude peptide, 55% crude peptide yield, and 29% pure peptide yield after purification. The mass spectrometry and chromatographic results are shown in Table 2.

[0169] Example 43. Synthesis of Compound 43

[0170] According to the same method as in Example 1, a linear peptide Val-His-2-Nal-Pra-Pro-Gln-Arg-Phe-NH2 was obtained as a white solid powder, 216 mg of crude peptide, 62% crude peptide yield, and 43% pure peptide yield after purification. The mass spectrometry and chromatographic results are shown in Table 2.

[0171] Example 44. Synthesis of Compound 44

[0172] According to the same method as Example 1, a linear peptide Val-His-4-Bpa-Pra-Pro-Gln-Arg-Phe-NH2 was obtained as a white solid powder, 340 mg of crude peptide, 93% crude peptide yield, and 66% pure peptide yield after purification. The mass spectrometry and chromatographic results are shown in Table 2.

[0173] Example 45. Synthesis of Compound 45

[0174] According to the same method as Example 1, a linear peptide Val-His-HomoPhe-Pra-Pro-Gln-Arg-Phe-NH2 was obtained as a white solid powder, 289 mg of crude peptide, 86% crude peptide yield, and 37% pure peptide yield after purification. The mass spectrometry and chromatographic results are shown in Table 2.

[0175] Example 46. Synthesis of Compound 46

[0176] According to the same method as Example 1, a linear peptide Val-His-Trp-Pra-Pro-Gln-Arg-Phe-NH2 was obtained as a white solid powder, 253 mg of crude peptide, 88% crude peptide yield, and 39% pure peptide yield after purification. The mass spectrometry and chromatographic results are shown in Table 2.

[0177] Example 47. Synthesis of Compound 47

[0178] According to the same method as in Example 1, a linear peptide Ala-2-Nal-Phe-Pra-Pro-Gln-Arg-Phe-NH2 was obtained as a white solid powder, 266 mg of crude peptide, 93% crude peptide yield, and 35% pure peptide yield after purification. The mass spectrometry and chromatographic results are shown in Table 2.

[0179] Example 48. Synthesis of Compound 48

[0180] Direct derivatization of peptides: The linear pure peptide synthesized in Example 33, 3-azidopropylamine, CuSO4·H2O, sodium ascorbate and THPTA were weighed and dissolved in a mixed solvent of DMSO and water (5:95) in a ratio of 1:2:2:10:2, and the reaction was oscillated for 2 hours at a rate of 500-700 rpm. Purity analysis was performed directly after the reaction, and the yield of pure peptide was 96.9%.

[0181] Example 49. Synthesis of Compound 49

[0182] Direct derivatization of peptides: The linear pure peptide synthesized in Example 33, 3-azidopropionic acid, CuSO4·H2O, sodium ascorbate and THPTA were weighed and dissolved in a mixed solvent of DMSO and water (5:95) in a ratio of 1:2:2:10:2, and the reaction was oscillated for 2 hours at a rate of 500-700 rpm. Purity analysis was performed directly after the reaction, and the yield of pure peptide was 97.4%.

[0183] Example 50. Synthesis of Compound 50

[0184] Direct derivatization of peptides: The linear pure peptide synthesized in Example 33, benzyl azide, CuSO4·H2O, sodium ascorbate and THPTA were weighed and dissolved in a mixed solvent of DMSO and water (5:95) in a ratio of 1:2:2:10:2, and the reaction was oscillated for 2 hours at a rate of 500-700 rpm. Purity analysis was performed directly after the reaction, and the yield of pure peptide was 100.0%.

[0185] Example 51. Synthesis of Compound 51

[0186] According to the same method as Example 1, a linear peptide Val-His-Phe-Ala(N3)-Pro-Gln-Arg-Phe-NH2 was obtained as a white solid powder, 182 mg of crude peptide, 65% crude peptide yield, and 19% pure peptide yield after purification. The mass spectrometry and chromatographic results are shown in Table 2.

[0187] Example 52. Synthesis of Compound 52

[0188] Direct derivatization of peptides: The linear pure peptide synthesized in Example 51, propargylamine, CuSO4·H2O, sodium ascorbate and THPTA were weighed and dissolved in a mixed solvent of DMSO and water (5:95) in a ratio of 1:2:2:10:2, and the reaction was oscillated for 2 hours at a rate of 500-700 rpm. Purity analysis was performed directly after the reaction, and the yield of pure peptide was 94.4%.

[0189] Example 53. Synthesis of Compound 53

[0190] Direct derivatization of peptides: The linear pure peptide synthesized in Example 51, propargyl acid, CuSO4·H2O, sodium ascorbate and THPTA were weighed and dissolved in a mixed solvent of DMSO and water (5:95) in a ratio of 1:2:2:10:2, and the reaction was oscillated for 2 hours at a rate of 500-700 rpm. Purity analysis was performed directly after the reaction, and the yield of pure peptide was 96.2%.

[0191] Example 54. Synthesis of Compound 54

[0192] Direct derivatization of peptides: The linear pure peptide synthesized in Example 51, phenylacetylene, CuSO4·H2O, sodium ascorbate and THPTA were weighed and dissolved in a mixed solvent of DMSO and water (5:95) in a ratio of 1:2:2:10:2, and the reaction was oscillated for 2 hours at a rate of 500-700 rpm. Purity analysis was performed directly after the reaction, and the yield of pure peptide was 95.2%.

[0193] Example 55. Synthesis of Compound 55

[0194] Direct derivatization of peptides: The linear pure peptide synthesized in Example 51, 4-pentyne-1-ethanol, CuSO4·H2O, sodium ascorbate and THPTA were weighed and dissolved in a mixed solvent of DMSO and water (5:95) in a ratio of 1:2:2:10:2, and the reaction was oscillated for 2 hours at a rate of 500-700 rpm. Purity analysis was performed directly after the reaction, and the yield of pure peptide was 91.9%.

[0195] Example 56. Synthesis of Compound 56

[0196] Direct derivatization of peptides: The linear pure peptide synthesized in Example 51, propargyl alcohol ethoxylate, CuSO4·H2O, sodium ascorbate and THPTA were weighed and dissolved in a mixed solvent of DMSO and water (5:95) in a ratio of 1:2:2:10:2, and the reaction was oscillated for 2 hours at a rate of 500-700 rpm. Purity analysis was performed directly after the reaction, and the yield of pure peptide was 97.6%.

[0197] Table 2 Mass spectrometry and purity analysis of analogs of the present invention

[0198]

[0199] Table 2 Mass spectra and purity analysis of analogs of the present invention

[0200]

[0201] Note: System: Gradient elution system 1 is: 10-80% acetonitrile / water (0.1% TFA) (30 min to complete), flow rate is: 1 mL / min, detection wavelength is 220 nm, analytical column is: XBridge TM BEH 130Prep C18, 4.6 mm × 250 mm;

[0202] Example 57. In vitro functional activity assay of NPFF receptor modulating peptide:

[0203] Using CHO-K1 / G cells that stably and highly express NPFF1 receptor α 15 cell line and HEK293 cell line of NPFF2 receptor, and the agonist activity of the compound on NPFF receptor was detected by detecting the regulation of intracellular calcium ion mobilization by NPFF receptor modulating peptide.

[0204] Experimental method: 96-well plates were seeded with 30,000 to 50,000 cells per well and cultured for more than 20 hours. At the beginning of the experiment, the culture medium in the culture dish was aspirated, and then 50 μL of calcium dye storage solution containing 2.5 mM probenecid, 0.9 μM Fluo-4-AM and F127 (0.03%) was added, and incubated at 37°C for 30 minutes. After the incubation, the dye was aspirated and 75 μL of HBSS buffer (containing 2.5 mM probenecid) was added. Then the 96-well drug addition plate, 96-well cell plate and automatic drug addition pipette tip were placed in the FlexStation 3 calcium flow workstation to detect changes in calcium signals after drug administration.

[0205] The calcium mobilization effect was expressed as the percentage increase of intracellular calcium ions by the drug (% control), % control = (detection value - basal value) / basal value * 100. The relevant % control data were expressed as mean ± standard error (Means ± SEM). The dose-effect relationship of the compound was statistically analyzed using a nonlinear regression model, and the EC values ​​of NPFF modulating peptides on intracellular calcium ion mobilization were calculated using the statistical software GraphPadPrism version 8.0.1. 50 The experimental results are shown in Table 3.

[0206] Table 3 Calcium mobilization experiment of NPFF regulatory peptides of the present invention on NPFF receptors

[0207]

[0208] Table 3 Calcium mobilization experiment of NPFF regulatory peptide of the present invention on NPFF receptor

[0209]

[0210] The experimental results showed that in cells stably expressing NPFF1 receptor, all compounds except compounds 8, 13, 15, 16, 22, 23, 24, 29, 30, 31 and 32 showed dose-dependent NPFF1 receptor agonist activity. In cells stably expressing NPFF2 receptor, all compounds except compounds 15, 16, 22, 23, 24, 30, 31 and 32 showed dose-dependent NPFF2 receptor agonist activity.

[0211] Among all the compounds, the agonist activity of NPFF1 receptor of compounds 4, 33, 35, 36, 37, 38, 41 and 50 increased by 30, 883, 631, 98, 40, 28, 23 and 14 times, respectively. Among all the compounds, only some of them had improved agonist activity on NPFF2 receptor. The agonist activity of NPFF2 receptor of compounds 13, 20, 25, 26, 39 and 40 increased by 2.4, 2.1, 2.1, 1.7, 6.4 and 9.9 times, respectively.

[0212] Example 58. In vitro antagonist activity assay of NPFF receptor modulating peptides:

[0213] Based on the results of Example 57, we evaluated the antagonist activity of all compounds that did not have agonist activity.

[0214] Experimental method: 96-well plates were seeded with 30,000 to 50,000 cells per well and cultured for more than 20 hours. At the beginning of the experiment, the culture medium in the culture dish was aspirated, and then 50 μL of calcium dye storage solution containing 2.5 mM probenecid, 0.9 μM Fluo-4-AM and F127 (0.03%) was added, and incubated at 37°C for 30 minutes. After the incubation, the dye was aspirated and 25 μL of the compound (antagonist, pretreated for 10 minutes) and 50 μL of HBSS buffer (containing 2.5 mM probenecid) were added. Then the 96-well drug addition plate, 96-well cell plate and automatic drug addition pipette tip were placed in the FlexStation 3 calcium flow workstation to detect changes in calcium signals after drug administration.

[0215] The calcium mobilization effect was expressed as the percentage increase of intracellular calcium ions by the drug (% control), % control = (test value - basal value) / basal value * 100. The relevant % control data were expressed as mean ± standard error (Means ± SEM). The antagonistic activity of the compound was calculated using the EC of Gaddum / Schild using the statistical software GraphPad Prism 8.0.1 version.50 The pA2 values ​​of NPFF antagonists on NPFF-mediated intracellular calcium ion mobilization were calculated. The experimental results are shown in Figure 1-2 And as shown in Table 4.

[0216] Table 4 pA2 values ​​of NPFF receptor peptide antagonists

[0217]

[0218] It can be seen from the experimental results that all compounds exhibit antagonist activity against NPFF receptors. Like the NPFF receptor non-selective antagonist RF9, compound 24 is also a NPFF receptor non-selective antagonist, but its antagonist activity against NPFF1 and NPFF2 receptors is increased by 21 and 73 times, respectively; like the NPFF1 receptor selective antagonist RF3286, compound 29 is also a NPFF1 receptor selective antagonist, but its antagonist activity against NPFF1 receptor is increased by 1.7 times; this patent has not previously reported peptide or small molecule selective antagonists for NPFF2 receptors, and this patent discloses a highly selective peptide antagonist compound 13 for NPFF2 receptors.

[0219] Example 59. Regulation of body temperature by the advantageous compound 35 and antagonists:

[0220] Rectal temperature measurement of mice is a common experimental operation used to detect changes in mouse body temperature. The following are the detailed steps of the mouse rectal temperature measurement method: Prepare tools and materials: temperature probe, gloves, sterile lubricant. Put on gloves and take the mouse out of the cage. Fix the mouse in the experimental fixture and gently grab the mouse's tail with one hand so that the mouse cannot move. Pick up the temperature probe with the other hand and apply an appropriate amount of sterile lubricant (vaseline) to it. Insert the temperature probe into the mouse's anus to a depth of about 2.5 cm.

[0221] The effect of drugs on body temperature was analyzed using one-way ANOVA (Dunnett's test). * P<0.05, ** P < 0.01 and *** P<0.001 indicates that the difference between the group only injected with the relevant drug and the saline group is significant. # P<0.05, ## P < 0.01 and ### P<0.001 indicates that the difference between the group only injected with related drugs and the NPFF group and the group only injected with NPFF is significant.

[0222] like Figure 3 As shown, intracerebral injection of NPFF (A), NPVF (B) and compound 35 (C) all produced a dose-dependent hypothermic response, with the ED50 They are 14.2, 12.3 and 6.6 nmol respectively.

[0223] like Figure 4 As shown, pretreatment of the third ventricle with RF9, compound 13, compound 24 and compound 29 had no effect on the body temperature of mice, while pretreatment with RF9, compound 13, compound 24 and compound 29 could significantly antagonize the cooling response caused by intracerebroventricular injection of 45 nmol NPFF. This study result shows that both NPFF1 receptor and NPFF2 receptor are involved in the cooling response mediated by the NPFF system.

[0224] Example 60. Antagonists Modulate Fentanyl-Induced Analgesia and Hyperalgesia:

[0225] Calcium mobilization experiments showed that compound 13 is a selective antagonist of NPFF2 receptors, compound 24 is a non-selective antagonist of NPFF1 and NPFF2 receptors, and compound 29 is a biased antagonist of NPFF1 receptors. Previous studies by our research group have shown that injection of NPFF1 receptor selective and NPFF1 receptor / NPFF2 receptor non-selective antagonists above the spinal cord can reverse acute fentanyl-induced hyperalgesia. Spinal injection of NPFF2 receptor antagonists can enhance the analgesic effect induced by low-dose fentanyl injection at the spinal cord level. Therefore, this patent conducts further research on the obtained antagonists.

[0226] The carrageenan-induced foot swelling model is used to evaluate the analgesic and allodynic effects of drugs in inflammatory pain. Carrageenan is a mucopolysaccharide isolated from the cell wall of algae, which causes an inflammatory response by upregulating the level of prostaglandin E2 (PGE2) at the injection site. Before the establishment of the inflammatory pain model, the paw withdrawal thresholds of the left and right hind paws of mice for mechanical stimulation were measured respectively, and each mouse was measured 5 times, with an interval of 2 minutes between each stimulation. Subsequently, the mice were anesthetized with 1.4% isoflurane, and 20 μL of 2% carrageenan solution was slowly injected into the subcutaneous part of the right hind paw of the mice using a microsyringe. After the injection, the needle was rotated and the needle hole was pressed to prevent the solution from overflowing. The mice were used for subsequent analgesic experiments 24 hours after the injection of the carrageenan solution.

[0227] The acute hyperalgesia model was induced by subcutaneous injection of 0.06 mg / kg fentanyl for 4 consecutive times with an interval of 15 min; 10 nmol of compound 13, compound 24 and compound 29 were injected 20 min before fentanyl to evaluate the regulation of acute fentanyl-induced analgesia and hyperalgesia. In addition, we chose to pre-treat RF9 with 5 mg / kg subcutaneously as a control. Hyperalgesia was measured for 5 consecutive days after administration.

[0228] The effect of drugs on analgesia was analyzed using two-way ANOVA (Bonferroni test).* P<0.05, ** P<0.01, *** P<0.001 indicates that the difference between the group injected with saline and fentanyl and the group injected with saline only and saline only is significant. # P<0.05, ## P < 0.01 and ### P<0.001 indicates that the difference between the group injected with only the relevant drugs and fentanyl and the group injected with fentanyl and saline is significant. The regulation of pain sensitivity by drugs was analyzed using two-way ANOVA (Tukey test). * P<0.05, ** P<0.01, *** P<0.001 indicates a significant difference from the baseline value on the first day. # P<0.05, ## P < 0.01 and ### P<0.001 indicates that the difference between the group injected with only the relevant drugs and fentanyl and the fentanyl group and the saline group is significant.

[0229] like Figure 5 As shown in A, subcutaneous injection of RF9 significantly enhanced the analgesic activity of multiple subcutaneous injections of fentanyl. On the second day, mice in the fentanyl-only group developed acute pain sensitivity, which did not return to baseline until the fifth day. Figure 5 As shown in B, subcutaneous injection of RF9 can significantly reverse fentanyl-induced acute hyperalgesia.

[0230] like Figure 6 As shown in A, intracerebroventricular injection of compounds 13, 24, and 29 had no significant modulation on the analgesic activity of multiple subcutaneous injections of fentanyl. On the second day, mice in the fentanyl-only group developed acute pain sensitivity, which did not return to baseline until the fifth day. Figure 6 As shown in Figure B, intracerebroventricular pretreatment with compounds 24 and 29 could reverse fentanyl-induced acute hyperalgesia, but compound 13 could not.

[0231] like Figure 7 As shown in A, intrathecal injection of compounds 13 and 24 significantly enhanced the analgesic activity of multiple subcutaneous injections of fentanyl, while compound 29 did not. On the second day, mice in the fentanyl-only group developed acute pain sensitivity, which did not return to baseline until the fifth day. Figure 7 As shown in B, intrathecal pretreatment with compounds 13, 24, and 29 could not reverse fentanyl-induced acute hyperalgesia.

[0232] The above specific description of the present invention does not limit the present invention, and those skilled in the art can make various Such changes and modifications, as long as they do not depart from the spirit of the present invention, should fall within the scope of the appended claims of the present invention.

Claims

1. A peptide ligand targeting neuropeptide FF receptor or a pharmaceutically acceptable salt thereof, wherein the molecular structure is as shown in Formula I, II, III, IV and V: Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2(I) The amino acids at positions 1-8 were scanned by Pra substitution; In another preferred embodiment, the neuropeptide FF receptor peptide ligand is a triazole derivative generated by a click chemistry reaction between Pra of structural formula (I) and azide compounds, and the general structural formula (II) only lists the general formula of Pra at position 1, and the general structural formulas of the other positions are basically the same as (II). Pra is introduced into different amino acid sites for replacement and derivatization. In the formula, R1 is R2 is R3 is In another preferred embodiment, the general structural formula of the neuropeptide FF receptor peptide ligand is shown as (III). Xaa1-Xaa2-Xaa3-Pra-Pro-Gln-Arg-Phe-NH2(Ⅲ) where, Xaa1 is selected from the group consisting of Val, Nva, Ala, Gly, AllylGly; Xaa2 is selected from Trp, Tpi, HomoPhe, 2-Nal, 4-Bpa; Xaa3 is selected from 2-Nal, 4-Bpa, HomoPhe, 2-Trp, 2-Nal; In another preferred embodiment, the general structural formula of the neuropeptide FF receptor peptide ligand is shown as (IV). In the formula, R1 is R2 is R3 is In another preferred embodiment, the general structural formula of the neuropeptide FF receptor peptide ligand is shown as (V). X1 is: X2 is: X3 is: X4 is: X5 is:

2. The peptide ligand for the neuropeptide FF receptor according to claim 1, characterized in that The peptide ligand for the neuropeptide FF receptor is selected from the following compounds: Compound 1: Pra-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2 Compound 2: Phe-Pra-Phe-Gln-Pro-Gln-Arg-Phe-NH2 Compound 3: Phe-Leu-Pra-Gln-Pro-Gln-Arg-Phe-NH2 Compound 4: Phe-Leu-Phe-Pra-Pro-Gln-Arg-Phe-NH2 Compound 5: Phe-Leu-Phe-Gln-Pra-Gln-Arg-Phe-NH2 Compound 6: Phe-Leu-Phe-Gln-Pro-Pra-Arg-Phe-NH2 Compound 7: Phe-Leu-Phe-Gln-Pro-Gln-Pra-Phe-NH2 Compound 8: Phe-Leu-Phe-Gln-Pro-Gln-Arg-Pra-NH2 Compound 9: Compound 10: Compound 11: Compound 12: Compound 13: Compound 14: Compound 15: Compound 16: Compound 17: Compound 18: Compound 19: Compound 20: Compound 21: Compound 22: Compound 23: Compound 24: Compound 25: Compound 26: Compound 27: Compound 28: Compound 29: Compound 30: Compound 31: Compound 32: Compound 33: Val-His-Phe-Pra-Pro-Gln-Arg-Phe-NH2 Compound 34: Nva-His-Phe-Pra-Pro-Gln-Arg-Phe-NH2 Compound 35: Ala-His-Phe-Pra-Pro-Gln-Arg-Phe-NH2 Compound 36: Gly-His-Phe-Pra-Pro-Gln-Arg-Phe-NH2 Compound 37: AllylGly-His-Phe-Pra-Pro-Gln-Arg-Phe-NH2 Compound 38: Val-Trp-Phe-Pra-Pro-Gln-Arg-Phe-NH2 Compound 39: Val-Tpi-Phe-Pra-Pro-Gln-Arg-Phe-NH2 Compound 40: Val-HomoPhe-Phe-Pra-Pro- Gln-Arg-Phe-NH2 Compound 41: Val-2-Nal-Phe-Pra-Pro-Gln-Arg-Phe-NH2 Compound 42: Val-4-Bpa-Phe-Pra-Pro-Gln-Arg-Phe-NH2 Compound 43: Val-His-2-Nal-Pra-Pro-Gln-Arg-Phe-NH2 Compound 44: Val-His-4-Bpa-Pra-Pro-Gln-Arg-Phe-NH2 Compound 45: Val-His-HomoPhe-Pra-Pro-Gln-Arg-Phe-NH2 Compound 46: Val-His-Trp-Pra-Pro-Gln-Arg-Phe-NH2 Compound 47: Ala-2-Nal-Phe-Pra-Pro-Gln-Arg-Phe-NH2 Compound 48: Compound 49: Compound 50: Compound 51: Val-His-Phe-Ala(N3)-Pro-Gln-Arg-Phe-NH2 Compound 52: Compound 53: Compound 54: Compound 55: Compound 56:

3. The method for synthesizing a peptide ligand targeting a neuropeptide FF receptor or a pharmaceutically acceptable salt thereof according to claim 1, characterized in that: Includes steps: (a) using the Fmoc solid phase synthesis method to synthesize a peptide chain according to the amino acid sequence of claim 1, thereby obtaining the peptide ligand for the neuropeptide FF receptor of claim 1 or a pharmaceutically acceptable salt thereof. (b) A linear peptide containing Pra is subjected to a cycloaddition reaction with 3-azidopropylamine, 3-azidopropionic acid and benzyl azide via a copper-catalyzed click chemistry reaction to directly derive a series of triazole-containing peptide ligands of the neuropeptide FF receptor or their pharmaceutically acceptable salts. (c) A linear peptide containing Ala(N3) is subjected to a cycloaddition reaction with propargylamine, propargyl acid, phenylacetylene, 4-pentyn-1-ol and propargyl alcohol ethoxylates via a copper-catalyzed click chemistry reaction to directly derive a series of peptide ligands of the neuropeptide FF receptor containing triazole or their pharmaceutically acceptable salts.

4. The method for preparing a peptide ligand targeting neuropeptide FF receptor or a pharmaceutically acceptable salt thereof according to claim 3, characterized in that: The Fmoc solid phase peptide synthesis method comprises the following process steps: swelling of resin, condensation of amino acids, extension of peptide chains, compression, drying and cutting of peptide chains, purification and chemical identification of peptides, or a combination thereof.

5. The method for preparing a peptide ligand targeting neuropeptide FF receptor or a pharmaceutically acceptable salt thereof according to claim 3, characterized in that: The copper-catalyzed click chemistry reaction includes copper sulfate pentahydrate, sodium ascorbate and tris(3-hydroxypropyltriazolemethyl)amine, and a mixture of water and dimethyl sulfoxide is used as a solvent.

6. The method for preparing a peptide ligand targeting neuropeptide FF receptor or a pharmaceutically acceptable salt thereof according to claim 3, characterized in that: The solid phase carrier is Rink-Amide-MBHA resin.

7. The method for preparing a peptide ligand targeting neuropeptide FF receptor or a pharmaceutically acceptable salt thereof according to claim 3, characterized in that: The condensation reagent used in the amino acid condensation step is a combination of HOBt, HBTU and DIEA.

8. A pharmaceutical composition comprising the peptide ligand for the neuropeptide FF receptor according to any one of claim 1, and a pharmaceutically acceptable carrier.

9. The pharmaceutical composition according to claim 8, further comprising an opioid.

10. The pharmaceutical composition according to claim 9, wherein the opioid comprises at least one selected from the group consisting of fentanyl, morphine, oxycodone, hydrocodone and buprenorphine.

11. The pharmaceutical composition according to claim 8, further comprising an antipsychotic drug.

12. The pharmaceutical composition according to claim 11, wherein the antipsychotic drug comprises at least one selected from the group consisting of haloperidol and aripiprazole.

13. The pharmaceutical composition according to claim 8, further comprising a monoamine reuptake inhibitor.

14. The pharmaceutical composition according to claim 11, wherein the antipsychotic drug comprises at least one selected from the group consisting of fluoxetine and sertraline.

15. A method for treating a subject suffering from or susceptible to a condition or disorder in which modulation of neuropeptide FF receptor activity has a therapeutic benefit, the method comprising administering to the subject suffering from or susceptible to the condition or disorder a therapeutically effective amount of a neuropeptide FF receptor peptide ligand according to any one of claim 1.

16. The method of claim 15, wherein the condition or disorder comprises the use or abuse of one or more opioid medications.

17. The method of claim 16, wherein the one or more opioid drugs include at least one selected from the group consisting of fentanyl, morphine, oxycodone, hydrocodone, buprenorphine, heroin, and opioid derivatives of the foregoing.

18. The method of claim 15, wherein the therapeutic benefit comprises at least partial alleviation of opioid-induced hyperalgesia and addiction.

19. The method of claim 18, wherein the opioid-induced hyperalgesia is induced by an opioid drug comprising at least one selected from the group consisting of fentanyl, morphine, oxycodone, hydrocodone, and buprenorphine.

20. The method of claim 15, wherein the administration of the neuropeptide FF receptor ligand of any one of claims 1 is performed in a therapeutic intervention comprising co-administering a drug that mitigates side effects of the neuropeptide FF receptor ligand.

21. The method of claim 20, wherein the drug whose side effects are alleviated by the neuropeptide FF receptor ligand is a drug that produces tolerance, addiction and hyperalgesia as side effects.

22. The method of claim 15, further comprising administering an effective amount of a second therapeutically effective dose.

23. The method of claim 15, wherein the condition or disorder in which modulation of neuropeptide FF receptor activity has a therapeutic benefit is selected from the group consisting of reduction of opioid tolerance, reduction of opioid addiction, and reduction of hyperalgesia.

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