WO2022188628A1 - 一类阿片/神经肽ff受体多靶点环肽分子及其制备和应用 - Google Patents

一类阿片/神经肽ff受体多靶点环肽分子及其制备和应用 Download PDF

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WO2022188628A1
WO2022188628A1 PCT/CN2022/077309 CN2022077309W WO2022188628A1 WO 2022188628 A1 WO2022188628 A1 WO 2022188628A1 CN 2022077309 W CN2022077309 W CN 2022077309W WO 2022188628 A1 WO2022188628 A1 WO 2022188628A1
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phe
cyclic peptide
receptors
compound
peptide molecule
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French (fr)
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方泉
张梦娜
许彪
张润
李宁
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上海天慈生命科学发展有限公司
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/64—Cyclic peptides containing only normal peptide links
    • 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
    • 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
    • A61K38/12—Cyclic peptides, e.g. bacitracins; Polymyxins; Gramicidins S, C; Tyrocidins A, B or C
    • 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
    • A61P25/04—Centrally acting analgesics, e.g. opioids
    • 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]
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/04—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length on carriers
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/06—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length using protecting groups or activating agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/16—Extraction; Separation; Purification by chromatography
    • C07K1/20—Partition-, reverse-phase or hydrophobic interaction chromatography
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/30—Extraction; Separation; Purification by precipitation
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/14—Extraction; Separation; Purification
    • C07K1/36—Extraction; Separation; Purification by a combination of two or more processes of different types
    • 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
    • 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 invention belongs to the technical field of biochemistry, and in particular relates to a class of multi-target cyclic peptide molecules targeting opioid receptors and neuropeptide FF receptors and their preparation and use.
  • Morphine and fentanyl are the most common opioid analgesics for the treatment of acute and chronic pain, but the side effects of long-term use severely limit the clinical application of these drugs, such as respiratory depression, tolerance, abuse and constipation Wait. Therefore, it is of great significance to explore and develop new opioid analgesics with high efficiency and low side effects.
  • the developed new opioid analgesic drugs can effectively reduce the side effects associated with traditional opioids, and have potential application prospects in the development of new analgesic drugs with high efficiency and low side effects.
  • the multi-targeted agonists BU08028, AT-121 and BU10038 developed targeting the opioid/nociceptin (NOP) receptor demonstrated potent analgesia in non-human primates with adverse drug effects Lower;
  • Cebranopadol, a multi-targeted agonist of opioid/NOR receptors is in phase II clinical study for the treatment of pathological pain such as postoperative pain, diabetes-induced peripheral neuropathy, and cancer pain.
  • Neuropeptide FF is an opioid-modulating peptide that exhibits anti-opioid activity at the supraspinal level, but produces opioid analgesia and potentiates opioid-induced analgesia at the spinal level.
  • NPFF neuropeptide FF
  • Patent 201610252648.7 discloses a new chimeric peptide DN-9 based on opioid peptides and NPFF. In vitro functional experiments show that the chimeric peptide acts as a multi-target agonist of opioid and NPFF receptors.
  • DN- 9 Can produce stronger analgesic effects than morphine at both the central and peripheral levels.
  • opioid side effects such as addiction, analgesic tolerance, and constipation were significantly reduced in DN-9 compared with morphine (J Med Chem. 2016, 59:10198-10208; J Pain. 2020, 21:477- 493; Br J Pharmacol. 2020, 177:93-109).
  • the reported druggability of DN-9 still has room for further improvement, such as in terms of analgesic activity and analgesic time.
  • Polypeptide drugs are a class of drugs with rapid growth in the market. Up to now, nearly 100 peptide drugs have been approved for the market worldwide, and about 200 new peptide drugs have entered the preclinical and clinical research stages. However, the instability of the polypeptide drug itself is a key technical problem that limits its further development. Studies have shown that chemical modification strategies such as non-natural amino acid substitution modification, cyclization, PEGylation and glycosylation can effectively improve the stability, receptor selectivity and bioavailability of polypeptide molecules, and further improve their druggability. .
  • the main purpose of the present invention is to provide a multi-targeted cyclic peptide molecule or a pharmaceutically acceptable salt thereof for opioid receptors and neuropeptide FF receptors.
  • Another object of the present invention is to provide a preparation method of the above-mentioned multi-target cyclic peptide molecule or a pharmaceutically acceptable salt thereof.
  • Another object of the present invention is to provide therapeutic use of the above-mentioned multi-target cyclic peptide molecule or a pharmaceutically acceptable salt thereof.
  • a multi-targeted cyclic peptide molecule or a pharmaceutically acceptable salt thereof for opioid receptors and neuropeptide FF receptors the structure of the cyclic peptide molecule is shown in formula I :
  • Xaa2 is Lys, D-Lys, D-Asp, D-Glu, D-Orn, D-Dab or D-Dap;
  • Xaa5 is Asp, D-Asp, Glu, Lys;
  • Xaa6 is Pro or Gly
  • Xaa7 is Gln, ⁇ -Ala or Aib;
  • Xaa9 is Phe or Cha
  • c [2,5] indicates that there is a cyclic covalent bond between the two amino acid residues Xaa2 and Xaa5 in the amino acid sequence.
  • the cyclic covalent bond between the two amino acid residues of Xaa2 and Xaa5 includes forming an amide bond through dehydration condensation.
  • Xaa2, Xaa5, Xaa6, Xaa7, and Xaa9 are as defined above;
  • ⁇ L0 ⁇ represents the cyclic covalent bond between the two amino acid residues of Xaa2 and Xaa5.
  • the cyclic peptide molecule is selected from the following compounds:
  • the cyclic peptide molecule shown in the formula I has one or more features selected from the following group:
  • the cyclic peptide molecule shown in the formula I is a dual-target agonist of opioid receptor and NPFF receptor;
  • the analgesic activity of the subcutaneous injection of the cyclic peptide molecule shown in the formula I is more than 10 times, preferably more than 100 times, more preferably more than 500 times, 1000 times, 10000 times or 10 times compared to DN-9;
  • the oral analgesic activity of the cyclic peptide molecule shown in the formula I is more than 3 times, preferably more than 10 times, more preferably more than 100 times, 500 times, 1000 times or 10000 times compared to DN-9;
  • the cyclic peptide molecule shown in the formula I has no analgesic tolerance after continuous oral administration for more than 5 days (preferably 8 days);
  • a method for preparing the multi-target cyclic peptide molecule or a pharmaceutically acceptable salt thereof comprising the steps of:
  • the method further comprises: separating the multi-target cyclic peptide molecule or a pharmaceutically acceptable salt thereof obtained in step (b), thereby obtaining a purified multi-target cyclic peptide molecule or its pharmacy acceptable salt.
  • step (b) the side chains of Xaa2 and Xaa5 are cyclized to form a cyclic covalent bond between the two amino acid residues of Xaa2 and Xaa5.
  • step (a) peptide chain synthesis is performed from the C-terminal to the N-terminal of the amino acid sequence.
  • step (a) peptide chain synthesis is performed from the N-terminus to the C-terminus of the amino acid sequence.
  • the solid-phase synthesis method includes process steps selected from the group consisting of: pretreatment of solid-phase support, condensation of amino acids, extension of peptide chains, compression and drying of polypeptides and cleavage, extraction, purification, or a combination thereof.
  • the solid phase carrier is an amino resin.
  • the condensation reagent used in the amino acid condensation step includes a combination of HOBt, HBTU and DIEA.
  • the cyclization reagent used in the cyclization step includes a combination of PyBOP and DIEA.
  • described preparation method comprises processing step:
  • Resin pretreatment a certain amount of Rink-Amide-MBHA resin was swollen in dichloromethane (DCM) for 30 min, drained, and the resin was rinsed with N,N-dimethylformamide (DMF).
  • DCM dichloromethane
  • the volume-to-mass ratio of DCM to resin is 8-12 mL/g. Ninhydrin test, under normal circumstances, the solution is light yellow and the resin is colorless.
  • removing the fluorenemethoxycarbonyl (Fmoc) protecting group adding 1,8-diazabicycloundec-7-ene ( DBU), piperidine and DMF, stir and react 3 times, the speed of stirring is 60 ⁇ 100rpm, the time of first two stirrings is 2 ⁇ 6min, and the time of last stirring is 8 ⁇ 12min; 10min at a time. Finally, DMF was added to wash. Ninhydrin test, under normal circumstances, the solution is blue-violet, and the resin is blue-violet.
  • DBU 1,8-diazabicycloundec-7-ene
  • DMF 1,8-diazabicycloundec-7-ene
  • N- ⁇ -Fmoc protected amino acid O-benzotriazole-N,N,N',N' in molar ratio of 1:0.2 ⁇ 1.5:0.2 ⁇ 1.5:1.5 ⁇ 3 - Tetramethylurea-hexafluorophosphate (HBTU), N-hydroxybenzotriazole (HOBt) and N,N-diisopropylethylamine (DIEA) were dissolved in a small amount of DMF, with DIEA added last,
  • the volume-to-mass ratio of DMF to the amino acid protected by N- ⁇ -Fmoc is 5-10 mL/g; then added to the resin obtained in step ii.i, the volume-to-mass ratio of the mixed solution to the resin is 4-6 mL/g; stirring The speed is 60-100 rpm; the reaction is at room temperature for 40-100 min. After the reaction was completed, it was washed with DMF. Ninhydrin test, under normal circumstances, the solution is
  • ii.iii Peptide chain extension According to the peptide sequence, sequentially insert different Fmoc-protected amino acids on the peptide resin obtained in step ii.ii, and the last amino acid usually uses a tert-butoxycarbonyl (Boc)-protected amino acid, omitting In the last step of removing Fmoc, the condensation method of all amino acids is the same as above. A peptide resin is obtained.
  • the resulting peptide resin was then transferred to a synthesizer, the resin was washed alternately with DCM and DMF, and benzotriazol-1-yl-oxytripyrrolidinophosphorus hexafluorophosphate (PyBOP) was added with 3 times the molar amount of the resin.
  • DIEA benzotriazol-1-yl-oxytripyrrolidinophosphorus hexafluorophosphate
  • the cyclization condensation reaction is carried out for 3-6 hours, and the stirring rate is 60-100 rpm.
  • Ninhydrin test under normal circumstances, the solution is light yellow and the resin is colorless. A cyclized peptide resin is obtained.
  • Peptide cleavage and precipitation extraction adding a cleavage agent (trifluoroacetic acid (TFA), triisopropylsilane (Tis) and water (H 2 O ) in a volume ratio of 95:2.5:2.5 to the resin obtained in step iii )).
  • a cleavage agent trifluoroacetic acid (TFA), triisopropylsilane (Tis) and water (H 2 O )
  • the reaction was carried out at room temperature for 1.5 to 4 hours, with stirring every 15 minutes.
  • the volume-to-mass ratio of the cutting agent to the cyclized peptide resin is 10-20 mL/g.
  • a pharmaceutical composition comprising:
  • the pharmaceutical composition is administered by an administration mode selected from the group consisting of oral administration, transdermal administration, intrathecal administration, intravenous administration, intramuscular administration and topical administration , nasal administration, etc.
  • the formulation of the pharmaceutical composition is selected from the group consisting of tablets, capsules, sugar-coated tablets, granules, oral solutions and syrups, aerosols, nasal sprays, dry powder injections, injections, Ointments and patches on the skin surface.
  • the preparation of the pharmaceutical composition is an injection.
  • the pharmaceutical injection can be used for parenteral administration.
  • the fourth aspect of the present invention there is provided a use of the multi-target cyclic peptide molecule or a pharmaceutically acceptable salt thereof according to the first aspect of the present invention, for preparing a medicament for relieving and and/or treat all types of pain, including acute pain and pathological pain.
  • a method for analgesia comprising the steps of: adding a safe and effective amount of the multi-targeted cyclic peptide molecules directed against opioid receptors and neuropeptide FF receptors according to the first aspect of the present invention Or a pharmaceutically acceptable salt thereof and/or the pharmaceutical composition described in the third aspect of the present invention is administered to a subject in need.
  • the method is non-therapeutic or therapeutic.
  • the method is in vitro or in vivo.
  • the subject in need is a subject in need of alleviation and/or treatment of various types of pain.
  • the subject is a mammal or a human.
  • the subject is a human.
  • Fig. 1 is the time-dose-effect curve of the dose-dependent analgesic effect produced by subcutaneous injection of compound 1 in mice;
  • Fig. 2 is the time-dose-effect curve of the dose-dependent analgesic effect produced by subcutaneous injection of compound 2 in mice;
  • Fig. 3 is the time-dose-effect curve of the dose-dependent analgesic effect produced by subcutaneous injection of compound 3 in mice;
  • Figure 4 is a time-dose-effect curve of the dose-dependent analgesic effect produced by subcutaneous injection of compound 4 in mice;
  • Figure 5 is a time-dose-effect curve of the dose-dependent analgesic effect produced by subcutaneous injection of compound 5 in mice;
  • Figure 6 is a time-dose-effect curve of the dose-dependent analgesic effect produced by subcutaneous injection of compound 6 in mice;
  • Figure 7 is a time-dose-effect curve of the dose-dependent analgesic effect produced by subcutaneous injection of compound 7 in mice;
  • Figure 8 is a time-dose-effect curve of the dose-dependent analgesic effect produced by subcutaneous injection of compound 8 in mice;
  • Figure 9 is a time-dose-effect curve of the dose-dependent analgesic effect produced by subcutaneous injection of compound 9 in mice;
  • Figure 10 is a time-dose-response curve of the dose-dependent analgesic effect produced by subcutaneous injection of compound 10 in mice;
  • Figure 11 is a time-dose-effect curve of the dose-dependent analgesic effect produced by subcutaneous injection of compound 11 in mice;
  • Figure 12 is a time-dose-effect curve of the dose-dependent analgesic effect produced by subcutaneous injection of compound 12 in mice;
  • Figure 13 is a time-dose-response curve of the dose-dependent analgesic effect produced by subcutaneous injection of compound 13 in mice;
  • Figure 14 is a time-dose-response curve of the dose-dependent analgesic effect produced by subcutaneous injection of compound 14 in mice;
  • Figure 15 is the time-dose-effect curve of analgesia produced by oral injection of parent DN-9 in mice;
  • Figure 16 is the time-dose-effect curve of the dose-dependent analgesic effect produced by oral injection of compound 1 in mice;
  • Figure 17 is the time-dose-effect curve of the dose-dependent analgesic effect produced by oral injection of compound 11 in mice;
  • Figure 18 shows the results of the blood-brain barrier permeability study of subcutaneous injection of compound 1 in mice injected with methyliodide naloxone and naloxone;
  • Figure 19 shows the results of blood-brain barrier permeability study of subcutaneous injection of compound 2 by injection of methyliodide naloxone and naloxone in mice;
  • Figure 20 shows the results of blood-brain barrier permeability study of subcutaneous injection of compound 3 by injection of methyliodide naloxone and naloxone in mice;
  • Figure 21 shows the results of blood-brain barrier permeability study of subcutaneous injection of compound 4 by injection of methyliodide naloxone and naloxone in mice;
  • Figure 22 shows the results of blood-brain barrier permeability study of subcutaneous injection of compound 5 by injection of methyliodide naloxone and naloxone in mice;
  • Figure 23 shows the results of blood-brain barrier permeability study of subcutaneous injection of compound 6 by injection of methyliodide naloxone and naloxone in mice;
  • Figure 24 shows the results of blood-brain barrier permeability study of subcutaneous injection of compound 7 by injection of methyliodide naloxone and naloxone in mice;
  • Figure 25 shows the results of blood-brain barrier permeability study of subcutaneous injection of compound 8 by injection of methyliodide naloxone and naloxone in mice;
  • Figure 26 shows the results of blood-brain barrier permeability study of subcutaneous injection of compound 9 by injection of methyliodide naloxone and naloxone in mice;
  • Figure 27 shows the results of blood-brain barrier permeability study of subcutaneous injection of compound 10 by injection of methyliodide naloxone and naloxone in mice;
  • Figure 28 shows the results of blood-brain barrier permeability study of subcutaneous injection of compound 11 by injection of methyliodide naloxone and naloxone in mice;
  • Figure 29 shows the results of blood-brain barrier permeability study of subcutaneous injection of compound 12 by injection of methyliodide naloxone and naloxone in mice;
  • Figure 30 shows the results of blood-brain barrier permeability study of subcutaneous injection of compound 13 by injection of methyliodide naloxone and naloxone in mice;
  • Figure 31 shows the results of blood-brain barrier permeability study of subcutaneous injection of compound 14 by injection of methyliodide naloxone and naloxone in mice;
  • Figure 32 shows the results of blood-brain barrier permeability study of oral injection of Compound 1 in mice injected with methyliodide naloxone and naloxone;
  • Figure 33 shows the results of the blood-brain barrier permeability study of oral injection of compound 11 in mice injected with methyliodide naloxone and naloxone;
  • Figure 34 shows the changes in the analgesic effect produced by oral injection of compounds 1 and 11 in mice for eight consecutive days;
  • Figure 35 is the effect of oral injection of compound 1 on gastrointestinal motility in mice
  • Figure 36 is the effect of oral injection of compound 11 on gastrointestinal motility in mice
  • Figure 37 is the regulatory effect of oral injection of compounds 1 and 11 on motor activity in mice
  • Figure 38 is the regulatory effect of oral injection of compounds 1 and 11 on conditioned position in mice
  • Figure 39 is the response of oral injection of compound 1 in mice to naloxone withdrawal
  • Figure 40 shows the response to oral injection of Compound 11 in mice after naloxone withdrawal.
  • the inventors after extensive and in-depth experimental research, the inventors, for the first time on the basis of the multi-target molecule DN-9 of the opioid/NPFF receptor, introduced the amino acid residue D-Dap containing amino and carboxyl structures in the side chain for the opioid pharmacophore. , D-Dab, Lys, D-Lys, D-Orn, Asp, D-Asp, D-Glu or Glu and cyclization modification, for NPFF pharmacophore by introducing Gly, ⁇ -Ala, Aib and Cha for amino acid Instead, a series of multi-targeted cyclic peptide molecules against opioid receptors and neuropeptide FF receptors or pharmaceutically acceptable salts thereof are obtained.
  • novel cyclic peptide molecule of the present invention or a pharmaceutically acceptable salt thereof overcomes the problems of short analgesic action time and weak peripheral analgesic activity of the parent DN-9 molecule, and has efficient peripheral analgesic activity and analgesic duration. Long-lasting, effective analgesic with low dose, no tolerance, low addiction, and low constipation, it can be used as a new generation of potential drugs for pain treatment for various types of pain. Based on the above findings, the inventors have completed the present invention.
  • multi-targeted cyclic peptide molecules against opioid receptors and neuropeptide FF receptors or pharmaceutically acceptable salts thereof refers to the opioid/neuropeptide FF receptor multi-target molecule DN-9 amide bond cyclized analog or a pharmaceutically acceptable salt thereof of the present invention, which is based on the multi-target opioid/NPFF receptor
  • the dot molecule DN-9 is a chemical template, targeting the opioid pharmacophore by introducing amino acid residues D-Dap, D-Dab, Lys, D-Lys, D-Orn, Asp, D-Asp containing amino and carboxyl structures in the side chain , D-Glu or Glu and modified by amide bond cyclization, and introduced Gly, ⁇ -Ala, Aib and Cha for amino acid substitution to obtain the NPFF pharmacophore.
  • the sequence of the multi-Nap, D-Dab, Lys, D-Lys, D-Orn targeting the opioid pharmacophore by
  • NMe-Phe N-Me-Phe
  • NMePhe N ⁇ -methylphenylalanine
  • amino acids described in the present invention are all L-type amino acids unless "D-" is specially indicated as D-type amino acids.
  • the term "pharmaceutically acceptable salt” refers to a salt of a cyclic peptide molecule of the invention synthesized with a non-toxic acid or base that retains the biological effectiveness of the cyclic peptide molecule of the invention without other side effects .
  • the multi-targeted cyclic peptide molecules for opioid receptors and neuropeptide FF receptors of the present invention or their pharmaceutically acceptable salts use DN-9 as a chemical template, and utilize polypeptide chemical strategies such as amino acid substitution and amide bond cyclization to modify
  • the structure optimization of opioid peptide and neuropeptide FF pharmacophore includes the following process steps:
  • Resin pretreatment Rink-Amide-MBHA resin was swollen in DCM, drained, washed with DMF, and tested for ninhydrin;
  • removing the Fmoc protecting group adding a mixed solution of DBU, piperidine and DMF with a volume ratio of 1:1:98 in the resin obtained in step i., stirring the reaction; adding DMF for washing, and indene detection;
  • ii.iii Peptide chain extension according to the polypeptide sequence, connect different Fmoc-protected amino acids in turn, the last amino acid uses a Boc-protected amino acid, and the condensation method of all amino acids is the same as above to obtain a peptide resin;
  • Cyclization of the polypeptide wash alternately with DCM and MeOH, compress and drain; under argon protection, add Pd(PPh 3 ) 4 and DABCO to the peptide resin obtained in step ii.iii, and then use a syringe Add a mixed solution of CHCl 3 , HAc and NMM to selectively remove Alloc and OAll protecting groups; indene detection, after washing, add PyBOP and DIEA, cyclization condensation reaction for 3-6 hours, stirring at a speed of 60-100 rpm, indene detection, get Cyclic peptide resin;
  • Peptide cleavage and precipitation extraction add a cleavage agent (TFA, Tis and H 2 O with a volume ratio of 95:2.5:2.5) to the peptide resin obtained in step iii, and react at room temperature for 1.5-4h; spin Evaporate, add pre-cooled ether to separate out the precipitate, extract the precipitate with 20% HAc aqueous solution, freeze-dry the extract to obtain the crude peptide;
  • TFA cleavage agent
  • compositions and methods of administration are provided.
  • the present invention also provides a pharmaceutical composition
  • a pharmaceutical composition comprising the cyclic peptide molecule of the present invention or a pharmaceutically acceptable salt thereof within a safe and effective amount, and a pharmaceutically acceptable carrier.
  • the cyclic peptide molecules of the present invention or their pharmaceutically acceptable salts and pharmaceutical compositions are used for preparing analgesic medicines.
  • the cyclic peptide molecules of the present invention or their pharmaceutically acceptable salts and pharmaceutical compositions can be used as analgesic drugs.
  • it is used to prepare analgesic drugs with high efficiency and low side effects.
  • it is used to relieve and treat various types of pain including acute pain and pathological pain.
  • a "safe and effective amount” refers to an amount of the cyclic peptide molecule of the present invention or a pharmaceutically acceptable salt thereof sufficient to produce a significant analgesic effect without causing serious side effects.
  • the pharmaceutical composition may be in any suitable form (depending on the method of administration desired by the patient). It can be presented in unit dosage form, usually in a sealed container, and can be presented as part of a kit. Such kits usually, but not necessarily, contain instructions for use. It may contain a plurality of said unit dosage forms.
  • the administration method of the pharmaceutical composition is not particularly limited, and representative administration methods include (but are not limited to): oral administration, transdermal administration, intrathecal administration, intravenous administration, intramuscular administration, topical administration administration, nasal administration, etc.
  • compositions of the present invention can be formulated into various suitable dosage forms, examples of suitable dosage forms being sterile solutions and dry powders for injection, tablets, capsules, sugar-coated tablets, granules , oral solutions and syrups, aerosols, nasal sprays, and ointments and patches applied to the skin.
  • suitable dosage forms being sterile solutions and dry powders for injection, tablets, capsules, sugar-coated tablets, granules , oral solutions and syrups, aerosols, nasal sprays, and ointments and patches applied to the skin.
  • the pharmaceutical composition containing the cyclic peptide molecule of the present invention or a pharmaceutically acceptable salt thereof can be prepared as a solution or lyophilized powder for parenteral administration, and a suitable solvent or other pharmaceutically acceptable carrier can be added to the solution before use.
  • the powder is reconfigured, and the solution formulation is generally a buffer, isotonic solution or an aqueous solution.
  • composition of the present invention can be administered alone or in combination with other analgesic drugs.
  • a safe and effective amount of the polypeptide of the present invention or a pharmaceutically acceptable salt thereof is administered to a mammal (such as a human) in need of treatment, wherein the dosage of the pharmaceutical composition of the present invention can be in a larger range
  • a mammal such as a human
  • the internal variation can be easily determined by those skilled in the art according to some objective factors, such as the type of the disease, the severity of the disease, the weight of the patient, the dosage form, and the route of administration.
  • the subcutaneous and oral analgesic activities of the cyclic peptide molecule of the present invention or a pharmaceutically acceptable salt thereof are significantly improved compared with parent DN-9, wherein the analgesic ED 50 (half effective dose) of subcutaneous injection is decreased by 100 times, preferably 1000 to 10000 times, and the oral analgesic activity is also improved compared to the parent body;
  • the effective action time of the cyclic peptide molecule of the present invention or its pharmaceutically acceptable salt is long, about 240min;
  • cyclic peptide molecules of the present invention or their pharmaceutically acceptable salts cannot penetrate the blood-brain barrier, have no analgesic tolerance, have no constipation or addictive side effects.
  • the solid-phase peptide synthesizer was independently designed by the present inventor, the rotary evaporator RE-5298A was purchased from Shanghai Yarong, the freeze dryer was purchased from VIRTIS Corporation of the United States, the mass spectrometer ESI-Q-TOF maXis-4G was purchased from Bruker Daltonics Germany Dalton Company, the circulating water pump SHB-III was purchased from Zhengzhou Great Wall, the high performance liquid chromatograph (RP-HPLC) was Delta 600 of Waters Company, and the analytical column: XBridge TM BEH 130Prep C18, 4.6mm ⁇ 250mm; preparation Column: XBridgeTM BEH 130Prep C18, 19mm x 250mm.
  • RP-HPLC high performance liquid chromatograph
  • resin is Rink-Amide-MBHA Resin (substitution value S is 0.4mmol/g) purchased from Tianjin Nankai Hecheng Company, N- ⁇ -Fmoc protected amino acid (Fmoc-Aa), O-benzotriazole -N,N,N',N'-tetramethylurea-hexafluorophosphate (HBTU) and N-hydroxybenzotriazole (HOBt) were purchased from Shanghai Jier Biochemical Co., Ltd., N,N-diisopropyl Ethylethylamine (DIEA) was purchased from Beijing Bailingwei, and 1,8-diazabicycloundec-7-ene (DBU) was purchased from Shanghai Merrell Company.
  • substitution value S is 0.4mmol/g
  • Fmoc-Aa N- ⁇ -Fmoc protected amino acid
  • HBTU O-benzotriazole -N,N,N',N'-tetramethylurea-hexaflu
  • Tetrakistriphenylphosphine palladium (Pd(PPh 3 ) 4 ), triethylenediamine (DABCO) and triisopropylsilane (Tis) were purchased from Shanghai Annaiji, and ninhydrin was a product of Shanghai Reagent No. 3 Factory.
  • Dichloromethane (DCM), N,N-dimethylformamide (DMF), hexahydropyridine (piperidine), methanol (MeOH) and pyridine were purchased from Tianjin Second Reagent Factory, trifluoroacetic acid (TFA) and phenol All are products of Tianjin Reagent Factory; the above organic reagents have been re-distilled before use.
  • Resin pretreatment Weigh 1 g of Rink-Amide-MBHA resin (substitution value is 0.4 mmol/g), add 10 mL of DCM, stir and swell at 80 rpm, stir and react for 30 min, drain and rinse the resin 3 times with DMF , 3min each time.
  • the ninhydrin test reagent is a phenol:pyridine:ninhydrin solution with a volume ratio of 1:2:1.
  • the phenol solution was prepared as 20g phenol in 5mL absolute ethanol
  • the pyridine solution was prepared as 0.05mL KCN (0.001M) in 2.5mL pyridine
  • the ninhydrin solution was prepared as 0.5g ninhydrin dissolved in 10mL absolute ethanol, Among them, phenol, pyridine and absolute ethanol were all re-distilled.
  • Ninhydrin test under normal circumstances, the solution is blue-violet, and the resin is blue-violet.
  • step (2) Condensation of amino acid: Weigh Fmoc-Phe-OH, HOBt and HBTU with a molar ratio of 1:1:1, then dissolve them in 5 mL of DMF, where DIEA is added last, and after stirring evenly, step (2) has been added.
  • the reaction is carried out for 60 min at a stirring rate of 80 rpm at room temperature, and the solvent is drained; after the reaction, indene detection is performed according to the method of step (3). Indicates that the amino acid is condensed onto the resin. Then remove the Fmoc protecting group according to step (2), and perform indene detection according to step (3). If both the solution and the resin are dark blue, it means that the removal of the Fmoc protecting group is complete, and the resin peptide without the Fmoc protecting group is obtained.
  • the peptide resin was then transferred to a round bottom flask, 255 mg of Pd(PPh 3 ) 4 , 225 mg of DABCO were added, under argon protection, and then 5 mL of CHCl 3 , HAc and NMM in a volume ratio of 37:2:1 were added by syringe. The reaction was stirred at 25 °C and 150 rpm for 4 h under argon protection, and the amino Alloc and carboxyl OAll were selectively removed.
  • the crude peptide was fully resolved as a white precipitate.
  • the crude peptide in ether was extracted with 20% HAc in water. Finally, the aqueous solution of the extracted peptide was freeze-dried to obtain 324 mg of white crude peptide solid powder, and the crude peptide yield was 70%.
  • the cyclized peptide Tyr-c [2,5] [D-Lys-Gly-NMe-Phe-Glu]-Pro-Gln-Arg-Phe-NH 2 was obtained as a white solid powder,
  • the crude peptide is 300 mg, the yield of crude peptide is 64%, and the yield of pure peptide after purification is 24%.
  • the detection results of mass spectrometry and chromatographic analysis are shown in Table 2.
  • the method was the same as that of Example 1, and the cyclized peptide Tyr-c [2,5] [D-Lys-Gly-NMe-Phe-D-Asp]-Pro-Gln-Arg-Phe-NH 2 was obtained as a white solid powder,
  • the crude peptide is 350 mg, the yield of crude peptide is 76%, and the yield of pure peptide after purification is 30%.
  • the detection results of mass spectrometry and chromatographic analysis are shown in Table 2.
  • the method is the same as in Example 1, and the cyclized peptide Tyr-c [2,5] [D-Asp-Gly-NMe-Phe-Lys]-Pro-Gln-Arg-Phe-NH 2 is obtained as white solid powder, crude peptide 330 mg, the yield of crude peptide is 72%, and the yield of pure peptide after purification is 60%.
  • the detection results of mass spectrometry and chromatographic analysis are shown in Table 2.
  • the method is the same as in Example 1, and the cyclized peptide Tyr-c [2,5] [D-Glu-Gly-NMe-Phe-Lys]-Pro-Gln-Arg-Phe-NH 2 is obtained as white solid powder, crude peptide 443 mg, the yield of crude peptide is 95%, and the yield of pure peptide after purification is 26%.
  • the detection results of mass spectrometry and chromatographic analysis are shown in Table 2.
  • the method is the same as in Example 1, and the cyclized peptide Tyr-c [2,5] [D-Orn-Gly-NMe-Phe-Asp]-Pro-Gln-Arg-Phe-NH 2 is obtained as white solid powder, crude peptide 285mg, the yield of crude peptide is 63%, and the yield of pure peptide after purification is 20%.
  • the detection results of mass spectrometry and chromatographic analysis are shown in Table 2.
  • the method is the same as in Example 1, and the cyclized peptide Tyr-c [2,5] [D-Dab-Gly-NMe-Phe-Asp]-Pro-Gln-Arg-Phe-NH 2 is obtained as white solid powder, crude peptide 290 mg, the yield of crude peptide is 65%, and the yield of pure peptide after purification is 40%.
  • the detection results of mass spectrometry and chromatographic analysis are shown in Table 2.
  • the method is the same as in Example 1, and the cyclized peptide Tyr-c [2,5] [D-Dap-Gly-NMe-Phe-Asp]-Pro-Gln-Arg-Phe-NH 2 is obtained as white solid powder, crude peptide 300mg, the yield of crude peptide is 68%, and the yield of pure peptide after purification is 24%.
  • the detection results of mass spectrometry and chromatographic analysis are shown in Table 2.
  • the method is the same as in Example 1, and the cyclized peptide Tyr-c [2,5] [D-Orn-Gly-NMe-Phe-Glu]-Pro-Gln-Arg-Phe-NH 2 is obtained as white solid powder, crude peptide 330mg, the yield of crude peptide is 72%, and the yield of pure peptide after purification is 38%.
  • the detection results of mass spectrometry and chromatographic analysis are shown in Table 2.
  • the method is the same as that of Example 1, and the cyclized peptide Tyr-c [2,5] [D-Lys-Gly-NMe-Phe-Asp]-Gly-Gln-Arg-Phe-NH 2 is obtained as white solid powder, crude peptide 307 mg, the yield of crude peptide is 69%, and the yield of pure peptide after purification is 50%.
  • the detection results of mass spectrometry and chromatographic analysis are shown in Table 2.
  • the method was the same as that of Example 1, and the cyclized peptide Tyr-c [2,5] [D-Lys-Gly-NMe-Phe-Asp]-Pro- ⁇ -Ala-Arg-Cha-NH 2 was obtained as a white solid powder,
  • the crude peptide is 329 mg, the yield of crude peptide is 74%, and the yield of pure peptide after purification is 28%.
  • the detection results of mass spectrometry and chromatographic analysis are shown in Table 2.
  • the method is the same as in Example 1, and the cyclized peptide Tyr-c [2,5] [D-Lys-Gly-NMe-Phe-Asp]-Pro-Aib-Arg-Cha-NH 2 is obtained as white solid powder, crude peptide 423 mg, the yield of crude peptide is 93%, and the yield of pure peptide after purification is 24%.
  • the detection results of mass spectrometry and chromatographic analysis are shown in Table 2.
  • the present invention has synthesized multi-target cyclic peptide molecules including opioid receptors and neuropeptide FF receptors listed in Table 1, and the chemical characterization results are shown in Table 2.
  • System 1 Gradient elution system 1 is: 10-80% acetonitrile/water (0.1% TFA) (completed in 30min), flow rate: 1mL/min, detection wavelength is 220nm, analytical chromatographic column: XBridge TM BEH 130Prep C 18 , 4.6 mm ⁇ 250 mm; system 2: gradient elution system 2: 10-100% acetonitrile/water (0.1% TFA) (completed in 30 min), flow rate: 1 mL/min, detection wavelength 220 nm, analytical chromatographic column As: XBridge TM BEH 130Prep C18 , 4.6mm x 250mm.
  • Example 15 In vitro functional activity assays at opioid and NPFF receptors
  • cyclic peptide molecules of the present invention By detecting the effect of the cyclic peptide molecules of the present invention on Forskolin-induced intracellular cyclic adenosine monophosphate (CAMP) in HEK293 cells stably expressing Mu-opioid receptor, Delta-opioid receptor, Kappa-opioid receptor, NPFF 1 and NPFF 2 receptors cAMP) accumulation to examine their agonistic activity at these five receptors.
  • CAMP forskolin-induced intracellular cyclic adenosine monophosphate
  • the medium in the petri dish was aspirated, and then 500 ⁇ L of pre-warmed serum-free medium containing 1 mM IBMX was added, and incubated at 37° C. for 10 min. Then, 10 ⁇ L of the test drug and 10 ⁇ M forskolin (final concentration) were added to each well, and incubated at 37° C. for 30 min. After the incubation, suck out all the liquid in the culture dish, add 500 ⁇ L of 0.2N hydrochloric acid to each well, and incubate at room temperature for 30 min to promote cell lysis. After the cleavage was completed, NaOH was added to neutralize the hydrochloric acid solution used for cleavage.
  • compounds 1-6 and 8-12 both inhibited forskolin-induced cAMP accumulation in a dose-dependent manner, indicating that these compounds also have Agonistic activity of NPFF 1 and NPFF 2 receptors.
  • compounds 1-6 and 8-12 can activate both opioid and NPFF receptors simultaneously, showing a class of multi-target agonists of opioid and NPFF receptors.
  • Example 16 In vivo analgesic activity assay
  • Subcutaneous administration (s.c.) The dorsal subcutaneous administration was selected. Administer with a 1 mL sterile syringe in a volume of 0.1 mL/10 g. Grab the back skin with the right hand, insert the needle obliquely, and inject the drug subcutaneously on the back of the mouse. After the needle is inserted, shake the needle left and right, and pay attention to observe to prove that the needle does enter the subcutaneous, so as to prevent the needle from piercing the skin and leaking the medicine.
  • a 1mL sterile syringe was used, and the needle was replaced with a mouse gavage needle, and the oral gavage was 0.1uL/10g.
  • the right hand holds a gavage needle (12-gauge needle and a 1mL syringe) from the corner of the mouse's mouth, close to the tongue surface, and goes into the esophagus along the upper palate.
  • the 2.5cm needle can be injected into the gavage solution.
  • the length of needle insertion should be determined by practice in advance, and there will be a sense of disappointment after needle insertion. If the needle insertion position is suitable, it should be smooth, otherwise it means that the needle insertion is not suitable, which means that the gavage needle may be inserted into the trachea of the mouse, which will cause the mouse to die immediately after gavage.
  • the present invention optimizes the experimental parameters based on the experimental method summarized by D'Amour and Smith.
  • male Kunming mice with a body weight of 21 ⁇ 2 g were selected, and the ambient temperature was controlled at 22 ⁇ 2 °C.
  • the experimental mice could drink water freely, and the mice were moved from the rearing room to the experimental area for 30 minutes before starting the experiment. Then grab the mouse with the right hand, the tail of the mouse hangs down freely, and then put the tail of the mouse on the radiation light source, and the position is 2-3 mm away from the tail of the decimal.
  • the intensity of radiant heat was adjusted to the tail flick time of mice for 3-5 s, which is the basic pain threshold of photothermal tail flick.
  • the irradiation time of the tail of the mice did not exceed 10s to prevent the tail of the mice from being scalded, that is, the maximum incubation period of the photothermal tail flick. Then after administration, the time points of 10, 20, 30, 45, 60, 90, 120, 180, 240, 300, 360 and 420 min were selected to measure the tail-flick latency of mice after administration.
  • the half effective dose ED 50 (50% effective dose, ED 50 ) refers to the corresponding drug dose that causes 50% of the effect. ED 50 and 95% confidence interval
  • the analgesic ED 50 of all cyclic peptide molecules and DN-9 parent molecule injected subcutaneously is shown in Table 5, and the analgesic ED 50 value of parent DN-9 is 228 ⁇ g/kg.
  • the ED 50 values of the 14 cyclic peptide molecules listed in Table 5 were all significantly lower than the analgesic ED 50 of the parent peptide, wherein the analgesic ED 50 of subcutaneous injection of compounds 11-14 was at least 10000 times lower than that of the parent DN-9 molecule.
  • the effective analgesic time was extended from 90min to 240min in the mother.
  • Subcutaneous analgesic dose-response curves of the compounds are shown in Figures 1-14.
  • Pharmacological evaluation of the permeability of the blood-brain barrier is performed by injecting methyl-iodide naloxone to detect whether the drug passes through the blood-brain barrier, which is a drug that cannot pass the blood-brain barrier.
  • methyl-iodide naloxone, naloxone and compounds were injected at different sites, and then the changes in the analgesic effects of the compounds were detected by a photothermal tail-flick experiment.
  • naloxone methyl iodide NALM
  • Nal naloxone
  • naloxone methyl iodide was administered 10 minutes in advance, and naloxone methyl iodide was administered in three ways: lateral ventricle (i.c.v.), subcutaneous (s.c.) and intraperitoneal (i.p.)
  • Naloxone is administered in two ways: subcutaneous (s.c.) and intraperitoneal (i.p.)
  • different compounds are mainly administered subcutaneously (s.c.) and oral (p.o.) at the peripheral level, and then the photothermal tail-flick test is used to detect the completion of administration. Changes in analgesia following antagonists and drugs.
  • the antagonism of the drugs was compared using the MPE values at the time points of the maximal analgesic effect of the relevant drugs.
  • MPE data are presented as mean ⁇ standard error (Means ⁇ SEM), differences in analgesic effects were statistically analyzed using one-way ANOVA (Bonferroni test for one-way ANOVA), * P ⁇ 0.05, ** P ⁇ 0.01 and * ** P ⁇ 0.001 indicates a significant difference between the group injected with related drugs only and the group injected with antagonists and related compounds simultaneously.
  • Blood-brain barrier permeability results for oral administration of Compounds 1 and 11 are shown in Figures 32 and 33.
  • Intracerebroventricular injection of methyl-iodide naloxone could not antagonize the analgesia induced by oral injection of compounds 1 and 11, while intraperitoneal injection of naloxone and methyl-iodide naloxone could antagonize the analgesia induced by oral injection of compounds 1 and 11, thus It is indicated that both compounds 1 and 11 can not penetrate the blood-brain barrier by oral injection, which suggests that the compounds of the present invention have low side effects on the central nervous system.
  • the analgesic tolerance test was performed by oral injection of Compounds 1 and 11 for 8 consecutive days, followed by a photothermal tail-flick test to identify changes in the analgesic tail-flick threshold from the first day to the eighth day.
  • a photothermal tail-flick test to identify changes in the analgesic tail-flick threshold from the first day to the eighth day.
  • mice 21 ⁇ 2g Kunming male mice were selected in the experiment, and the mice could eat freely.
  • the basal pain threshold of mice was measured on the first day, and then compounds 1 and 11 were orally injected for 8 consecutive days.
  • the pain threshold at different time points was measured on the first day, and only the pain threshold at the highest analgesic point was measured on the next 7 days.
  • Mice injected with traditional opioids will generally experience a decrease in the analgesic threshold on the 3rd or 4th day, that is, analgesic tolerance.
  • the experimental data are represented by tail-flick time.
  • the analgesic tolerance of drugs was compared using the tail-flick latency at the time point of maximal analgesic effect of different compounds.
  • Tail-flick latency data are expressed as mean ⁇ standard error (Means ⁇ SEM).
  • Differences in analgesic effects of mice administered subcutaneously for eight consecutive days were analyzed using one-way ANOVA (Tukey HSD test for one-way ANOVA), * P ⁇ 0.05, ** P ⁇ 0.01, *** P ⁇ 0.001 indicates a very significant difference compared to the analgesic effect of the first day injection of the drug.
  • Figure 34 There was no change in the saline group after 8 consecutive oral injections, and no analgesic tolerance was observed with 8 consecutive oral injections of Compounds 1 and 11.
  • Constipation is a common side effect of opioids. Therefore, the effect of drugs on gastrointestinal motility is generally evaluated by the side effects of constipation.
  • mice 26 ⁇ 2g male Kunming mice were selected for the experiment.
  • the mice were starved, and the mice were placed in a box without bedding. The mice could not eat or drink but could drink water freely, and were starved for 16 hours.
  • pre-prepared activated charcoal suspension a saline suspension containing 5% activated charcoal and 10% gum arabic
  • the volume was perfused orally into the stomach.
  • the mice were sacrificed by cervical dislocation. Immediately dissect, take the full length of the small intestine, the distance from the gastric pylorus to the cecum. The total length of the small intestine and the length of the toner travel are then measured.
  • the experimental data of gastrointestinal motility is expressed by the percentage of gastrointestinal motility, and the specific calculation method is the percentage of the distance moved by the toner divided by the total length of the small intestine. Data are presented as mean ⁇ standard error of gastrointestinal motility percentage (Means ⁇ SEM). Differences between compounds and saline were performed with one-way ANOVA (Dunnett's test for one-way ANOVA). * P ⁇ 0.05, ** P ⁇ 0.01, and *** P ⁇ 0.001 indicate significant differences between saline injections alone and related compounds. The results of gastrointestinal motility experiments for compounds 1 and 11 are shown in Figures 35-36.
  • Addiction evaluation of compounds 1 and 11 was performed by open field test, conditioned place preference (CPP) and naloxone withdrawal test. Injection of opioids promotes the release of dopamine, and the motor activity of mice is enhanced, so it is often said that the motor activity of mice is linked to the evaluation of addiction.
  • CPP conditioned place preference
  • the open field experiment consisted of a topless 50 ⁇ 50 ⁇ 40cm black plexiglass box and a motion monitoring system. 21 ⁇ 2 g of Kunming male mice were selected for the experiment, and the room temperature was controlled between 22 ⁇ 1 °C, otherwise the movement of the mice would be affected if the temperature was too high or too low.
  • the box was wiped with alcohol to remove the odor in the box, so as to prevent the odor in the box from affecting the motor activity of the next mouse.
  • the basal motor activity of the mice was recorded for 30 min, and then the mice were orally (po) injected with normal saline, 1000 ⁇ g/kg of compound 1, and 100 ⁇ g/kg of compound 11.
  • mice The locomotor activity of the mice was recorded within 150 min.
  • Mouse locomotor activity was represented by the total movement distance, that is, the mouse movement total distance ⁇ standard error (Means ⁇ SEM), and the difference between the compound and the saline control group was expressed by one-way ANOVA (Bonferroni test of one-way ANOVA). ) for data statistics and analysis, * P ⁇ 0.05, ** P ⁇ 0.01 and *** P ⁇ 0.001 indicate significant differences between saline and compounds. The results are shown in Figure 37.
  • CPP Conditioned place preference experiment
  • the experiment was done in a device consisting of three plexiglass boxes, with two large boxes (20 ⁇ 20 ⁇ 20 cm) separated by a small box (5 ⁇ 20 ⁇ 20 cm).
  • a 5 ⁇ 5 small door is opened at the bottom of the two large grids for the mice to enter and exit, and the small door can be closed.
  • the box next to it is white, and the bottom of the box is a rough barbed wire with a light intensity of 50lux.
  • a box is black, the bottom is glossy, the light intensity is 20lux.
  • 25 ⁇ 5g male mice were selected for the experiment, and the room temperature was 22 ⁇ 1°C.
  • the first day of the experiment was to screen the mice. The mice were free to shuttle between the two boxes and recorded for 15 minutes.
  • mice The time that the mice stayed in one box was recorded, and the mice that stayed for more than 9 minutes were excluded. Mice with no preference/aversion were picked.
  • mice were placed in the white box for administration, and the mice were adapted to the box for 45 minutes and trained for 3 consecutive days.
  • CPP performance was determined.
  • the stay time of mice in each grid after administration was measured, and the duration was 15 min.
  • the group differences between different drug treatments in the CPP experiment and the number of jumps in the naloxone withdrawal experiment were calculated using paired t-tests, * P ⁇ 0.05, ** P ⁇ 0.01 and *** P ⁇ 0.001 for drug treatment There was a significant difference between the group and the saline group. The results are shown in Figure 38.
  • the naloxone withdrawal test is a classic test for evaluating the physiological addiction of drugs.
  • the experimental method refers to Venetia Zachariou (2003).
  • the specific experimental method is as follows: Compounds 1 and 10 are administered orally (po) once every 8 hours, and the dose of the experimental drugs is gradually increased.
  • the analgesic ED 50 of morphine is 1.68 mg/kg, that is, the administered dose is 10, 20, 30, and 40 of the analgesic ED 50 , respectively. , 50, 50 and 50 times.
  • the oral ED 50 of compound 1 is 1.37 ⁇ g/kg, so the low dose of oral administration is about 20, 40, 60, 80, 100, 100, 100 ⁇ g/kg in sequence; in addition, the high dose group was given analgesia 100, 200, 300, 400, 500, 500 and 500 times the ED50 of Compound 1, the doses were approximately 200, 400, 600, 800, 1000, 1000, 1000 ⁇ g/kg, respectively.
  • the analgesic ED 50 of Compound 11 was 0.14 ⁇ g/kg, so the oral administration dose was about 2, 4, 6, 8, 10, 10, and 10 ⁇ g/kg in sequence.
  • mice were injected intraperitoneally with 10 mg/kg naloxone. Then, the mice were immediately placed in an opaque barrel structure with an inner diameter of 9 cm and a height of 32 cm, and the number of jumps of the mice within 30 min was recorded.
  • the experimental results were expressed by the number of jumps of the mice, that is, the number of jumps ⁇ standard error (Means ⁇ SEM), and the difference between the compound and the saline control group was analyzed by one-way analysis of variance (Bonferroni test of one-way ANOVA). Statistics and Analysis, * P ⁇ 0.05, ** P ⁇ 0.01 and *** P ⁇ 0.001 indicate significant differences between saline and compounds. The results are shown in Figures 39-40.

Abstract

提供了一类新型外周限制性的针对阿片受体和神经肽FF受体的多靶点环肽分子或其药学上可接受的盐。即以DN-9为化学模板，利用氨基酸替换和环化修饰等多肽化学策略对阿片肽和神经肽FF药效团进行结构优化，获得一系列环肽分子。该环肽分子能够同时激活阿片受体和NPFF受体，其镇痛活性、镇痛时长与母体DN-9分子相比均大幅度提高且镇痛耐受、便秘和成瘾等阿片样副作用都较低。

Description

一类阿片/神经肽FF受体多靶点环肽分子及其制备和应用 技术领域
本发明属于生物化学技术领域，具体地涉及一类针对阿片受体和神经肽FF受体的多靶点环肽分子及其制备和用途。
背景技术
吗啡和芬太尼是治疗急、慢性疼痛最常见的阿片类镇痛药物，但长期使用过程中产生的副作用严重限制了这类药物在临床中的应用，如呼吸抑制、耐受、滥用和便秘等。因此，探索开发新型高效低副作用的阿片类镇痛新药具有重要的意义。
近年来，利用多靶点、外周限制性分子等策略，所开发的阿片类镇痛新药能有效降低传统阿片类药物相关的副作用，在高效、低副作用镇痛新药研发中具有潜在的应用前景。例如，靶向阿片/痛敏肽(NOP)受体开发的多靶点激动剂BU08028、AT-121和BU10038证明在非人灵长类动物体内可产生有效的镇痛作用，并且药物的不良反应较低；阿片/NOR受体的多靶点激动剂Cebranopadol正处于临床Ⅱ期研究阶段，用于术后痛、糖尿病诱导的周围神经病变和癌痛等病理性疼痛的治疗，其耐受、成瘾和呼吸抑制等副作用相比于常见阿片类药物明显降低；外周限制性Kappa-阿片受体激动剂阿西马朵林和CR845目前正处于临床研究阶段，通过静脉注射的给药途径用于中重度慢性肾病相关瘙痒症和急性术后疼痛的治疗，由于这类分子不能通过血脑屏障激活中枢的阿片受体，因而其中枢副作用明显降低。
神经肽FF(NPFF)是一种阿片调节肽，其在脊髓以上水平表现出抗阿片活性，但在脊髓水平可产生阿片样镇痛作用且可加强阿片类药物诱导的镇痛作用。此外，NPFF还参与阿片类药物的镇痛耐受和成瘾等副作用的调节(Brain Res.1999,848:191-196)。专利201610252648.7中公开了一种基于阿片肽和NPFF的全新嵌合肽DN-9，体外功能实验表明该嵌合肽表现为阿片和NPFF受体的多靶点激动剂，体内药理学数据发现DN-9在中枢和外周水平均可产生强于吗啡的镇痛作用。此外，与吗啡相比，DN-9的成瘾性、镇痛耐受和便秘等阿片样副作用均显著降低(J Med Chem.2016,59:10198-10208；J Pain.2020,21:477-493；Br J Pharmacol.2020,177:93-109)。然而，已报道的DN-9的成药性仍有进一步提升的空间，如在镇痛活性和镇痛时间等方面。
多肽类药物是一类市场增长极快的药物，截止目前，全球已经批准了近100个多肽药物上市，并约有200种左右的多肽类新药已进入临床前和临床研 究阶段。然而，多肽药物自身的不稳定性是限制其进一步开发的关键性技术难题。已有研究表明，非天然氨基酸替换修饰、环化、聚乙二醇化和糖基化等化学修饰策略能有效提高多肽分子的稳定性、受体选择性和生物利用度，并进一步提高其成药性。
因此，开发一种高效、低副作用、具有长效镇痛效果的多肽类镇痛药物是非常有必要的。
发明内容
本发明的主要目的在于提供一种针对阿片受体和神经肽FF受体的多靶点环肽分子或其药学上可接受的盐。
本发明的另一目的是提供一种上述多靶点环肽分子或其药学上可接受的盐的制备方法。
本发明还有一个目的在于提供上述多靶点环肽分子或其药学上可接受的盐的治疗用途。
在本发明的第一方面，提供了一种针对阿片受体和神经肽FF受体的多靶点环肽分子或其药学上可接受的盐，所述环肽分子的结构如式I所示：
Tyr-c [2,5][Xaa2-Gly-NMe-Phe-Xaa5]-Xaa6-Xaa7-Arg-Xaa9-NH 2    (I)
式中，
Xaa2为Lys、D-Lys、D-Asp、D-Glu、D-Orn、D-Dab或D-Dap；
Xaa5为Asp、D-Asp、Glu、Lys；
Xaa6为Pro或Gly；
Xaa7为Gln、β-Ala或Aib；
Xaa9为Phe或Cha；
c [2,5]表示所述氨基酸序列中Xaa2和Xaa5两个氨基酸残基之间存在成环共价键。
在另一优选例中，在Xaa2和Xaa5两个氨基酸残基之间的成环共价键包括通过脱水缩合形成酰胺键。
在另一优选例中，所述环肽分子的结构如式II所示：
Figure PCTCN2022077309-appb-000001
式中，
Xaa2、Xaa5、Xaa6、Xaa7、和Xaa9如上定义；
“～L0～”表示Xaa2和Xaa5两个氨基酸残基之间的成环共价键。
在另一优选例中，所述环肽分子选自下述化合物：
化合物1：Tyr-c [2,5][D-Lys-Gly-NMe-Phe-Asp]-Pro-Gln-Arg-Phe-NH 2
化合物2：Tyr-c [2,5][D-Lys-Gly-NMe-Phe-Glu]-Pro-Gln-Arg-Phe-NH 2
化合物3：Tyr-c [2,5][Lys-Gly-NMe-Phe-Asp]-Pro-Gln-Arg-Phe-NH 2
化合物4：Tyr-c [2,5][Lys-Gly-NMe-Phe-D-Asp]-Pro-Gln-Arg-Phe-NH 2
化合物5：Tyr-c [2,5][D-Lys-Gly-NMe-Phe-D-Asp]-Pro-Gln-Arg-Phe-NH 2
化合物6：Tyr-c [2,5][D-Asp-Gly-NMe-Phe-Lys]-Pro-Gln-Arg-Phe-NH 2
化合物7：Tyr-c [2,5][D-Glu-Gly-NMe-Phe-Lys]-Pro-Gln-Arg-Phe-NH 2
化合物8：Tyr-c [2,5][D-Orn-Gly-NMe-Phe-Asp]-Pro-Gln-Arg-Phe-NH 2
化合物9：Tyr-c [2,5][D-Dab-Gly-NMe-Phe-Asp]-Pro-Gln-Arg-Phe-NH 2
化合物10：Tyr-c [2,5][D-Dap-Gly-NMe-Phe-Asp]-Pro-Gln-Arg-Phe-NH 2
化合物11：Tyr-c [2,5][D-Orn-Gly-NMe-Phe-Glu]-Pro-Gln-Arg-Phe-NH 2
化合物12：Tyr-c [2,5][D-Lys-Gly-NMe-Phe-Asp]-Gly-Gln-Arg-Phe-NH 2
化合物13：Tyr-c [2,5][D-Lys-Gly-NMe-Phe-Asp]-Pro-β-Ala-Arg-Cha-NH 2
化合物14：Tyr-c [2,5][D-Lys-Gly-NMe-Phe-Asp]-Pro-Aib-Arg-Cha-NH 2。
在另一优选例中，所述式I所示的环肽分子具有选自下组的一个或多个特征：
(1)所述式I所示的环肽分子为阿片受体和NPFF受体的双靶点激动剂；
(2)所述式I所示的环肽分子皮下注射的镇痛活性相比DN-9超过10倍，优选超过100倍，更优选超过500倍、1000倍、10000倍或10 6倍；
(3)所述式I所示的环肽分子的口服镇痛活性相比DN-9超过3倍，优选超过10倍，更优选超过100倍、500倍、1000倍或10000倍；
(4)所述式I所示的环肽分子不会通过血脑屏障；
(5)所述式I所示的环肽分子连续口服5天以上(优选8天)未出现镇痛耐受；
(6)所述式I所示的环肽分子不具有便秘副作用；
(7)所述式I所示的肽分子不具有成瘾性副作用。
在本发明的第二方面，提供了一种所述的多靶点环肽分子或其药学上可接受的盐的制备方法，包括步骤：
(a)采用液相合成法和/或固相合成法，按照对应于式I结构式的氨基酸序列，进行肽链合成，从而获得线性肽链；和
(b)对所述线性肽链进行环化，从而获得所述的针对阿片受体和神经肽FF受体的多靶点环肽分子或其药学上可接受的盐。
在另一优选例中，所述方法还包括：对于步骤(b)中获得多靶点环肽分子或其药学上可接受的盐进行分离，从而获得纯化的多靶点环肽分子或其药学上可接受的盐。
在另一优选例中，步骤(b)中，对Xaa2和Xaa5的侧链进行环化，从而在Xaa2和Xaa5两个氨基酸残基之间形成成环共价键。
在另一优选例中，在步骤(a)中，从氨基酸序列的C端向N端进行进行肽链合成。
在另一优选例中，在步骤(a)中，从氨基酸序列的N端向C端进行进行肽链合成。
在另一优选例中，在步骤(a)中，所述的固相合成法包括选自下组的工艺步骤：固相载体的预处理、氨基酸缩合、肽链的延伸、多肽的压缩抽干和切割、萃取、纯化，或其组合。
在另一优选例中，所述的固相载体是氨基树脂。
在另一优选例中，所述的氨基酸缩合步骤中使用的缩合试剂包括HOBt、HBTU和DIEA的组合。
在另一优选例中，所述的环化步骤中使用的环化试剂包括PyBOP和DIEA的组合。
在另一优选例中，所述的制备方法包括工艺步骤：
i.树脂预处理：将一定量的Rink-Amide-MBHA树脂在二氯甲烷(DCM)中溶胀30min，抽干，用N,N-二甲基甲酰胺(DMF)润洗树脂。DCM与树脂的体积质量比为8～12mL/g。茚三酮检验，正常情况下，溶液淡黄色，树脂无色。
ii.循环缩合氨基酸
ii.i脱除芴甲氧羰基(Fmoc)保护基：在步骤i.所得的树脂中加入体积比为1：1：98的1,8-二氮杂二环十一碳-7-烯(DBU)、哌啶和DMF，搅拌反应3次，搅拌的速率为60～100rpm，前两次搅拌的时间为2～6min，最后一次搅拌的时间为8～12min；时间优选前两次5min，最后一次10min。最后加入DMF洗涤。茚三酮检验，正常情况下，溶液蓝紫色，树脂蓝紫色。
ii.ii氨基酸缩合：将摩尔比为1：0.2～1.5：0.2～1.5：1.5～3的N-α-Fmoc保护的氨基酸、O-苯并三氮唑-N,N,N',N'-四甲基脲-六氟磷酸盐(HBTU)、N-羟基苯并三氮唑(HOBt)和N,N-二异丙基乙胺(DIEA)溶解于少量DMF中，其中DIEA最后加入，DMF与N-α-Fmoc保护的氨基酸的体积质量比为5～10mL/g；然后加入到步骤的ii.i所得的树脂中，混合溶液与树脂的体积质量比为4～6mL/g；搅拌速率为60～100rpm；所述的反应为在室温下反应40～100min。反应结束后用DMF洗涤。茚三酮检验，正常情况下，溶液淡黄色，树脂无色，得到无Fmoc保护基团的肽树脂。
ii.iii肽链的延长：根据多肽序列，依次在步骤ii.ii所得的肽树脂上接入不同的Fmoc保护氨基酸，最后一个氨基酸通常使用叔丁氧羰基(Boc)保护的氨 基酸，省去了最后一步脱Fmoc的步骤，所有氨基酸的缩合方法同上所述。得到肽树脂。
ii.iv多肽的环化：用DCM和甲醇(MeOH)交替洗树脂，压缩抽干。在氩气保护下，将步骤的ii.iii所得的树脂加入树脂摩尔量0.55倍的四三苯基膦钯(Pd(PPh 3) 4)和树脂摩尔量5倍的三乙烯二胺(DABCO)；用注射器加入体积比为37：2：1的氯仿(CHCl 3)、醋酸(HAc)和N-甲基吗啉(NMM)，混合液与树脂的体积质量比为3～6mL/g；25℃缓慢搅拌反应3～6h，搅拌速率为100～200rpm，选择性脱去氨基烯丙氧羰基(Alloc)和羧基烯丙基酯(OAll)保护基。茚三酮检验，正常情况下，溶液蓝紫色，树脂蓝紫色。然后将得到的肽树脂转移到合成仪中，用DCM和DMF交替洗树脂，再加入树脂摩尔量3倍的六氟磷酸苯并三唑-1-基-氧基三吡咯烷基磷(PyBOP)和树脂摩尔量6倍的DIEA，环化缩合反应3～6h，搅拌速率为60～100rpm。茚三酮检验，正常情况下，溶液淡黄色，树脂无色。得到环化肽树脂。
iii.压缩树脂：依次用DCM和MeOH交替洗环化肽树脂，洗完之后移去搅拌棒，抽干3～5h。
iv.多肽的切割和沉淀萃取：在步骤iii所得的树脂中加入切割剂(体积比为95：2.5：2.5的三氟乙酸(TFA)、三异丙基硅烷(Tis)和水(H 2O))。在室温条件下反应1.5～4h，每15min搅拌一次。切割剂与环化肽树脂的体积质量比为10～20mL/g。用旋转蒸发仪将切割液蒸干，温度不高于40℃。将剩余的液体置于冰箱预冷。加入预冷的乙醚，静置沉淀，沉淀用20％的HAc水溶液萃取。然后将萃取液冻干，得到粗肽。
v.多肽的纯化和分析：用反相高效液相色谱(RP-HPLC)半制备柱分离纯化粗肽，经分离后收集主峰，冻干后得到纯肽。用RP-HPLC分析柱鉴定纯度，然后用电喷雾质谱(ESI)鉴定多肽的分子量。
在本发明的第三方面，提供了一种药物组合物，所述的药物组合物包括：
(a)本发明第一方面所述的针对阿片受体和神经肽FF受体的多靶点环肽分子或其药学上可接受的盐作为活性成分；
(b)药学上可接受的载体和/或辅料。
在另一优选例中，所述药物组合物通过选自下组的施用方式进行给药：口服给药、经皮给药、鞘内给药、静脉给药、肌肉内给药、局部给药、经鼻给药等。
在另一优选例中，所述药物组合物的制剂选自下组：片剂、胶囊、糖衣片剂、粒剂、口服溶液和糖浆、气雾剂、鼻喷剂、干粉针剂、注射剂、用于皮肤表面的油膏和药贴。
在另一优选例中，所述药物组合物的制剂为注射剂。
在另一优选例中，所述的药物注射剂可用于胃肠外给药。
在本发明的第四方面，提供了一种本发明第一方面所述的多靶点环肽分子或其药学上可接受的盐的用途，用于制备一药物，所述药物用于缓解和/或治疗各类疼痛，包括急性痛和病理性疼痛。
在本发明的第五方面，提供了一种镇痛的方法，包括步骤：将安全有效量的本发明第一方面所述的针对阿片受体和神经肽FF受体的多靶点环肽分子或其药学上可接受的盐和/或本发明第三方面所述的药物组合物施用于需要的对象。
在另一优选例中，所述方法是非治疗性或治疗性的。
在另一优选例中，所述方法是体外的或体内的。
在另一优选例中，所述的需要的对象为需要缓解和/或治疗各类疼痛的对象。
在另一优选例中，所述的对象是哺乳动物或人。
在另一优选例中，所述的对象是人。
应理解，在本发明范围内中，本发明的上述各技术特征和在下文(如实施例)中具体描述的各技术特征之间都可以互相组合，从而构成新的或优选的技术方案。限于篇幅，在此不再一一累述。
附图说明
图1为小鼠皮下注射化合物1所产生的剂量依赖性镇痛作用的时间-量效曲线；
图2为小鼠皮下注射化合物2所产生的剂量依赖性镇痛作用的时间-量效曲线；
图3为小鼠皮下注射化合物3所产生的剂量依赖性镇痛作用的时间-量效曲线；
图4为小鼠皮下注射化合物4所产生的剂量依赖性镇痛作用的时间-量效曲线；
图5为小鼠皮下注射化合物5所产生的剂量依赖性镇痛作用的时间-量效曲线；
图6为小鼠皮下注射化合物6所产生的剂量依赖性镇痛作用的时间-量效曲线；
图7为小鼠皮下注射化合物7所产生的剂量依赖性镇痛作用的时间-量效曲线；
图8为小鼠皮下注射化合物8所产生的剂量依赖性镇痛作用的时间-量效曲线；
图9为小鼠皮下注射化合物9所产生的剂量依赖性镇痛作用的时间-量效曲线；
图10为小鼠皮下注射化合物10所产生的剂量依赖性镇痛作用的时间-量效曲线；
图11为小鼠皮下注射化合物11所产生的剂量依赖性镇痛作用的时间-量效曲线；
图12为小鼠皮下注射化合物12所产生的剂量依赖性镇痛作用的时间-量效曲线；
图13为小鼠皮下注射化合物13所产生的剂量依赖性镇痛作用的时间-量效曲线；
图14为小鼠皮下注射化合物14所产生的剂量依赖性镇痛作用的时间-量效曲线；
图15为小鼠口服注射母体DN-9所产生的镇痛作用时间-量效曲线；
图16为小鼠口服注射化合物1所产生的剂量依赖性镇痛作用的时间-量效曲线；
图17为小鼠口服注射化合物11所产生的剂量依赖性镇痛作用的时间-量效曲线；
图18为小鼠注射甲碘化纳洛酮和纳洛酮对皮下注射化合物1的血脑屏障通透性研究结果；
图19为小鼠注射甲碘化纳洛酮和纳洛酮对皮下注射化合物2的血脑屏障通透性研究结果；
图20为小鼠注射甲碘化纳洛酮和纳洛酮对皮下注射化合物3的血脑屏障通透性研究结果；
图21为小鼠注射甲碘化纳洛酮和纳洛酮对皮下注射化合物4的血脑屏障通透性研究结果；
图22为小鼠注射甲碘化纳洛酮和纳洛酮对皮下注射化合物5的血脑屏障通透性研究结果；
图23为小鼠注射甲碘化纳洛酮和纳洛酮对皮下注射化合物6的血脑屏障通透性研究结果；
图24为小鼠注射甲碘化纳洛酮和纳洛酮对皮下注射化合物7的血脑屏障通透性研究结果；
图25为小鼠注射甲碘化纳洛酮和纳洛酮对皮下注射化合物8的血脑屏障通透性研究结果；
图26为小鼠注射甲碘化纳洛酮和纳洛酮对皮下注射化合物9的血脑屏障通透性研究结果；
图27为小鼠注射甲碘化纳洛酮和纳洛酮对皮下注射化合物10的血脑屏障通透性研究结果；
图28为小鼠注射甲碘化纳洛酮和纳洛酮对皮下注射化合物11的血脑屏障通透性研究结果；
图29为小鼠注射甲碘化纳洛酮和纳洛酮对皮下注射化合物12的血脑屏障通透性研究结果；
图30为小鼠注射甲碘化纳洛酮和纳洛酮对皮下注射化合物13的血脑屏障通透性研究结果；
图31为小鼠注射甲碘化纳洛酮和纳洛酮对皮下注射化合物14的血脑屏障通透性研究结果；
图32为小鼠注射甲碘化纳洛酮和纳洛酮对口服注射化合物1的血脑屏障通透性研究结果；
图33为小鼠注射甲碘化纳洛酮和纳洛酮对口服注射化合物11的血脑屏障通透性研究结果；
图34为小鼠连续八天口服注射化合物1和11所产生的镇痛作用的变化；
图35为小鼠口服注射化合物1对胃肠运动的作用；
图36为小鼠口服注射化合物11对胃肠运动的作用；
图37为小鼠口服注射化合物1和11对运动活性的调节作用；
图38为小鼠口服注射化合物1和11对条件位置的调节作用；
图39为小鼠口服注射化合物1对纳洛酮戒断后的反应；
图40为小鼠口服注射化合物11对纳洛酮戒断后的反应。
具体实施方式
本发明人经过广泛而深入的实验研究，首次在阿片/NPFF受体的多靶点分子DN-9基础上，针对阿片药效团通过引入侧链含氨基和羧基结构的氨基酸残基D-Dap、D-Dab、Lys、D-Lys、D-Orn、Asp、D-Asp、D-Glu或Glu并进行环化修饰，针对NPFF药效团通过引入Gly、β-Ala、Aib和Cha进行氨基酸替换，获得了一系列针对阿片受体和神经肽FF受体的多靶点环肽分子或其药学上可接受的盐。研究表明本发明的新型环肽分子或其药学上可接受的盐克服了母体DN-9分子镇痛作用时间短以及外周镇痛活性弱等问题，具有高效的外周镇痛活性、镇痛持续时间长、有效镇痛剂量低，同时无耐受、低成瘾性、低便秘，可作为新一代治疗疼痛的潜在药物用于各类疼痛的治疗。基于上述发现，发明人完成了本发明。
术语
如本文所用，术语“针对阿片受体和神经肽FF受体的多靶点环肽分子或其药学上可接受的盐”、“本发明的环肽分子或其药学上可接受的盐”可互换使用，是指本发明所述的阿片/神经肽FF受体多靶点分子DN-9酰胺键环化类似物或其药学上可接受的盐，其以阿片/NPFF受体的多靶点分子DN-9为化学模板，针对阿片药效团通过引入侧链含氨基和羧基结构的氨基酸残基D-Dap、D-Dab、Lys、D-Lys、D-Orn、Asp、D-Asp、D-Glu或Glu并进行酰胺键环化修饰，针对NPFF药效团引入Gly、β-Ala、Aib和Cha进行氨基酸替换从而得到。其中，多靶点分子DN-9母体肽的序列如下：
Tyr-D-Ala-Gly-NMe-Phe-Gly-Pro-Gln-Arg-Phe-NH 2。
在另一优选例中，所述环肽分子的具体序列如表1所示：
表1.针对阿片受体和神经肽FF受体的多靶点环肽分子的氨基酸序列
Figure PCTCN2022077309-appb-000002
如本文所用，术语“NMe-Phe”、“N-Me-Phe”、或“NMePhe”可互换使用，指N α-甲基苯丙氨酸，其结构式如下所示：
Figure PCTCN2022077309-appb-000003
如本文所用，采用常规的三字母代码代表天然氨基酸，并采用公认的三字母代码代表其他氨基酸，如NMe-Phe(N α-甲基苯丙氨酸)、D-Lys(D型赖氨 酸)、D-Orn(D型鸟氨酸)、D-Dab(D型2,4-二氨基丁酸)、D-Dap(D型2,4-二氨基丙酸)、D-Asp(D型天冬氨酸)、D-Glu(D型谷氨酸)、β-Ala(β-丙氨酸)、Aib(氨基异丁酸)、Cha(β-环已基-L-丙氨酸)。此外，本发明所述的氨基酸，除特别注明“D-”表示为D型氨基酸，其余均为L型氨基酸。
如本文所用，术语“药学上可接受的盐”是指能够保留本发明所述环肽分子的生物有效性而无其他副作用的，与无毒的酸或碱合成的本发明环肽分子的盐。
制备方法
本发明的针对阿片受体和神经肽FF受体的多靶点环肽分子或其药学上可接受的盐以DN-9为化学模板，利用氨基酸替换和酰胺键环化修饰等多肽化学策略对阿片肽和神经肽FF药效团进行结构优化，具体包括以下工艺步骤：
i.树脂预处理：将Rink-Amide-MBHA树脂在DCM中溶胀，抽干，用DMF洗涤树脂，茚三酮检验；
ii.循环缩合氨基酸
ii.i脱除Fmoc保护基：在步骤i.所得的树脂中加入体积比为1：1：98的DBU、哌啶和DMF的混合溶液，搅拌反应；加入DMF洗涤，茚检；
ii.ii氨基酸缩合：将摩尔比为1：0.2～1.5：0.2～1.5：1.5～3的Fmoc-Aa、HBTU、HOBt和DIEA溶解于DMF中，然后加入到步骤的ii.i所得的树脂中；用DMF洗涤，茚检，得到无Fmoc保护基团的肽树脂；
ii.iii肽链的延长：根据多肽序列，依次接入不同的Fmoc保护氨基酸，最后一个氨基酸使用Boc保护的氨基酸，所有氨基酸的缩合方法同上所述，得到肽树脂；
ii.iv多肽的环化：用DCM和MeOH交替洗涤，压缩抽干；在氩气保护下，在步骤的ii.iii所得的肽树脂中加入Pd(PPh 3) 4和DABCO，之后再用注射器加入CHCl 3、HAc和NMM的混合溶液，选择性脱去Alloc和OAll保护基；茚检，洗涤后加入PyBOP和DIEA，环化缩合反应3～6h，搅拌速率为60～100rpm，茚检，得到环化肽树脂；
iii.压缩树脂：依次用DCM和MeOH交替洗环化肽树脂，抽干；
iv.多肽的切割和沉淀萃取：在步骤iii所得的的肽树脂中加入切割剂(体积比为95：2.5：2.5的TFA、Tis和H 2O)，在室温条件下反应1.5～4h；旋蒸，加入预冷的乙醚析出沉淀，沉淀用20％的HAc水溶液萃取，冻干萃取液，得到粗肽；
v.多肽的纯化和分析：用反相高效液相色谱(RP-HPLC)半制备柱分离纯化粗肽，经分离后收集主峰，冻干后得到纯肽。然后用电喷雾质谱(ESI)鉴定 多肽的分子量。
药物组合物和施用方法
本发明还提供了一种药物组合物，其包含安全有效量范围内的本发明的环肽分子或其药学上可接受的盐，以及药学上可接受的载体。
所述的本发明的环肽分子或其药学上可接受的盐和药物组合物用于制备镇痛的药物。所述的本发明的环肽分子或其药学上可接受的盐和药物组合物可用作镇痛药物。
在另一优选例中，用于制备高效、低副作用的镇痛药物。
在另一优选例中，用于缓解和治疗包括急性痛和病理性疼痛在内的各类疼痛。
“安全有效量”是指本发明环肽分子或其药学上可接受的盐的量足以明显产生镇痛效果，而不至于产生严重的副作用。
所述的药物组合物可以是任何合适的形式，(取决于患者需要的施用方法)。其可以以单位剂型的形式提供，通常置于密封容器中，并且可以作为试剂盒的一部分提供。此类试剂盒通常(但不是必须)包含使用说明。其可以包含多个所述的单位剂型。
所述的药物组合物的施用方法没有特别限制，代表性的施用方法包括(但并不限于)：口服给药、经皮给药、鞘内给药、静脉给药、肌肉内给药、局部给药、经鼻给药等。
根据所采用的施用方法，可将本发明的药物组合物制成各种合适的剂型，适当剂型的实例为可用于注射的无菌溶液和干粉针剂、片剂、胶囊、糖衣片剂、粒剂、口服溶液和糖浆、气雾剂、鼻喷剂，以及用于皮肤表面的油膏和药贴。
含有本发明环肽分子或其药学上可接受的盐的药物组合物可以制成溶液或者冻干粉末以用于胃肠外给药，在使用前可加入适当溶剂或者其他可药用的载体将粉末重新配置，溶液配方一般是缓冲液、等渗溶液或水溶液。
本发明的药物组合物可以单独给药，或者与其他镇痛药物联合给药。
使用药物组合物时，是将安全有效量的本发明的多肽或其药学上可接受的盐施用于需要治疗的哺乳动物(如人)，其中本发明药物组合物的剂量可以在一个较大范围内变动，本领域技术人员可以根据一些客观的因素，如根据疾病的种类、病情严重程度、病人体重、剂型、给药途径等因素很容易地加以确定。
与现有技术相比，本发明的主要优点有：
(1)本发明的环肽分子或其药学上可接受的盐的皮下和口服镇痛活性相比母体DN-9有显著提高，其中皮下注射的镇痛ED 50(半数有效剂量)下降了 上百倍，更佳地可达1000～10000倍，并且口服镇痛活性相对母体也均有所提高；
(2)本发明的环肽分子或其药学上可接受的盐的有效作用时间长，约为240min；
(3)本发明的环肽分子或其药学上可接受的盐不能穿透血脑屏障、无镇痛耐受、无便秘或成瘾性副作用。
下面结合具体实施例，进一步阐述本发明。应理解，这些实施例仅用于说明本发明而不用于限制本发明的范围。下列实施例中未注明具体条件的实验方法，通常按照常规条件或按照制造厂商所建议的条件。除非另外说明，否则百分比和份数是重量百分比和重量份数。
除非另行定义，文中所使用的所有专业与科学用语与本领域熟练人员所熟悉的意义相同。此外，任何与所记载内容相似或均等的方法及材料皆可应用于本发明方法中。文中所述的较佳实施方法与材料仅作示范之用。
所用仪器及主要实验材料如下：
实验仪器：固相多肽合成仪由本发明人自主设计，旋转蒸发仪RE-5298A购自上海亚荣，冷冻干燥机购自美国VIRTIS公司，质谱仪ESI-Q-TOF maXis-4G，Bruker Daltonics购自德国道尔顿公司，循环水泵SHB-Ⅲ型购自郑州长城，高效液相色谱仪(RP-HPLC)为Waters公司的Delta 600，其中分析柱：XBridge TM BEH 130Prep C18，4.6mm×250mm；制备柱：XBridge TM BEH 130Prep C18，19mm×250mm。
实验试剂：树脂为Rink-Amide-MBHA Resin(取代值S为0.4mmol/g)购自天津南开和成公司，N-α-Fmoc保护的氨基酸(Fmoc-Aa)，O-苯并三氮唑-N,N,N',N'-四甲基脲-六氟磷酸盐(HBTU)和N-羟基苯并三氮唑(HOBt)购自上海吉尔生化有限公司，N,N-二异丙基乙胺(DIEA)购自北京百灵威，1,8-二氮杂二环十一碳-7-烯(DBU)购自上海迈瑞尔公司。四三苯基膦钯(Pd(PPh 3) 4)，三乙烯二胺(DABCO)和三异丙基硅烷(Tis)购自上海安耐吉，茚三酮为上海试剂三厂产品。二氯甲烷(DCM)、N,N-二甲基甲酰胺(DMF)、六氢吡啶(哌啶)、甲醇(MeOH)和吡啶购自天津第二试剂厂，三氟乙酸(TFA)和苯酚均为天津试剂厂产品；以上有机试剂使用前均经过重蒸处理。
实施例1.化合物1的合成
(1)树脂预处理：称取1g的Rink-Amide-MBHA树脂(取代值为0.4mmol/g)，加入10mL的DCM，80rpm搅拌溶胀，搅拌反应30min，抽干，用DMF润洗树脂 3次，每次3min。
(2)脱除Fmoc保护基团：在树脂中加入10mL体积比为1：1：98的DBU、哌啶和DMF；按照80rpm搅拌5min，重复2次。抽干后，再次加入上述混合溶液，按照80rpm搅拌10min。最后加入DMF洗涤4次，每次3min。
(3)茚三酮检验：茚检试剂为体积比为1：2：1的苯酚：吡啶：茚三酮溶液。其中苯酚溶液的配制为20g苯酚于5mL无水乙醇，吡啶溶液的配制为0.05mL KCN(0.001M)于2.5mL吡啶，茚三酮溶液的配制为0.5g茚三酮溶于10mL无水乙醇，其中苯酚、吡啶和无水乙醇均经重蒸处理。茚三酮检验，正常情况下，溶液蓝紫色，树脂蓝紫色。
(4)氨基酸的缩合：称取摩尔比为1：1：1的Fmoc-Phe-OH、HOBt和HBTU，然后溶解在5mL的DMF中，其中DIEA最后加入，搅拌均匀之后加入步骤(2)已经脱除Fmoc保护基团的树脂中，在氩气保护下，室温80rpm搅拌速率下反应60min，抽干溶剂；反应完之后按步骤(3)的方法茚检，若溶液淡黄色树脂为无色则表明氨基酸缩合到树脂上。然后再按照步骤(2)脱去Fmoc保护基，按步骤(3)再茚检，若溶液、树脂均为深蓝色表示Fmoc保护基团脱除完全，得到无Fmoc保护基团的树脂肽。
(5)肽链的延伸：将步骤(4)所获得的肽树脂按照步骤(2-4)的方法依次将Fmoc-Arg(pbf)-OH、Fmoc-Gln(Trt)-OH、Fmoc-Pro-OH、Fmoc-Asp(OAll)-OH、Fmoc-NMe-Phe-OH、Fmoc-Gly-OH、Fmoc-D-Lys(Alloc)-OH和Boc-Tyr(tBu)-OH依次缩合到肽树脂上。得到肽树脂Boc-Tyr(tBu)-D-Lys(Alloc)-Gly-NMe-Phe-Asp(OAll)-Pro-Gln(Trt)-Arg(Pbf)-Phe-Resin。
(6)多肽的环化：将得到的肽树脂Boc-Tyr(tBu)-D-Lys(Alloc)-Gly-NMe-Phe-Asp(OAll)-Pro-Gln(Trt)-Arg(Pbf)-Phe-Resin依次用DCM(2×3min)，MeOH(1×3min)，DCM(1×3min)，MeOH(2×3min)交替洗，压缩，抽干4h。然后将肽树脂转移至圆底烧瓶，加入255mg的Pd(PPh 3) 4，225mg的DABCO，充氩气保护，之后用注射器加入5mL体积比为37：2：1的CHCl 3、HAc和NMM。在氩气保护条件下25℃和150rpm搅拌反应4h，选择性脱去氨基Alloc和羧基OAll。茚三酮检验，正常情况下，溶液蓝紫色，树脂蓝紫色；然后加入625mg的PyBOP和396μL的DIEA，80rpm搅拌反应4h。茚三酮检验，溶液淡黄色，树脂无色。得到环化肽Boc-Tyr(tBu)-c [2,5][D-Lys(Alloc)-Gly-NMe-Phe-Asp(OAll)]-Pro-Gln(Trt)-Arg(Pbf)-Phe-Resin。
(7)肽链的压缩抽干：依次用DCM(2×3min)，MeOH(1×3min)，DCM(1×3min)，MeOH(2×3min)交替洗树脂，之后移去搅拌棒抽干4h。
(8)肽链的切割：在抽干的肽树脂Boc-Tyr(tBu)-c [2,5][D-Lys(Alloc)-Gly-NMe-Phe-Asp(OAll)]-Pro-Gln(Trt)-Arg(Pbf)-Phe-Resin中加入15mL的切割剂 (TFA：H 2O：Tis＝95：2.5：2.5)。于室温下搅拌反应3h，每15min于50rpm搅拌速率下搅拌1min。滤液在不高于40℃的条件下充分减压旋干，加入冰乙醚，充分振荡混匀。粗肽以白色沉淀的形式充分析出。20％的HAc水溶液萃取乙醚中的粗肽。最后将萃取到的肽的水溶液通过冷冻干燥得到白色的粗肽固体粉末324mg，粗肽产率为70％。
(9)粗肽的纯化：用反相高效液相色谱(RP-HPLC)C18柱(XBridge TM BEH 130Prep C18，19mm×250mm)对上述粗肽化合物进行分离纯化，乙腈(含0.1％TFA)和水(含0.1％TFA)，经分离后收集主峰样品，得到纯化的化合物1样品，上样量为50mg，通冷冻干燥得到白色的纯肽固体粉末15mg，纯肽产率30％。质谱和色谱分析检测结果如表2所示。
实施例2.化合物2的合成
按照同实施例1的方法，得到环化肽Tyr-c [2,5][D-Lys-Gly-NMe-Phe-Glu]-Pro-Gln-Arg-Phe-NH 2，为白色固体粉末，粗肽300mg，粗肽产率为64％，精制后纯肽产率24％。质谱和色谱分析检测结果如表2所示。
实施例3.化合物3的合成
按照同实施例1的方法，得到环化肽Tyr-c [2,5][Lys-Gly-NMe-Phe-Asp]-Pro-Gln-Arg-Phe-NH 2，为白色固体粉末，粗肽320mg，粗肽产率为69％，精制后纯肽产率20％。质谱和色谱分析检测结果如表2所示。
实施例4.化合物4的合成
按照同实施例1的方法，得到环化肽Tyr-c [2,5][Lys-Gly-NMe-Phe-D-Asp]-Pro-Gln-Arg-Phe-NH 2，为白色固体粉末，粗肽325mg，粗肽产率为71％，精制后纯肽产率26％。质谱和色谱分析检测结果如表2所示。
实施例5.化合物5的合成
方法同实施例1，得到环化肽Tyr-c [2,5][D-Lys-Gly-NMe-Phe-D-Asp]-Pro-Gln-Arg-Phe-NH 2，为白色固体粉末，粗肽350mg，粗肽产率为76％，精制后纯肽产率30％。质谱和色谱分析检测结果如表2所示。
实施例6.化合物6的合成
方法同实施例1，得到环化肽Tyr-c [2,5][D-Asp-Gly-NMe-Phe-Lys]-Pro-Gln-Arg-Phe-NH 2，为白色固体粉末，粗肽330mg，粗肽产率为72％，精制后纯肽产率60％。质谱和色谱分析检测结果如表2所示。
实施例7.化合物7的合成
方法同实施例1，得到环化肽Tyr-c [2,5][D-Glu-Gly-NMe-Phe-Lys]-Pro-Gln-Arg-Phe-NH 2，为白色固体粉末，粗肽443mg，粗肽产率为95％，精制后纯肽产率26％。质谱和色谱分析检测结果如表2所示。
实施例8.化合物8的合成
方法同实施例1，得到环化肽Tyr-c [2,5][D-Orn-Gly-NMe-Phe-Asp]-Pro-Gln-Arg-Phe-NH 2，为白色固体粉末，粗肽285mg，粗肽产率为63％，精制后纯肽产率20％。质谱和色谱分析检测结果如表2所示。
实施例9.化合物9的合成
方法同实施例1，得到环化肽Tyr-c [2,5][D-Dab-Gly-NMe-Phe-Asp]-Pro-Gln-Arg-Phe-NH 2，为白色固体粉末，粗肽290mg，粗肽产率为65％，精制后纯肽产率40％。质谱和色谱分析检测结果如表2所示。
实施例10.化合物10的合成
方法同实施例1，得到环化肽Tyr-c [2,5][D-Dap-Gly-NMe-Phe-Asp]-Pro-Gln-Arg-Phe-NH 2，为白色固体粉末，粗肽300mg，粗肽产率为68％，精制后纯肽产率24％。质谱和色谱分析检测结果如表2所示。
实施例11.化合物11的合成
方法同实施例1，得到环化肽Tyr-c [2,5][D-Orn-Gly-NMe-Phe-Glu]-Pro-Gln-Arg-Phe-NH 2，为白色固体粉末，粗肽330mg，粗肽产率为72％，精制后纯肽产率38％。质谱和色谱分析检测结果如表2所示。
实施例12.化合物12的合成
方法同实施例1，得到环化肽Tyr-c [2,5][D-Lys-Gly-NMe-Phe-Asp]-Gly-Gln-Arg-Phe-NH 2，为白色固体粉末，粗肽307mg，粗肽产率为69％，精制后纯肽产率50％。质谱和色谱分析检测结果如表2所示。
实施例13.化合物13的合成
方法同实施例1，得到环化肽Tyr-c [2,5][D-Lys-Gly-NMe-Phe-Asp]-Pro-β-Ala-Arg-Cha-NH 2，为白色固体粉末，粗肽329mg，粗肽产率为74％，精制后纯肽产率28％。质谱和色谱分析检测结果如表2所示。
实施例14.化合物14的合成
方法同实施例1，得到环化肽Tyr-c [2,5][D-Lys-Gly-NMe-Phe-Asp]-Pro-Aib-Arg-Cha-NH 2，为白色固体粉末，粗肽423mg，粗肽产率为93％，精制后纯肽产率24％。质谱和色谱分析检测结果如表2所示。
基于以上合成步骤，本发明合成了包括如表1所列的针对阿片受体和神经肽FF受体的多靶点环肽分子，其化学表征结果如表2所示。
表2.本发明的环肽分子的化学表征
Figure PCTCN2022077309-appb-000004
注：体系1：梯度洗脱体系1为：10-80％乙腈/水(0.1％TFA)(30min完成)，流速为：1mL/min，检测波长为220nm，分析色谱柱为：XBridge TM BEH 130Prep C 18，4.6mm×250mm；体系2：梯度洗脱体系2为：10-100％乙腈/水(0.1％TFA)(30min完成)，流速为：1mL/min，检测波长为220nm，分析色谱柱为：XBridge TM BEH 130Prep C 18，4.6mm×250mm。
实施例15.对阿片受体和NPFF受体的体外功能活性测定
通过检测本发明的环肽分子对稳定表达Mu-阿片受体、Delta-阿片受体、Kappa-阿片受体、NPFF 1与NPFF 2受体的HEK293细胞中Forskolin引起的细胞内环磷酸腺苷(cAMP)积累的调节来检测它们对这五种受体的激动活性。实验方法为：24孔板种细胞，每孔种12万，培养20h以上。实验开始时，先吸出培养皿中的培养 基，然后加入500μL预热的含1mM的IBMX的无血清培养基，37℃孵育10min。然后每孔再加入待测药物与10μM forskolin(终浓度)各10μL，37℃共孵育30min。孵育结束后，吸出培养皿中的全部液体，每孔加入500μL 0.2N的盐酸，室温下孵育30min，促进细胞裂解。裂解完成后加入NaOH中和用于裂解的盐酸溶液。然后将培养皿中的液体全部吸出到离心管中，12000rpm，离心2min。取50μL于干净的离心管，再加入100uL 60ug/uL的PKA，而空白对照组(B)中加入100μL TE cAMP缓冲液。以上离心管中每管再加入50μL 0.5μCi[ 3H]cAMP，迅速混匀后，4℃孵育大于2h。孵育结束后，每管再加入100μL活性炭悬浮液，涡旋震荡均匀后冰浴放置1min，5000rpm离心4min。每管吸取离心后的上清液200μL加入到24孔板中，每孔再加入700μL闪烁液，之后用胶膜封闭24孔板，放置3h后将其置于闪烁仪上测量。
cAMP的抑制效应用药物对Forskolin诱导的胞内cAMP积累的抑制百分比(％control)表示，％control＝(Forskolin处理的cAMP含量－待测药物与Forskolin共处理的cAMP含量)/(Forskolin处理的cAMP含量－溶剂处理的cAMP含量)。相关的％control数据用平均值±标准误差(Means±S.E.M.)表示。药物剂量效应关系用非线性回归模型统计，利用统计软件GraphPad Prism 5.0版本，分别计算出该多靶点环肽分子对Forskolin引起的细胞内cAMP的积累抑制的IC 50值，实验结果见表3和表4。
表3 本发明的环肽分子在阿片受体上的cAMP功能实验
Figure PCTCN2022077309-appb-000005
表4 本发明的环肽分子在NPFF受体上的cAMP功能实验
Figure PCTCN2022077309-appb-000006
Figure PCTCN2022077309-appb-000007
如表3所示，在稳定表达Mu-和Delta-阿片受体的HEK293细胞系中，化合物1-6和8-12都剂量依赖地抑制forskolin引起的cAMP积累，从而说明这些化合物均表现为Mu-和Delta-阿片受体的激动活性。在稳定表达Kappa-阿片受体的HEK293细胞系中，化合物1-3、6和8-12都剂量依赖地抑制forskolin引起的cAMP积累，从而说明化合物1-3、6和8-12均表现为Kappa-阿片受体的激动活性。并且，如表4所示，在稳定表达NPFF 1和NPFF 2受体的HEK293细胞系中，化合物1-6和8-12都剂量依赖地抑制forskolin引起的cAMP积累，从而说明这些化合物同时还具有NPFF 1和NPFF 2受体的激动活性。综上所述，化合物1-6和8-12能同时激活阿片和NPFF受体，表现为一类阿片和NPFF受体的多靶点激动剂。
实施例16.体内镇痛活性测定
采取外周皮下和口服给药两种给药方式，之后通过小鼠光热甩尾的急性痛模型进行药物镇痛活性的研究。
皮下给药(s.c.)选择背部皮下给药。用1mL的无菌注射器给药，按0.1mL/10g的体积注射给药。右手抓起背部皮肤，倾斜进针，将药物注射于小鼠背部皮下。进针之后左右晃动针，注意观察证明针确实进入皮下，防止针戳穿皮肤漏药。
口服给药(p.o.)选用1mL的无菌注射器，将针头换成小鼠灌胃针，口服灌胃，0.1uL/10g。左手抓住小鼠的颈背部皮肤，腹部朝上，注意小鼠的整个身子固定垂直，利于口服灌胃。右手拿着灌胃针(12号针头和1mL的注射器)从小鼠的口角紧贴舌面沿上颚进去食管，进针2.5cm可注射灌胃液。进针长度应该提前练习确定，进针之后有落空感。进针位置如果合适，应该很顺利，否则说明进针不合适，说明灌胃针可能插入到小鼠的气管中，这样灌胃之后，会导致小鼠马上死亡。
光热甩尾实验，本发明基于D’Amour和Smith总结的实验方法对实验参数 进行了优化。实验选择体重21±2g昆明系雄性小鼠，环境温度控制在22±2℃。实验小鼠可以自由饮水，开始实验之前将小鼠从饲养室移至实验区适应30min。然后右手抓住小鼠，小鼠的尾巴自由下垂，然后将小鼠的尾巴放在辐射光源上，放置的位置距离小数的尾部2～3mm。辐射热的强度调至小鼠的甩尾时间3～5s，即光热甩尾的基础痛阈值。小鼠尾部的照射时间不超过10s，以防烫伤小鼠的尾部，即光热甩尾的最大潜伏期。然后给药之后，选取10、20、30、45、60、90、120、180、240、300、360和420min的时间点测量小鼠给药之后的甩尾潜伏期。
药物的镇痛效果一般用最大镇痛效应MPE(％)来评价，MPE(％)＝100×[(给药后的痛阈－基础痛阈)/(10秒－基础痛阈)]。半数有效剂量ED 50(50％effective dose,ED 50)指引起50％的作用效果时对应的药物剂量。ED 50和95％的置信区间
通过统计学软件GraphPad Prism 5.0利用MPE(％)以及达到最大镇痛效应MPE(％)的药物浓度来计算。镇痛作用的差异使用单因素方差分析(one-way ANOVA的Dunnett检验)进行统计， *P<0.05、 **P<0.01以及 ***P<0.001表示只注射相关药物组与盐水组差异显著性。
在小鼠的光热甩尾实验中，皮下注射所有环肽分子和DN-9母体分子的镇痛ED 50如表5所示，母体DN-9镇痛ED 50值为228μg/kg。表5所列14个环肽分子的ED 50值均显著低于母肽的镇痛ED 50，其中，化合物11-14的皮下注射镇痛ED 50低于母体DN-9分子至少10000倍。并且有效镇痛作用时间从母体的90min均延长至240min。化合物的皮下镇痛量效曲线如图1-14所示。
在小鼠的光热甩尾实验中，口服注射化合物1-6和8-12的镇痛最高剂量下的镇痛活性如表6所示。DN-9在40mg/kg的较高剂量下才会产生较强的镇痛活性，化合物1-6和8-12的口服镇痛效果且镇痛活性显著优于母体(DN-9)，不仅半数镇痛剂量下降了上百倍，而且有效镇痛作用时间从母体的90min均延长至240min。尤其是化合物1和化合物11的ED 50值分别为1.37和0.14μg/kg，其镇痛效价均远远优于母体。母肽DN-9，化合物1和11的口服镇痛图如图15-17所示。
表5.皮下注射多靶点环肽分子所产生的镇痛活性
Figure PCTCN2022077309-appb-000008
Figure PCTCN2022077309-appb-000009
表6.口服注射多靶点环肽分子所产生的镇痛活性
Figure PCTCN2022077309-appb-000010
实施例17.血脑屏障通透性测定
血脑屏障通透性的药理学评价实验通过注射甲碘化纳洛酮来检测药物是否通过血脑屏障，甲碘化纳洛酮是一种不能通过血脑屏障的药物。实验中通过不同位点注射甲碘化纳洛酮、纳洛酮和化合物，然后通过光热甩尾实验来检测化合物镇痛效果的改变。
血脑屏障渗透性机制实验选用21±2g的昆明雄系小鼠，环境温度控制在22±2℃，小鼠可自由进食、饮水。甲碘化纳洛酮(NALM)和纳洛酮(Nal)都是提前10min给药，甲碘化纳洛酮选择侧脑室(i.c.v.)、皮下(s.c.)和腹腔(i.p.)三种给药方式，纳洛酮选择皮下(s.c.)和腹腔(i.p.)两种给药方式，不同的化合物主要选择皮下(s.c.)和口服(p.o.)外周水平给药，然后通过光热甩尾实验来检测给完拮抗剂和药物之后镇痛的改变。
实验数据用MPE(maximum possible effect)表示，MPE(％)＝100×[(给药后的痛阈－基础痛阈)/(10秒－基础痛阈)]。药物的拮抗作用利用相关药物最大镇痛效应时间点的MPE值来进行比较。MPE数据用平均值±标准误(Means±S.E.M.)来表示，镇痛作用的差异使用单因素方差分析(one-way ANOVA的Bonferroni检验)进行统计， *P<0.05、 **P<0.01以及 ***P<0.001表示只注射相关药物组与同时注射拮抗剂组和相关化合物的差异显著性。
血脑屏障渗透性检测结果如表7所示。侧脑室注射甲碘化纳洛酮不能拮抗皮下注射化合物1-14所引起的镇痛，皮下注射纳洛酮和甲碘化纳洛酮能拮抗皮下注射化合物1-14所引起的镇痛，从而说明化合物1-14均不能穿透血脑屏障。如图18-31所示，化合物1-14的血脑屏障通透性研究的具体结果。
表7.本发明的环肽分子的血脑屏障通透性测定
Figure PCTCN2022077309-appb-000011
化合物1和11口服给药的血脑屏障渗透性结果如图32和33所示。侧脑室注射甲碘化纳洛酮不能拮抗口服注射化合物1和11所引起的镇痛，腹腔注射纳洛酮和甲碘化纳洛酮能拮抗口服注射化合物1和11所引起的镇痛，从而说明口服注射化合物1和11均不能穿透血脑屏障，这提示本发明化合物的对中枢神经系统的副作用低。
实施例18.化合物1和11的镇痛耐受实验测定
镇痛耐受实验是通过连续8天口服注射化合物1和11，然后通过光热甩尾实 验鉴定从第一天到第八天镇痛甩尾阈值的变化。从而评价本发明中化合物1和11在镇痛耐受方面的药理活性。
实验选取21±2g的昆明系雄性小鼠，小鼠可以自由饮食。一般是第一天先测定小鼠的基础痛阈值，然后口服注射化合物1和11，连续注射8天，第一天测定不同时间点的痛阈值，随后7天只测定最高镇痛点的痛阈值。小鼠注射传统阿片类药物一般会在第3或者第4天，会出现镇痛阈值的下降，即出现镇痛耐受。
实验数据用甩尾时间表示。药物的镇痛耐受作用利用不同化合物的最大镇痛效应时间点的甩尾潜伏期来进行比较。甩尾潜伏期数据用平均值±标准误(Means±S.E.M.)来表示，小鼠皮下连续给药八天镇痛作用的差异使用单因素方差分析(one-way ANOVA的Tukey HSD检验)进行统计， *P<0.05、 **P<0.01， ***P<0.001表示与第一天注射该药物的镇痛作用相比有极显著性差异。如图34所示。口服连续注射8天之后盐水组没有任何变化，口服连续8天注射化合物1和11也没有出现镇痛耐受。
实施例19.口服注射化合物1和11的便秘副作用测定
便秘是阿片类药物常见的副作用。因此一般通过便秘副作用来评价药物对胃肠运动的影响。
实验选取26±2g的昆明系雄性小鼠，首先饥饿小鼠，将小鼠放于不放垫料的盒子，小鼠不能饮食但是可以自由饮水，饥饿处理16h。16h之后称重，然后口服给药(p.o.)，给药15min之后将预先准备好的活性炭悬浮液(一种含5％活性炭和10％阿拉伯树胶的生理盐水悬浮液)以每0.1mL/10g的体积经口灌注到胃。灌胃30min之后，颈部脱臼处死小鼠。马上解剖，取小肠全长，从胃幽门到盲肠这段距离。然后测量小肠总长以及碳粉移动的长度。
胃肠运动实验数据用胃肠运动百分比来表示，具体计算方法为碳粉移动的距离除以小肠总长之后的百分比来表示。数据用胃肠运动百分比的平均值±标准误(Means±S.E.M.)来表示，化合物与盐水之间的差异用单因素方差分析(one-way ANOVA的Dunnett检验)进行数据统计和分析， *P<0.05、 **P<0.01以及 ***P<0.001表示只注射盐水与注射相关化合物的差异显著性。化合物1和11的胃肠运动实验结果如图35-36所示。
在图35中，口服分别注射生理盐水，100，1000和10000μg/kg的化合物1，生理盐水组的胃肠抑制百分比为84.54％，给药组的胃肠抑制百分比分别为87.82％，86.46％和62.25％。胃肠抑制的ED 50为37050μg/kg。化合物1口服镇痛ED 50为1.37μg/kg，即便秘副作用的胃肠抑制ED 50是镇痛ED 50的27044倍。即在镇痛的有效剂量范围内没有出现便秘的副作用。
在图36中，口服分别注射生理盐水，100，1000和10000μg/kg的化合物11， 生理盐水组的胃肠抑制百分比为80.54％，给药组的胃肠抑制百分比分别为65.42％，56.35％和32.12％，胃肠抑制的ED 50为1239μg/kg。化合物11口服镇痛ED 50为0.14μg/kg，即便秘副作用的胃肠抑制ED 50是镇痛ED 50的8850倍。即在镇痛的有效剂量范围内没有出现便秘的副作用。
实施例20.口服注射化合物1和11的成瘾性副作用测定
化合物1和11的成瘾性评价通过旷场实验，条件位置偏爱(CPP)和纳洛酮戒断实验进行。注射阿片类药物促进多巴胺的释放，小鼠的运动活性加强，因此经常讲小鼠的运动活性与成瘾性评价联系起来。
旷场实验，由一个无顶的50×50×40cm黑色有机玻璃盒及一套运动监测系统组成。实验选取21±2g的昆明系雄性小鼠，室温控制在22±1℃之间，否则温度过高或者过低都会影响小鼠的运动。实验开始前，先用酒精擦拭盒子，除去盒子中的气味，避免盒子中的气味影响下一只小鼠的运动活性。实验开始前，首先记录小鼠30min的基础运动活性，然后口服(p.o.)注射生理盐水，1000μg/kg的化合物1，以及100μg/kg的化合物11。记录150min内小鼠的运动活性。小鼠运动活性用运动总路程来表示，即小鼠运动总路程±标准误(Means±S.E.M.)来表示，化合物与盐水对照组之间的差异用单因素方差分析(one-way ANOVA的Bonferroni检验)进行数据统计和分析， *P<0.05、 **P<0.01以及 ***P<0.001表示盐水与化合物之间具有差异显著性。结果如图37所示。
条件位置偏爱实验(CPP)，实验是在三个有机玻璃盒子组成的装置中完成的，旁边两个大盒子(20×20×20cm)中间被一个小盒子(5×20×20cm)分开。两个大格子底部开一个5×5的小门供小鼠出入，小门可以关闭。旁边的盒子一个是白色的，盒子的底部是糙面的铁丝网，光强50lux。一个盒子是黑色的，底部是光面的，光强20lux。实验选用25±5g的雄性小鼠，室温22±1℃。实验的第一天首先是筛选小鼠，小鼠在两个盒子中可以自由穿梭，记录15min，记录小鼠在一个盒子逗留的时间，将逗留时间超过9min的小鼠剔除。挑选出没有偏爱/厌恶的小鼠。后面的3天连续口服(p.o.)生理盐水或者口服化合物1(10000μg/kg)和11(10000μg/kg)。中间的小门关闭，将给药组与生理盐水组都分成两组，一组小鼠早上放在白盒子给盐水，下午放在黑盒子给药，一组小鼠早上放在黑盒子给盐水，下午放在白盒子给药，小鼠在盒子适应45min，连续训练3天。第5天，测定CPP表现。同第一天一样，测定给药后小鼠在每个格子中的逗留时间，时长为15min。CPP实验中不同药物处理的组间差异和纳洛酮戒断实验中跳跃次数的差异使用成对T检验进行统计， *P<0.05、 **P<0.01以及 ***P<0.001表示药物处理组与生理盐水组之间存在显著性差异。结果如图38所示。
纳洛酮戒断实验是一种经典的评价药物生理成瘾的实验。实验方法参考Venetia Zachariou(2003)。具体的实验方法为：每隔8h口服(p.o.)给化合物1和10一次，实验药物的剂量是逐渐增加的。参考吗啡的(20、40、60、80、100、100、100mg/kg)吗啡的镇痛ED 50为1.68mg/kg，即给药剂量分别是镇痛ED 50的10、20、30、40、50、50和50倍。因此参考实验方法，化合物1的口服ED 50为1.37μg/kg，因此口服给药低剂量依次约为20、40、60、80、100、100、100μg/kg；此外高剂量组给了镇痛ED 50的100、200、300、400、500、500和500倍的化合物1，剂量依次约为200、400、600、800、1000、1000、1000μg/kg。化合物11的镇痛ED 50为0.14μg/kg，因此口服给药剂量依次约为2、4、6、8、10、10、10μg/kg。同样给了高剂量的化合物11，给药剂量依次为20、40、60、80、100、100、100μg/kg。最后一次给药2h后，小鼠腹腔注射10mg/kg的纳洛酮。然后立即将小鼠放于内径9cm、高32cm的不透明桶状结构中，记录30min内小鼠的跳跃次数。实验结果用小鼠的跳跃次数来表示，即跳跃次数±标准误(Means±S.E.M.)来表示，化合物与盐水对照组之间的差异用单因素方差分析(one-way ANOVA的Bonferroni检验)进行数据统计和分析， *P<0.05、 **P<0.01以及 ***P<0.001表示盐水与化合物之间具有差异显著性。结果如图39-40所示。
如图37所示，在旷场实验中，口服注射生理盐水，1000μg/kg化合物1以及100μg/kg化合物11。生理盐水组的运动路程为131.1±33.93m。化合物1的运动路程分别为109.4±32.16m。化合物11的运动路程分别为127.9±23.49m。与生理盐水对照组相比，小鼠的运动活性没有任何改变。
如图38所示，在小鼠位置偏爱实验(CPP)中，口服注射生理盐水，10000μg/kg化合物1以及10000μg/kg化合物11。都没有出现条件位置偏爱现象。
如图39和40所示，在纳洛酮戒断实验中，口服注射生理盐水，低剂量以及高剂量的化合物1和化合物11。盐水组的跳跃次数为5次。低剂量和高剂量的化合物1的跳跃次数都为1次。低剂量和高剂量的化合物11的跳跃次数分别为9次和4次。与生理盐水对照组相比，小鼠没有出现戒断现象。
在本发明提及的所有文献都在本发明中引用作为参考，就如同每一篇文献被单独引用作为参考那样。此外应理解，在阅读了本发明的上述讲授内容之后，本领域技术人员可以对本发明作各种改动或修改，这些等价形式同样落于本发明所附权利要求书所限定的范围。

Claims (14)

  1. 一种针对阿片受体和神经肽FF受体的多靶点环肽分子或其药学上可接受的盐，其特征在于，所述环肽分子的结构如式I所示：
    Tyr-c [2,5][Xaa2-Gly-NMe-Phe-Xaa5]-Xaa6-Xaa7-Arg-Xaa9-NH 2     (I)
    式中，
    Xaa2为Lys、D-Lys、D-Asp、D-Glu、D-Orn、D-Dab或D-Dap；
    Xaa5为Asp、D-Asp、Glu、Lys；
    Xaa6为Pro或Gly；
    Xaa7为Gln、β-Ala或Aib；
    Xaa9为Phe或Cha；
    c [2,5]表示所述氨基酸序列中Xaa2和Xaa5两个氨基酸残基之间存在成环共价键。
  2. 根据权利要求1所述的针对阿片受体和神经肽FF受体的多靶点环肽分子或其药学上可接受的盐，其特征在于，在Xaa2和Xaa5两个氨基酸残基之间的成环共价键包括通过脱水缩合形成酰胺键。
  3. 根据权利要求1所述的针对阿片受体和神经肽FF受体的多靶点环肽分子或其药学上可接受的盐，其特征在于，所述环肽分子的结构如式II所示：
    Figure PCTCN2022077309-appb-100001
    式中，
    Xaa2、Xaa5、Xaa6、Xaa7、和Xaa9如权利要求1定义；
    “～L0～”表示Xaa2和Xaa5两个氨基酸残基之间的成环共价键。
  4. 根据权利要求1所述的针对阿片受体和神经肽FF受体的多靶点环肽分子或其药学上可接受的盐，其特征在于，所述环肽分子选自下述化合物：
    化合物1：Tyr-c [2,5][D-Lys-Gly-NMe-Phe-Asp]-Pro-Gln-Arg-Phe-NH 2
    化合物2：Tyr-c [2,5][D-Lys-Gly-NMe-Phe-Glu]-Pro-Gln-Arg-Phe-NH 2
    化合物3：Tyr-c [2,5][Lys-Gly-NMe-Phe-Asp]-Pro-Gln-Arg-Phe-NH 2
    化合物4：Tyr-c [2,5][Lys-Gly-NMe-Phe-D-Asp]-Pro-Gln-Arg-Phe-NH 2
    化合物5：Tyr-c [2,5][D-Lys-Gly-NMe-Phe-D-Asp]-Pro-Gln-Arg-Phe-NH 2
    化合物6：Tyr-c [2,5][D-Asp-Gly-NMe-Phe-Lys]-Pro-Gln-Arg-Phe-NH 2
    化合物7：Tyr-c [2,5][D-Glu-Gly-NMe-Phe-Lys]-Pro-Gln-Arg-Phe-NH 2
    化合物8：Tyr-c [2,5][D-Orn-Gly-NMe-Phe-Asp]-Pro-Gln-Arg-Phe-NH 2
    化合物9：Tyr-c [2,5][D-Dab-Gly-NMe-Phe-Asp]-Pro-Gln-Arg-Phe-NH 2
    化合物10：Tyr-c [2,5][D-Dap-Gly-NMe-Phe-Asp]-Pro-Gln-Arg-Phe-NH 2
    化合物11：Tyr-c [2,5][D-Orn-Gly-NMe-Phe-Glu]-Pro-Gln-Arg-Phe-NH 2
    化合物12：Tyr-c [2,5][D-Lys-Gly-NMe-Phe-Asp]-Gly-Gln-Arg-Phe-NH 2
    化合物13：Tyr-c [2,5][D-Lys-Gly-NMe-Phe-Asp]-Pro-β-Ala-Arg-Cha-NH 2
    化合物14：Tyr-c [2,5][D-Lys-Gly-NMe-Phe-Asp]-Pro-Aib-Arg-Cha-NH 2。
  5. 根据权利要求1所述的针对阿片受体和神经肽FF受体的多靶点环肽分子或其药学上可接受的盐，其特征在于，所述式I所示的环肽分子具有选自下组的一个或多个特征：
    (1)所述式I所示的环肽分子为阿片受体和NPFF受体的双靶点激动剂；
    (2)所述式I所示的环肽分子皮下注射的镇痛活性相比DN-9超过10倍，优选超过100倍，更优选超过500倍、1000倍、10000倍或10 6倍；
    (3)所述式I所示的环肽分子的口服镇痛活性相比DN-9超过3倍，优选超过10倍，更优选超过100倍、500倍、1000倍或10000倍；
    (4)所述式I所示的环肽分子不会通过血脑屏障；
    (5)所述式I所示的环肽分子连续口服5天以上(优选8天)未出现镇痛耐受；
    (6)所述式I所示的环肽分子不具有便秘副作用；
    (7)所述式I所示的肽分子不具有成瘾性副作用。
  6. 一种权利要求1所述的针对阿片受体和神经肽FF受体的多靶点环肽分子或其药学上可接受的盐的制备方法，其特征在于，包括步骤：
    (a)采用液相合成法和/或固相合成法，按照对应于式I结构式的氨基酸序列，进行肽链合成，从而获得线性肽链；和
    (b)对所述线性肽链进行环化，从而获得权利要求1所述的针对阿片受体和神经肽FF受体的多靶点环肽分子或其药学上可接受的盐。
  7. 根据权利要求6所述的针对阿片受体和神经肽FF受体的多靶点环肽分子或其药学上可接受的盐的制备方法，其特征在于，所述步骤(b)包括：对Xaa2和Xaa5的侧链进行环化，从而在Xaa2和Xaa5两个氨基酸残基之间形成成环共价键。
  8. 根据权利要求6所述的针对阿片受体和神经肽FF受体的多靶点环肽分子或其药学上可接受的盐的制备方法，其特征在于，所述的固相合成法包括选自下组的工艺步骤：固相载体的预处理、氨基酸缩合、肽链的延伸、多肽的压缩抽干和切割、萃取、纯化，或其组合。
  9. 根据权利要求8所述的针对阿片受体和神经肽FF受体的多靶点环肽分子或其药学上可接受的盐的制备方法，其特征在于，所述的固相载体是氨基树 脂。
  10. 根据权利要求8所述的针对阿片受体和神经肽FF受体的多靶点环肽分子或其药学上可接受的盐的制备方法，其特征在于，所述的氨基酸缩合步骤中使用的缩合试剂包括HOBt、HBTU和DIEA的组合。
  11. 根据权利要求6所述的针对阿片受体和神经肽FF受体的多靶点环肽分子或其药学上可接受的盐的制备方法，其特征在于，所述的环化步骤中使用的环化试剂包括PyBOP和DIEA的组合。
  12. 一种药物组合物，其中，所述的药物组合物包括：
    (a)权利要求1所述的针对阿片受体和神经肽FF受体的多靶点环肽分子或其药学上可接受的盐作为活性成分；
    (b)药学上可接受的载体和/或辅料。
  13. 根据权利要求1所述的针对阿片受体和神经肽FF受体的多靶点环肽分子或其药学上可接受的盐的用途，其特征在于，用于制备一药物，所述药物用于缓解和/或治疗各类疼痛，包括急性痛和病理性疼痛。
  14. 一种镇痛的方法，其特征在于，包括步骤：将安全有效量的权利要求1所述的针对阿片受体和神经肽FF受体的多靶点环肽分子或其药学上可接受的盐和/或权利要求12所述的药物组合物施用于需要的对象。
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