CN112457369A

CN112457369A - Preparation and application of long-acting analgesic peptide compounds acting on multiple targets

Info

Publication number: CN112457369A
Application number: CN202010943836.0A
Authority: CN
Inventors: 董守良; 贺春波; 王豪; 赵耀峰; 王小丽; 苏文婷
Original assignee: Lanzhou University
Current assignee: Lanzhou University
Priority date: 2019-09-09
Filing date: 2020-09-09
Publication date: 2021-03-09
Anticipated expiration: 2040-09-09
Also published as: CN112457369B

Abstract

The invention belongs to the technical field of biochemistry, and particularly relates to a modification preparation method and application of a multi-target peptide compound acting on opioid receptors and neuropeptide FF receptors. The invention takes multi-target chimeric peptide MCRT as a matrix, obtains a series of compounds through systematic peptide terminal modification, and screens out two compounds with significantly improved analgesic effects, namely HD-10 and HD-11, by utilizing a mouse radiant heat tail-flick acute pain model. The compound HD-10 has the most excellent analgesic effect and has the characteristics of quick response, long half-life period, strong drug effect and the like. Preliminary pharmacological studies show that in a mouse radiant heat tail-flick acute pain model, the lateral ventricle is injected with the HD-10 multi-target peptide compound, which shows the analgesic activity with quicker, more lasting and more powerful effect than morphine, and has potential application value for clinical pain treatment.

Description

Preparation and application of long-acting analgesic peptide compounds acting on multiple targets

Technical Field

The invention belongs to the technical field of biochemistry, relates to a multi-target peptide compound acting on opioid receptors and neuropeptide FF receptors and a synthesis method thereof, and also relates to application of the multi-target peptide compound in preparation of analgesic drugs.

Background

Pain is a painful sensation resulting from actual or potential tissue damage, often accompanied by unpleasant mood or cardiovascular and respiratory changes. Clinically, analgesic drugs are mainly classified into opioids, non-steroids, anticonvulsants, antidepressants, and the like. Among them, opioid is the most important analgesic in clinical treatment of moderate to severe pain, and produces analgesic effect mainly by binding with central opioid receptors, and has significant therapeutic effect on pain. However, these drugs are usually accompanied by addiction, tolerance, respiratory depression and other toxic and side effects (Annu Rev Pharmacol Toxicol,2018,58:143), which seriously affect the clinical application of opioid drugs. With the development of multi-target drugs in recent years, multi-target opioid drugs represented by dezocine are widely applied clinically, and a brand new idea is provided for drug developers to develop safe and efficient novel opioid drugs (Anesthesiology,2014,120: 714; Curr Med Chem,2016,23: 4506).

The invention patent "chimeric peptide based on endomorphin 2 and neuropeptide FF and synthesis and application thereof" (ZL201110097843.4)The peptide EN-9 is kappa opioid receptor, NPFF₁Receptor, NPFF₂The multi-target agonist of the receptor shows stronger analgesic activity than that of a parent body, and has the advantages of no tolerance, low addiction and the like. The invention patent "chimeric peptide based on opioid peptide Biphalin and neuropeptide FF and its synthesis and application" (ZL201210098832.2) discloses a chimeric peptide BN-9 which is a mu opioid receptor, a delta opioid receptor, a kappa opioid receptor and NPFF₁Receptor, NPFF₂The multi-target agonist of the receptor shows stronger central analgesic activity than morphine, and effectively overcomes adverse reactions such as tolerance, constipation and the like. The invention discloses a chimeric peptide DN-9 disclosed by a class of multi-target peptide molecules of opioid and neuropeptide FF receptors and a preparation method and application thereof (ZL201610252648.7), which is obtained by optimizing the chimeric peptide BN-9, has analgesic activity far higher than that of a parent body, and has broad-spectrum analgesic effect on various types of pain such as inflammatory pain, neuralgia and the like.

The subject group designed a synthetic chimeric peptide MCRT in 2012, which is a multi-target ligand for the mu-, delta-and NPFF receptors, and which generates half the effective dose of the gastrointestinal motility inhibitory Effect (ED) at the central level compared to the analgesic dose(s) of MCRT₅₀) More than 33-fold higher, with a larger therapeutic window (Regul Peptides,2012,179: 23; j Pharm Pharmacol,2017,69: 1244; neuropeptides,2019, 74: 82). The related research of multi-target novel opioid molecules has a plurality of defects while making great progress. For example, the chimeric Peptides EN-9, BN-9, DN-9 and MCRT have short half-lives and effective analgesic periods of less than 90min (Neuropharmacology,2016,108: 364; Brit J Pharmacol,2016, 173: 1864; J Med Chem,2016,59: 10198; Regul Peptides,2012,179:23), which somewhat limit the clinical utility of peptide drugs.

Amino acid deletions, changes in configuration, side chain modifications, and C-or N-terminal modifications of the polypeptide backbone can all unexpectedly alter the structure, biological activity, and pharmacokinetics of the polypeptide. Aiming at the technical problems of toxic and side effects caused by systemic administration of opiates, short half-life period of multi-target opiate peptide compounds and the like, the invention carries out a series of structural modification and optimization based on chimeric peptide MCRT, firstly obtains HD-3 with N-terminal acetylation and C-terminal ending with free carboxyl, and shows excellent analgesic activity in a mouse reserved nerve injury model (SNI) by adopting a mode of plantar subcutaneous local administration. On the basis, the inventor carries out terminal systematic modification on MCRT to obtain a series of MCRT analogues, and selects and obtains two new compounds HD-10 and HD-11 with analgesic effect far superior to that of parent MCRT and morphine, wherein the analgesic effect of HD-10 is most obvious, and the compounds have the characteristics of quick response, long half-life period, strong analgesic effect and the like. Has potential application value for treating clinical pain.

Disclosure of Invention

Aiming at the technical problems of opioid drugs, the invention designs, synthesizes and screens a multi-target peptide compound acting on an opioid receptor and a neuropeptide FF receptor, and has the advantages of quick response, long half-life and good analgesic effect.

The structural general formula of the multi-target peptide compound is shown as the formula (I):

wherein R1 is-NH₂or-NHCOCH₃or-NHCOCH₂O(CH₂CH₂O)_nH, R2 is-COOH or-CONH₂or-COO (CH)₂CH₂O)_nH or-COOCH₃or-COOCH₂CH₃Furthermore, R1 is-NH₂When R2 cannot be-CONH₂，(CH₂CH₂O)_nMolecular weight range 500-.

Specific modifications of the compounds are shown below:

HD-1, in the structural general formula, R1 is-NH₂R2 is-COOH

HD-2, in the structural general formula, R1 is-NHCOCH₃R2 is-CONH₂

HD-3, in the structural general formula, R1 is-NHCOCH₃R2 is-COOH

HD-7, in the above general formula, R1 is-PEG, R2 is-COOH

HD-9, in the above general formula, R1 is-PEG, R2 is-CONH₂

HD-10, in the above structural general formula, R1 is-NHCOCH₃R2 is-COOCH₃

HD-11, in the above general formula, R1 is-PEG, R2 is-COOCH₃

HD-13 in the structural formula, R1 is-NH₂R2 is-COOCH₃

HD-14, in the above structural general formula, R1 is-NHCOCH₃R2 is-COOCH₂CH₃

HD-15, in the above general formula, R1 is-PEG, R2 is-COOCH₂CH₃

HD-16, in the above structural general formula, R1 is-NH₂R2 is-COOCH₂CH₃

HD-26, in the above structural general formula, R1 is-NHCOCH₃R2 is-PEG

HD-27, the general formula of the structure is that R1 is-PEG and R2 is-PEG

HD-28, in the above structural general formula, R1 is-NH₂R2 is-PEG

It is an object of the present invention to provide a pharmaceutical composition comprising a compound as described above or a pharmaceutically acceptable salt or stereoisomer thereof, and at least one pharmaceutically acceptable excipient.

Preferably, the pharmaceutical composition is selected from tablets, capsules, oral solutions, injections, powder injections, sustained-release agents, dropping pills, granules, sustained-release pellets and any pharmaceutically-realizable dosage form.

One of the objects of the present invention is to provide the use of a compound of formula as described above for the preparation of an analgesic drug.

It is an object of the present invention to provide the use of a compound of formula as described above for the manufacture of a medicament for the treatment of acute pain.

One of the objects of the present invention is to provide the use of the compound represented by the formula as above for the preparation of an analgesic drug for the treatment of postherpetic neuralgia, diabetic peripheral neuralgia, central neuralgia after stroke, cancerous neuralgia, trigeminal neuralgia, post-operative pain in the mouth, and the like.

Preparation of the compound of the general formula

1. The synthesis method of the compound HD-1 comprises the following process steps:

(1) resin pretreatment: pouring 500mg of 2-Chlorotrityl-Chloride-Resin into a syringe, adding 5ml of DCM, shaking for 30min, fully swelling the Resin, and then decompressing and draining the solvent;

(2) removing Fmoc protecting groups: adding 5ml of 20% piperidine/DMF solution into the resin with the swelling and solvent-draining functions, shaking for 15min, and draining; adding 5ml DMF, washing for 1min, and draining; adding 5ml of 20% piperidine/DMF solution, shaking for 5min, draining, and repeating twice to completely remove the Fmoc group; finally washing piperidine with DMF;

(3) condensation: 2.5 times of amino acid (Fmoc-AA) protected by N-alpha-Fmoc, N-hydroxybenzotriazole (HOBt) and O-benzotriazol-N, N, N ', N' -tetramethylurea-Hexafluorophosphate (HBTU) are completely dissolved in DMF in sequence, and 3 times of Diisopropylethylamine (DIEA) is added, so that the resin with the Fmoc protecting group removed is subjected to oscillation reaction in the mixed solution for 60 min; after the solvent is drained, DMF and DCM are used for alternately washing to remove redundant unreacted substances;

(4) elongation of peptide chain: repeating the steps (2) and (3), sequentially condensing Fmoc-AA to the resin one by one according to the sequence of the polypeptide structure from the C-terminal to the N-terminal until all amino acid residues are condensed;

(5) cleavage of peptide chain from resin: completely removing the Fmoc protecting group of the last amino acid according to the method in the step (2), washing the Fmoc protecting group for 3 times by shaking DMF, alternately washing the resin by using DCM and MeOH, and fully pumping out the solvent; adding 10-15 ml of cutting agent (TFA: TIS: water: EDT: 92.5:2.5:2.5:2.5) into each gram of resin, and cutting for 2 hours with shaking; filtering and collecting lysate, fully decompressing and spin-drying, separating out precipitate in ice methyl tert-butyl ether, and air-drying overnight to obtain crude peptide solid powder;

(6) purification of the crude peptide: dissolving the crude peptide in 20% acetonitrile/water solution, adopting reversed phase high performance liquid chromatography (RP-HPLC) to prepare a C18 column (Nacalai Tesque 20 x 250mm), separating, collecting a main peak, and freeze-drying to obtain pure peptide white solid powder.

2. The synthesis method of the compound HD-2 comprises the following process steps:

(1) resin pretreatment: pouring 500mg Rink-Amide-Resin into a syringe, adding 5ml DCM, oscillating for 30min, fully swelling the Resin, and then decompressing and draining the solvent;

(2) removing Fmoc protecting groups: the same as 1- (2);

(3) condensation: the same as 1- (3);

(4) elongation of peptide chain: the same as 1- (4);

(5) acetylation of the N-terminus: completely removing the Fmoc group of the last amino acid of the peptide resin according to the operation of the step (2), and washing with DMF and DCM for 3 times in a shaking way respectively; then, adding 5ml of acetic anhydride/pyridine mixed solution (3:2), and carrying out shaking reaction for 30-60 min; after the solvent is drained, the mixture is respectively shaken and washed for 3 times by DMF and DCM, and the N-terminal is confirmed to be completely acetylated through indene detection;

(6) cleavage of the peptide chain from the resin: the same as 1- (5);

(7) purification of the crude peptide: the same as in 1- (6).

3. The synthesis method of the compound HD-3 comprises the following process steps:

(1) resin pretreatment: the same as 1- (1);

(2) removing Fmoc protecting groups: the same as 1- (2);

(3) condensation: the same as 1- (3);

(4) elongation of peptide chain: the same as 1- (4);

(5) acetylation of the N-terminus: the same as 2- (5);

(6) cleavage of peptide chain from resin: the same as 1- (5);

(7) purification of the crude peptide: the same as in 1- (6).

4. The synthesis method of the compound HD-7 comprises the following process steps:

(1) resin pretreatment: the same as 1- (1);

(2) removing Fmoc protecting groups: the same as 1- (2);

(3) condensation: the same as 1- (3);

(4) elongation of peptide chain: the same as 1- (4);

(5) n-terminal PEGylation: completely removing the Fmoc group of the last amino acid of the peptide resin according to the operation of the step (2), and washing the peptide resin by oscillation of DMF for 5 times; then, 2.5 times of mPEG-COOH and HATU are completely dissolved in DCM: DMF (1:9), and then 5 times of DIEA is added to obtain the peptide resin with Fmoc protecting groups removed, and the peptide resin is subjected to oscillation reaction in the mixed solution for 60 min; after the solvent was drained, the mixture was washed with DMF for 5 times with shaking, and then subjected to indene examination to confirm that the N-terminus was completely PEGylated;

(6) cleavage of peptide chain from resin: the same as 1- (5);

(7) purification of the crude peptide: dissolving the crude peptide in 30% acetic acid/water solution, performing RP-HPLC to obtain C18 column (HPLCONE-10C18A 20X 250mm), separating, collecting main peak, and freeze drying to obtain white solid powder of pure peptide.

5. The synthesis method of the compound HD-9 comprises the following process steps:

(1) resin pretreatment: the same as 2- (1);

(2) removing Fmoc protecting groups: the same as 1- (2);

(3) condensation: the same as 1- (3);

(4) elongation of peptide chain: the same as 1- (4);

(5) n-terminal PEGylation: the same as 4- (5);

(6) cleavage of peptide chain from resin: the same as 1- (5);

(7) purification of the crude peptide: the same as in 4- (7).

6. The synthesis method of the compounds HD-10, HD-11, HD-13, HD-14, HD-15, HD-16, HD-26, HD-27 and HD-28 comprises the following process steps, and the common synthesis steps comprise the following parts:

(1) resin pretreatment: pouring 3g of 2-Chlorotrityl-Chloride-Resin into a syringe, adding 5ml of DCM, oscillating for 30min, fully swelling the Resin, and then decompressing and draining the solvent;

(2) removing Fmoc protecting groups: adding 30ml of 20% piperidine/DMF solution into the resin with the swelling and solvent draining functions, shaking for 15min, and draining; adding 30ml DMF, washing for 1min, and draining; adding 30ml of 20% piperidine/DMF solution, shaking for 5min, pumping, and repeating twice to completely remove the Fmoc group; finally washing piperidine with DMF;

(3) condensation: the same as 1- (3);

(4) elongation of peptide chain: repeating the steps (2) and (3), sequentially condensing Fmoc-AA to the resin one by one according to the sequence of the polypeptide structure from the C-terminal to the N-terminal until the condensation of a sixth amino acid residue is completed;

(5) drying and partitioning of peptide resin: the peptide resin was washed five times with DMF, then with alternating washes of methanol and DCM and thoroughly drained of solvent. The peptide resin was divided into 9 parts on average by weight and distributed in 9 reactors.

7. The subsequent synthesis step of compound HD-10 comprises the following processes:

(1) linkage of Fmoc-Tyr (tBu) -OH: firstly, removing Fmoc protective groups according to the method of the common synthesis step (2), completely dissolving 2.5 times of Fmoc-Tyr (tBu) -OH, HOBt and HBTU in DMF, and then adding 5 times of DIEA to ensure that the hexapeptide resin with the Fmoc protective groups removed is subjected to oscillation reaction in the mixed solution for 60 min; after the solvent is pumped out, washing the solution by DMF to remove redundant unreacted substances;

(2) acetylation of the N-terminus: the same as 2- (5);

(3) cleavage of peptide chain from resin: the resin was washed alternately with DCM and MeOH and the solvent was thoroughly drained; adding 10-15 ml of cutting agent (TFA: TES: DCM: 1:3:96) into each gram of resin, and cutting for 1h with shaking; filtering and collecting lysate, fully decompressing and spin-drying, separating out precipitate in ice water, centrifuging, removing supernatant, and drying in a vacuum drying oven to obtain crude peptide solid powder;

(4) methyl esterification of carboxyl terminal: dissolving polypeptide in DCM, adding 10 times of anhydrous methanol, 5 times of DIC and 0.5 times of DMAP, stirring at room temperature for reaction for 3h, removing the reaction solvent by rotary evaporation, and terminating the reaction;

(5) and (3) removing a protecting group: the product after rotary evaporation was added with a deprotection solution (TFA: TIS: water: EDT: phenol: 90.0:2.5:2.5:2.5:2.5), and reacted for 1h with stirring. Fully decompressing, spin-drying, separating out precipitate in glacial methyl tert-butyl ether, centrifuging, removing supernatant, and drying in a vacuum drying oven to obtain crude peptide solid powder;

(6) purification of the crude peptide: the same as in 4- (7).

8. The subsequent synthesis step of compound HD-11 comprises the following processes:

(1) linkage of Fmoc-Tyr (tBu) -OH: the same as 7- (1);

(2) n-terminal PEGylation: the same as 4- (5);

(3) cleavage of peptide chain from resin: the same as 7- (3);

(4) methyl esterification of carboxyl terminal: the same as 7- (4);

(5) and (3) removing a protecting group: the same as 7- (4);

(6) purification of the crude peptide: dissolving the crude peptide in 20% acetonitrile/water solution, performing RP-HPLC to obtain C18 column (HPLCONE-10C18A 20X 250mm), separating, collecting main peak, and lyophilizing to obtain white solid powder of pure peptide. .

9. The subsequent synthesis step of compound HD-13 comprises the following processes:

(1) Boc-Tyr (tBu) -OH attachment: firstly, removing Fmoc protective groups according to the method of the common synthesis step (2), completely dissolving 2.5 times of Boc-Tyr (tBu) -OH, HOBt and HBTU in DMF, and then adding 5 times of DIEA to ensure that the hexapeptide resin from which the Fmoc protective groups are removed is subjected to oscillation reaction in the mixed solution for 60 min; after the solvent is pumped out, washing the solution by DMF to remove redundant unreacted substances;

(2) cleavage of peptide chain from resin: the same as 7- (3);

(3) methyl esterification of carboxyl terminal: the same as 7- (4);

(4) and (3) removing a protecting group: the same as 7- (5);

(5) purification of the crude peptide: the same as in 4- (7).

10. The subsequent synthesis step of compound HD-14 comprises the following processes:

(1) linkage of Fmoc-Tyr (tBu) -OH: the same as 7- (1);

(2) acetylation of the N-terminus: the same as 2- (5);

(3) cleavage of peptide chain from resin: the same as 7- (3);

(4) and (3) carboxyl terminal ethylation: dissolving polypeptide in DCM, adding 10 times of anhydrous methanol, 5 times of DIC and 0.5 times of DMAP, stirring at room temperature for reaction for 3h, removing the reaction solvent by rotary evaporation, and terminating the reaction;

(5) and (3) removing a protecting group: the same as 7- (5);

(6) purification of the crude peptide: the same as in 4- (7).

11. The subsequent synthesis step of compound HD-15 comprises the following processes:

(1) linkage of Fmoc-Tyr (tBu) -OH: the same as 7- (1);

(2) n-terminal PEGylation: the same as 4- (5);

(3) cleavage of peptide chain from resin: the same as 7- (3);

(4) and (3) carboxyl terminal ethylation: the same as 10- (4);

(5) and (3) removing a protecting group: the same as 7- (5);

(6) purification of the crude peptide: the same as 8- (6).

12. The subsequent synthesis step of compound HD-16 comprises the following processes:

(1) Boc-Tyr (tBu) -OH attachment: the same as 9- (1);

(2) cleavage of peptide chain from resin: the same as 7- (3);

(3) and (3) carboxyl terminal ethylation: the same as 10- (4);

(4) and (3) removing a protecting group: the same as 7- (5);

(5) purification of the crude peptide: the same as in 4- (7).

13. The subsequent synthesis step of compound HD-26 comprises the following processes:

(1) linkage of Fmoc-Tyr (tBu) -OH: the same as 7- (1);

(2) acetylation of the N-terminus: the same as 2- (5);

(3) cleavage of peptide chain from resin: the same as 7- (3);

(4) pegylation at the carboxy terminus: dissolving the polypeptide in DCM, adding 2.5 times of mPEG-NH2, 2.5 times of PyBop and 5 times of DIEA, stirring at room temperature for 1h, removing the reaction solvent by rotary evaporation, and terminating the reaction;

(5) and (3) removing a protecting group: the same as 7- (5);

(6) purification of the crude peptide: the same as 8- (6).

14. The subsequent synthesis step of compound HD-27 comprises the following processes:

(1) linkage of Fmoc-Tyr (tBu) -OH: the same as 7- (1);

(2) n-terminal PEGylation: the same as 4- (5);

(3) cleavage of peptide chain from resin: the same as 7- (3);

(4) pegylation at the carboxy terminus: the same as 13- (4);

(5) and (3) removing a protecting group: the same as 7- (5);

(6) purification of the crude peptide: the same as 8- (6).

15. The subsequent synthesis step of compound HD-28 comprises the following processes:

(1) Boc-Tyr (tBu) -OH attachment: the same as 9- (1);

(2) cleavage of peptide chain from resin: the same as 7- (3);

(3) pegylation at the carboxy terminus: the same as 13- (4);

(4) and (3) removing a protecting group: the same as 7- (5);

(5) purification of the crude peptide: the same as 8- (6).

If compounds containing other substituents than the above compounds are synthesized, those skilled in the art can also prepare them using the corresponding starting materials and preparation methods, as will be understood by those skilled in the art.

(II) advantageous effects of the invention

The invention carries out a series of structural modification and optimization based on the chimeric peptide MCRT, and 2 compounds HD-10 and HD-11 with analgesic effect far exceeding that of the parent MCRT are obtained by screening, wherein the analgesic effect of HD-10 is most obvious, and the invention has the characteristics of quick effect, long half-life period, strong analgesic effect and the like.

In a mouse radiation heat acute pain model, MCRT and HD-10 can quickly take effect under the same administration dosage, the maximum analgesic effect is achieved within 5min, however, the analgesic effect of MCRT is only maintained for about 15min, rapid attenuation begins to occur later, almost no effect is achieved until 90min, the analgesic effect of HD-10 is maintained for 210min, and the duration of analgesia is remarkably prolonged. Although the maximal analgesic effect of HD-11 (65.4953 + -12.78339) was slightly reduced compared to MCRT, the duration of analgesia was significantly prolonged and was not reduced by 180 min. The positive control morphine with the same administration dose starts to take effect within 20min, the duration of the drug effect is short, the drug effect begins to weaken after 20min, the effect is almost ineffective until 90min, the analgesic effect is the area under the curve, HD-10 is 2 times of that of morphine within 0-90 min, and the analgesic effect of HD-10 is continuous, so that the analgesic effect of the new drug HD-10 is far stronger than that of morphine. In conclusion, the invention provides a multi-target peptide terminal modified compound acting on opioid receptors and neuropeptide FF receptors, which has the advantages of quick response, long half-life, good analgesic effect and the like, and can be clinically popularized and applied.

Drawings

FIG. 1 time course of lateral ventricle injection of mice with morphine (morphine), MCRT, HD-1, HD-2, HD-3 in a tail flick model with radiant heat.

FIG. 2 lateral ventricle injection of mice with 2% DMSO, MCRT, HD-2, HD-13, HD-10, aging curves in the radiant heat tail flick model.

FIG. 3 lateral ventricle injection of mice with 2% DMSO, MCRT, HD-2, HD-16, HD-14, aging curves in a radiant heat tail flick model.

FIG. 4 is a graph showing the aging curves of mice injected with MCRT, HD-7, HD-9, HD-11, and HD-15 into the lateral ventricles of the brain in the tail flick model of radiant heat.

FIG. 5 aging curves of mice injected with MCRT, HD-26, HD-27, HD-28 in the lateral ventricle of the brain in the radiant heat tail flick model.

FIG. 6 shows comparison of analgesic effects (area under curve) of mice injected with 2% DMSO, morphine (morphine), MCRT, HD-1, HD-2, HD-3, HD-13, HD-10, HD-14, HD-16, HD-7, HD-9, HD-11, HD-15, HD-26, HD-27, and HD-28 into lateral ventricle of the tail in a radiant heat tail flick model for 0-90 min.

Detailed Description

The synthesis of the multi-target peptide compounds and analgesic studies claimed by the present invention are described in further detail by way of specific implementation below, but the scope of the present invention is not limited by the following examples.

Experimental reagent: 2-Chlorotrityl-Chloride-Resin (substitution S ═ 0.97mmol/g) available from gill biochemical (shanghai) ltd; Rink-Amide-Resin (substitution value S ═ 0.627mmol/g), available from Shanghai vast chemical technology Limited; ethanedithiol (EDT), trifluoroacetic acid (TFA), 4-Dimethylaminopyridine (DMAP) were purchased from Bailingwei technologies, Beijing; N-alpha-Fmoc protectantProtected amino acids (Fmoc-AA), N-hydroxybenzotriazole (HOBt), O-benzotriazol-N, N, N ', N' -tetramethyluronium-Hexafluorophosphate (HBTU) were purchased from Gill Biochemical (Shanghai) Co., Ltd.; methoxypolyethylene glycol-carboxyl (mPEG-COOH) was purchased from Shanghai coconut Biotech, Inc.; methoxy polyethylene glycol-amino (mPEG-NH)₂) Purchased from Shenzhen charm technology ltd; acetic anhydride was purchased from Riranbohua (Tianjin) pharmaceutical chemistry, Inc.; pyridine, N-Diisopropylcarbodiimide (DIC), benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate (PyBop) are available from Annaige GmbH-Saen chemical technology (Shanghai) Limited; methanol, ethanol, and piperidine were purchased from Dalochi chemical industries, Tianjin.

Example one Synthesis method of Compound HD-1

1. Resin pretreatment: 500mg of 2-Chlorotrityl-Chloride-Resin was poured into a syringe, 5ml of DCM was added and the mixture was shaken for 30min, after the Resin had fully swelled, the solvent was drained under reduced pressure, washed with DMF and the solvent was drained.

2. Removing Fmoc protecting groups: adding 5ml of 20% piperidine/DMF solution into the resin with the swelling and solvent-draining functions, shaking for 15min, and draining; adding 5ml DMF, washing for 1min, and draining; 5ml of 20% piperidine/DMF solution is added, shaken for 5min and then pumped out, and the operation is repeated twice, so that the Fmoc group is completely removed. Then, 5ml of DMF is added, the mixture is shaken for 1min and then is pumped dry, and the operation is repeated for three times; adding 5ml of DCM, shaking for 1min, draining, and repeating for three times; adding 1ml DMF, shaking, and draining after 1min to clean piperidine. Finally, a portion of the resin was subjected to indene detection following the procedure of step 3, and the solution turned blue indicating complete deprotection.

3. And (4) indene detection: during the synthesis, ninhydrin reagent test was used to determine whether condensation and deprotection were complete at each step. Formulation of three reagents for indene detection: reagent I20 g phenol/5 ml ethanol; reagent 0.05 ml 0.001M potassium cyanide (water)/2.5 ml pyridine; reagent 3 0.5g ninhydrin/10 ml absolute ethanol. Adding the components in the following sequence: a peptide resin sample, 50 mu l of reagent, 100 mu l of reagent and 50 mu l of reagent are put in boiling water bath for 3-5 min, and then the color of the solution is observed.

4. Condensation: 2.5 times of N-alpha-Fmoc protected amino acid (Fmoc-AA), N-hydroxy benzotriazole (HOBt) and O-benzotriazole-N, N, N ', N' -tetramethylurea-Hexafluorophosphate (HBTU) are completely dissolved in 5ml of DMF in a 25ml round-bottom flask, the mixed solution is added into a syringe filled with the Fmoc protected resin removed, 3 times of Diisopropylethylamine (DIEA) is added, and the mixture is subjected to shaking reaction for 60 min. Then, the solvent is pumped out, 5ml of DMF is added, the mixture is pumped out after shaking for 1min, and the process is repeated for three times; 1ml of DCM was added again, shaken for 1min and then drained and repeated three times. Finally, the indene detection is carried out according to the step 3, the indene detection solution is light yellow, and the resin is colorless and shows complete condensation.

5. Elongation of peptide chain: and (3) repeating the steps 2 and 4, sequentially condensing Fmoc-AA to the resin one by one according to the sequence of the polypeptide structure from the C-terminal to the N-terminal until all amino acid residues are condensed.

6. Cleavage of peptide chain from resin: after the peptide chain condensation is completely finished, the Fmoc group of the last amino acid of the peptide resin is completely removed according to the operation in the step 2. Washing with 5ml DMF for 1min under shaking, draining, and repeating for three times; the resin was washed alternately with 1min × 2 times of DCM, 1min × 1 times of MeOH, 1min × 1 times of DCM, 1min × 2 times of MeOH, and the solvent was drained off by suction with a water pump under reduced pressure (minimum 1 h). Then, 5ml of a cleavage agent (TFA: TIS: water: EDT: 92.5:2.5:2.5:2.5) was added, and the cleavage reaction was shaken for 2 hours; the lysate was collected by filtration into a round bottom flask and thoroughly dried under reduced pressure at a temperature not higher than 39 ℃. Finally, precipitating in glacial methyl tert-butyl ether, standing, centrifuging (4000rpm for 5min), and discarding the supernatant; adding glacial methyl tert-butyl ether again, ultrasonically mixing the precipitate uniformly to obtain a suspension, centrifuging (4000rpm for 5min), discarding the supernatant, and repeating for three times; the precipitate was air dried overnight to give a crude peptide solid powder.

7. Purification of the crude peptide: dissolving the crude peptide in 20% acetonitrile/water solution, performing reverse phase high performance liquid chromatography (RP-HPLC) with C18 column (Nacalai Tesque 20 × 250mm), separating, collecting main peak, and lyophilizing to obtain white solid powder of pure peptide. The purity of the purified sample is more than 95%. The mass spectrometry and chromatography results are shown in table 1.

EXAMPLE II Synthesis of Compound HD-2

1. Resin pretreatment: adding 500mg Rink-Amide-Resin into a syringe, adding 5ml DCM, oscillating for 30min to fully swell the Resin, decompressing, draining the solvent, washing with DMF, and draining the solvent;

2. removing Fmoc protecting groups: the same as 2 in the first embodiment;

3. and (4) indene detection: the same as 3 in the first embodiment;

4. condensation: the same as 4 in the first embodiment;

5. elongation of peptide chain: the same as 5 in the first embodiment;

acetylation of the N-terminus: after the peptide chain condensation is completely completed, the Fmoc group of the last amino acid of the peptide resin is completely removed according to the operation of the step 2. Then, shaking and washing the mixture for 1min by using 5ml of DMF, then pumping the mixture to dry the mixture, and repeating the washing for three times; washing with 5ml DCM for 1min under shaking, draining, and repeating for three times; adding 5ml of acetic anhydride/pyridine mixed solution (3:2), and carrying out shaking reaction for 30-60 min. Finally, pumping out the solvent, adding 5ml of DMF, shaking and washing for 1min, pumping out, and repeating for three times; adding 5ml DCM, shaking and washing for 1min, draining, and repeating for three times; the indene detection confirms that the amino has reacted completely and the N-terminal is all acetylated;

7. cleavage of the peptide chain from the resin: the same as 6 in the first embodiment;

8. purification of the crude peptide: the same as 7 in the first embodiment.

EXAMPLE III Synthesis of Compound HD-3

1. Resin pretreatment: the same as 1 in the first embodiment;

2. removing Fmoc protecting groups: the same as 2 in the first embodiment;

3. and (4) indene detection: the same as 3 in the first embodiment;

4. condensation: the same as 4 in the first embodiment;

5. elongation of peptide chain: the same as 5 in the first embodiment;

acetylation of the N-terminus: the same as 6 in the second embodiment;

8. purification of the crude peptide: the same as 6 in the first embodiment.

Example four, synthesis of compound HD-7:

1. resin pretreatment: the same as 1 in the first embodiment;

2. removing Fmoc protecting groups: the same as 2 in the first embodiment;

3. and (4) indene detection: the same as 3 in the first embodiment;

4. condensation: the same as 4 in the first embodiment;

5. elongation of peptide chain: the same as 5 in the first embodiment;

n-terminal PEGylation: the same as 6 in the fourth embodiment;

8. purification of the crude peptide: the same as 7 in the first embodiment.

Example five, synthesis of compound HD-9:

1. resin pretreatment: the same as example 1;

2. removing Fmoc protecting groups: the same as 2 in the first embodiment;

3. and (4) indene detection: in embodiment one, 3;

4. condensation: the same as 4 in the first embodiment;

5. elongation of peptide chain: the same as 5 in the first embodiment;

n-terminal PEGylation: the same as 6 in the fourth embodiment;

8. purification of the crude peptide: the same as 7 in the first embodiment.

Example six, the synthesis of the compounds HD-10, HD-11, HD-13, HD-14, HD-15, HD-16, HD-26, HD-27, HD-28:

1. resin pretreatment: pouring 3g of 2-Chlorotrityl-Chloride-Resin into a syringe, adding 30ml of DCM, oscillating for 30min, fully swelling the Resin, draining the solvent under reduced pressure, washing with DMF, and draining the solvent;

2. removing Fmoc protecting groups: adding 30ml of 20% piperidine/DMF solution into the resin with the swelling and solvent-draining functions, shaking for 5min, and draining; this was repeated three times to completely remove the Fmoc group. Then, 30ml of DMF is added, and after shaking for 1min, the mixture is drained and repeated for five times; to wash the piperidine. Finally, a part of resin is subjected to indene detection according to the operation of the step 3, and the solution turns blue to indicate that the deprotection is completely carried out;

3. and (4) indene detection: the same as 3 in the first embodiment;

4. condensation: 2.5 times of Fmoc-AA, HOBt and HBTU are completely dissolved in 25ml of DMF in a 25ml round-bottom flask, the mixed solution is added into a syringe filled with the removed Fmoc protection resin, 5 times of DIEA is added, and the mixture is shaken for 60 min. Then, the solvent was drained, 5ml of DMF was added, shaken for 1min and then drained, and the process was repeated five times. Finally, performing indene detection according to the step 3, wherein the indene detection solution is light yellow, and the resin is colorless and represents complete condensation;

5. elongation of peptide chain: repeating the steps 2 and 4, sequentially condensing Fmoc-AA to the resin one by one according to the sequence of the polypeptide structure from the C-terminal to the N-terminal until the condensation of the first six amino acid residues is completed;

6. drying and partitioning of peptide resin: the peptide resin was washed five times with DMF, the resin was washed alternately with DCM 1min × 2 times, MeOH 1min × 1 times, DCM 1min × 1 times, MeOH 1min × 2 times, and the solvent was pumped off with a water pump under reduced pressure (minimum 1 h). The peptide resin is evenly divided into 9 parts by weight and distributed in 9 reactors;

example seven, subsequent Synthesis procedure for Compound HD-10:

linkage of Fmoc-Tyr (tBu) -OH: firstly, removing Fmoc protective groups according to the method of the joint synthesis step 2, completely dissolving 2.5 times of Fmoc-Tyr (tBu) -OH, HOBt and HBTU in a 25ml round-bottom flask by using 5ml of DMF, adding the mixed solution into a syringe filled with the removed Fmoc protective resin, then adding 5 times of DIEA, and carrying out oscillation reaction for 60 min. Then, the solvent was drained, 5ml of DMF was added, and after shaking for 1min, the draining was repeated five times. Finally, performing indene detection according to the common synthesis step 3, wherein an indene detection solution is light yellow, and the resin is colorless and represents complete condensation;

acetylation of the N-terminus: the same as 6 in the second embodiment;

3. cleavage of peptide chain from resin: the resin was washed alternately with 1min × 2 times of DCM, 1min × 1 times of MeOH, 1min × 1 times of DCM, 1min × 2 times of MeOH, and the solvent was drained off by suction with a water pump under reduced pressure (minimum 1 h). Adding 5ml of a cutting agent (TFA: TES: DCM 1:3:96), and carrying out shake-cut reaction for 1 h; the lysate was collected by filtration into a round bottom flask and thoroughly dried under reduced pressure at a temperature not higher than 39 ℃. Finally, precipitating in ice water, standing, centrifuging (4000rpm for 5min), discarding supernatant, and drying in a vacuum drying oven to obtain crude peptide solid powder;

4. methyl esterification of carboxyl terminal: dissolving the polypeptide in 5ml DCM, adding 35 μ L of anhydrous methanol, 65 μ L of DIC and 5mg of DMAP, stirring at room temperature for reaction for 3h, removing the reaction solvent by rotary evaporation, and stopping the reaction;

5. and (3) removing a protecting group: to the rotary evaporated product was added 5ml of deprotection solution (TFA: TIS: water: EDT: phenol: 90.0:2.5:2.5:2.5:2.5), stirred for 1 hour, and sufficiently dried under reduced pressure at not higher than 39 ℃. Finally, precipitating in glacial methyl tert-butyl ether, standing, centrifuging (4000rpm for 5min), and discarding the supernatant; adding glacial methyl tert-butyl ether again, ultrasonically mixing the precipitate uniformly to obtain a suspension, centrifuging (4000rpm for 5min), discarding the supernatant, and repeating for three times; drying the precipitate by using a vacuum drying oven to obtain crude peptide solid powder;

6. purification of the crude peptide: dissolving the crude peptide in 30% acetic acid/water solution, performing RP-HPLC with C18 column (HPLCONE-10C18A 20X 250mm), separating, collecting main peak, and freeze drying to obtain pure peptide white solid powder. The purity of the purified sample is more than 95%. The mass spectrometry and chromatography results are shown in table 1.

Example eight subsequent Synthesis procedure for Compound HD-11:

linkage of Fmoc-Tyr (tBu) -OH: the same as 1 in the seventh embodiment;

n-terminal PEGylation: the same as 6 in the fourth embodiment;

3. cleavage of peptide chain from resin: the same as 3 in the seventh embodiment;

4. methyl esterification of carboxyl terminal: the same as example 4;

5. and (3) removing a protecting group: the same as 5 in the seventh embodiment;

6. purification of the crude peptide: dissolving the crude peptide in 20% acetonitrile/water solution, performing RP-HPLC with C18 column (HPLCONE-10C18A 20X 250mm), separating, collecting main peak, and freeze drying to obtain pure peptide white solid powder. The purity of the purified sample is more than 95%. The mass spectrometry and chromatography results are shown in table 1.

EXAMPLE nine subsequent Synthesis procedure for Compound HD-13:

linkage of Boc-Tyr (tBu) -OH: firstly, removing Fmoc protective groups according to the method of the joint synthesis step 2, completely dissolving 2.5 times of Boc-Tyr (tBu) -OH, HOBt and HBTU in a 25ml round-bottom flask by using 5ml of DMF, adding the mixed solution into a syringe filled with the removed Fmoc protective resin, then adding 5 times of DIEA, and carrying out shaking reaction for 60 min. Then, the solvent was drained, 5ml of DMF was added, and after shaking for 1min, the draining was repeated five times. Finally, indene detection is carried out according to the common synthesis step 3, the indene detection solution is light yellow, and the resin is colorless and shows complete condensation.

2. Cleavage of peptide chain from resin: the same as 3 in the seventh embodiment;

3. methyl esterification of carboxyl terminal: the same as example 4;

4. and (3) removing a protecting group: the same as 5 in the seventh embodiment;

5. purification of the crude peptide: the same as 6 in example eight.

Example ten, subsequent Synthesis procedure for Compound HD-14:

linkage of Fmoc-Tyr (tBu) -OH: the same as 1 in the seventh embodiment;

acetylation of the N-terminus: the same as 6 in the second embodiment;

4. and (3) carboxyl terminal ethylation: dissolving the polypeptide in 4ml DCM, adding 95 mul absolute ethyl alcohol, 125 mul DIC and 7.5mg DMAP, stirring at room temperature for reaction for 3h, removing the reaction solvent by rotary evaporation, and stopping the reaction;

6. purification of the crude peptide: the same as 6 in the seventh embodiment.

Example eleven, subsequent Synthesis procedure for Compound HD-15:

linkage of Fmoc-Tyr (tBu) -OH: the same as 1 in the seventh embodiment;

n-terminal PEGylation: the same as 6 in the fourth embodiment;

3. cleavage of peptide chain from resin: the same procedure as in example seven, 3;

4. and (3) carboxyl terminal ethylation: the same as 4 in the tenth embodiment;

6. purification of the crude peptide: the same as 6 in example eight.

Example twelve, subsequent Synthesis procedure for Compound HD-16:

linkage of Boc-Tyr (tBu) -OH: the same as example 1;

3. and (3) carboxyl terminal ethylation: the same as 4 in the tenth embodiment;

5. purification of the crude peptide: the same as example seven, 6.

Example thirteen, subsequent synthetic procedure for compound HD-26:

linkage of Fmoc-Tyr (tBu) -OH: the same as 1 in the seventh embodiment;

acetylation of the N-terminus: the same as 6 in the second embodiment;

4. pegylation at the carboxy terminus: the polypeptide was dissolved in 2ml DCM and 122mg mPEG-NH was added₂115 mg PyBop, 59 mu L DIEA, stirring and reacting for 1h at room temperature, removing the reaction solvent by rotary evaporation, and terminating the reaction;

6. purification of the crude peptide: the same as 6 in example eight.

Example fourteen subsequent synthetic procedures for compound HD-27:

linkage of Fmoc-Tyr (tBu) -OH: the same as 1 in the seventh embodiment;

n-terminal PEGylation: the same as 6 in the fourth embodiment;

4. pegylation at the carboxy terminus: the same as in 4 of the thirteenth embodiment;

6. purification of the crude peptide: the same as 6 in example eight.

Example fifteen, subsequent Synthesis procedure for Compound HD-28:

linkage of Boc-Tyr (tBu) -OH: the same as example 1;

3. pegylation at the carboxy terminus: the same as in 4 of the thirteenth embodiment;

5. purification of the crude peptide: the same as 6 in example eight.

TABLE 1 Mass spectrometric and chromatographic assay results for the above synthetic compounds

Note:

(1) the method comprises the following steps: gradient elution was 10% -90% acetonitrile/water (0.1% TFA) completed 15 min; the flow rate is 1 ml/min; the detection wavelength is 220 nm; the analytical chromatographic column is Phenomenex Synergi^TM 4μm Max-RP

250×4.6mm。

(2) The method 2 comprises the following steps: gradient elution was 10% -90% acetonitrile/water (0.1% TFA) completed in 16 min; the flow rate is 1 ml/min; the detection wavelength is 220 nm; the analytical column was Kromasil 100-5-C184.6X 250 mm.

(3) The method 3 comprises the following steps: gradient elution was 0-16min, 10% -90% acetonitrile/water (0.1% TFA), 16-18min, 90% -100% acetonitrile/water (0.1% TFA); the flow rate is 1 ml/min; the detection wavelength is 220 nm; the analytical column was Kromasil 100-5-C184.6X 250 mm.

EXAMPLE sixteen analgesic experiments

The central analgesic effect of The HD-series compounds was evaluated by a lateral ventricle dosing mode using The mouse radiant heat tail-flick test (The radial heat tail-flick test) acute pain model.

1. Laboratory animal

The experimental animals used in the patent are all male Kunming mice provided by animal experiment center of Lanzhou university. All animals were housed in a 22 + -1 deg.C, 12h light, 12h dark quiet animal house with adequate food and water supply, mice were freely available, all experimental animals were used once, and the experimental procedures were approved by the animal ethics committee of Lanzhou university.

2. Lateral ventricle buried tube

1) Pre-operation pain threshold primary screening: a male Kunming mouse (19 +/-1 g) is selected, and is adapted for 3 days at least in an ethological determination laboratory, and the mouse is stroked and lightly held for 3 times every day to be adapted to a hot tail flicking operation. A mark is made at one third of the tail part of the mouse from the tip of the tail, and the radiant heat stimulation is carried out near the mark so as to avoid the damage of the tail tissue of the mouse caused by repeated stimulation at the same site. The method comprises the steps of measuring the radiant heat stimulation latency (Tail-flash latency) of a mouse by using a mouse radiant heat measuring instrument which is debugged in advance, selecting the mouse with the latency of 2-5 s to carry out subsequent experiments (part of mice fail to measure the latency for the first time and can carry out secondary measurement, the time interval of the two measurements is more than 5min, and the mice which fail to measure for the second time are eliminated), and preventing the Tail of the mouse from being scalded by using cut-off time of 10 s.

2) Lateral ventricle intubation: selecting a mouse with a proper basal pain threshold, carrying out intraperitoneal injection (i.p.) on pentobarbital sodium solution (65mg/kg) for anesthesia, fixing the mouse on a mouse brain stereotaxic apparatus, scratching the scalp, finding a Bregma point as a reference, moving the mouse backwards by 0.6mm, moving the midline by 1.1mm, leading the skull to have a depth of 2.0mm downwards, implanting a 26G stainless steel sleeve with a length of 8mm into the lateral ventricle of the mouse, and then sealing the sleeve by using a self-made string. Injecting the solution of penicillin into the abdominal cavity, and placing the mouse in an electric blanket for recovery. After the lateral ventricular intubation operation, the experimental mice are fed in a single cage, the mice recover for at least 3 days, and subsequent experiments are carried out when the weights of the mice are proper (22 +/-1 g).

3. Dosing assay

1) Measuring a basic pain threshold value: selecting mice (22 +/-1 g) with proper weight, determining three tail flick incubation periods, taking an average value at least for more than 10min at each determination interval, taking the average value as a basic pain threshold value of each mouse, selecting the mice with the basic threshold value of 2-5 s, and administering to perform radiant heat tail flick determination.

2) And (3) administration determination: a25 μ l Hamilton microsampler was used to administer the drug into the lateral ventricle using a micro syringe pump at a rate of 2.5 μ l/min for each mouse at 2.5 μ l/min (except for the experiment in which HD-3 was administered at a low concentration of 0.25nmol/mouse and the other drugs were administered at a dose of 2.5 nmol/mouse; HD-10 and HD-14 were administered with 2% DMSO as a solvent and a set of 2% DMSO solution experimental groups were added as a blank, while the other drugs were administered with 0.9% physiological saline as a solvent and physiological saline without drug as a blank). And stopping the needle for 1-1.5 min after the medicine injection is finished, and then pulling out the inner tube to prevent the medicine from flowing back and overflowing. Holding the mouse by hand lightly to fix the mouse, and measuring tail flick incubation period of the mouse by using a mouse radiant heat measuring instrument, wherein cut-off time is 10s to prevent the tail of the mouse from being scalded. The measurement process is recorded into a video, and the final result, namely the tail flick latency of the mouse is determined by the video. After the experiment is finished, injecting blue ink into the lateral ventricle to check whether the position of the embedded pipe of the experimental animal is correct or not, and rejecting experimental data of a mouse with an improper embedded pipe position. All experimental designs followed a blind design, i.e. dosing, measurement, number video and location detection were done by different personnel to prevent the experimenter from the expected impact.

4. The grouping situation is as follows

a) Blank control group 1, 0.9% physiological saline, n ═ 15;

b)

blank control

2, 2% DMSO + 0.9% saline, n ═ 7

c) Positive control group 1, morphine, 2.5nmol/mouse, n-9

d) Positive control group 2, parental MCRT, 2.5nmol/mouse, n ═ 8

e) Compound HD-1, 2.5nmol/mouse, n ═ 7;

f) compound HD-2, 2.5nmol/mouse, n ═ 8;

g) compound HD-3, 0.25nmol/mouse, n ═ 7;

h) compound HD-7, 2.5nmol/mouse, n ═ 7;

i) compound HD-9, 2.5nmol/mouse, n ═ 8;

j) compound HD-10, 2.5nmol/mouse, n ═ 9;

k) compound HD-11, 2.5nmol/mouse, n ═ 9;

l) compound HD-13, 2.5nmol/mouse, n ═ 6;

m) compound HD-14, 2.5nmol/mouse, n-8;

n) compound HD-15, 2.5nmol/mouse, n ═ 6;

o) compound HD-16, 2.5nmol/mouse, n-7;

p) compound HD-26, 2.5nmol/mouse, n-8;

q) compound HD-27, 2.5nmol/mouse, n ═ 8;

r) compound HD-28, 2.5nmol/mouse, n-5;

5. data processing

1) Data represent: the analgesic effect of the drug is expressed as MPE (maximum possible effect) calculated as MPE (%) ═ threshold value-base value)/(cutoff value-base value after administration (x 100).

2) Data processing was performed using OriginPro 2017(OriginLab Corporation, USA) and expressed as mean ± standard error (M ± s.e.m.). In addition, the time-lapse data for each mouse was converted to Area under the curve (AUC) to evaluate and compare the analgesic effect of different drugs in the acute pain model.

6. Results of the experiment

MCRT is a heptapeptide which is formed by embedding opioid peptides Morpheceptin and NPFF analog PFRTic-NH2 in the laboratory by sharing one proline, wherein the N end is a free amino group, the C end is a tail in an amide form, and the central administration mode shows a better analgesic effect in a mouse hot water bath tail flick model, but the duration of the analgesic effect is short, and the maximum limiting factor for limiting the application is the Regul Pept, 2012.179 (1-3): p.23-8). In the experiment, MCRT is used as a parent body, and a series of modifications are carried out on the tail end of the MCRT to obtain a series of compounds named as HD-series. HD-1 is a compound ending with free carboxyl group and C end is deacylated and aminated, while HD-2 is a compound obtained by acetylating N end under the condition that MCRT C end is unchanged, and the two modifications are combined, i.e. MCRT C end is ended with free carboxyl group and N end is acetylated to obtain compound HD-3. The 3 compounds show remarkably enhanced analgesic effect in a retained nerve injury model (SNI) by means of local subcutaneous administration compared with the parent peptide MCRT and positive control morphine (morphine), and still show better analgesic effect at lower use dose, so that the opioid analgesic effect and the side effect thereof are possibly separated to form drug molecules, and the superiority of the peptide terminal modification is also shown.

The patent screens the series of compounds by adopting a lateral ventricle administration mode and utilizing a mouse radiant heat tail-flick acute pain model. As shown in FIG. 1, injection of physiological Saline (salt) did not substantially alter tail flick latency in mice, while the positive control Morphine (Morphine) produced a more pronounced analgesic effect, reaching maximum analgesic effect 20min after administration, followed by onset of decay, with MPE of only 18.6260 + -11.71143 at 90min, consistent with the results published by the former (Journal of Medicinal Chemistry, 2016.59(22): p.10198-10208). At the same dose (2.5nmol/mouse), MCRT reached maximum analgesic effect 5min after administration and this effect was maintained for 15min, after which a slow decay began to occur. The N-terminal acetylated product, HD-2, produced almost the same peak shape as MCRT, but its maximum analgesic effect was weaker than MCRT, its duration of analgesia was not prolonged, HD-1 activity ending with free carboxyl groups was more attenuated, and produced more specific peak shapes, probably related to metabolism in vivo. HD-3 produced a very significant analgesic effect at lower concentrations (0.3 nmol/mouse) in the SNI model by topical plantar subcutaneous administration, and we expected that the lower concentrations (0.25nmol/mouse) in the acute pain radiation heat tail flick model by lateral ventricle administration still produced a more significant analgesic effect, but unfortunately the expected results did not appear, and the expected results did not appear at the lower concentrations (0.025nmol/mouse) (results not shown). This may be associated with the indication for which each drug corresponds, i.e. with a different analgesic mechanism, which requires more intensive subsequent research.

The compound HD-13 is obtained by converting MCRT C-terminal amide into methyl esterification, the analgesic aging curve of the compound is shown in figure 3, HD-13 generates strong and stable (stronger than MCRT) analgesic effect within 0-30 min, the compound starts to decay after 30min, and the compound is only slightly higher than normal saline at 90 min. The modification of HD-2 and HD-13 is integrated to obtain the compound HD-10 with acetylated N-terminal and methyl esterified C-terminal, the maximum analgesic activity of which is slightly reduced compared with HD-13, and the maximum analgesic activity of which is not significantly different from that of MCRT, but the duration of the analgesic effect is remarkably prolonged to 210min, and the plateau period is maintained for at least 120min, so that the compound HD-10 has strong analgesic activity. And (3) calculating the area under the curve (figure 6) within 0-90 min, wherein HD-10 is 2 times of morphine and 2 times of MCRT respectively, and the analgesic activity is not low at this moment, so that the significantly enhanced analgesic activity is shown.

Based on successful modification of HD-10, the compound HD-16 is expanded, the HD-13C-terminal methylation modification is changed into ethyl ester to obtain the compound HD-16, the modification enables the maximum analgesic activity (86.3839 +/-13.61607) of HD-16 to be slightly reduced compared with HD-13(100 +/-0), but the analgesic activity of the compound HD-16 is enhanced within 20-45 min, namely the analgesic activity is attenuated slowly, but the activity is rapidly attenuated after 45 min. The modification of HD-2 and HD-16 is integrated, namely, the C-terminal methyl ester of HD-10 is changed into ethyl ester to obtain a compound HD-14, and the result shows (figure 5) that the maximum analgesic activity of HD-14 is lower than that of MCRT, although the duration of the analgesic activity of HD-14 is slightly prolonged and is obviously weakened after 60min, the maximum analgesic effect and the duration of the drug effect of HD-14 cannot be comparable to that of HD-10, and the area under the curve is not obviously increased.

The pegylation of drug molecules can significantly enhance the solubility and stability of drugs, so that drugs with good analgesic effect are likely to be produced, and then we design a series of compounds modified by PEG at the tail end, and expect to obtain drug molecules with higher solubility and stronger analgesic effect. First we PEGylate the N-terminus of the parent MCRT, then let its C-terminus be free carboxyl (HD-7), amide (HD-9), methyl esterified (HD-11), and ethyl esterified (HD-15), respectively. Overall, N-terminal PEGylation resulted in a reduction in the maximum analgesic activity of the drug molecule (FIG. 4), and comparison of MCRT with HD-9 best demonstrated this problem, probably due to the PEG molecule

Meanwhile, PEG modification is converted to C terminal (figure 5), N terminal is modified, and parent (MCRT) is subjected to pure C terminal PEGylation to obtain HD-28, although the curve has an analgesic aging curve similar to MCRT, the analgesic activity of the parent is obviously reduced, on the basis, N terminal acetylation is carried out to obtain HD-26, and N terminal PEGylation is carried out to obtain HD-27, the two modifications do not improve the analgesic activity of HD-28, but reduce the activity of the parent again, and the area under the curve is smaller than MCRT.

In conclusion, a series of drug molecules with enhanced effects are obtained by systematically modifying MCRT terminals, wherein the drug molecules are particularly shown as HD-10, have the advantages of rapid onset of action, strong analgesic effect and remarkably prolonged analgesic duration, and provide a prodrug molecule for treating clinical acute pain. Meanwhile, a peptide terminal modification method for effectively enhancing the analgesic activity of the opioid chimeric peptide is also provided, in general, the analgesic activity of a drug molecule is weakened by terminal PEGylation, the maximum analgesic activity of the chimeric peptide cannot be significantly enhanced or the analgesic duration cannot be prolonged by only modifying one terminal, the effect can be significantly achieved by properly modifying two terminals, and HD-10 is a good example.

Claims

1. A multi-target peptide compound acting on opioid receptors and neuropeptide FF receptors has a structural general formula shown in formula (I):

wherein R1 is-NH₂、-NHCOCH₃、-NHCOCH₂O(CH₂CH₂O)_nAny one of H, R2 is-COOH, -CONH₂、-COO(CH₂CH₂O)_nH、-CHO、-COOCH₃、-COOCH₂CH₃、-COOC(CH₃)₃Any of the above, wherein R1 is-NH₂When R2 cannot be-CONH₂，(CH₂CH₂O)_nMolecular weight range 500-.

2. The compound of claim 1, wherein R1 is-NH₂And R2 is-COOH.

3. The compound of claim 1, wherein R1 is-NHCOCH₃R2 is-CONH₂。

4. The compound of claim 1, wherein R1 is-NHCOCH₃And R2 is-COOH.

5. A pharmaceutical composition comprising a therapeutically effective amount of a compound according to any one of claims 1 to 4, or a pharmaceutically acceptable salt or stereoisomer thereof, and at least one pharmaceutically acceptable excipient.

6. The pharmaceutical composition of claim 5, wherein the pharmaceutical composition is selected from the group consisting of tablets, capsules, oral solutions, injections, powder injections, sustained-release preparations, dripping pills, granules, sustained-release pellets and any pharmaceutically acceptable dosage form.

7. Use of a compound according to claims 1-4 for the preparation of an analgesic medicament.

8. Use of a compound according to claims 1-4 for the preparation of a medicament for the treatment of sciatica.

9. Use of a compound according to claims 1-4 for the manufacture of a medicament for the treatment of neuropathic pain.

10. Use of a compound according to claims 1-4 for the preparation of an analgesic for the treatment of postherpetic neuralgia, diabetic peripheral neuralgia, poststroke central neuralgia, cancerous neuralgia, trigeminal neuralgia, post-operative pain.