Heteropeptides based on small molecule opioids and neuropeptide ff and preparation and application thereof

Abstract

This invention provides a class of multi-target heteropeptides based on N-4-piperidinyl-N-phenylpropionamide analogs and neuropeptide FF. These heteropeptides use the small-molecule N-4-piperidinyl-N-phenylpropionamide and the polypeptide-structured neuropeptide FF as chemical templates. A series of peptide-small-molecule hybrid peptides are obtained by chemically coupling the pharmacophores of the N-4-piperidinyl-N-phenylpropionamide analog and the neuropeptide FF. In vivo pharmacological activity identification in animals shows that these heteropeptides possess analgesic activity without opioid-like side effects such as addiction and respiratory depression. Furthermore, compounds 1-4 and compounds 7-9 do not exhibit analgesic tolerance and can be used for long-term analgesic treatment of chronic pain. Therefore, these heteropeptides based on N-4-piperidinyl-N-phenylpropionamide analogs and neuropeptide FF can be used to prepare analgesic drugs.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology, specifically the field of pain treatment, and relates to a class of heteropeptides based on small molecule structures of N-4-piperidinyl-N-phenylpropionamide analogs and polypeptide structures of neuropeptide FF, as well as their synthesis methods and the application of such heteropeptide molecules in the preparation of analgesic drugs. Background Technology

[0002] Pain is one of the most common clinical diseases, with a very high incidence rate. More than one billion people worldwide suffer from chronic pain, severely impacting their quality of life. Global aging accelerates the onset of pain (Nature 2016, 535:S4). Currently, pain is mainly treated with medication, especially opioid analgesics such as morphine and fentanyl. These analgesics are first-line drugs for treating moderate to severe pain due to their significant clinical efficacy. However, long-term use of opioid analgesics can cause side effects such as respiratory depression, constipation, tolerance, and addiction, along with adverse reactions such as nausea, vomiting, muscle rigidity, and itching, thus greatly limiting their clinical application. In recent years, in the United States, the number of people abusing fentanyl-based opioids has increased dramatically, leading to a continuous rise in the number of deaths from opioid overdoses, resulting in an "opioid crisis" (Science 2018, 361:831).

[0003] However, fentanyl-based analgesics generally possess advantages such as good efficacy, high bioavailability, and low production costs. Their clinical efficacy is significant, with analgesic effects 50 to 100 times stronger than morphine, and their constipation side effect is weaker. They are among the most widely used opioid analgesics, used to relieve severe pain during and after surgery, treat chronic pain such as cancer pain, and as an adjunct to anesthesia (Neuropharmacology 2018, 134:121). Therefore, fentanyl-based small-molecule opioid ligands themselves have good efficacy and their structures are easily modified. Through effective drug design strategies, chemical modification of these ligands and reduction of their side effects can promote the development of new analgesics with higher safety and drug-like properties.

[0004] Patent applications ZL201110098832.2, ZL201110097843.4, and CN 106084001 A (patent application number 201610252648.7) disclose a series of multi-target peptide ligands chemically constructed based on opioid peptides and neuropeptide FF. These ligands produce tolerance-free analgesia and reduce constipation side effects, indicating that the multi-target strategy of the opioid / neuropeptide FF system can effectively reduce the side effects of opioid peptides. However, the druggability of the above-disclosed compounds remains unclear. This invention employs a multi-target molecular design strategy based on the opioid / neuropeptide FF system, using opioid small molecule N-4-piperidinyl-N-phenylpropionamide analogues and neuropeptide FF as chemical template molecules to chemically construct peptide-small molecule hybrid peptides. These ligands exhibit high druggability while reducing opioid-like side effects such as analgesia tolerance, addiction, and respiratory depression. Summary of the Invention

[0005] The purpose of this invention is to address the significant opioid-like side effects of existing small molecule analgesics such as fentanyl by providing a class of heteropeptide molecules with good analgesic effects and low side effects, based on N-4-piperidinyl-N-phenylpropionamide analogs and neuropeptide FF.

[0006] The heteropeptide molecules of this invention are a series of peptide-small molecule hybrid structures constructed by chemically intercalating an N-4-piperidinyl-N-phenylpropionamide analog with a neuropeptide FF pharmacophore. Their general structural formula is as follows:

[0007]

[0008] in:

[0009] R is selected from phenyl or thiophene group;

[0010] Xaa1-Xaa2-Xaa3-Xaa4-Arg-Xaa5-NH2 is the key pharmacophore of the neuropeptide FF (Phe-Leu-Phe-Gln-Pro-Gln-Arg-Phe-NH2). Xaa1, Xaa2, Xaa3, Xaa4, and Xaa5 are selected from natural or non-natural amino acids; Xaa1 is selected from Phe or NMe-Phe; Xaa2 is selected from Gln or β-Ala; Xaa3 is selected from Pro or D-Pro; Xaa4 is selected from Gln, β-Ala, or Aib; Xaa5 is selected from Phe, Phg, Ala, or Cha.

[0011] Y is selected from -CH2C(=O)-, -C2H5C(=O)-, or -OCH2C(=O)-, wherein the corresponding carboxylic acid of -CH2C(=O)-, -C2H5C(=O)-, or -OCH2C(=O)- is linked to the amino group of Xaa1 by forming a peptide bond.

[0012] Throughout this specification, conventional three-letter codes are used to represent natural amino acids, and recognized codes are used to represent other amino acids, such as NMe-Phe (N α β-methylphenylalanine, β-Ala (β-alanine), D-Pro (D-proline), Aib (2-aminoisobutyric acid), Phg (phenylglycine) and Cha (cyclohexylalanine).

[0013] When R is phenyl, Xaa1 is Phe, Xaa2 is Gln, Xaa3 is Pro, Xaa4 is Gln, Xaa5 is Phe, and Y is -CH2C(=O)-, the heteropeptide is labeled as compound 1.

[0014] When R is phenyl, Xaa1 is Phe, Xaa2 is Gln, Xaa3 is Pro, Xaa4 is Gln, Xaa5 is Phe, and Y is -C2H5C(=O)-, the heteropeptide is labeled as compound 2.

[0015] When R is phenyl, Xaa1 is Phe, Xaa2 is Gln, Xaa3 is Pro, Xaa4 is Gln, Xaa5 is Phe, and Y is -OCH2C(=O)-, the heteropeptide is labeled as compound 3.

[0016] When R is thiophene, Xaa1 is Phe, Xaa2 is Gln, Xaa3 is Pro, Xaa4 is Gln, Xaa5 is Phe, and Y is -CH2C(=O)-, the heteropeptide is labeled as compound 4.

[0017] When R is phenyl, Xaa1 is Phe, Xaa2 is Gln, Xaa3 is Pro, Xaa4 is Gln, Xaa5 is Phg, and Y is -CH2C(=O)-, the heteropeptide is labeled as compound 5.

[0018] When R is phenyl, Xaa1 is Phe, Xaa2 is Gln, Xaa3 is Pro, Xaa4 is Gln, Xaa5 is Ala, and Y is -CH2C(=O)-, the heteropeptide is labeled as compound 6.

[0019] When R is phenyl, Xaa1 is Phe, Xaa2 is Gln, Xaa3 is Pro, Xaa4 is β-Ala, Xaa5 is Cha, and Y is -CH2C(=O)-, the heteropeptide is labeled as compound 7.

[0020] When R is phenyl, Xaa1 is Phe, Xaa2 is Gln, Xaa3 is Pro, Xaa4 is Aib, Xaa5 is Cha, and Y is -CH2C(=O)-, the heteropeptide is labeled as compound 8.

[0021] When R is phenyl, Xaa1 is NMe-Phe, Xaa2 is β-Ala, Xaa3 is D-Pro, Xaa4 is β-Ala, Xaa5 is Cha, and Y is -CH2C(=O)-, the heteropeptide is labeled as compound 9.

[0022] The amino acid sequences of the above compounds are shown in Table 1:

[0023] Table 1. Amino acid sequence structures of heteropeptides based on N-4-piperidinyl-N-phenylpropionamide analogs and neuropeptide FF

[0024]

[0025] Another object of the present invention is to provide a chemical synthesis method for the above-mentioned heteropeptides based on N-4-piperidinyl-N-phenylpropionamide analogs and neuropeptide FF, comprising the following process steps:

[0026] Step 1, Resin Pretreatment: Soak the amide resin in an appropriate amount of dichloromethane, stir at low speed for 30-40 minutes to fully swell, remove the solvent under reduced pressure, then add DMF to wash, and remove from heat.

[0027] Step 2, Deprotection of Fmoc protection: Add a deprotection agent to the resin pretreated in Step 1, stir and react for 3-7 minutes, then dry it, repeat 3 times; then add N,N-dimethylformamide to wash, and check with indene to obtain resin with Fmoc group protection removed;

[0028] Step 3, amino acid condensation: N-α-Fmoc-protected amino acids, N-hydroxybenzotriazole, and O-benzotriazole-N,N,N',N'-tetramethylurea-hexafluorophosphate were completely dissolved in N,N-dimethylformamide, and then diisopropylethylamine was added and mixed to obtain a mixed solution; then added to the resin obtained in Step 2 with Fmoc-protected groups removed, and stirred and condensed for 60 min under argon protection, and the solvent was dried; the resin was repeatedly washed with N,N-dimethylformamide and dried, and after indene testing, the Fmoc-protected groups were removed according to the process in Step 2 to obtain peptide resin with Fmoc-protected groups removed;

[0029] Step 4, peptide chain elongation: Based on the structural design of the peptide, starting from the C-terminus, N-α-Fmoc and the side-chain protected amino acids are selected sequentially and coupled one by one. The resin obtained in Step 2 with the Fmoc protecting group removed is used to complete the peptide chain condensation according to the process in Step 3 to obtain a peptide resin with fully protected side chains.

[0030] Step 5, peptide chain cleavage: Wash the peptide resin obtained in Step 4 with dichloromethane and methanol alternately, and after thoroughly drying the solvent, add the cleavage agent and cleave the reaction at room temperature for 2-3 hours; filter, and evaporate the filtrate under reduced pressure at a temperature below 40°C, then precipitate the precipitate with ice-cold diethyl ether; remove the supernatant, dissolve the precipitate thoroughly with water, and then separate the diethyl ether from the aqueous phase. The aqueous phase is freeze-dried to obtain a white powdery solid crude peptide;

[0031] Step 6, purification of crude peptide: Purify using high performance liquid chromatography, collect the main peak, and freeze-dry to obtain the heteropeptide product.

[0032] In a preferred embodiment of the present invention, the deprotecting agent in step 2 is a mixed solution composed of hexahydropyridine, 1,8-diazabicycloundec-7-ene and N,N-dimethylformamide in a volume ratio of 1:1:98.

[0033] In a preferred embodiment of the present invention, in the condensation of the amino acid in step 3, the molar ratio of N-α-Fmoc and the side-chain protected amino acid, N-hydroxybenzotriazole, O-benzotriazole-N,N,N',N'-tetramethylurea-hexafluorophosphate and diisopropylethylamine is 1:1:1:2, and the N-α-Fmoc protected amino acid is in excess by 2 to 5 times compared with the resin after the Fmoc group protection is removed.

[0034] In a preferred embodiment of the present invention, in the elongation of the peptide chain in step 4, the amino acids N-α-Fmoc protected at the C-terminus are sequentially Fmoc-Xaa5-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Xaa4-OH, Fmoc-Xaa3-OH, Fmoc-Xaa2-OH, Fmoc-Xaa1-OH, and the general formula of the N-terminal structure is as follows: Carboxylic acid analogues.

[0035] In a preferred embodiment of the present invention, the cutting agent in step 5 is a solution formed by mixing trifluoroacetic acid, triisopropylsilane, and water in a volume ratio of 95:2.5:2.5.

[0036] The series of heteropeptides prepared by the above method were identified by mass spectrometry and chromatographic analysis, and their structures were consistent with the designed chemical compounds. This indicates that heteropeptides based on N-4-piperidinyl-N-phenylpropionamide analogs and neuropeptide FF were successfully synthesized, and the purity of the purified heteropeptides reached over 98%.

[0037] Another objective of this invention is to provide the application of the above-mentioned heteropeptide based on N-4-piperidinyl-N-phenylpropionamide analog and neuropeptide FF in the preparation of analgesic drugs. Attached Figure Description

[0038] Figure 1 The time-dose response curve of the dose-dependent analgesic effect of compound 1 on acute pain in mice after subcutaneous injection.

[0039] Figure 2 Evaluation of analgesic tolerance of compounds 1-9 in acute pain in mice after eight consecutive days of subcutaneous injection.

[0040] Figure 3 Analgesic effect of subcutaneous injection of compound 1 on inflammatory pain in mice.

[0041] Figure 4 Effects of subcutaneous injection of compound 1 on the respiratory system of mice

[0042] Figure 5 Evaluation of somatic dependence of compound 1 in mice by subcutaneous injection

[0043] Figure 6 Evaluation of the psychological addiction of compound 1 in mice after subcutaneous injection Detailed Implementation

[0044] The following examples illustrate the application of the heteropeptide based on N-4-piperidinyl-N-phenylpropionamide analog and neuropeptide FF in pain treatment, intended to demonstrate that the invention is not limited in scope. Those skilled in the art can make various changes or modifications to the invention under the guidance of this specification, and these changes or modifications are also within the scope of protection of the invention.

[0045] The instruments used in this invention are as follows:

[0046]

[0047] The carboxylic acid analogues of the known starting material N-4-piperidinyl-N-phenylpropionamide can be synthesized according to methods known in the art (Future Med Chem, 2014, 6:385-412; PLoS One, 2014, 9:e108250). Other raw materials can be purchased from companies such as Jier Biochemical (Shanghai) Co., Ltd., Tianjin Nankai Hecheng Co., Ltd., Beijing Bailingwei Technology Co., Ltd., and Saen Chemical Technology (Shanghai) Co., Ltd.

[0048] Example 1: Synthesis of Compound 1

[0049] (1) Resin pretreatment: Weigh 500 mg of Rink-Amide-MBHA resin with a substitution value of 0.4 mmol / g and place it in a synthesizer. Soak it in an appropriate amount of dichloromethane and stir at low speed for 30 minutes to fully swell the resin. Then, remove the solvent under reduced pressure and add N,N-dimethylformamide to wash and remove it under reduced pressure.

[0050] (2) Removal of Fmoc protection: In the pretreated resin, add hexahydropyridine: 1,8-diazabicycloundec-7-ene: N,N-dimethylformamide deprotecting agent in a volume ratio of 1:1:98, stir for 5 minutes and then dry, repeat 3 times; then add N,N-dimethylformamide to wash and obtain resin with Fmoc group protection removed.

[0051] (3) Ninhydrin test: Pick out a small amount of resin and add it to a test tube. Add 1 drop of formula ①, 2 drops of formula ② and 1 drop of formula ③ in sequence at a ratio of 1:2:1. Boil for 3 minutes and observe the color of the solution and resin. If the Fmoc protection is completely removed, both the solution and the resin will be blue. The ninhydrin reagent formula is: ① 20 g phenol / 5 mL ethanol; ② 0.05 mL 0.001 mol / L potassium cyanide (water) / 2.5 mL pyridine; (3) 0.5 g ninhydrin / 10 mL ethanol.

[0052] (4) Condensation of amino acids: N-α-Fmoc-protected Fmoc-Phe-OH, N-hydroxybenzotriazole, O-benzotriazole-N,N,N',N'-tetramethylurea-hexafluorophosphate and diisopropylethylamine were dissolved in N,N-dimethylformamide in a molar ratio of 1:1:1:2 and mixed to obtain a mixed solution, wherein the N-α-Fmoc-protected amino acid was in 2.5 times excess compared to the resin with the Fmoc group removed. Then the mixed solution was added to the resin with the Fmoc group removed obtained in step (2) above, and stirred and condensed for 60 min under argon protection, and the solvent was dried. The resin was washed and dried repeatedly with N,N-dimethylformamide. After drying, the resin was tested using the method in step (3) above. If the condensation was complete, the solution was pale yellow and the resin was colorless. Then the Fmoc group protection was removed according to the process in step (2) to obtain the peptide resin with the Fmoc group removed.

[0053] (5) Elongation of peptide chain: Starting from the C-terminus of the peptide chain sequence, Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Pro-OH, Fmoc-Gln(Trt)-OH, Fmoc-Phe-OH and the N-4-piperidinyl-N-phenylpropionamide analog 4-oxonyl-4-{phenyl[1-(2-phenylethyl)piperidin-4-yl]amino}butyric acid are coupled one by one to complete the peptide chain condensation of the peptide resin obtained in step (4) according to the process in step (4), so as to obtain a peptide resin with fully protected side chains. .

[0054] (6) Peptide cleavage: The peptide resin obtained in step (4) was washed alternately with dichloromethane and methanol. After the solvent was completely removed, 10 mL of a cleaving agent consisting of trifluoroacetic acid, triisopropylsilane, and water in a volume ratio of 95:2.5:2.5 was added, and the cleavage reaction was carried out at room temperature for 3 hours. The filtrate was filtered, and the filtrate was evaporated under reduced pressure at a temperature below 40°C. The precipitate was then precipitated with ice-cold diethyl ether. The supernatant was removed, and the precipitate was completely dissolved in 20% acetic acid aqueous solution. The diethyl ether was then separated from the aqueous phase. The aqueous phase was freeze-dried to obtain 170.1 mg of white powdery solid crude peptide, with a crude peptide yield of 71.9%.

[0055] (7) Purification of crude peptides: Crude peptides were purified using a reverse-phase high-performance liquid chromatography (RP-HPLC) system with a preparative column XBridge™ BEH 130Prep C. 18 The crude peptide obtained in step (6) was purified using a 619 mm × 250 mm column. Acetonitrile (containing 0.1% TFA) and water (containing 0.1% TFA) were selected as the mobile phase. The main peak was collected, and the sample was freeze-dried to obtain compound 1. An XBridge™ BEH 130 Prep C column was used for chromatographic analysis. 18 The purity of compound 1 was 98.2% as measured by a 4.6 mm × 250 mm sample, and the total yield was calculated to be 25.0%.

[0056] Example 2: Synthesis of Compound 2

[0057] (1) Resin pretreatment: Weigh 500 mg of Rink-Amide-MBHA resin with a substitution value of 0.4 mmol / g and place it in a synthesizer. Soak it in an appropriate amount of dichloromethane and stir at low speed for 30 minutes to fully swell the resin. Then, remove the solvent under reduced pressure and add N,N-dimethylformamide to wash and remove it under reduced pressure.

[0058] (2) Remove Fmoc protection: The method is the same as in Example 1.

[0059] (3) Indene detection: The method is the same as in Example 1.

[0060] (4) Condensation of amino acids: N-α-Fmoc-protected Fmoc-Phe-OH, N-hydroxybenzotriazole, O-benzotriazole-N,N,N',N'-tetramethylurea-hexafluorophosphate and diisopropylethylamine were dissolved in N,N-dimethylformamide in a molar ratio of 1:1:1:2 and mixed to obtain a mixed solution, wherein the N-α-Fmoc-protected amino acid was in 2.5 times excess compared to the resin with the Fmoc group removed. Then the mixed solution was added to the resin with the Fmoc group removed obtained in step (2) above, and stirred and condensed for 60 min under argon protection, and the solvent was dried. The resin was washed and dried repeatedly with N,N-dimethylformamide. After drying, the resin was tested using the method in step (3) above. If the condensation was complete, the solution was pale yellow and the resin was colorless. Then the Fmoc group protection was removed according to the process in step (2) to obtain the peptide resin with the Fmoc group removed.

[0061] (5) Elongation of peptide chain: Starting from the C-terminus of the peptide chain sequence, Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Pro-OH, Fmoc-Gln(Trt)-OH, Fmoc-Phe-OH and the N-4-piperidinyl-N-phenylpropionamide analog 5-oxo-5-{phenyl[1-(2-phenylethyl)piperidin-4-yl]amino}valeric acid are coupled one by one to complete the peptide chain condensation of the peptide resin obtained in step (4) according to the process in step (4), so as to obtain a peptide resin with fully protected side chains. .

[0062] (6) Peptide cleavage: The peptide resin obtained in step (4) was washed alternately with dichloromethane and methanol. After the solvent was completely removed, 10 mL of a cleaving agent consisting of trifluoroacetic acid, triisopropylsilane, and water in a volume ratio of 95:2.5:2.5 was added, and the cleavage reaction was carried out at room temperature for 3 hours. The filtrate was filtered, and the filtrate was evaporated under reduced pressure at a temperature below 40°C. The precipitate was then precipitated with ice-cold diethyl ether. The supernatant was removed, and the precipitate was completely dissolved in 20% acetic acid aqueous solution. The diethyl ether was then separated from the aqueous phase. The aqueous phase was freeze-dried to obtain 160.6 mg of white powdery solid crude peptide, with a crude peptide yield of 67.1%.

[0063] (7) Purification of crude peptides: Crude peptides were purified using a reverse-phase high-performance liquid chromatography (RP-HPLC) system with a preparative column XBridge™ BEH 130Prep C. 18 The crude peptide obtained in step (6) was purified using a 619 mm × 250 mm column. Acetonitrile (containing 0.1% TFA) and water (containing 0.1% TFA) were selected as the mobile phase. The main peak was collected, and the sample was freeze-dried to obtain compound 2. An XBridge™ BEH 130 Prep C column was used for chromatographic analysis.18 The purity of compound 2 was 99.3% as measured by a 4.6 mm × 250 mm sample, and the total yield was calculated to be 31.3%.

[0064] Example 3: Synthesis of Compound 3

[0065] (1) Resin pretreatment: Weigh 500 mg of Rink-Amide-MBHA resin with a substitution value of 0.4 mmol / g and place it in a synthesizer. Soak it in an appropriate amount of dichloromethane and stir at low speed for 30 minutes to fully swell the resin. Then, remove the solvent under reduced pressure and add N,N-dimethylformamide to wash and remove it under reduced pressure.

[0066] (2) Remove Fmoc protection: The method is the same as in Example 1.

[0067] (3) Indene detection: The method is the same as in Example 1.

[0068] (4) Condensation of amino acids: N-α-Fmoc-protected Fmoc-Phe-OH, N-hydroxybenzotriazole, O-benzotriazole-N,N,N',N'-tetramethylurea-hexafluorophosphate and diisopropylethylamine were dissolved in N,N-dimethylformamide in a molar ratio of 1:1:1:2 and mixed to obtain a mixed solution, wherein the N-α-Fmoc-protected amino acid was in 2.5 times excess compared to the resin with the Fmoc group removed. Then the mixed solution was added to the resin with the Fmoc group removed obtained in step (2) above, and stirred and condensed for 60 min under argon protection, and the solvent was dried. The resin was washed and dried repeatedly with N,N-dimethylformamide. After drying, the resin was tested using the method in step (3) above. If the condensation was complete, the solution was pale yellow and the resin was colorless. Then the Fmoc group protection was removed according to the process in step (2) to obtain the peptide resin with the Fmoc group removed.

[0069] (5) Elongation of peptide chain: Starting from the C-terminus of the peptide chain sequence, Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Pro-OH, Fmoc-Gln(Trt)-OH, Fmoc-Phe-OH and N-4-piperidinyl-N-phenylpropionamide analog [(2-oxo-2-{phenyl[1-(2-phenylethyl)hexahydropyridin-4-yl]amino}ethyl)oxy]acetic acid are coupled one by one to complete the peptide chain condensation of the peptide resin obtained in step (4) after removing the Fmoc protecting group, and obtain a peptide resin with fully protected side chains. .

[0070] (6) Peptide cleavage: The peptide resin obtained in step (4) was washed alternately with dichloromethane and methanol. After the solvent was completely removed, 10 mL of a cleaving agent consisting of trifluoroacetic acid, triisopropylsilane, and water in a volume ratio of 95:2.5:2.5 was added, and the cleavage reaction was carried out at room temperature for 3 hours. The filtrate was filtered, and the filtrate was evaporated under reduced pressure at a temperature below 40°C. The precipitate was then precipitated with ice-cold diethyl ether. The supernatant was removed, and the precipitate was completely dissolved in 20% acetic acid aqueous solution. The diethyl ether was then separated from the aqueous phase. The aqueous phase was freeze-dried to obtain 185.6 mg of white powdery solid crude peptide, with a crude peptide yield of 77.4%.

[0071] (7) Purification of crude peptides: Crude peptides were purified using a reverse-phase high-performance liquid chromatography (RP-HPLC) system with a preparative column XBridge™ BEH 130Prep C. 18 The crude peptide obtained in step (6) was purified using a 619 mm × 250 mm column. Acetonitrile (containing 0.1% TFA) and water (containing 0.1% TFA) were selected as the mobile phase. The main peak was collected, and the sample was freeze-dried to obtain compound 3. An XBridge™ BEH 130 Prep C column was used for chromatographic analysis. 18 The purity of compound 3 was 99.1% as measured by a 4.6 mm × 250 mm sample, and the total yield was calculated to be 44.2%.

[0072] Example 4: Synthesis of Compound 4

[0073] (1) Resin pretreatment: Weigh 500 mg of Rink-Amide-MBHA resin with a substitution value of 0.4 mmol / g and place it in a synthesizer. Soak it in an appropriate amount of dichloromethane and stir at low speed for 30 minutes to fully swell the resin. Then, remove the solvent under reduced pressure and add N,N-dimethylformamide to wash and remove it under reduced pressure.

[0074] (2) Remove Fmoc protection: The method is the same as in Example 1.

[0075] (3) Indene detection: The method is the same as in Example 1.

[0076] (4) Condensation of amino acids: N-α-Fmoc-protected Fmoc-Phe-OH, N-hydroxybenzotriazole, O-benzotriazole-N,N,N',N'-tetramethylurea-hexafluorophosphate and diisopropylethylamine were dissolved in N,N-dimethylformamide in a molar ratio of 1:1:1:2 and mixed to obtain a mixed solution, wherein the N-α-Fmoc-protected amino acid was in 2.5 times excess compared to the resin with the Fmoc group removed. Then the mixed solution was added to the resin with the Fmoc group removed obtained in step (2) above, and stirred and condensed for 60 min under argon protection, and the solvent was dried. The resin was washed and dried repeatedly with N,N-dimethylformamide. After drying, the resin was tested using the method in step (3) above. If the condensation was complete, the solution was pale yellow and the resin was colorless. Then the Fmoc group protection was removed according to the process in step (2) to obtain the peptide resin with the Fmoc group removed.

[0077] (5) Elongation of peptide chain: Starting from the C-terminus of the peptide chain sequence, Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Pro-OH, Fmoc-Gln(Trt)-OH, Fmoc-Phe-OH and the N-4-piperidinyl-N-phenylpropionamide analog 4-oxonyl-4-(phenyl{1-[2-(thiophen-2-yl)ethyl]piperidin-4-yl}amino)butyric acid are coupled one by one to complete the peptide chain condensation of the peptide resin obtained in step (4) by removing the Fmoc protecting group, and obtain a peptide resin with fully protected side chains. .

[0078] (6) Peptide cleavage: The peptide resin obtained in step (4) was washed alternately with dichloromethane and methanol. After the solvent was completely removed, 10 mL of a cleaving agent consisting of trifluoroacetic acid, triisopropylsilane, and water in a volume ratio of 95:2.5:2.5 was added, and the cleavage reaction was carried out at room temperature for 3 hours. The filtrate was filtered, and the filtrate was evaporated under reduced pressure at a temperature below 40°C. The precipitate was then precipitated with ice-cold diethyl ether. The supernatant was removed, and the precipitate was completely dissolved in 20% acetic acid aqueous solution. The diethyl ether was then separated from the aqueous phase. The aqueous phase was freeze-dried to obtain 174.1 mg of white powdery solid crude peptide, with a crude peptide yield of 73.1%.

[0079] (7) Purification of crude peptides: Crude peptides were purified using a reverse-phase high-performance liquid chromatography (RP-HPLC) system with a preparative column XBridge™ BEH 130Prep C. 18The crude peptide obtained in step (6) was purified using a 619 mm × 250 mm column. Acetonitrile (containing 0.1% TFA) and water (containing 0.1% TFA) were selected as the mobile phase. The main peak was collected, and the sample was freeze-dried to obtain compound 4. An XBridge™ BEH 130 Prep C column was used for chromatographic analysis. 18 The purity of compound 4 was 99.4% as measured by a 4.6 mm × 250 mm sample, and the total yield was calculated to be 16.8%.

[0080] Example 5: Synthesis of Compound 5

[0081] (1) Resin pretreatment: Weigh 500 mg of Rink-Amide-MBHA resin with a substitution value of 0.4 mmol / g and place it in a synthesizer. Soak it in an appropriate amount of dichloromethane and stir at low speed for 30 minutes to fully swell the resin. Then, remove the solvent under reduced pressure and add N,N-dimethylformamide to wash and remove it under reduced pressure.

[0082] (2) Remove Fmoc protection: The method is the same as in Example 1.

[0083] (3) Indene detection: The method is the same as in Example 1.

[0084] (4) Condensation of amino acids: Fmoc-Phg-OH, N-hydroxybenzotriazole, O-benzotriazole-N,N,N',N'-tetramethylurea-hexafluorophosphate and diisopropylethylamine protected by N-α-Fmoc were completely dissolved in N,N-dimethylformamide in a molar ratio of 1:1:1:2 and mixed to obtain a mixed solution, wherein the N-α-Fmoc protected amino acid was in 2.5 times excess compared to the resin with the Fmoc group removed. Then the mixed solution was added to the resin with the Fmoc group removed obtained in step (2) above, and stirred and condensed for 60 min under argon protection, and the solvent was dried; the resin was washed and dried repeatedly with N,N-dimethylformamide. After drying, the resin was tested by the method in step (3) above. If the condensation was complete, the solution was pale yellow and the resin was colorless. Then the Fmoc group protection was removed according to the process in step (2) to obtain the peptide resin with the Fmoc group removed.

[0085] (5) Elongation of peptide chain: Starting from the C-terminus of the peptide chain sequence, Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Pro-OH, Fmoc-Gln(Trt)-OH, Fmoc-Phe-OH and the N-4-piperidinyl-N-phenylpropionamide analog 4-oxonyl-4-{phenyl[1-(2-phenylethyl)piperidin-4-yl]amino}butyric acid are coupled one by one to complete the peptide chain condensation of the peptide resin obtained in step (4) according to the process in step (4), so as to obtain a peptide resin with fully protected side chains. .

[0086] (6) Peptide cleavage: The peptide resin obtained in step (4) was washed alternately with dichloromethane and methanol. After the solvent was completely removed, 10 mL of a cleaving agent consisting of trifluoroacetic acid, triisopropylsilane, and water in a volume ratio of 95:2.5:2.5 was added, and the cleavage reaction was carried out at room temperature for 3 hours. The filtrate was filtered, and the filtrate was evaporated under reduced pressure at a temperature below 40°C. The precipitate was then precipitated with ice-cold diethyl ether. The supernatant was removed, and the precipitate was completely dissolved in 20% acetic acid aqueous solution. The diethyl ether was then separated from the aqueous phase. The aqueous phase was freeze-dried to obtain 185.1 mg of white powdery solid crude peptide, with a crude peptide yield of 80.0%.

[0087] (7) Purification of crude peptides: Crude peptides were purified using a reverse-phase high-performance liquid chromatography (RP-HPLC) system with a preparative column XBridge™ BEH 130Prep C. 18 The crude peptide obtained in step (6) was purified using a 619 mm × 250 mm column. Acetonitrile (containing 0.1% TFA) and water (containing 0.1% TFA) were selected as the mobile phase. The main peak was collected, and the sample of compound 5 was obtained by freeze-drying. An XBridge™ BEH 130 Prep C column was used for chromatographic analysis. 18 The purity of compound 5 was 98.9% as measured by a 4.6 mm × 250 mm sample, and the total yield was calculated to be 23.6%.

[0088] Example 6: Synthesis of Compound 6

[0089] (1) Resin pretreatment: Weigh 500 mg of Rink-Amide-MBHA resin with a substitution value of 0.4 mmol / g and place it in a synthesizer. Soak it in an appropriate amount of dichloromethane and stir at low speed for 30 minutes to fully swell the resin. Then, remove the solvent under reduced pressure and add N,N-dimethylformamide to wash and remove it under reduced pressure.

[0090] (2) Remove Fmoc protection: The method is the same as in Example 1.

[0091] (3) Indene detection: The method is the same as in Example 1.

[0092] (4) Condensation of amino acids: Fmoc-Ala-OH, N-hydroxybenzotriazole, O-benzotriazole-N,N,N',N'-tetramethylurea-hexafluorophosphate and diisopropylethylamine protected by N-α-Fmoc were completely dissolved in N,N-dimethylformamide in a molar ratio of 1:1:1:2 and mixed to obtain a mixed solution, wherein the N-α-Fmoc protected amino acid was in 2.5 times excess compared to the resin with the Fmoc group removed. Then the mixed solution was added to the resin with the Fmoc group removed obtained in step (2) above, and stirred and condensed for 60 min under argon protection. The solvent was then dried. The resin was washed and dried repeatedly with N,N-dimethylformamide. After drying, the resin was tested using the method in step (3) above. If the condensation was complete, the solution was pale yellow and the resin was colorless. Then the Fmoc group protection was removed again according to the process in step (2) to obtain the peptide resin with the Fmoc group removed.

[0093] (5) Elongation of peptide chain: Starting from the C-terminus of the peptide chain sequence, Fmoc-Arg(Pbf)-OH, Fmoc-Gln(Trt)-OH, Fmoc-Pro-OH, Fmoc-Gln(Trt)-OH, Fmoc-Phe-OH and the N-4-piperidinyl-N-phenylpropionamide analog 4-oxonyl-4-{phenyl[1-(2-phenylethyl)piperidin-4-yl]amino}butyric acid are coupled one by one to complete the peptide chain condensation of the peptide resin obtained in step (4) according to the process in step (4), so as to obtain a peptide resin with fully protected side chains. .

[0094] (6) Peptide cleavage: The peptide resin obtained in step (4) was washed alternately with dichloromethane and methanol. After the solvent was completely removed, 10 mL of a cleaving agent consisting of trifluoroacetic acid, triisopropylsilane, and water in a volume ratio of 95:2.5:2.5 was added, and the cleavage reaction was carried out at room temperature for 3 hours. The filtrate was filtered, and the filtrate was evaporated under reduced pressure at a temperature below 40°C. The precipitate was then precipitated with ice-cold diethyl ether. The supernatant was removed, and the precipitate was completely dissolved in 20% acetic acid aqueous solution. The diethyl ether was then separated from the aqueous phase. The aqueous phase was freeze-dried to obtain 157.5 mg of white powdery solid crude peptide, with a crude peptide yield of 71.1%.

[0095] (7) Purification of crude peptides: Crude peptides were purified using a reverse-phase high-performance liquid chromatography (RP-HPLC) system with a preparative column XBridge™ BEH 130Prep C. 18The crude peptide obtained in step (6) was purified using a 619 mm × 250 mm column. Acetonitrile (containing 0.1% TFA) and water (containing 0.1% TFA) were selected as the mobile phase. The main peak was collected, and the sample of compound 6 was obtained by freeze-drying. An XBridge™ BEH 130 Prep C column was used for chromatographic analysis. 18 The purity of compound 6 was 99.1% as measured by a 4.6 mm × 250 mm sample, and the total yield was calculated to be 23.4%.

[0096] Example 7: Synthesis of Compound 7

[0097] (1) Resin pretreatment: Weigh 500 mg of Rink-Amide-MBHA resin with a substitution value of 0.4 mmol / g and place it in a synthesizer. Soak it in an appropriate amount of dichloromethane and stir at low speed for 30 minutes to fully swell the resin. Then, remove the solvent under reduced pressure and add N,N-dimethylformamide to wash and remove it under reduced pressure.

[0098] (2) Remove Fmoc protection: The method is the same as in Example 1.

[0099] (3) Indene detection: The method is the same as in Example 1.

[0100] (4) Condensation of amino acids: Fmoc-Cha-OH protected by N-α-Fmoc, N-hydroxybenzotriazole, O-benzotriazole-N,N,N',N'-tetramethylurea-hexafluorophosphate and diisopropylethylamine were dissolved in N,N-dimethylformamide in a molar ratio of 1:1:1:2 and mixed to obtain a mixed solution, wherein the N-α-Fmoc protected amino acid was in 2.5 times excess compared with the resin without Fmoc group protection. Then the mixed solution was added to the resin without Fmoc group protection obtained in step (2) above, and stirred and condensed for 60 min under argon protection, and the solvent was dried; the resin was washed and dried repeatedly with N,N-dimethylformamide. After drying, the resin was tested by the method in step (3) above. If the condensation was complete, the solution was pale yellow and the resin was colorless. Then the Fmoc group protection was removed according to the process in step (2) to obtain the peptide resin without Fmoc protection group.

[0101] (5) Elongation of peptide chain: Starting from the C-terminus of the peptide chain sequence, Fmoc-Arg(Pbf)-OH, Fmoc-β-Ala-OH, Fmoc-Pro-OH, Fmoc-Gln(Trt)-OH, Fmoc-Phe-OH and the N-4-piperidinyl-N-phenylpropionamide analog 4-oxonyl-4-{phenyl[1-(2-phenylethyl)piperidin-4-yl]amino}butyric acid are coupled one by one to complete the peptide chain condensation of the peptide resin obtained in step (4) by removing the Fmoc protecting group, and obtain a peptide resin with fully protected side chains. .

[0102] (6) Peptide cleavage: The peptide resin obtained in step (4) was washed alternately with dichloromethane and methanol. After the solvent was completely removed, 10 mL of a cleaving agent consisting of trifluoroacetic acid, triisopropylsilane, and water in a volume ratio of 95:2.5:2.5 was added, and the cleavage reaction was carried out at room temperature for 3 hours. The filtrate was filtered, and the filtrate was evaporated under reduced pressure at a temperature below 40°C. The precipitate was then precipitated with ice-cold diethyl ether. The supernatant was removed, and the precipitate was completely dissolved in 20% acetic acid aqueous solution. The diethyl ether was then separated from the aqueous phase. The aqueous phase was freeze-dried to obtain 179 mg of white powdery solid crude peptide, with a crude peptide yield of 98.9%.

[0103] (7) Purification of crude peptides: Crude peptides were purified using a reverse-phase high-performance liquid chromatography (RP-HPLC) system with a preparative column XBridge™ BEH 130Prep C. 18 The crude peptide obtained in step (6) was purified using a 619 mm × 250 mm column. Acetonitrile (containing 0.1% TFA) and water (containing 0.1% TFA) were selected as the mobile phase. The main peak was collected, and the sample was freeze-dried to obtain compound 7. An XBridge™ BEH 130 Prep C column was used for chromatographic analysis. 18 The purity of compound 7 was 97.6% as measured by a 4.6 mm × 250 mm sample, and the total yield was calculated to be 13.0%.

[0104] Example 8: Synthesis of Compound 8

[0105] (1) Resin pretreatment: Weigh 500 mg of Rink-Amide-MBHA resin with a substitution value of 0.4 mmol / g and place it in a synthesizer. Soak it in an appropriate amount of dichloromethane and stir at low speed for 30 minutes to fully swell the resin. Then, remove the solvent under reduced pressure and add N,N-dimethylformamide to wash and remove it under reduced pressure.

[0106] (2) Remove Fmoc protection: The method is the same as in Example 1.

[0107] (3) Indene detection: The method is the same as in Example 1.

[0108] (4) Condensation of amino acids: Fmoc-Cha-OH protected by N-α-Fmoc, N-hydroxybenzotriazole, O-benzotriazole-N,N,N',N'-tetramethylurea-hexafluorophosphate and diisopropylethylamine were dissolved in N,N-dimethylformamide in a molar ratio of 1:1:1:2 and mixed to obtain a mixed solution, wherein the N-α-Fmoc protected amino acid was in 2.5 times excess compared with the resin without Fmoc group protection. Then the mixed solution was added to the resin without Fmoc group protection obtained in step (2) above, and stirred and condensed for 60 min under argon protection, and the solvent was dried; the resin was washed and dried repeatedly with N,N-dimethylformamide. After drying, the resin was tested by the method in step (3) above. If the condensation was complete, the solution was pale yellow and the resin was colorless. Then the Fmoc group protection was removed according to the process in step (2) to obtain the peptide resin without Fmoc protection group.

[0109] (5) Peptide chain elongation: Starting from the C-terminus of the peptide chain sequence, Fmoc-Arg(Pbf)-OH, Fmoc-Aib-OH, Fmoc-Pro-OH, Fmoc-Gln(Trt)-OH, Fmoc-Phe-OH and the N-4-piperidinyl-N-phenylpropionamide analog 4-oxonyl-4-{phenyl[1-(2-phenylethyl)piperidin-4-yl]amino}butyric acid are coupled one by one to complete the peptide chain condensation of the peptide resin obtained in step (4) according to the process in step (4), so as to obtain a peptide resin with fully protected side chains. .

[0110] (6) Peptide cleavage: The peptide resin obtained in step (4) was washed alternately with dichloromethane and methanol. After the solvent was completely removed, 10 mL of a cleaving agent consisting of trifluoroacetic acid, triisopropylsilane, and water in a volume ratio of 95:2.5:2.5 was added, and the cleavage reaction was carried out at room temperature for 3 hours. The filtrate was filtered, and the filtrate was evaporated under reduced pressure at a temperature below 40°C. The precipitate was then precipitated with ice-cold diethyl ether. The supernatant was removed, and the precipitate was completely dissolved in 20% acetic acid aqueous solution. The diethyl ether was then separated from the aqueous phase. The aqueous phase was freeze-dried to obtain 180.0 mg of white powdery solid crude peptide, with a crude peptide yield of 98.4%.

[0111] (7) Purification of crude peptides: Crude peptides were purified using a reverse-phase high-performance liquid chromatography (RP-HPLC) system with a preparative column XBridge™ BEH 130Prep C. 18The crude peptide obtained in step (6) was purified using a 619 mm × 250 mm column. Acetonitrile (containing 0.1% TFA) and water (containing 0.1% TFA) were selected as the mobile phase. The main peak was collected, and the sample was freeze-dried to obtain compound 8. An XBridge™ BEH 130 Prep C column was used for chromatographic analysis. 18 The purity of compound 8 was 95.7% as measured by a 4.6 mm × 250 mm sample, and the total yield was calculated to be 21.0%.

[0112] Example 9: Synthesis of Compound 9

[0113] (1) Resin pretreatment: Weigh 500 mg of Rink-Amide-MBHA resin with a substitution value of 0.43 mmol / g and place it in a synthesizer. Soak it in an appropriate amount of dichloromethane and stir at low speed for 30 minutes to fully swell the resin. Then, remove the solvent under reduced pressure and add N,N-dimethylformamide to wash and remove it under reduced pressure.

[0114] (2) Remove Fmoc protection: The method is the same as in Example 1.

[0115] (3) Indene detection: The method is the same as in Example 1.

[0116] (4) Condensation of amino acids: Fmoc-Cha-OH protected by N-α-Fmoc, N-hydroxybenzotriazole, O-benzotriazole-N,N,N',N'-tetramethylurea-hexafluorophosphate and diisopropylethylamine were dissolved in N,N-dimethylformamide in a molar ratio of 1:1:1:2 and mixed to obtain a mixed solution, wherein the N-α-Fmoc protected amino acid was in 2.5 times excess compared with the resin without Fmoc group protection. Then the mixed solution was added to the resin without Fmoc group protection obtained in step (2) above, and stirred and condensed for 60 min under argon protection, and the solvent was dried; the resin was washed and dried repeatedly with N,N-dimethylformamide. After drying, the resin was tested by the method in step (3) above. If the condensation was complete, the solution was pale yellow and the resin was colorless. Then the Fmoc group protection was removed according to the process in step (2) to obtain the peptide resin without Fmoc protection group.

[0117] (5) Peptide chain elongation: Starting from the C-terminus of the peptide chain sequence, Fmoc-Arg(Pbf)-OH, Fmoc-β-Ala-OH, Fmoc-D-Pro-OH, Fmoc-β-Ala-OH, Fmoc-NMe-Phe-OH and the N-4-piperidinyl-N-phenylpropionamide analog 4-oxonyl-4-{phenyl[1-(2-phenylethyl)piperidin-4-yl]amino}butyric acid are coupled one by one to complete the peptide chain condensation of the peptide resin obtained in step (4) according to the process in step (4), so as to obtain a peptide resin with fully protected side chains. .

[0118] (6) Peptide cleavage: The peptide resin obtained in step (4) was washed alternately with dichloromethane and methanol. After the solvent was completely removed, 10 mL of a cleaving agent consisting of trifluoroacetic acid, triisopropylsilane, and water in a volume ratio of 95:2.5:2.5 was added, and the cleavage reaction was carried out at room temperature for 3 hours. The filtrate was filtered, and the filtrate was evaporated under reduced pressure at a temperature below 40°C. The precipitate was then precipitated with ice-cold diethyl ether. The supernatant was removed, and the precipitate was completely dissolved in 20% acetic acid aqueous solution. The diethyl ether was then separated from the aqueous phase. The aqueous phase was freeze-dried to obtain 187.0 mg of white powdery solid crude peptide, with a crude peptide yield of 80.0%.

[0119] (7) Purification of crude peptides: Crude peptides were purified using a reverse-phase high-performance liquid chromatography (RP-HPLC) system with a preparative column XBridge™ BEH 130Prep C. 18 The crude peptide obtained in step (6) was purified using a 619 mm × 250 mm column. Acetonitrile (containing 0.1% TFA) and water (containing 0.1% TFA) were selected as the mobile phase. The main peak was collected, and the sample was freeze-dried to obtain compound 9. An XBridge™ BEH 130 Prep C column was used for chromatographic analysis. 18 The purity of compound 9 was 97.5% as measured by a 4.6 mm × 250 mm sample, and the total yield was calculated to be 15.8%.

[0120] The chemical characterization results of the series of heteropeptides synthesized above are shown in Table 2:

[0121] Table 2 Chemical characterization of heteropeptides

[0122]

[0123] Note: System 1: Gradient elution system 1 is: 10-80% acetonitrile / water (0.1% TFA) (completed in 30 minutes), flow rate: 1 mL / min, detection wavelength: 220 nm, analytical column: XBridge TM BEH 130 Prep C 184.6 mm × 250 mm; System 2: Gradient elution system 2 is: 10-100% acetonitrile / water (0.1% TFA) (completed in 30 minutes), flow rate: 1 mL / min, detection wavelength: 220 nm, analytical column: XBridge TM BEH 130 Prep C 18 , 4.6 mm × 250 mm.

[0124] Example 10: Activity Test

[0125] (1) Mouse radiant heat tail flick analgesia experiment

[0126] Using a mouse photothermal tail-flick experiment, the analgesic effect of heteropeptides on acute pain was studied via subcutaneous administration.

[0127] 1) Drug injection method

[0128] For subcutaneous administration in mice, use a 1 mL syringe. Insert the needle subcutaneously into the mouse's back and gently wiggle the needle tip from side to side; easy wiggle indicates successful subcutaneous penetration. Then inject the drug at a volume of 10 μl / g body weight. After injection, press or pinch the injection site to prevent leakage.

[0129] 2) Radiative thermal tail flicking experiment method

[0130] The mouse radiative heat tail-flick experiment was conducted using a self-made photothermal tail-flick device. Adult male Kunming mice weighing 18-22g were used as experimental animals. Operators needed prior training to handle the mice gently and skillfully, placing the outer third of the animal's tail on the beam of the photothermal tail-flick device during the experiment. Before drug injection, the baseline tail-flick latency was determined, and to minimize tissue damage, the maximum tail-flick latency was set to 10 seconds. After drug injection, the tail-flick latency at different time points was recorded. The recorded data were expressed as the maximum probable analgesic effect (MPE): MPE (%) = 100 × [(post-injection pain threshold - baseline pain threshold) / (10 seconds - baseline pain threshold)]. 50 The p-value represents the dose at which the drug produces half of its maximum analgesic effect, calculated using Graphpad PRISM 5 software. A p-value less than 0.05 indicates statistical significance compared to the saline group based on one-way ANOVA and Dunnett's test.

[0131] 3) Results and conclusions of the radiant heat tail-flick analgesia experiment

[0132] The experimental results of the mouse radiant heat tail-flick analgesia experiment are as follows: Figure 1 As shown in Table 3, subcutaneous injection of compound 1 produced dose-dependent analgesia, and its analgesic effect had an ED50 value. 50The values ​​were 0.024 mg / kg, and the analgesic effect lasted for 90 minutes. Similar to compound 1, subcutaneous injection of compounds 2-9 also produced dose-dependent analgesia. These experimental results indicate that the heteropeptide based on N-4-piperidinyl-N-phenylpropionamide analog and neuropeptide FF described in this patent has potential therapeutic value for acute pain.

[0133] Table 3. Analgesic activity of subcutaneously injected heteropeptides

[0134]

[0135] (2) Analgesic tolerance test of compounds 1-9

[0136] Mice were subcutaneously injected for 8 consecutive days, and the analgesic tolerance of compounds 1-9 was evaluated using the above-mentioned photothermal tail-flick experiment.

[0137] 1) Analgesia tolerance test method

[0138] Mice were subcutaneously injected with the drug once daily for eight consecutive days. The tail-flick latency was determined using a photothermal tail-flick test. The maximum analgesic effect of the control compound fentanyl and compounds 1-9 occurred within 30 minutes after injection; therefore, the tail-flick latency at 30 minutes after administration on days 1 and 8 was recorded. On day 1, the baseline tail-flick latency was determined first. To minimize tissue damage in mice, the maximum tail-flick latency was set to 10 seconds. Recorded data were expressed as maximum probable analgesic effect (MPE), calculated as: MPE (%) = 100 × [(pain threshold after drug injection - baseline pain threshold) / (10 seconds - baseline pain threshold)]. A p-value less than 0.05 indicated statistical significance compared to the analgesic effect on day 1, based on one-way ANOVA and Tukey's HSD test.

[0139] 2) Results and conclusions of the analgesic tolerance experiment

[0140] The experimental results of analgesic tolerance in acute pain caused by radiative heat tail flicking after 8 days of continuous drug injection are as follows: Figure 2 As shown in the figure, compared to the first day, subcutaneous injection of fentanyl almost lost its analgesic effect on the 8th day, indicating that subcutaneous fentanyl injection produces analgesia tolerance. Subcutaneous injection of compounds 5-6 for 8 consecutive days produced analgesia tolerance similar to that of fentanyl. Conversely, subcutaneous injection of compounds 1-4 and 7-9 maintained equivalent analgesic effects on the 8th day. In addition, subcutaneous injection of saline alone did not produce analgesia. The above experimental results indicate that compounds 1-4 and 7-9 do not produce analgesia tolerance in acute pain, suggesting that they exert a highly effective analgesic effect without the opioid side effects of analgesia tolerance, and have the potential to treat long-term pain such as chronic pain, while compounds 5-6 can be used for short-term pain such as acute pain.

[0141] (3) Analgesic effect of compound 1 in carrageenan inflammatory pain model

[0142] Using a carrageenan-induced inflammatory pain model in mice, the analgesic effect of injected heteropeptides on inflammatory pain was examined.

[0143] 1) Carrageenan-induced inflammatory pain model and Von Frey experiment

[0144] Carrageenan-induced inflammatory pain in mice was detected using an electronic Von Frey analgesia analyzer. First, the baseline pain threshold (g) was determined in normal Kunming mice. Then, 20 μl of a 2% carrageenan solution was subcutaneously injected into the plantar surface of the mice, inducing mechanorepression in the injected paw. The mechanorepression value (g) of the injected paw was recorded 24 hours later. Subsequently, compound 1 was injected subcutaneously, and the mechanorepression threshold (g) was recorded at different time points after drug injection. Mechanorepression values ​​(g) are expressed as mean ± standard error. 50 The p-value represents the dose at which the drug produces half of its maximum analgesic effect, calculated using Graphpad PRISM 5 software. A p-value less than 0.05 indicates statistical significance compared to the saline group based on one-way ANOVA and Dunnett's test.

[0145] 2) Results and conclusions of the inflammatory pain analgesia experiment

[0146] The experimental results of compound 1 on the analgesic effect of carrageenan-induced inflammatory pain in mice are as follows: Figure 3 As shown. Subcutaneous injection of compound 1 produces dose-dependent analgesia for inflammatory pain, and its analgesic effect has an ED50 value. 50 The effective dose was 0.042 mg / kg, and the analgesic effect lasted for 90 minutes. These experimental results indicate that compound 1 has potential medicinal value in the treatment of inflammatory pain.

[0147] (4) Effects of compound 1 on the respiratory system

[0148] After administration, blood was drawn from the heart of mice at specific time points for blood gas analysis to detect the effect of compound 1 on the respiratory system of mice.

[0149] 1) Blood gas analysis experiment

[0150] Blood gas analysis was performed by collecting blood from mice and then analyzing it using a blood gas analyzer. After subcutaneous injection of the drug, 0.2 mL of blood was collected from the mouse heart at specific time points (5, 15, and 30 minutes) using a syringe containing anticoagulant; each mouse underwent this procedure only once. Immediately after collection, the blood was analyzed using a Medica EasyBloodGas blood gas analyzer, and pH, pCO2, and pO2 values ​​were recorded. Values ​​are expressed as mean ± standard error. A p-value less than 0.05 was considered statistically significant compared to the saline group based on one-way ANOVA and Dunnett's test.

[0151] 2) Experimental results and conclusions regarding the effects on the respiratory system

[0152] Experimental results on the effects of drugs on the respiratory system, such as Figure 4 As shown in the figure, compared with the saline group, subcutaneous injection of the control compound fentanyl resulted in a significant decrease in pH at 15 minutes, a significant increase in pCO2 at 15 minutes, and a significant decrease in pO2 at 5 minutes, indicating that fentanyl has a significant respiratory depressant effect. However, after subcutaneous injection of compound 1, the recorded pH, pCO2, and pO2 values ​​remained unchanged compared with the saline group. These experimental results demonstrate that compound 1 does not have the opioid side effect of respiratory depression.

[0153] (5) Evaluation of the physical dependence of compound 1

[0154] The physical dependence of mice after injection of compound 1 was detected by naloxone withdrawal test.

[0155] 1) Naloxone withdrawal test

[0156] Mice were administered the drug subcutaneously every 8 hours at increasing doses (0.2, 0.4, 0.6, 0.8, 1, 1, 1 mg / kg) for seven consecutive times. Two hours after the last injection, naloxone was injected subcutaneously to induce opioid withdrawal. The number of jumps by mice in an opaque observation barrel (32 cm high, 9 cm in diameter) was recorded via video monitoring to evaluate the drug's physical dependence, i.e., physiological addiction. The number of jumps is expressed as mean ± standard error. A p-value less than 0.05 was considered statistically significant compared to the saline group according to one-way ANOVA and Dunnett's test.

[0157] 2) Experimental results and conclusions of physical dependence assessment

[0158] Experimental results for evaluating drug physical dependence, such as Figure 5As shown in the figure, compared with the saline group, subcutaneous injection of the positive control fentanyl significantly increased the number of jumps in mice, indicating the presence of naloxone withdrawal symptoms. However, subcutaneous injection of compound 1 did not change the number of jumps in mice compared with the saline group. These experimental results indicate that compound 1 does not have the physical opioid-like side effects of dependence.

[0159] (6) Evaluation of the psychological addictiveness of compound 1

[0160] The psychological addictiveness of mice after injection of compound 1 was evaluated using a conditional place preference assay.

[0161] 1) Conditional location preference experiment

[0162] The Conditional Place Preference (CPP) experiment was conducted using a CPP box, which consisted of three parts: two large compartments measuring 20 cm × 20 cm × 20 cm, and a smaller compartment measuring 5 cm × 20 cm × 20 cm connecting the large compartments. The two large compartments were visually different: one was covered with black wallpaper, and the other with white wallpaper. Small doors were located at the bottom of both large compartments, which could be closed or opened to control mouse entry and exit. The experiment lasted five days and was conducted in three phases: screening, training, and testing. In the screening phase, on day 1, mice were allowed free movement throughout the device for 15 minutes, and the time spent in each large compartment was recorded. Mice that spent no more than 60% of their total time in a single compartment were selected. In the training phase, mice were injected with saline solution and then confined to one compartment for 40 minutes of training; six hours later, they were injected with the drug and confined to the other compartment for another 40 minutes of training. Training continued for three consecutive days, from day 2 to day 4. The final day was the testing phase. Mice were allowed free movement throughout the device for 15 minutes, and the time spent in each large compartment was recorded. Data were expressed as conditional position preference scores, defined as the time spent in the drug-associated compartment on day 5 minus the time spent on day 1. Data are expressed as mean ± standard error. A p-value less than 0.05 was considered statistically significant compared to the saline group based on one-way ANOVA and Tukey's HSD test.

[0163] 2) Experimental Results and Conclusions of Psychological Addiction Assessment

[0164] Experimental results of drug psycho-addictive assessment, such as Figure 6 As shown in the figure, compared with the saline group, subcutaneous injection of the positive control fentanyl significantly increased the time mice spent in the drug-associated compartment, indicating the presence of conditioned place preference. However, subcutaneous injection of high doses (1 and 10 mg / kg) of compound 1 did not induce significant conditioned place preference in mice compared with the saline group. These experimental results indicate that compound 1 has no psychoaddictive opioid side effects.

Claims

1. A kind of heteropolymer based on N-4-piperidyl-N-phenylpropionamide analogue and neuropeptide FF, which structure is as follows: Compound 1: Compound 2: Compound 3: Compound 7: Compound 8: Compound 9: .

2. The method for synthesizing hetero-peptides based on N-4-piperidyl-N- phenylpropionamide analogues and neuropeptide FF according to claim 1, characterized by that, The synthesis method comprises the following process steps: Step 1, resin pretreatment: the amide resin is soaked in an appropriate amount of dichloromethane, stirred at low speed for 30-40 minutes to swell fully, and the solvent is extracted under reduced pressure. Then, N, N-dimethylformamide is added for washing, and the solvent is extracted. Step 2, removal of 9-fluorenylmethoxycarbonyl (Fmoc) protection: in the resin pretreated in step 1, a deprotection reagent is added, stirred for 3-7 minutes, and then extracted. The process is repeated 3 times. Then, N, N-dimethylformamide is added for washing, and indene is checked to obtain the resin with Fmoc group protection removed. Step 3, condensation of amino acid: N-α-Fmoc and side chain protected amino acid, N-hydroxybenzotriazole, O-benzotriazole-N, N, N', N'-tetramethyluronium hexafluorophosphate are completely dissolved in N, N-dimethylformamide, and then mixed with diisopropyl ethylamine to obtain a mixed solution. Then, it is added to the resin with Fmoc group protection removed obtained in step 2, stirred for 60 minutes under argon protection, and the solvent is extracted. N, N-dimethylformamide is used for repeated washing, extraction, and indene checking. Then, the Fmoc group protection is removed according to the process of step 2 to obtain the peptide resin with Fmoc protection group removed. Step 4, extension of peptide chain: according to the structure design of polypeptide, N-α-Fmoc protected amino acid is selected from C terminal, and the resin with Fmoc protection group removed obtained in step 2 is used for condensation of peptide chain according to the process of step 3 to obtain the side chain fully protected peptide resin. Step 5, cleavage of peptide chain: the peptide resin obtained in step 4 is washed with dichloromethane and methanol alternately, and the solvent is fully extracted. Then, a cleavage agent is added, and the cleavage reaction is carried out at room temperature for 2-3 hours. After filtration, the filtrate is fully rotary evaporated under reduced pressure at below 40℃, and then the precipitate is separated out with ice-cold ether. The supernatant is removed, and the precipitate is fully dissolved in water. Then, ether is separated from the water phase, and the water phase is freeze-dried to obtain white powdery crude peptide. Step 6, purification of crude peptide: high performance liquid chromatograph is used for purification, and the main peak is collected. After freeze-drying, the heteropolymer product is obtained.

3. The method of claim 2, wherein the heteropolymeric peptide is synthesized by the method of, In step 2, the deprotection reagent is a mixed solution composed of hexahydropyridine, 1, 8-diazabicyclo [5.4.0] undec-7-ene and N, N-dimethylformamide in a volume ratio of 1:1:

98.

4. The method of claim 2, wherein the heteropolymeric peptide is synthesized by the method of, In step 3, the molar ratio of N-α-Fmoc protected amino acid, N-hydroxybenzotriazole, O-benzotriazole-N, N, N', N'-tetramethyluronium hexafluorophosphate and diisopropyl ethylamine is 1:1:1:2, and N-α-Fmoc protected amino acid is 2-5 times excess compared with the resin with Fmoc group protection removed.

5. The method for synthesizing heteropeptides according to claim 2, characterized in that, In the step 4, the C-terminal starting N-α-Fmoc and side chain protected amino acids are in turn Fmoc-Xaa5-OH, Fmoc-Arg(Pbf)-OH, Fmoc-Xaa4-OH, Fmoc-Xaa3-OH, Fmoc-Xaa2-OH, Fmoc-Xaa1-OH and N-terminal structural general formula is a carboxylic acid analog, wherein Xaa1 is Phe or NMe-Phe; Xaa2 is Gin or β-Ala; Xaa3 is Pro or D-Pro; Xaa4 is Gin, β-Ala or Aib; Xaa5 is Phe or Cha; Y is -CH2COOH, -C2H5COOH - or -OCH2COOH.

6. The method for synthesizing heteropeptides according to claim 2, characterized in that, In step 5, the cleavage agent is a mixed solution formed by trifluoroacetic acid, triisopropylsilane and water in a volume ratio of 95:2.5:2.

5.

7. Use of the heteropolypeptide based on N-4-piperidyl-N-phenylpropionamide analogues and the neuropeptide FF according to claim 1 for the manufacture of an analgesic medicament.

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