CN109021117B

CN109021117B - Opioid and neuropeptide FF receptor multi-target molecule BN-9 analogue, and preparation method and application thereof

Info

Publication number: CN109021117B
Application number: CN201810950987.1A
Authority: CN
Inventors: 方泉; 张梦娜; 王锐; 许彪; 石学睿
Original assignee: Lanzhou University
Current assignee: Lanzhou University
Priority date: 2018-08-20
Filing date: 2018-08-20
Publication date: 2020-05-08
Anticipated expiration: 2038-08-20
Also published as: CN109021117A

Abstract

The invention relates to analogues of multi-target molecule BN-9 of opioid and neuropeptide FF receptors, and a preparation method and application thereof, namely cysteine (Cys) or D-type cysteine (D-Cys) containing sulfhydryl side chains are introduced into an amino acid sequence of BN-9, and in-vitro function experiment results show that the four brand-new BN-9 analogues have multi-target agonistic activity of the opioid and neuropeptide FF receptors, the central and peripheral analgesic efficacy and action time of part of analogues are improved, and the analogues can mediate intolerant analgesic effect. Therefore, the brand new BN-9 analogues can be used for preparing analgesic drugs.

Description

Opioid and neuropeptide FF receptor multi-target molecule BN-9 analogue, and preparation method and application thereof

Technical Field

The invention belongs to the technical field of biochemistry, and relates to an analog of a multi-target peptide molecule BN-9 of an opioid/neuropeptide FF receptor, a preparation method and an application thereof.

Background

Pain management is a major global challenge and the international society for pain research defines pain as "unpleasant sensation of perception and psychological sensation caused by actual or potential damage to body tissues" for the needs of clinical research. Pain under physiological conditions serves as a protection mechanism to prevent the body from being injured, but pain under pathological conditions not only brings pain to patients, but also causes adverse effects on systems of central nerves, circulation, respiration, endocrine, digestion, autonomic nerves and the like of human bodies, and even certain severe pain threatens the lives of the patients. In particular, chronic pain is a clinically unsolved problem and significantly affects the quality of life of people. Although the opioid analgesic drugs at the first line of the clinic play an important role in the treatment of moderate or severe pain, they are often accompanied by a series of opioid side effects such as constipation, vomiting, respiratory depression, sedation, drug dependence and the like, thus greatly limiting their use. Studies have shown that opioid peptides mediate highly potent analgesic effects and have reduced opioid side effects compared to conventional opioid analgesics. Therefore, the opioid peptide can be used as a chemical template molecule for developing a novel opioid analgesic with high efficiency and low side effect.

Recent studies show that multi-target drugs can act on multiple targets in a disease network simultaneously, and the action on each target can generate synergistic effect, so that better curative effect and lower side effect are generated, and the curative effect is verified in the treatment of cancers, diabetes, virus and bacterial infection diseases (Neurotheliaceae, 2009, 6 (1): 152-162). Recent research finds that the multi-target drug has potential application prospect in the research and development of novel analgesic drugs, particularly in the research and development of complex pain treatment drugs. Opioid multi-target molecules have potent analgesic activity with reduced side effects such as tolerance and addiction (Br J Pharmacol.2018, 175 (14): 2857; Curr Pharm Des.2013, 19 (42): 7435). The multi-target molecules such as opium-NK 1, opium-CB 1, opium-Nociceptin (NOR) and the like all have no tolerance analgesic activity, and particularly, the experimental result of the multi-target drug BU08028 of an opium-Nociceptin (NOR) receptor on macaques shows that the multi-target molecules have long-acting analgesic effect, but the side effects such as addiction, pruritus, respiratory depression, hypotension and the like are greatly reduced (Proc Natl Acad Sci U SA.2016, 113 (37): E5511).

Neuropeptide FF (NPFF) was isolated from bovine brain in 1985, and is now considered to be a class of endogenous opioid regulatory peptides that activate two G protein-coupled receptors NPFF₁And NPFF₂Has a regulating effect on the biological activities of analgesic, tolerance and addiction of the opioid (Protein & Peptide Letters, 2011, 18 (4): 403-409). The subject group disclosed in patent ZL201210098832.2 an entirely new chimeric peptide BN-9 (Tyr-D-Ala-Gly-Phe-Gln-Pro-Gln-Arg-Phe-NH) based on opioid peptides Biphalin and NPFF₂) The chimeric peptide can simultaneously activate Mu-, Delta-, Kappa-opioid receptor and NPFF₁And NPFF₂Receptor and in vivo side effect researches show that the opioid side effect of BN-9 in tolerance, constipation, addiction and the like is remarkably reduced compared with morphine. However, the analgesic effect of the chimeric peptide BN-9 was comparable to that of morphine (Br JPharmacol.2016, 173 (11): 1864; Eur J Pharmacol.2017, 813: 122). Also, linear analogs of BN-9, each of which does not contain Cys or amino acid residues having a thiol structure in the side chain such as D-Cys, are disclosed in patent 201610252648.7, wherein DN-9 is the most analgesic compound in this class of BN-9 analogs, and the analgesic activity at a level above the spinal cord is increased by 32.70 times as compared with BN-9 (J Med chem.2016, 59 (22): 10198). However, the reported pharmaceutical properties of the BN-9 analogues still have room for further improvement, such as in terms of analgesic dose, analgesic time, etc.

Disclosure of Invention

The invention aims to provide a more medicinal multi-target molecule BN-9 analogue. Through a large number of experimental researches, the inventor introduces amino acid residues with sulfhydryl structures on side chains into parent BN-9 molecules to chemically modify multi-target molecule BN-9 of an opioid/NPFF receptor, and the results show that the novel BN-9 analogue has the advantages of longer analgesic action time, lower effective analgesic dose and no tolerance analgesia. Compared with the reported BN-9 and the analogues thereof, the in-vivo analgesic efficacy and action time of the medicament are improved to a certain extent, and the medicament can be used for treating various diseases such as acute pain, pathological pain and the like.

The invention also aims to provide a preparation method of the multi-target peptide molecule BN-9 analogue.

The invention also aims to provide the therapeutic application of the multi-target peptide molecule BN-9 analogue, and the novel BN-9 analogue can be used as a new generation of potential medicament for treating pain, can be used for relieving various pain symptoms such as acute pain or pathological pain and the like, and simultaneously reduces the side effect of the traditional opioid analgesic medicament.

In order to achieve the purpose, the invention adopts the following technical scheme:

the amino acid sequence of the analogues of opioid and neuropeptide FF receptor multi-target molecule BN-9 is shown as follows:

Tyr-Xaa2-Gly-Phe-Xaa5-Pro-Gln-Arg-Phe-NH₂

wherein Xaa2 is Cys or D-Cys with a side chain containing a sulfydryl structure;

xaa5 is Cys or D-Cys with side chain containing sulfhydryl structure.

Preferably, the analogue of the multi-target molecule BN-9 is selected from one of the following compounds:

compound 1: Tyr-Cys-Gly-Phe-Cys-Pro-Gln-Arg-Phe-NH₂

Compound 2: Tyr-D-Cys-Gly-Phe-Cys-Pro-Gln-Arg-Phe-NH₂

Compound 3: Tyr-Cys-Gly-Phe-D-Cys-Pro-Gln-Arg-Phe-NH₂

Compound 4: Tyr-D-Cys-Gly-Phe-D-Cys-Pro-Gln-Arg-Phe-NH₂。

Based on the research results of the structure-activity relationship between the existing opioid peptide and NPFF, the compound of the invention reserves the amino acid residue which plays a key role in activating opioid and NPFF receptors, such as Tyr¹、Gly³、Arg⁸And Phe⁹. On the basis of BN-9, amino acid residues with sulfhydryl structures in side chains are introduced, and a novel linear peptide analogue is obtained through amino acid substitution modification.

The preparation method of the opioid and neuropeptide FF receptor multi-target molecule BN-9 analogue comprises the steps of sequentially coupling amino acids on a solid phase carrier one by a solid phase synthesis method, and then cracking to obtain the target peptide.

Wherein, the solid phase carrier is preferably amino resin.

Wherein, in the solid phase synthesis, the used amino deprotection reagent is preferably a DMF solution of piperidine with the volume percentage of 20 percent or a DMF solution of DBU with the volume percentage of 1 percent; the coupling agent used is a combination of HBTU and HOBt and DIEA, or a combination of DIC and HOBt, or a combination of PyBOP and HOBt and DIEA. The amount, ratio, timing and treatment method of the amino group deprotecting reagent and the coupling agent are known to those skilled in the art.

Wherein, in the cleavage process, the cleavage agent used is preferably TFA and H₂Mixed solution of O in the volume ratio of 95:5, or TFA, EDT, TIS, PhOH and H₂Mixed solution of O in the volume ratio of 80:5, or TFA and EDT and TIS and H₂O is mixed solution according to the volume ratio of 92.5: 2.5. The amount, ratio, timing and treatment method of the above-mentioned cracking agent are known to those skilled in the art.

In the preparation method, more preferable process steps are as follows:

i. polypeptide synthesis:

i.i. resin pretreatment: connecting and fixing a manual glass polypeptide synthesizer with an electric stirrer with a stirring rod, adding a certain amount of resin into the synthesizer, adding Dichloromethane (DCM) for swelling, stirring for 30min, draining for 30min, and washing with N, N-Dimethylformamide (DMF) for 3 times for 3min each time. Finally, performing indene detection on the resin, wherein the resin is colorless under normal conditions, and the solution is light yellow;

i.ii sequential cyclic condensation of amino acids

i.ii.i. Fmoc-protecting group removal: adding mixed solution of piperidine, 1, 8-diazabicycloundecen-7-ene and DMF in a volume ratio of 1: 98, stirring and reacting for 3 times, the last time for 5min of the first two times for 10min, and then washing for 4 times for 3min each time by using DMF. The indene test is usually: resin blue, solution dark blue;

i.ii.ii condensed amino acids: sequentially dissolving Fmoc-AA-OH, O-benzotriazole-N, N, N ', N' -tetramethylurea-Hexafluorophosphate (HBTU) and N-hydroxybenzotriazole (HOBt) in DMF, finally adding N, N-Diisopropylethylamine (DIEA), stirring uniformly, directly adding into a synthesizer, and stirring under argon protection for reaction for 1 h. Then, the tube was dried by suction and washed with DMF 3 times for 3min each, and then subjected to indene detection: the resin is colorless, and the solution is light yellow;

the dosage ratio of the amino acid and the condensing agent thereof is Fmoc-AA-OH/HOBt/HBTU/DIEA 1/1/1/2

iii peptide chain extension: sequentially inoculating different Fmoc protected amino acids according to the polypeptide sequence of the linear peptide, wherein the last amino acid usually uses tyrosine protected by Boc, so that the last deprotection step is omitted, and the amino acid condensation method is as described above;

cleavage of peptide resin

ii.i compressed resin: after washing with DCM (2X 3min), MeOH (1X 3min), DCM (1X 3min), MeOH (2X 3min) in succession, the stir bar was removed and the tube was drained for 4 h.

ii cleavage and precipitation extraction of the polypeptide: adding a cutting agent into the obtained resin, wherein 15mL of the cutting agent is added into every 1g of the resin, and the ratio of the cutting agent is as follows: trifluoroacetic acid, triisopropylsilane, 1, 2-ethanedithiol, water (94/1/2.5/2.5), the reaction is generally carried out at room temperature for 3 h. The cleavage solution was rotary evaporated using a rotary evaporator (less than 40 ℃) to remove TFA. And then precooled for 15min in a refrigerator. Adding precooled ether, standing and precipitating. The ether phase is extracted with 20% acetic acid solution, the precipitate is dissolved with water, and the precipitate is extracted several times with 20% acetic acid solution. The extract was lyophilized to give crude peptide.

Purification of the polypeptide: and (3) separating and purifying the crude peptide by using a reversed-phase high performance liquid chromatography column, and collecting a main peak of a sample after separation to obtain a multi-target polypeptide product.

Of course, the present invention is not limited to the above specific process steps, and those skilled in the art can adapt and modify the above process steps based on the concept of solid phase synthesis to finally obtain the target peptide product of the present invention.

A pharmaceutical composition comprising an analog of the class of opioid and neuropeptide FF receptor multi-target molecule BN-9 of claim 1. For example, the BN-9 analogue is used as an active ingredient, and a pharmaceutically acceptable carrier and/or an auxiliary material is added to prepare the pharmaceutical composition.

The application of the opioid and neuropeptide FF receptor multi-target molecule BN-9 analogue in the preparation of the pain treatment medicine is also within the protection scope of the invention. The pain includes, but is not limited to, acute pain or chronic pain, such as postoperative incision pain, inflammatory pain (including arthritis pain), neuralgia, cancer pain, and other pathological pain. Research experiments show that the BN-9 analogue has high-efficiency analgesia, reduces tolerance side effects of traditional opioid analgesic drugs, and has potential application in the aspect of high-efficiency and low-side-effect drugs for treating various pains.

It will be understood by those skilled in the art that the pharmaceutical composition containing the BN-9 analogue of the present invention can be suitably used for various administration modes, such as oral administration, transdermal administration, intrathecal administration, intravenous administration, intramuscular administration, topical administration, nasal administration, etc. Depending on the mode of administration used, the polypeptide pharmaceutical composition of the present invention may be formulated into various suitable dosage forms comprising at least one effective amount of the polypeptide of the present invention and at least one pharmaceutically acceptable carrier. Examples of suitable dosage forms are sterile solutions and dry injections which can be used for injection, tablets, capsules, sugar-coated tablets, granules, oral solutions and syrups, aerosols, nasal sprays, and also ointments and patches for the skin surface.

Pharmaceutical compositions containing a polypeptide of the invention may be formulated as solutions or lyophilized powders for parenteral administration, the powders being reconstituted with a suitable solvent or other pharmaceutically acceptable carrier prior to use, the solution formulation typically being a buffer, an isotonic solution or an aqueous solution.

The pharmaceutical dosage of the polypeptide of the present invention may vary over a wide range and can be readily determined by one skilled in the art based on objective factors, such as the type of disease, the severity of the condition, the weight of the patient, the dosage form, the route of administration, and the like.

In this specification, the conventional three-letter code is used to represent the natural amino acids, and the accepted three-letter code is used to represent other amino acids, such as D-Cys (D-cysteine).

The material abbreviations used in the present invention are well known in the art, and the specific English abbreviations and Chinese full name are shown in Table 1.

TABLE 1 comparison table of actual English abbreviation and Chinese full name

Has the advantages that: the opioid and neuropeptide FF receptor multi-target molecule BN-9 analogue disclosed by the invention has multi-target agonistic activity of opioid and neuropeptide FF receptors, the central and peripheral analgesic efficacy and action time of part of analogues are improved, and the central and peripheral analgesic efficacy and action time of the analogues are improved, and the central and peripheral analgesic efficacy and the intolerant analgesic action can be mediated. Therefore, the brand new BN-9 analogues can be used for preparing analgesic drugs. The concrete advantages are as follows:

(1) compared with the parent BN-9, the analgesic activity of the compound is improved to a certain extent when the molecular number is the same;

(2) the analgesic effect of the partial analogs can be extended to 180 or 240 minutes compared to the parent BN-9.

In particular embodiments, the following multi-target molecule BN-9 analogs are contemplated, the specific sequences of which are shown in table 1:

drawings

FIG. 1 is a time-dose response curve of dose-dependent analgesia produced by lateral ventricle BN-9 injection in mice;

FIG. 2 is a time-dose response curve of dose-dependent analgesia produced by lateral ventricle injection of Compound 1 in mice;

FIG. 3 is a time-dose response curve of dose-dependent analgesia produced by lateral ventricle injection of Compound 2 in mice;

FIG. 4 is a time-dose response curve of dose-dependent analgesia produced by lateral ventricle injection of Compound 3 in mice;

FIG. 5 is a time-dose response curve of dose-dependent analgesia produced by lateral ventricle injection of Compound 4 in mice;

FIG. 6 is a time-dose response curve of dose-dependent analgesia produced by subcutaneous injection of BN-9 in mice;

FIG. 7 is a time-dose response curve of dose-dependent analgesia produced by subcutaneous injection of Compound 1 in mice;

FIG. 8 is a time-dose response curve of dose-dependent analgesia produced by mice injected subcutaneously with Compound 2;

FIG. 9 is a time-dose response curve of dose-dependent analgesia produced by mice injected subcutaneously with Compound 3;

FIG. 10 is a time-dose response curve of dose-dependent analgesia produced by mice injected subcutaneously with Compound 4;

figure 11 is a graph of the change in analgesic effect of mice injected laterally with morphine and compounds 1, 2, 3, 4 for eight consecutive days.

Detailed Description

Embodiments of the present invention will be described in detail below with reference to examples, but those skilled in the art will appreciate that the following examples are only illustrative of the present invention and should not be construed as limiting the scope of the present invention. The examples, in which specific conditions are not specified, were conducted under conventional conditions or conditions recommended by the manufacturer. The reagents or instruments used are not indicated by the manufacturer, and are all conventional products commercially available.

Example 1 synthesis of compounds 1-4:

experimental reagent: the Resin was Rink-Amide-MBHA-Resin (substitution value S of 0.43 mmol/g; Tianjin Nankai Kangcheng Co., Ltd.), O-benzotriazole-N, N, N ', N' -tetramethyluronium-Hexafluorophosphate (HBTU) (Shanghai Gill Biochemical Co., Ltd.), N-hydroxybenzotriazole (HOBt) (Shanghai Gill Biochemical Co., Ltd.), N, N-Diisopropylethylamine (DIEA) (Beijing Bailingwei), 1, 8-diazabicycloundecan-7-ene (DBU) (Beijing Bailingwei), ninhydrin being a product of Shanghai reagent III, methylene chloride (DCM), N, N-Dimethylformamide (DMF), hexahydropyridine (piperidine), methanol (MeOH) and pyridine all being available from Tianjin second reagent factory, trifluoroacetic acid (TFA), phenol (PhOH) and pyridine all being Tianjin reagent first factory products, and the organic reagents are all subjected to redistilling treatment before use.

An experimental instrument: a solid phase polypeptide synthesizer (independently designed by the laboratory), a rotary evaporator (RE-5298A, Shanghai Yangrong), a freeze dryer (VIRTIS, USA), a mass spectrometer (ESI-Q-TOF maXis-4G, Bruker Daltonics, Germany Dalton), a circulating water pump (SHB-III, Zheng City), and a High Performance Liquid Chromatography (HPLC) is Delta 600 of Waters; and (3) analyzing the column: xbridge TM BEH 130 Prep C18, 4.6mm × 250 mm; preparing a column: xbridge TMBEH 130 Prep C18, 19 mm. times.250 mm.

The synthesis method comprises the following steps:

1. synthesis of Compound 1

(1) Resin pretreatment: 600mg of Rink MBHA resin (substitution value 0.43mmol/g) was weighed out and then added with an appropriate amount of DCM to swell the resin, stirred for 30min and then drained for 30 min.

(2) Removing Fmoc protecting groups: and adding a proper amount of DMF into the swelled resin to wash the resin for three times, wherein each time lasts for 3 min. Then adding appropriate amount of (completely submerged resin) deprotection reagents piperidine: DBU: DMF (1: 98), repeating the deprotection for 3 times, the first two times for 5min, and the last time stirring and reacting for 10 min. After which it was washed 4 times with DMF, 3min each time.

(3) Indene test A small amount of resin and indene test reagent (phenol, pyridine and ninhydrin volume ratio is 1: 2: 1. the test tube is placed in boiling water for 3min, if the solution and the resin are dark blue, the Fmoc group is removed. the indene test reagent is prepared according to the formula of ① 20g of phenol/5 ml of absolute ethyl alcohol, ② 0.05ml of KCN (0.001M)/2.5ml of pyridine and ③ 0.5.5 g of ninhydrin/10 ml of absolute ethyl alcohol, and the prepared phenol, pyridine and absolute ethyl alcohol need to be used after being redistilled.

(4) Condensation of amino acids: Fmoc-Phe-OH, HOBt and HBTU were weighed in a molar ratio of 1: 1, dissolved in a small amount of DMF and stirred to dissolve. Then adding 2 times of molar amount of DIEA, stirring uniformly, adding the mixture into the resin with the Fmoc protecting group removed, and stirring and reacting for 60min in a synthesizer under the protection of argon. And (4) after the reaction is finished, performing indene detection according to the method of the step (3), and if the light yellow resin of the solution is colorless, indicating that the amino acid is condensed onto the resin. And (3) removing the protecting group according to the step (2), performing indene detection according to the step (3), and if the solution and the resin are bluish purple, completely removing the Fmoc protecting group to obtain the resin peptide without the Fmoc protecting group.

(5) Extension of peptide chain: condensing Fmoc-Arg (pbf) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Pro-OH, Fmoc-Cys (Trt) -OH, Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-Cys (Trt) -OH and Boc-Tyr (tBu) -OH sequentially onto the peptide resin according to the method of step (4). The peptide Resin Boc-Tyr (tBu) -Cys (Trt) -Gly-Phe-Cys (Trt) -Pro-Gln (Trt) -Arg (Pbf) -Phe-Resin was obtained.

(6) Compression and draining of peptide chains: the peptide resin synthesized in step (5) was washed with DCM (2X 3min), MeOH (1X 3min), DCM (1X 3min), MeOH (2X 3min) alternately, the stir bar was removed and the synthesizer was sealed and pumped dry with circulating water for 4 h.

(7) Cleavage of peptide chain: adding a cleavage agent (TFA: EDT: H) to the drained peptide Resin Boc-Tyr (tBu) -Cys (Trt) -Gly-Phe-Cys (Trt) -Pro-Gln (Trt) -Arg (Pbf) -Phe-Resin₂Tis 94: 2.5: 1, 15ml of cleavage agent per gram of peptide resin). The reaction was stirred at room temperature for 3h, every 15 min. Then fully decompressing and spin-drying at the temperature of not higher than 40 ℃, then adding the ethyl acetate, fully shaking and mixing uniformly. Shaking caused the crude peptide to precipitate sufficiently as a white precipitate. The crude peptide in ether was then extracted with 20% aqueous acetic acid. The extracted peptide aqueous solution was then lyophilized to give 260.3mg of a white crude peptide solid powder with a crude peptide yield of 90.16%.

(8) Purification of the crude peptide: the crude peptide compound was separated and purified by reverse phase High Performance Liquid Chromatography (HPLC) using C18 column (Xbridge TM BEH 130 PrepC18, 19 mm. times.250 mm) with mobile phase of acetonitrile (containing 0.1% TFA) and water (containing 0.1% TFA), and after separation, a main peak sample was collected to obtain a purified compound 1 sample with a loading of 53.5 mg. The pure peptide was obtained as a white solid powder (20.1 mg) by freeze-drying, and the yield of the pure peptide was 37.57%. The results of mass spectrometry and chromatography are shown in Table 2.

2. Synthesis of Compound 2

(1) Resin pretreatment: 800mg of Rink MBHA resin (substitution value 0.43mmol/g) was weighed, and then an appropriate amount of DCM was added to swell the resin, and after stirring for 30min, the resin was drained for 30 min.

(2) Removing Fmoc protecting groups: and (3) removing the Fmoc protecting group in the synthesis process of the compound 1.

(3) And (4) indene detection: this was followed by an indene detection operation during the synthesis of Compound 1.

(4) Condensation of amino acids: Fmoc-Phe-OH, HOBt and HBTU were weighed in a molar ratio of 1: 1, dissolved in a small amount of DMF and stirred to dissolve. Then adding 2 times of molar amount of DIEA, stirring uniformly, adding the mixture into the resin with the Fmoc protecting group removed, and stirring and reacting for 60min in a synthesizer under the condition of argon protection. After the reaction is finished, the reaction is performed according to the method of the step (3), and if the light yellow resin in the solution is colorless, the amino acid is condensed on the resin. And (3) removing the protecting group according to the step (2), performing indene detection according to the step (3), and if the solution and the resin are dark blue, completely removing the Fmoc protecting group to obtain the resin peptide without the Fmoc protecting group.

(5) Extension of peptide chain: condensing Fmoc-Arg (pbf) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Pro-OH, Fmoc-Cys (Trt) -OH, Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-D-Cys (Trt) -OH and Boc-Tyr (tBu) -OH sequentially onto the peptide resin according to the method of step (4). Obtaining the peptide Resin Boc-Tyr (tBu) -D-Cys (Trt) -Gly-Phe-Cys (Trt) -Pro-Gln (Trt) -Arg (Pbf) -Phe-Resin.

(6) Compression and draining of peptide chains: the compound 1 is subjected to compression and suction drying in the synthesis process.

(7) Cleavage of peptide chain: adding a cut (TFA: EDT: H) to the drained peptide Resin Boc-Tyr (tBu) -D-Cys (Trt) -Gly-Phe-Cys (Trt) -Pro-Gln (Trt) -Arg (Pbf) -Phe-Resin₂Tis 94: 2.5: 1, 15ml of cleavage agent per 1g of peptide resin). Stirring and reacting for 3h at room temperature, and stirring once every 15min. Then fully decompressing and spin-drying at the temperature of not higher than 40 ℃, then adding the ethyl acetate, fully shaking and mixing uniformly. Shaking caused the crude peptide to precipitate sufficiently as a white precipitate. The crude peptide in ether was then extracted with 20% aqueous acetic acid. The aqueous solution of the extracted peptide was then lyophilized to give 321.6mg of a white crude peptide solid powder with a crude peptide yield of 83.52%.

(8) Purification of the crude peptide: the crude peptide compound was separated and purified by reverse phase High Performance Liquid Chromatography (HPLC) using C18 column (Xbridge TM BEH 130 PrepC18, 19 mm. times.250 mm) with mobile phase of acetonitrile (containing 0.1% TFA) and water (containing 0.1% TFA), and after separation, a main peak sample was collected to give a purified compound 2 sample with a loading of 107.4mg, and freeze-dried to give 17.8mg of white pure peptide solid powder with a pure peptide yield of 16.57%. The results of mass spectrometry and chromatography are shown in Table 2.

3. Synthesis of Compound 3

(1) Resin pretreatment: 600mg of Rink MBHA resin (substitution value 0.43mmol/g) was weighed, and then an appropriate amount of DCM was added to swell the resin, and after stirring for 30min, the resin was drained for 30 min.

(5) Extension of peptide chain: condensing Fmoc-Arg (pbf) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Pro-OH, Fmoc-D-Cys (Trt) -OH, Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-Cys (Trt) -OH and Boc-Tyr (tBu) -OH sequentially onto the peptide resin according to the method of step (4). The peptide Resin Boc-Tyr (tBu) -Cys (Trt) -Gly-Phe-D-Cys (Trt) -Pro-Gln (Trt) -Arg (Pbf) -Phe-Resin was obtained.

(7) Cleavage of peptide chain: adding a cleavage agent (TFA: EDT: H) to the drained peptide Resin Boc-Tyr (tBu) -Cys (Trt) -Gly-Phe-D-Cys (Trt) -Pro-Gln (Trt) -Arg (Pbf) -Phe-Resin₂Tis 94: 2.5: 1, 15ml of cleavage agent per 1g of peptide resin). The reaction was stirred at room temperature for 3h, every 15 min. Then fully decompressing and spin-drying at the temperature of not higher than 40 ℃, then adding the ethyl acetate, fully shaking and mixing uniformly. Shaking caused the crude peptide to precipitate sufficiently as a white precipitate. The crude peptide in ether was then extracted with 20% aqueous acetic acid. The aqueous solution of the extracted peptide was then lyophilized to give 272.4mg of a white crude peptide solid powder with a crude peptide yield of 94.35%.

(8) Purification of the crude peptide: the crude peptide compound was separated and purified by reverse phase High Performance Liquid Chromatography (HPLC) using C18 column (Xbridge TM BEH 130 PrepC18, 19 mm. times.250 mm) with mobile phase of acetonitrile (containing 0.1% TFA) and water (containing 0.1% TFA), and after separation, a main peak sample was collected to obtain a purified compound 3 sample with a loading of 102.0 mg. Freeze-drying gave 25.0mg of a white pure peptide solid powder with a pure peptide yield of 24.51%. The results of mass spectrometry and chromatography are shown in Table 2.

4. Synthesis of Compound 4

(5) Extension of peptide chain: condensing Fmoc-Arg (pbf) -OH, Fmoc-Gln (Trt) -OH, Fmoc-Pro-OH, Fmoc-D-Cys (Trt) -OH, Fmoc-Phe-OH, Fmoc-Gly-OH, Fmoc-D-Cys (Trt) -OH and Boc-Tyr (tBu) -OH sequentially onto the peptide resin according to the method of step (4). Obtaining the peptide Resin Boc-Tyr (tBu) -D-Cys (Trt) -Gly-Phe-D-Cys (Trt) -Pro-Gln (Trt) -Arg (Pbf) -Phe-Resin.

(7) Cleavage of peptide chain: adding a cleavage agent (TFA: EDT: H) to the drained peptide Resin Boc-Tyr (tBu) -D-Cys (Trt) -Gly-Phe-D-Cys (Trt) -Pro-Gln (Trt) -Arg (Pbf) -Phe-Resin₂Tis 94: 2.5: 1, 15ml of cleavage agent per 1g of peptide resin). The reaction was stirred at room temperature for 3h, every 15 min. Then fully decompressing and spin-drying at the temperature of not higher than 40 ℃, then adding the ethyl acetate, fully shaking and mixing uniformly. Shaking caused the crude peptide to precipitate sufficiently as a white precipitate. The crude peptide in ether was then extracted with 20% aqueous acetic acid. The aqueous solution of the extracted peptide was then lyophilized to give 163.8mg of a white crude peptide solid powder with a crude peptide yield of 56.73%.

(8) Purification of the crude peptide: the crude peptide compound was separated and purified by reverse phase High Performance Liquid Chromatography (HPLC) using C18 column (XBridge TM BEH 130 PrepC18, 19 mm. times.250 mm) with mobile phase of acetonitrile (containing 0.1% TFA) and water (containing 0.1% TFA), and after separation, a main peak sample was collected to obtain purified compound 4 sample with a loading of 69.0mg, and by freeze-drying, white pure peptide solid powder of 21.2mg was obtained with a pure peptide yield of 30.72%. The results of mass spectrometry and chromatography are shown in Table 2.

Based on the above synthetic steps, the synthesis of the invention comprises the multi-target molecule BN-9 analogues as listed in Table 1, and the chemical characterization results are shown in Table 2.

TABLE 2 chemical characterization of Multi-target molecule BN-9 analogs

Note: system 1: gradient elution system 1 was: 10-80% acetonitrile/water (0.1% TFA) (30 min complete) at flow rates: 1mL/min, the detection wavelength is 220nm, and the analytical chromatographic column is as follows: xbridge^TMBEH 130 Prep C₁₈4.6mm × 250 mm; system 2: the gradient elution system 2 was: 10-100% acetonitrile/water (0.1% TFA) (30 min complete) at flow rates: 1mL/min, the detection wavelength is 220nm, and the analytical chromatographic column is as follows: xbridge^TMBEH 130 Prep C₁₈，4.6mm×250mm。

Example 2 in vitro functional activity assay of compounds 1-4 at opiate and NPFF receptors:

stably expressing Mu-, Delta-, Kappa-opium and NPFF₁And NPFF₂In HEK293 cells of the receptor, the agonist activity of the opioid and NPFF receptors on five receptors is detected by detecting the regulation of intracellular cyclic adenosine monophosphate (cAMP) accumulation caused by Forskolin by multi-target cyclized polypeptides of the opioid and NPFF receptors. The specific method comprises the following steps: will stably express Mu-, Delta-, Kappa-opium and NPFF₁And NPFF₂Recipient HEK293 cells were seeded in 24-well plates at 12 million cells per well and cultured in an incubator for over 20 hours. At the start of the experiment, the cell culture medium was replaced with pre-warmed serum-free medium containing 1mM IBMX and incubated at 37 ℃ for 10 min. Then, 10. mu.l each of the test drug and 10. mu.M Forskolin (final concentration) was added to each well, and incubated at 37 ℃ for 30 min. After the incubation was completed, the solution in each well was aspirated and the cells were lysed by incubating for 30min at room temperature using 500 μ L of 0.2M hydrochloric acid. After completion of the lysis, 10. mu.L of 10M NaOH solution was added per well to neutralize the hydrochloric acid solution used for lysis. The solution in each hole is blown and beaten by a pipetteAfter being homogenized, the mixture is transferred into a centrifuge tube and centrifuged at 12000rpm for 2 min. 50 μ L of supernatant was pipetted into each tube and 100 μ L of 60ug/μ L of PKA was added to the clear tube, while 100 μ LTE cAMP buffer was added to the blank. Adding 50 μ L of 0.5 μ Ci [ H ] into each centrifugal tube³]cAMP, mixed rapidly and incubated at 4 ℃ for more than 2 hours. After the incubation is finished, adding 100 mu L of activated carbon suspension into each tube, uniformly vortexing and shaking, standing in an ice bath for 1min, and centrifuging at 5000rpm for 4 min. And sucking 200 mu L of centrifuged supernatant in each tube, adding the supernatant into a 24-well plate, adding 700 mu L of scintillation fluid into each well, sealing the 24-well plate by using a glue film, standing for 3 hours, and then placing the plate on a scintillation instrument for measurement.

Inhibitory effect of cAMP percent (%) control of Forskolin-induced intracellular cAMP accumulation using drugs is expressed as% control (Forskolin-treated cAMP content-cAMP content co-treated with drug to be tested and Forskolin)/(Forskolin-treated cAMP content-solvent-treated cAMP content). The relevant% control data are expressed as mean ± standard error (Means + s.e.m.). The relationship of drug dose effect is counted by a nonlinear regression model, and the IC of the inhibition of the multi-target dotted linear polypeptide of the opioid and NPFF receptor on the accumulation of cAMP in cells caused by Forskolin is respectively calculated by using the version 5.0 of a statistical software GraphPad Prism₅₀The values, experimental results are shown in tables 3 and 4.

TABLE 3 cAMP function assay of multi-target molecule BN-9 analogs on opioid receptors

As shown in Table 3, compounds 1-4 all dose-dependently inhibited Forskolin-induced cAMP accumulation in the HEK293 cell line stably expressing Mu-, Delta-and Kappa-opioid receptors, indicating that these compounds all exhibit Mu-, Delta-and Kappa-opioid receptor agonistic activity.

TABLE 4 cAMP function assay of multiple target molecule BN-9 analogs on NPFF receptors

As shown in Table 4, NPFF was stably expressed₁And NPFF₂Compounds 1-4 all dose-dependently inhibited Forskolin-induced cAMP accumulation in HEK293 cell lines at the receptor, indicating that these compounds also have NPFF₁And NPFF₂Agonistic activity of the receptor. In summary, compounds 1-4 are capable of activating both opioid and NPFF receptors, and are characterized as multi-target agonists of both opioid and NPFF receptors.

Example 3 in vivo analgesic activity assay of compounds 1-4:

the in-vivo analgesic activity of the BN-9 analogue is detected by adopting two different levels of administration modes of central administration (lateral ventricle administration) and peripheral administration (subcutaneous administration) and utilizing an acute pain model mouse photothermal tail flick experiment.

Lateral ventricle administration at the central level requires a lateral ventricle catheterization procedure to be performed in advance to ensure accuracy of the administration site and minimal injury to the mice. Using a mouse stereotaxic apparatus to carry out lateral ventricle tube burying, wherein a Kunming male mouse is in a weight of 21 +/-2 g; mice were first anesthetized by intraperitoneal injection of sodium pentobarbital (80mg/kg), and all surgical instruments were prepared and sterilized. The hair in the operated area of the top of the mouse head was then cut clean. The head of the mouse is fixed on a stereotaxic instrument, then an iodophor is used for disinfecting an operation area, the scalp is cut along a sagittal suture, the skull is exposed, and then the bregma position is found. 3mm from bregma and 1mm from bregma and marked by puncturing with a needle, which is the upper part of lateral ventricle. The self-made stainless steel tube with PE10 was then moved 3mm down at the needle insertion, here the lateral ventricle. The tube is then secured with glue and tray powder, and after the tray powder has set, it is sutured with surgical thread while the strings are inserted over the stainless steel tube to prevent foreign objects from entering the tube and clogging the stainless steel tube. Mice were rested for 3-4 days after surgery and day 4/5 was used for dosing experiments. Mice were injected with 4. mu.l of the drug each time in the lateral ventricles, and then the steel tubes were rinsed with 1. mu.l of physiological saline, and the total volume of administration was 5. mu.L.

The skin was administered subcutaneously on the back of the neck using a 1mL sterile syringe and injected at a volume of 0.1mL/10 g. The left hand picks up the skin and the right hand holds the syringe. When the needle head is punctured into the subcutaneous tissue, the needle head swings left and right, and if the needle head is easy to swing, the hypodermic injection is indicated.

Photothermal tail flick experiments, the experimental procedure originally summarized by D' Amour and Smith, have been slightly modified to date. The male Kunming mouse is used in the experiment, and the mouse can freely drink water before the experiment and is placed on an experiment table to adapt for 30 min. In the photothermal tail flick experimental determination, the mouse is tightly held by the right hand, and the radiant heat is intensively irradiated at the position 2-3cm away from the tail of the mouse. The intensity of the radiant heat is adjusted to the basal threshold of the tail flick of the mouse to be between 3 and 5 s. After administration of the mice, the tail flick latency of the mice was determined at different time points according to different drug design. 10s was set as the maximum latency period because too long a radiant heat exposure time would scald the mouse's tail. The tail flick times exceeding 10s were each counted as 10 s.

Following administration to mice, the analgesic effect of the drug is generally evaluated using the maximum analgesic effect MPE (%); the calculation method of MPE (%) is: MPE (%) ═ 100 × [ (pain threshold after administration-basal pain threshold)/(10 sec-basal pain threshold)]。ED₅₀Half effective dose (ED, 50% effective dose)₅₀) In a dose-effect response, refers to the amount of drug that causes 50% of the maximal response intensity. Calculating related statistical data ED by using MPE% and the dosage of the corresponding medicine reaching MPE% through statistical software GraphPad prism5.0₅₀And a 95% confidence interval.

In the experiment of acute pain of photothermal tail flick of a mouse, analgesic ED of all analogs is injected into a lateral ventricle₅₀As shown in table 5. Central analgesic ED of all analogues₅₀All values were lower than the parent peptide BN-9(390pmol), and the central analgesic effect of the analogs were 2.6, 736, 70 and 346 fold stronger than that of BN-9, respectively. The central analgesic effect time curves of BN-9 and 4 compounds are shown in figures 1, 2, 3, 4 and 5. As shown in the figure, the central analgesic time of the compound 1 can reach 90min, the central analgesic time of the

compounds

2, 3 and 4 can be prolonged to 180-240min, and the analgesic effect time of the parent BN-9 is less than 90 min.

TABLE 5 analgesic Activity of lateral ventricle and subcutaneous injection of multiple target molecule BN-9 analogs

Note that DN-9 is compound 9 disclosed in chinese patent 201610252648.7, and it is referred to in the following examples that DN-9 is the same compound as DN-9 used in this example.

Also in the photothermal tail flick acute analgesia experiment of mice, peripheral analgesia ED of all analogs was injected subcutaneously₅₀As shown in table 5. BN-9 peripheral analgesic ED₅₀ED with a value of 1.461mg/kg, 4 analogues listed in Table 5₅₀The values are all below 1 mg/kg. These analogs have 6, 10, 13 and 25 times stronger peripheral analgesia than BN-9, respectively. The peripheral analgesic effect time profiles of BN-9 and 4 compounds are shown in FIGS. 6, 7, 8, 9 and 10. As shown in the figure, the peripheral analgesic time of the compound 1 can reach 90min, the peripheral analgesic time of the

compounds

2, 3 and 4 can be prolonged to 180min, and the analgesic action time of the parent BN-9 is only 60 min.

Example 4 determination of analgesic tolerance phenomena for BN-9 analogues:

the analgesic tolerance experiment is to evaluate the pharmacological activity of the analogue in the analgesic tolerance aspect by observing the analgesic effect of the drug in a photothermal tail flick experiment after continuous 8-day administration.

21 plus or minus 2g of Kunming male mice, the environmental temperature is controlled at 22 plus or minus 2 ℃, and the mice can freely eat and drink water. The mouse is injected with the drug with the same concentration in the lateral ventricle at the same time every day for 1 day/time and 8 days continuously, and the photo-thermal tail flicking instrument is utilized to detect the influence on the analgesic effect of the drug after the continuous injection of the drug. The tail-flick latency (3-5s) and analgesia at different time points after administration were measured on the first day, and the analgesic activity at the highest analgesic effect point was generally measured on the following days. Mice injected with conventional opioid analgesics generally develop a tolerance event, i.e., a downregulation of analgesic activity, on day 4.

The lateral ventricles were injected with the positive control morphine, as well as

compounds

1, 2, 3 and 4, and the analgesic activity of the highest analgesic effect site of each compound was determined.

Experimental data are expressed in tail flick time. Analgesic tolerance of drugs using different compoundsThe tail flick latencies at the time points of maximal analgesic effect were compared. Tail flick latency data are expressed as mean ± standard error (Means ± s.e.m.), and the difference in analgesia over 8 consecutive days of lateral ventricle administration in mice was counted using one-way analysis of variance (Tukey HSD test by one-way ANOVA),^***p < 0.001 shows that the analgesic effect of the medicine is very different from that of the medicine injected on the first day. The results of the experiment are shown in FIG. 11.

Figure 11 is a graph of the tolerability results of lateral ventricle injections of saline, morphine and 4 analogs, with 7-10 mice per group. After 8 days of continuous injection, the positive control medicament morphine has obvious analgesic effect down-regulated in the fourth day, the tail-flick threshold value is reduced from 9.25s to 7.00s, and the BN-9 analogue has no tail-flick threshold value reduction, namely, the analgesic tolerance phenomenon is not generated.

Sequence listing

<110> Lanzhou university

<120> analogues of opioid and neuropeptide FF receptor multi-target molecule BN-9, preparation method and application thereof

<130>SG180820001

<160>4

<170>SIPOSequenceListing 1.0

<210>1

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>1

Tyr Cys Gly Phe Cys Pro Gln Arg Phe

1 5

<210>2

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>2

Tyr Cys Gly Phe Cys Pro Gln Arg Phe

1 5

<210>3

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>3

Tyr Cys Gly Phe Cys Pro Gln Arg Phe

1 5

<210>4

<211>9

<212>PRT

<213> Artificial Sequence (Artificial Sequence)

<400>4

Tyr Cys Gly Phe Cys Pro Gln Arg Phe

1 5

Claims

1. The amino acid sequence of the analogues of opioid and neuropeptide FF receptor multi-target molecule BN-9 is shown as follows:

Tyr-Xaa2-Gly-Phe-Xaa5-Pro-Gln-Arg-Phe-NH₂

xaa5 is Cys or D-Cys with side chain containing sulfhydryl structure.

2. The preparation method of the analogues of the opioid and neuropeptide FF receptor multi-target molecule BN-9 as claimed in claim 1, wherein the objective peptide is obtained by coupling amino acids on a solid phase carrier one by one in sequence by using a solid phase synthesis method and then cracking.

3. The method according to claim 2, wherein the solid support is an amino resin.

4. The method according to claim 2, wherein the amino deprotection reagent used in the solid phase synthesis is a 20% by volume solution of piperidine in DMF or a 1% by volume solution of DBU in DMF;

the coupling agent used is one of three combinations:

combination of HBTU, HOBt and DIEA,

Or a combination of DIC and HOBt,

Or a combination of PyBOP and HOBt and DIEA.

5. The method of claim 2, wherein the cleavage step uses a cleavage agent comprising TFA and H₂Mixed solution of O in a volume ratio of 95:5, or TFA and EDT and TIS and PhOH and H₂Mixed solution of O in a volume ratio of 80:5:5:5:5, or TFA and EDT and TIS and H₂And O is mixed solution according to the volume ratio of 92.5:2.5:2.5: 2.5.

6. A pharmaceutical composition comprising an analog of the class of opioid and neuropeptide FF receptor multi-target molecule BN-9 of claim 1.

7. Use of a class of opioid and neuropeptide FF receptor multi-target molecule analogues of BN-9 as claimed in claim 1 in the preparation of a medicament for the treatment of pain.

8. The use of claim 7, wherein the pain is acute pain or chronic pain.