Peptide multi-target molecules based on kappa-opioid / sigma 1 receptor system and preparation and application thereof

Abstract

The application provides a novel peptide multi-target ligand molecule based on a kappa-opioid and sigma 1 receptor system, the ligand is a multi-target ligand based on a kappa-opioid / sigma 1 receptor system obtained by using a kappa-opioid receptor agonist CR845 and a sigma 1 receptor antagonist S1RA as chemical template molecules and sharing a pharmacophore. Animal in-vivo pharmacological identification shows that the multi-target peptide ligand of the application can produce analgesic effect in various preclinical pain models, the kappa-like side effects of sedation and depression are reduced compared with the template molecule CR845, and there is no side effect of analgesic tolerance. Therefore, the multi-target ligand based on the kappa-opioid and / or sigma 1 receptor system provided by the application has the value of developing a new analgesic drug with low side effects.

Description

Technical Field

[0001] This invention belongs to the field of biochemistry technology, specifically relating to a novel peptide multi-target molecule based on the κ-opioid receptor system and the σ1 receptor system, as well as its preparation and uses. Background Technology

[0002] Pain management, especially chronic pain, is a major clinical challenge. Chronic pain is defined as pain that persists or recurs for more than three months. Furthermore, chronic pain is often accompanied by other complications, such as depression, severely impacting patients' quality of life. Opioid receptors have long been the primary targets of drugs used to treat different types of acute and chronic pain. However, while these opioids effectively relieve pain, they are often accompanied by a series of serious adverse reactions (Science 2018, 361:831). Therefore, the development of novel opioids with lower side effects has potential application value in the field of clinical pain management.

[0003] κ-opioid receptor agonists are an important research area for novel analgesics and have been used in drug development for various diseases, including pain, inflammation, itching, and addiction. However, studies have shown that while κ-opioid receptor activation mediates effective analgesia, it can also produce side effects such as sedation, depression, and diuresis. Therefore, employing effective drug design strategies to reduce the side effects of κ-opioid receptor agonists is beneficial for developing novel analgesics with lower side effects.

[0004] In recent years, multi-target molecule design strategies have made positive progress, and opioid multi-target molecules have potential applications in new drug development due to their reduced side effects (Pharmacol Ther, 2020, 210:107519). The σ1 receptor is a unique ligand-regulated chaperone protein expressed in key regions responsible for pain control. Studies have shown that the σ1 receptor system is involved in the regulation of κ-opioid receptor-related analgesia and side effects (Neurosci Lett, 1995, 190:137-139; Eur J Pharmacol, 2013, 716:78-93). The κ-opioid receptor agonist CR845 has entered Phase III clinical trials as an analgesic. However, studies have shown that while CR845 produces potent analgesia, it also has certain side effects such as sedation, depression, and diuresis, often accompanied by adverse reactions such as vomiting and diarrhea, greatly limiting its application prospects in the field of pain treatment (Front Pharmacol, 2021, 12:773204). Existing research has confirmed that multi-target molecules of the μ-opioid / σ1 receptor system possess highly effective analgesic effects with low side effects (Eur J Med Chem, 2021, 223:113658), suggesting that the σ1 receptor system can be applied to the construction strategy of low-side-effect opioid multi-target molecules. Therefore, this invention uses the pharmacophores of the κ-opioid agonist CR845 and the σ1 receptor antagonist S1RA as chemical template molecules. By sharing the pharmacophores, a peptide multi-target molecule KSP001 based on the κ-opioid / σ1 receptor system is chemically constructed, and its preparation and activity identification are performed. Summary of the Invention

[0005] The main objective of this invention is to provide a novel peptide multi-target molecule with κ-opioid receptor agonism / σ1 receptor antagonism.

[0006] Another object of the present invention is to provide a method for preparing the above-mentioned peptide multi-target molecules.

[0007] Another objective of this invention is to provide therapeutic uses for the aforementioned peptide multi-target molecules.

[0008] In a first aspect of the invention, a novel κ-opioid / σ1 receptor peptide multi-target molecule is provided, the structure of which is shown below:

[0009]

[0010] The compounds of this invention are based on existing structure-activity relationship studies of CR845 and S1RA. By replacing the C-terminal [ω(4-aminopiperidine-4-carboxylic acid)] of CR845 and the morpholine ring at the terminal of S1RA with a piperazine ring, a multi-target peptide molecule based on the κ-opioid / σ1 receptor is chemically constructed through the sharing of the piperazine ring.

[0011] In a second aspect of the present invention, a method for preparing the above-mentioned peptide multi-target molecule is provided, characterized in that the preparation method includes the following process steps:

[0012] (a) The N-terminal tetrapeptide (side chain protected) Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)-OH of the multi-target ligand molecule was synthesized by the Fmoc solid-phase synthesis method.

[0013] (b) The condensation of the piperazine-modified S1RA analog 1-(2-{[5-methyl-1-(naphth-2-yl)pyrazol-3-yl]oxy}ethyl)piperazine with the above peptide was performed by liquid-phase synthesis to obtain the multi-target ligand molecule.

[0014] In another preferred embodiment, in step (a), the solid-phase synthesis method includes the following process steps: resin swelling, amino acid condensation, peptide chain elongation, peptide chain compression and drying and half-cutting, crude peptide extraction and precipitation and chemical identification.

[0015] In another preferred embodiment, in step (b), the analog synthesis method includes the following steps: synthesis of N-(naphthyl-2-yl)acetylhydrazine, synthesis of 5-methyl-1-(naphthyl-2-yl)pyrazol-3-phenol and 1-(2-{[5-methyl-1-(naphthyl-2-yl)pyrazol-3-yl]oxy}ethyl)piperazine.

[0016] In another preferred embodiment, in step (b), the multi-target molecule synthesis method is carried out by the following process steps: the carboxyl group and the terminal amino group of the piperazine group in the CR845 structural formula are dehydrated and condensed to form an amide bond.

[0017] In another preferred embodiment, in step (a), the method includes the following process steps:

[0018] i. Resin pretreatment: A certain amount of 2-chlorotriphenylmethyl chloride resin is swollen in dichloromethane for 30 min, and then dried. The swelling described herein refers to placing the resin in dichloromethane and stirring at a rate of 60-100 rpm for 10-40 min, preferably 80 rpm, for 30 min, to allow the resin to swell fully; the volume-to-mass ratio of dichloromethane to resin is 8-12 mL / g, preferably 10 mL.

[0019] ii. Loading of the first amino acid: Weigh a certain amount of the first amino acid protected by the fluorenemethyloxycarbonyl (Fmoc) group, dissolve the amino acid in dichloromethane, and then add six times the excess of N,N-diisopropylethylamine. The mixture is then poured into dried 2-chlorotriphenylmethyl chloride resin. The volume-to-mass ratio of the mixed solution to the 2-chlorotriphenylmethyl chloride resin is 4–6 mL / g. The mixture is stirred at room temperature for 1 h. Unreacted sites are blocked by adding methanol at a ratio of 0.8 mL / g to the resin and reacting for 30 min. After the reaction, wash three times with DMF for 3 min each time. Ninhydrin test: Under normal conditions, the solution is pale yellow, and the resin is colorless.

[0020] iii. Cyclic condensation of amino acids

[0021] iii.i. Removal of the Fmoc protecting group from the first amino acid: Add a deprotecting agent mixture of 1,8-diazabicycloundec-7-ene (DBU), piperidine, and DMF in a volume ratio of 1:1:98 to the resin. Shake the resin three times, the first two times for 5 min each, and the last time for 10 min. The volume ratio of the mixed solution to the resin is 8–12 mL / g. The stirring speed for the first time is 60–100 rpm, and the stirring time is 2–6 min; the stirring speed for the second time is 60–100 rpm, and the stirring time is 8–12 min. After deprotection, wash four times with DMF, 3 min each time. Ninhydrin test: Under normal conditions, both the solution and the resin will be blue-purple.

[0022] iii.ii Amino acid condensation: Fmoc-amino acids, O-benzotriazole-N,N,N',N'-tetramethylurea-hexafluorophosphate (HBTU), N-hydroxybenzotriazole (HOBT), and N,N-diisopropylethylamine (DIEA) in a molar ratio of 1:0.2–1.5:0.2–1.5:1.5–3 are dissolved in a small amount of DMF. Then, N,N-diisopropylethylamine is added to the mixture, and the mixture is shaken three times. The solution is then poured into the deprotected resin. The volume-to-mass ratio of DMF to N-α-Fmoc-protected amino acids is 5–10 mL / g; the volume-to-mass ratio of the mixed solution to the deprotected resin is 4–6 mL / g; the stirring rate is 60–100 rpm; the reaction is carried out at room temperature for 40–100 min. After the reaction, the solution is washed three times with DMF for three minutes each time. Ninhydrin test: Under normal conditions, the solution is pale yellow, and the resin is colorless.

[0023] iii.iii Peptide chain elongation: Based on the sequence of the polypeptide chain, we sequentially insert different Fmoc-protected amino acids into the sequence. If the last amino acid inserted into the sequence is Fmoc-protected, we need to remove the terminal Fmoc protecting group. If the last amino acid in the sequence is protected by a tert-butyloxycarbonyl (Boc) group, the final step of removing Fmoc is omitted. The remaining amino acids in the peptide chain are condensed as described above.

[0024] iv. Resin compression: The resin was washed alternately with dichloromethane and anhydrous methanol. The specific compression process was as follows: first, wash twice with dichloromethane for three minutes each time, then wash once with anhydrous methanol for three minutes each time, followed by another wash with dichloromethane for three minutes each time, and finally wash twice with anhydrous methanol for three minutes each time. After washing, the stirring rod was removed, and the resin was dried for 3-5 hours.

[0025] v. Peptide cleavage and extraction precipitation: Dichloromethane (DCM), triisopropylsilane (Tis), and trifluoroacetic acid (TFA) in a volume ratio of 98:1:1 are added to the dried resin. Cleavage typically requires 3-4 hours at room temperature. The volume-to-mass ratio of the cleaving agent to the peptide resin is 10-20 mL / g. The cleavage agent is then evaporated to dryness using a rotary evaporator. A small amount of DMF is used to dissolve the residue after evaporation. The DMF solution is pipetted and then added dropwise to ice-cold deionized water, with slow stirring until precipitation occurs. The precipitate is filtered through a sintered glass funnel and washed with ice-cold deionized water. The precipitate is then dried for later use.

[0026] In another preferred embodiment, in step (b), the method includes the following process steps:

[0027] Synthesis of iN-(naphthyl-2-yl)acetylhydrazine: A certain amount of 2-naphthylhydrazine hydrochloride was weighed and dissolved in deionized water to form a suspension. The volume-to-mass ratio of 2-naphthylhydrazine hydrochloride to deionized water was 1:10. Potassium carbonate was added to the suspension in portions, with a mass ratio of 2-naphthylhydrazine hydrochloride to potassium carbonate of 1:1. The reaction was stirred at room temperature for 15–30 min. After the reaction was complete, ethyl acetate (EtoAc) was added to the system in an equal volume ratio to deionized water, the organic phase was extracted and dried with anhydrous sodium sulfate (Na2SO4). The ethyl acetate was evaporated to dryness using a rotary evaporator, and the residue was diluted with ultra-dry toluene (or redistilled toluene) and ethyl acetate in a volume ratio of 3:2. The volume-to-mass ratio of ultra-dry toluene to 2-naphthylhydrazine hydrochloride was 12:1, and the volume-to-mass ratio of ethyl acetate to 2-naphthylhydrazine hydrochloride was 8:1. Acetic anhydride was then added to the system, with a mass ratio of acetic anhydride to 2-naphthylhydrazine hydrochloride of 13.1:25. The reaction was stirred at room temperature for 60–90 min. Add an appropriate amount of petroleum ether, cool the solution to 7°C, and then filter and wash the precipitate with petroleum ether in a sintered glass funnel to obtain N-(naphthyl-2-yl)acetylhydrazine (molecular weight 200.24).

[0028] ii. Synthesis of 5-methyl-1-(naphthyl-2-yl)pyrazole-3-phenol: First, prepare a reaction system with N-(naphthyl-2-yl)acetylhydrazine, ethyl acetoacetate, and phosphorus trichloride (PCl3) in a molar ratio of 1:1:1. Add ethyl acetoacetate to N-(naphthyl-2-yl)acetylhydrazine, and then slowly add phosphorus trichloride dropwise to the mixture. Heat the mixture at 50°C for 2–3 h. After the reaction is complete, slowly add the reaction product to ice water and stir for 60–90 min. Add ethyl acetate to the suspension, filter to remove the precipitate, separate the filtrate, and extract the organic phase. Add anhydrous sodium sulfate to the organic phase and dry it. Rotate the organic phase to dryness using a rotary evaporator. Add a small amount of anhydrous ethanol to the residue for recrystallization. Filter and wash the crystallized product with ice-cold anhydrous ethanol to obtain 5-methyl-1-(naphthyl-2-yl)pyrazole-3-phenol (molecular weight 224.26).

[0029] iii. Synthesis of 1-(2-{[5-methyl-1-(naphthyl-2-yl)pyrazol-3-yl]oxy}ethyl)piperazine: 5-methyl-1-(naphthyl-2-yl)pyrazol-3-phenol, 1-Boc-4-(2-chloroethyl)piperazine, potassium carbonate, and sodium iodide were dissolved in anhydrous DMF in a molar ratio of 1:1:2:1, and the mixture was stirred overnight at 70°C. After the reaction was complete, the reaction mixture was poured into an appropriate amount of deionized water, and the solution was extracted four times with diethyl ether. The organic phase was then washed with saturated NaCl and dried over anhydrous sodium sulfate. The sample was purified by column chromatography (4:1 ratio of petroleum ether and ethyl acetate), followed by rotary evaporation to remove the developing solvent. The residue in the round-bottom flask was purified Boc-protected 1-(2-{[5-methyl-1-(naphth-2-yl)pyrazol-3-yl]oxy}ethyl)piperazine. The Boc protecting group in the molecule was further removed using DCM and TFA in a 1:1 volume ratio. The residual TFA was removed by toluene suspension evaporation to obtain 1-(2-{[5-methyl-1-(naphth-2-yl)pyrazol-3-yl]oxy}ethyl)piperazine.

[0030] In another preferred embodiment, in step (b), the method includes the following process steps:

[0031] iv. Synthesis of multi-target molecules: DCM and TFA in a volume ratio of 1:1 were added to purified 1-(2-{[5-methyl-1-(naphth-2-yl)pyrazol-3-yl]oxy}ethyl)piperazine. Peptides, O-benzotriazole-N,N,N',N'-tetramethylurea-hexafluorophosphate (HBTU), N-hydroxybenzotriazole (HOBT), and N,N-diisopropylethylamine were dissolved in a small amount of DMF. The synthesized peptides were then added to the DMF, and the Boc-protected 1-(2-{[5-methyl-1-(naphth-2-yl)pyrazol-3-yl]oxy}ethyl)piperazine was stirred at room temperature for 3 h. After the reaction was complete, the DMF in the reaction system was removed by rotary evaporation. A TFA:Tis:H2O mixture with a volume ratio of 98:1:1 was added to the residue to remove the Boc protecting group from the multi-target peptide moiety of this invention. Subsequently, TFA was removed by rotary evaporation, and immediately ice-cold diethyl ether was added to the residue, causing precipitation.

[0032] v. Purification and analysis of the multi-target molecule: The multi-target molecule was separated and purified using a reversed-phase high-performance liquid chromatography (RP-HPLC) semi-preparative column. After separation, the main peak was collected, and the peptide was lyophilized to obtain pure peptide. The purity was identified using an RP-HPLC analytical column, and the molecular weight of the multi-target molecule was identified using electrospray ionization mass spectrometry (ESI).

[0033] A third aspect of this invention provides the pharmaceutical use of the multi-target peptide molecule of this invention. In vivo animal experiments show that the multi-target molecule of this invention has good analgesic activity, and significantly reduces sedative and depressive side effects. It has potential use in the development of highly effective, low-side-effect analgesic drugs, and can relieve and treat various types of pain, including pathological pain.

[0034] The pharmaceutical dosage of the polypeptide of the present invention can vary within a wide range, and those skilled in the art can easily determine it based on some objective factors, such as the type of disease, the severity of the disease, the patient's weight, the dosage form, the route of administration, etc. Attached Figure Description

[0035] Figure 1 Analgesic effect of intraperitoneal injection of multi-target molecules in formalin pain assay in mice;

[0036] Figure 2 Analgesic effect of intraperitoneal injection of multi-target molecules in acetic acid writhing test in mice;

[0037] Figure 3 The analgesic time and dose-response curve of intraperitoneal injection of multi-target molecules in a CFA-induced inflammatory pain model in mice;

[0038] Figure 4Pharmacological mechanism of the multi-target molecular analgesia effect of injecting the κ-opioid receptor selective antagonist nor-BNI into mice;

[0039] Figure 5 Pharmacological mechanism of the multi-target molecular analgesia effect of the σ1 receptor selective agonist PRE-084 injected into mice;

[0040] Figure 6 The effect of intraperitoneal injection of multi-target molecules on sedation behavior in mice;

[0041] Figure 7 The effect of intraperitoneal injection of multi-target molecules on depressive-like behavior in mice;

[0042] Figure 8 Changes in the analgesic effect of intraperitoneal injection of multi-target molecules into mice over eight consecutive days. Detailed Implementation

[0043] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0044] the term

[0045] This invention uses the κ-opioid receptor agonist CR845 and the σ1 receptor antagonist S1RA as chemical template molecules. The C-terminal [ω(4-aminopiperidine-4-carboxylic acid)] of CR845 and the morpholine ring at the end of S1RA are replaced with piperazine rings. By sharing the piperazine ring, a multi-target molecule based on κ-opioid / σ1 receptor peptides is chemically constructed.

[0046] In another preferred embodiment, the specific chemical structure of the κ-opioid / σ1 receptor peptide multi-target molecule is shown below:

[0047]

[0048] Compared with the prior art, the main advantages of the present invention are:

[0049] (1) The multi-target molecule in this invention is a multi-target ligand that acts on both κ-opioid receptors and σ1 receptors. It has κ-opioid receptor agonist activity and σ1 receptor antagonist activity. It has shown dose-dependent analgesic effects in CFA-induced inflammatory pain, acetic acid-induced visceral pain and formalin pain in mice.

[0050] (2) The multi-target molecules in this invention produce fewer sedative and depressive kappa-like side effects in mice than CR845, and have no analgesic tolerance side effects.

[0051] The experimental instruments and main experimental materials are as follows:

[0052] Experimental instruments: Solid-phase peptide synthesizer (BD Medical Instruments Shanghai Co., Ltd.), rotary evaporator (RE-5298A, Shanghai Yarong), freeze dryer (Ningbo Shuangjia Instrument Co., Ltd.), mass spectrometer (ESI-Q-TOF maXis-4G, Bruker Daltonics, Germany), circulating water pump (SHB-Ⅲ, Zhengzhou Great Wall), preparative high-performance liquid chromatograph (LC-3050N, Shandong Runyang Instrument Co., Ltd.), preparative column (C18 reverse-phase column, 19mm×250mm, Suzhou McWon), analytical high-performance liquid chromatograph (Waters, Delta 600), analytical column (XBridge™ BEH 130PrepC18, 4.6mm×250mm).

[0053] Experimental reagents: Complete Freund's adjuvant and formalin were from Sigma-Aldrich, USA; S1RA was from DCC Chemicals; PRE-084 was from Beijing Puxitang Biotechnology Co., Ltd.; nor-BNI was from Tocris Bioscience, UK; acetic acid was from Kaitong Chemical Reagent Co., Ltd. Fmoc / Boc protecting amino acids, 2-chlorotriphenylmethyl chloride resin (100-200 mesh, 1% crosslinking degree, substitution value 0.55 mmol / g), N-hydroxybenzotriazole (HOBt), O-benzotriazole-tetramethylurea hexafluorophosphate (HBTU), and N,N-diisopropylethylamine (DIEA) were all purchased from Jier Biochemical (Shanghai) Co., Ltd. Dichloromethane (DCM) and N,N-dimethylformamide (DMF) were both from Tianjin Chemical Reagent Factory No. 2. Phthalimide (DBU), triisopropylsilane (Tis), and trifluoroacetic acid were from Bailingwei (Beijing) Technology Co., Ltd. 4-(2-hydroxyethyl)morpholine, 2-hydrazinonaphthalene hydrochloride, ethyl acetoacetate, potassium carbonate, sodium iodide, and 1-Boc-4-(2-chloroethyl)piperazine are all from Shaoyuan Technology (Shanghai) Co., Ltd.; 4-(2-chloroethyl)morpholine hydrochloride is from Bid Pharmaceutical (Shanghai) Technology Co., Ltd.

[0054] Example 1. Synthesis of multi-target peptide molecules

[0055] (1) Resin pretreatment: Weigh 1g of 2-chlorotriphenylmethyl chloride resin (substitution value 0.55mmol / g), add 10mL of DCM, stir at 80rpm to swell, stir for 30min, and then dry.

[0056] (2) Loading of the first amino acid: Weigh a certain amount of Fmoc-D-Lys(Boc)-OH, dissolve the amino acid in DCM, and then add six times the excess of N,N-diisopropylethylamine. Then pour the mixture into the dried 2-chlorotriphenylmethyl chloro resin. The volume-to-mass ratio of the mixed solution to the 2-chlorotriphenylmethyl chloro resin is 4-6 mL / g. Under argon protection, react at room temperature and stirring rate of 80 rpm for 60 min.

[0057] (3) Methanol capping: Subsequently, methanol at a ratio of 0.8 ml / g resin was added to the reaction system of step (2) and reacted for 30 min to seal the unreacted sites on the 2-chlorotriphenylmethyl chloride resin. After the reaction was completed, the resin was washed three times with DMF for 3 min each time.

[0058] (4) Ninhydrin test: The ninhydrin test reagent is a phenol:pyridine:ninhydrin solution with a volume ratio of 1:2:1. The phenol solution is prepared by dissolving 20g of phenol in 5mL of anhydrous ethanol; the pyridine solution is prepared by dissolving 0.05mL of KCN (0.001M) in 2.5mL of pyridine; and the ninhydrin solution is prepared by dissolving 0.5g of ninhydrin in 10mL of anhydrous ethanol. All phenol, pyridine, and anhydrous ethanol are redistilled. Under normal conditions, the ninhydrin solution is pale yellow, and the resin is colorless.

[0059] (5) Removal of Fmoc protecting groups: 10 mL of a DBU, piperidine, and DMF solution with a volume ratio of 1:1:98 was added to the peptide resin (Fmoc-D-Lys(Boc)-Resin) obtained in step (3); the mixture was stirred at 80 rpm for 5 min, and repeated twice. After drying, the above mixed solution was added again, and the mixture was stirred at 80 rpm for 10 min. Finally, DMF was added and the mixture was washed 4 times, 3 min each time.

[0060] (6) Ninhydrin test: Using the test reagent prepared in step (4), if the Fmoc group is removed, the solution will be blue-purple and the resin will be blue-purple.

[0061] (7) Amino acid condensation: Weigh Fmoc-D-Leu-OH, HOBt, and HBTU in a molar ratio of 1:1:1, and dissolve them in 5 mL of DMF. N,N-diisopropylethylamine is added last. After stirring evenly, add the mixture to the resin peptide in step (5) where the Fmoc protecting group has been removed. React for 60 min at room temperature and stirring at 80 rpm under argon protection. After the reaction is complete, perform an indole test as described in step (4). If the solution is pale yellow and the resin is colorless, it indicates that the amino acid has condensed onto the resin. Then, remove the Fmoc protecting group as described in step (5) and perform an indole test as described in step (4). If both the solution and the resin are dark blue, it indicates that the Fmoc protecting group has been completely removed, resulting in a resin peptide without the Fmoc protecting group.

[0062] (8) Peptide chain extension: The resin peptide obtained in step (7) is sequentially condensed onto the peptide resin using the method in steps (5-7) with Fmoc-D-Phe-OH and Boc-D-Phe-OH. The resin peptide Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)-Resin is obtained.

[0063] (9) Compression and drying of peptide chains: The resin was washed alternately with dichloromethane (2×3min), anhydrous methanol (1×3min), dichloromethane (1×3min), and anhydrous methanol (2×3min), and then the stirring rod was removed and the resin was dried for 4 hours.

[0064] (10) Peptide cleavage: 15 mL of cleavage agent (TFA:DCM:Tis = 1:98:1) was added to the dried resin peptide Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)-Resin. The reaction was stirred at room temperature for 3 h, with stirring at 50 rpm for 1 min every 15 min. The filtrate was evaporated to dryness under reduced pressure at a temperature not exceeding 40 °C, dissolved in a small amount of DMF, and slowly added dropwise to ice water with stirring until homogeneous. The crude peptide was fully separated as a white precipitate. The precipitate was then filtered through a sintered glass funnel, sealed with sealing film, and dried for later use. The crude peptide yield was 70%.

[0065] (11) Synthesis of N-(naphthyl-2-yl)acetylhydrazine: 2.5 g of 2-naphthylhydrazine hydrochloride was weighed and dissolved in 25 mL of deionized water to form a suspension. 2.5 g of potassium carbonate was added to the suspension in portions. The mixture was stirred at room temperature for 15 min. After the reaction was complete, ethyl acetate was added to the system in an equal volume ratio to deionized water. The organic phase was extracted and dried over anhydrous sodium sulfate. The ethyl acetate was evaporated to dryness using a rotary evaporator. The residue was diluted with 30 mL of ultra-dry toluene and 20 mL of ethyl acetate. Then, 1.2 mL of acetic anhydride was added to the system, and the mixture was stirred at room temperature for 60 min. An appropriate amount of petroleum ether was added, and the solution was cooled to 7 °C. The precipitate was then filtered and washed with petroleum ether in a sintered glass funnel to obtain 1.64 g of N-(naphthyl-2-yl)acetylhydrazine, with a yield of 63.8%.

[0066] (12) Synthesis of 5-methyl-1-(naphthyl-2-yl)pyrazole-3-phenol: 510 μL of ethyl acetoacetate was added to 800 mg of N-(naphthyl-2-yl)acetylhydrazine, followed by slow dropwise addition of 352 μL of phosphorus trichloride to the mixture. The reaction was heated at 50 °C for 2 h. After the reaction was completed, the reaction product was slowly added to ice water and stirred for 60 min. Ethyl acetate (EtoAc) was added to the suspension, the precipitate was filtered, and the organic phase was extracted after separation of the filtrate. The organic phase was dried with anhydrous sodium sulfate and evaporated to dryness using a rotary evaporator. A small amount of anhydrous ethanol was added to the residue for recrystallization. The crystallized product was washed with ice-cold anhydrous ethanol to obtain 240 mg of 5-methyl-1-(naphthyl-2-yl)pyrazole-3-phenol, with a yield of 27%.

[0067] (13) Synthesis of the piperazinyl-modified S1RA analog 1-(2-{[5-methyl-1-(naphthyl-2-yl)pyrazol-3-yl]oxy}ethyl)piperazine: 200 mg of 5-methyl-1-(naphthyl-2-yl)pyrazol-3-phenol, 222 mg of 1-Boc-4-(2-chloroethyl)piperazine, 246.6 mg of potassium carbonate, and 133.7 mg of sodium iodide were dissolved in 5 mL of anhydrous DMF and reacted overnight at 70 °C with stirring. After the reaction was completed, the reaction mixture was poured into 20 mL of deionized water. The solution was extracted four times with diethyl ether, followed by washing with saturated NaCl on the organic phase and drying with anhydrous sodium sulfate. The solution was purified by column chromatography (4:1 petroleum ether:ethyl acetate), and then the solvent was removed by rotary evaporation to obtain Boc-protected 1-(2-{[5-methyl-1-(naphthyl-2-yl)pyrazol-3-yl]oxy}ethyl)piperazine. The Boc protecting group in the molecule was further removed by using 30 mL each of DCM and TFA at a volume ratio of 1:1. The residual TFA was removed by toluene evaporation to obtain 207 mg of 1-(2-{[5-methyl-1-(naphth-2-yl)pyrazol-3-yl]oxy}ethyl)piperazine, with a yield of 69%.

[0068] (14) Synthesis of multi-target molecules: The peptide obtained in step (10) in a molar ratio of 1:1.5:1.5:3, O-benzotriazole-N,N,N',N'-tetramethylurea-hexafluorophosphate (HBTU), N-hydroxybenzotriazole (HOBT), and N,N-diisopropylethylamine (DIEA) were dissolved in a small amount of DMF, and 100 mg of 1-(2-{[5-methyl-1-(naphthyl-2-yl)pyrazol-3-yl]oxy}ethyl)piperazine obtained in the previous step was added. The mixture was stirred at room temperature for 3 h. After the reaction was completed, the DMF in the reaction system was removed by rotary evaporation, and TFA:Tis:H2O in a volume ratio of 98:1:1 was added to the residue to remove the Boc protecting group of the peptide portion of the multi-target molecules. Subsequently, TFA was removed by rotary evaporation, and ice-cold diethyl ether was immediately added to the residue to precipitate the product.

[0069] (15) Purification and analysis of the multi-target molecule: The multi-target molecule was separated and purified using a reversed-phase high-performance liquid chromatography (RP-HPLC) semi-preparative column. After separation, the main peak was collected and lyophilized to obtain pure peptide. 100 mg of sample was loaded, and 48 mg of white pure peptide solid powder was obtained by lyophilization, with a pure peptide yield of 48%. The purity was identified using an RP-HPLC analytical column, and the molecular weight (MW: 871.5) of the multi-target molecule of this invention was then identified using electrospray mass spectrometry (ESI).

[0070] Based on the above synthesis steps, the present invention synthesizes as follows: Figure 1 The chemical characterization results of the multi-target molecules shown are shown in Table 1.

[0071] Table 1 Purity and mass spectrometry analysis of the multi-target molecules of this invention

[0072]

[0073] Note: System 1: Gradient elution system 1 is 10-80% acetonitrile / water (0.1% TFA) (completed in 30 min), flow rate is 1 mL / min, detection wavelength is 220 nm, and analytical column is XBridge™ BEH 130Prep C18, 4.6 mm × 250 mm; System 2: Gradient elution system 2 is 10-100% acetonitrile / water (0.1% TFA) (completed in 30 min), flow rate is 1 mL / min, detection wavelength is 220 nm, and analytical column is XBridge™ BEH 130Prep C18, 4.6 mm × 250 mm.

[0074] Example 2. In vitro functional activity assay of multi-target molecules against κ-opioid receptors

[0075] Co-transfection with GPCRs (MOR, DOR, and KOR) and the Gα15 / 16 subunit was performed. When the compounds activated the receptor, the GDP bound to Gα15 / 16 was replaced by GTP. The Gα15 / 16 subunit subsequently dissociated from the Gβγ complex and activated phospholipase Cβ, which in turn hydrolyzed phosphatidylinositol diphosphate to produce diacylglycerol and inositol triphosphate. Inositol triphosphate activated inositol triphosphate-dependent calcium channels present in the endoplasmic reticulum membrane, inducing calcium release into the cytoplasm. The interaction of calcium with fluorescent dyes generated a fluorescent signal, which was used to detect the agonistic activity of multi-target molecules on opioid receptors.

[0076] Experimental Methods: Cells were seeded in 96-well plates at a rate of 30,000–50,000 cells per well and cultured for at least 20 hours. At the start of the experiment, the culture medium was aspirated from the culture dishes, and then 50 μL of a calcium dye stock solution containing 2.5 mM probenecid, 0.9 μM Fluo-4-AM, and F127 (0.03%) was added. The plates were incubated at 37°C for 30 min. After incubation, the dye was aspirated, and 75 μL of HBSS buffer (containing 2.5 mM probenecid) was added. The 96-well drug delivery plates, 96-well cell culture plates, and automated drug delivery pipette tips were then placed into a FlexStation 3 calcium flow workstation to detect changes in calcium signal after drug administration.

[0077] The calcium mobilization effect was expressed as the percentage increase in intracellular calcium ions due to the drug (%control), calculated as %control = (detected value - baseline value) / baseline value × 100. Relevant %control data are expressed as mean ± standard error (Means ± SEM). The dose-response relationship of the compound was statistically analyzed using a nonlinear regression model. The EC50 of the regulation of intracellular calcium ion mobilization by multi-target molecules was calculated using the statistical software GraphPadPrism version 8.0.1. 50 The values ​​are shown in Table 2.

[0078] Experimental results showed that the positive control drugs corresponding to the receptors all activated IP3-dependent calcium channels on the endoplasmic reticulum membrane, inducing the release of calcium from the endoplasmic reticulum into the cytoplasm, thereby increasing the calcium ion concentration in the cytoplasm. Compared with the positive control drugs DAMGO and DPDPE, the multi-target molecule of this patent showed only weak agonistic activity against MOR and DOR. However, KSP001 has a potent agonistic activity against KOR, with its agonistic activity being approximately 13 times stronger than that of the KOR agonist U69,593. Therefore, KSP001 is a potent agonist of KOR. Further in vivo activity studies of KSP001 were subsequently conducted.

[0079] Table 2. Effects of KSP001 and opioid receptor agonists on G protein-Ca. 2+ agonist activity assay

[0080]

[0081] Example 3. Evaluation of the analgesic activity of multi-target molecules in a formalin pain model

[0082] The formalin model produces both acute and persistent pain responses. Acute pain occurs within 0-5 minutes after formalin injection, while persistent inflammatory pain occurs within 15-30 minutes, caused by activation of peripheral nociceptors and induction of inflammatory cytokine release, respectively. In this experiment, male Kunming mice weighing 22±2g were selected, and the experimental temperature was maintained at room temperature (22±2℃). Two days prior to the experiment, mice were moved from the animal storage room to the experimental area for 1 hour each day to acclimatize. On the day of the experiment, mice were moved from the animal storage room to the experimental area for 30 minutes to acclimatize. Subsequently, the mice were placed in a device made of acrylic sheet measuring (20×20×30cm) and allowed to acclimatize to the experimental environment for 10-15 minutes. A mirror tilted at 30-45° was placed under this device to observe the mouse's paw response to formalin injection. At the start of the experiment, physiological saline or the test drug was injected intraperitoneally. Five minutes later, 20 μL of 1.25% formalin solution was subcutaneously injected into the middle of the paw of the mouse model. The mouse was then returned to the experimental setup and the timing was immediately recorded. The time for biting and shaking the right hind paw was recorded at the corresponding time points of pain after formalin injection.

[0083] The analgesic effect of a drug is generally evaluated using the maximum analgesic effect (MPE) (%), calculated as follows: MPE (%) = 100 × [(time of corresponding phase pain in the saline group - time of corresponding phase pain after drug administration) / (time of corresponding phase pain in the saline group)]. The median effective dose (ED) is also used. 50 This refers to the drug dose required to achieve 50% of the maximum effect. ED 50 The 95% confidence intervals were calculated using the statistical software GraphPad Prism 5.0 with MPE (%) and the drug concentration at which the maximum analgesic effect (MPE) was achieved (%). Differences in analgesic effect were statistically analyzed using one-way ANOVA (Dunnett's test). * P<0.05, ** P<0.01 and *** P<0.001 indicates a significant difference between the group that only received the relevant drug and the saline group.

[0084] This invention uses a formalin pain model to evaluate the analgesic activity of intraperitoneally injected multi-target molecules. For example... Figure 1 As shown, intraperitoneal injection of the multi-target molecule at concentrations of 1, 3, and 10 mg / kg produced dose-dependent analgesia in a mouse formalin-induced pain model. The multi-target molecule of this invention effectively inhibited phase I acute pain (F... 4,35 =40.48, P<0.001) and phase II inflammatory pain (F 4,35=40.48, P<0.001), ED for analgesia 50 The values ​​are ED 50 =4.620 mg / kg and ED 50 = 3.349 mg / kg. Therefore, the multi-target molecule of this invention exhibits significant analgesic effects in formalin pain.

[0085] Example 4. Evaluation of the analgesic activity of multi-target molecules in an acetic acid torsional visceral pain model

[0086] The acetic acid writhing test was performed using male Kunming mice weighing 22–25 g, with the ambient temperature controlled at 18±2℃. To avoid researchers getting too close and affecting the mice's behavior, the mice were placed in a device made of acrylic sheet (20×20×30cm), with a mirror tilted at 30–45° placed underneath to observe the mice's writhing behavior. The pain induced in the experiment was a 0.6% acetic acid solution, prepared from glacial acetic acid and ultrapure water. Two days before the experiment, the mice were moved from the animal storage room to the experimental area for 1 hour each day to acclimatize. On the day of the experiment, the mice were moved from the animal room to the experimental area for 30 minutes to acclimatize. Then, the mice were placed in the experimental device and allowed to acclimatize to the environment for 10–15 minutes. Subsequently, the test drug was injected intraperitoneally, followed by an intraperitoneal injection of 10 ml / kg (mouse body weight) of 0.6% acetic acid solution 5 minutes later. The mice were then immediately returned to the peritoneum and a stopwatch was started. When the experimental mice exhibited characteristic behaviors such as body stretching, abdominal twitching and retraction, and hind limb extension, it was recorded as a standard visceral pain response. The experimenters needed to record the number of writhing movements of the mice within 5-15 minutes after the injection of the pain-inducing agent.

[0087] The analgesic effect of a drug is generally evaluated using the maximum analgesic effect (MPE) (%), calculated as: MPE (%) = 100 × [(number of writhing movements in the saline group - number of writhing movements in the drug group) / (number of writhing movements in the saline group)]. The median effective dose (ED) is... 50 This refers to the drug dose required to achieve 50% of the maximum effect. ED 50 The 95% confidence intervals were calculated using the statistical software GraphPad Prism 5.0 with MPE (%) and the drug concentration at which the maximum analgesic effect (MPE) was achieved (%). Differences in analgesic effect were statistically analyzed using one-way ANOVA (Dunnett's test). * P<0.05, ** P<0.01 and *** P<0.001 indicates a significant difference between the group that only received the relevant drug and the saline group.

[0088] This invention uses an acetic acid-induced visceral pain model to evaluate the analgesic activity of intraperitoneally injected multi-target molecules. For example... Figure 2As shown, intraperitoneal injection of multi-target molecules at concentrations of 1, 3, and 10 mg / kg produced dose-dependent analgesia in a mouse model of visceral pain, significantly inhibiting the number of writhing movements (F) in the experimental mice. 4,25 =42.82, P<0.001), ED for analgesia 50 The value is ED 50 = 3.12 mg / kg. The multi-target molecules of this invention all exhibit significant analgesic effects in visceral pain.

[0089] Example 5. Evaluation of the analgesic activity of multi-target molecules in a CFA-induced inflammatory pain model

[0090] For the photothermal paw withdrawal experiment, male Kunming mice weighing 20-25g were selected. Two days before the experiment, the mice were moved from the animal storage room to the experimental area for 1 hour each day to acclimatize. On the day of the experiment, the animals were placed in the experimental environment for 2-3 hours to acclimatize. During the preparation period, the PL-200 thermal analgesia device was turned on and the light intensity was adjusted to 20%, and the laboratory temperature was controlled at 22±1℃. The mice were then placed in an experimental device made of glued acrylic plates. After the mice's excrement was cleaned, the baseline paw withdrawal value of the right hind paw was measured (five sets of data were measured, and the intervals between each measurement should not be too close to avoid lowering the threshold) as the baseline paw withdrawal latency of the mice. The baseline paw withdrawal latency of normal male mice that had not yet been modeled should be between 13-16 seconds. After the baseline value was measured, unqualified experimental mice were removed according to the above standard. Subsequently, 20μL of CFA was injected into the right paw of the mice, and they were then housed in separate cages for three days. Three days later, the paw withdrawal time of the right hind paw of the experimental mice was measured again, and this time was the mouse's pain sensitivity value. The pain sensitivity of mice after normal CFA modeling was between 3 and 6 seconds. Subsequently, the paw retraction time of mice under photothermal stimulation was measured at time points of 15 min, 30 min, 45 min, 60 min, 90 min, 120 min, 180 min, 240 min, and 360 min after drug administration. The cut-off time of the light source in the experiment was set to 25 seconds.

[0091] The analgesic effect of a drug is generally evaluated using the maximum analgesic effect (MPE) (%), MPE (%) = 100 × [(post-dose withdrawal threshold - baseline withdrawal threshold) / (25 - baseline withdrawal threshold)]. The median effective dose (ED) is... 50 This refers to the drug dose required to achieve 50% of the maximum effect. ED 50 The 95% confidence intervals were calculated using the statistical software GraphPad Prism 5.0 with MPE (%) and the drug concentration at which the maximum analgesic effect (MPE) was achieved (%). Differences in analgesic effect were statistically analyzed using one-way ANOVA (Dunnett's test). * P<0.05, **P<0.01 and *** P<0.001 indicates a significant difference between the group that only received the relevant drug and the saline group.

[0092] This invention uses a CFA-induced inflammatory pain model to evaluate the analgesic activity and duration of analgesia of intraperitoneally injected multi-target molecules. For example... Figure 3 As shown, intraperitoneal injection of 0.1, 0.3, and 1.0 mg / kg of the multi-target molecule produced dose-dependent analgesia in a mouse CFA-induced inflammatory pain model (F...). 3,22 =8.334, P=0.007). The analgesic effect reached its maximum at 45 min after administration, and the analgesic duration lasted for about 4 hours. The analgesic effect of the multi-target molecules of this invention... 50 The value was 0.1659 mg / kg. Intraperitoneal injection of maternal doses of 0.01, 0.03, and 0.1 mg / kg CR845 also produced dose-dependent analgesic effects in a mouse CFA-induced inflammatory pain model (F...). 3,20 =16.11, P<0.001), its analgesic effect reached its maximum 60 min after administration and the analgesic duration lasted for about 6 h, analgesic ED 50 The value was 0.01197 mg / kg. In summary, intraperitoneal injection of this multi-target molecule produced dose-dependent analgesia in the CFA-induced inflammatory pain model.

[0093] Example 6. Analgesic Pharmacological Study of Multi-Target Molecules

[0094] The analgesic targets of the multi-target molecule of this invention were evaluated using the κ-opioid receptor selective antagonist nor-BNI and the σ1 receptor selective agonist PRE-084. The analgesic targets of the compound were determined by detecting changes in the analgesic activity of the multi-target molecule under pretreatment with nor-BNI and PRE-084 using a formalin analgesia assay.

[0095] In the experiment, we selected male Kunming mice weighing 22±2g, and the experimental temperature was controlled at room temperature (22±2℃). Mice were allowed free access to food and water. Pretreatment with a κ-opioid receptor selective antagonist and a σ1 receptor selective agonist, PRE-084, was performed, followed by a formalin analgesia test to detect changes in the analgesic activity of the drugs after antagonist / agonist and saline pretreatment.

[0096] Differences in analgesic effects were statistically analyzed using one-way ANOVA (Bonferroni test). ** P<0.01 and *** P<0.001 indicates a significant difference between the group that received only the relevant drug and the saline group. # P<0.05, ##P<0.01 and ### P<0.001 indicates a significant difference between the group that received only the relevant drug and the group that received co-injection of nor-BNI or PRE-084.

[0097] like Figure 4 and Figure 5 As shown. Intraperitoneal nor-BNI pretreatment completely blocked the analgesic effect of the 1 mg / kg CR845 group, while also significantly antagonizing the analgesic effect of the multi-target molecule of this invention in phase II. This indicates that κ-opioid receptors are indeed involved in the analgesic effect of intraperitoneal injection of this multi-target molecule and CR845. Subcutaneous σ1 receptor agonist PRE-084 pretreatment significantly blocked the analgesic effect of the 80 mg / kg S1RA group, while also significantly attenuating the analgesic effect of the highest intraperitoneal dose of the multi-target molecule in both phases.

[0098] Example 7. Determination of sedative behavior of multi-target molecules administered intraperitoneally.

[0099] Rotarod assays are commonly used to detect sedative side effects in mice within the κ-opioid receptor system. Therefore, we used a balanced rotarod apparatus to determine whether intraperitoneal injection of the highest analgesic dose of a multi-target molecule would produce sedative side effects. The sedative behavior in mice was measured by intraperitoneal injection of the multi-target molecule, followed by a rotarod assay to measure the time mice remained on the fatigue rotarod apparatus 15 min, 30 min, 45 min, and 60 min after administration. This evaluation assessed the effect of the multi-target molecule in this patent on sedative side effects.

[0100] Male Kunming mice weighing 21±2g were used in the experiment and were allowed free access to food. Before the formal experiment, two consecutive days of training and animal selection were conducted. On the first day, the subjects were trained for 5 minutes continuously. If a mouse fell off the rotisserie, it was immediately placed back in the rotisserie and training continued. After the 5-minute training period, a 15-minute interval was observed before training resumed, and this process was repeated three times. On the second day, qualified subjects were selected, and the training was repeated twice more. For the third training, only mice that could remain on the rotisserie for ≥180 seconds were selected as subjects. On the third day, the formal experiment was conducted. The maximum analgesic dose of the multi-target molecule was injected intraperitoneally, and the time the mice remained on the corresponding rotisserie at different time points was recorded to evaluate the effect of the multi-target molecule on the sedation behavior of mice.

[0101] Experimental data are expressed as the time mice spend on the rotarod. The effects of the multi-target molecules of this invention on sedation behavior, in relation to the parent compound CR845, were compared by examining the duration of residence on the rotarod at the same administration time for different compounds. Drop latency data are expressed as mean ± standard error (Means ± SEM), and differences were statistically analyzed using one-way ANOVA (Bonferroni's test).*** P < 0.001 indicates a significant difference between the drug group and the saline group. ## P<0.01 and ### P<0.001 indicates a significant difference between the multi-target molecular group and the CR845 group.

[0102] like Figure 6 As shown, compared with the saline group, intraperitoneal injection of CR845 significantly affected the sedative behavior of mice at 15, 30, 45, and 60 minutes after administration. While intraperitoneal injection of the multi-target molecule of this invention reduced the time mice spent on the balance rotarod in the first 60 minutes after administration, this difference was not statistically significant compared to the saline group, meaning the multi-target molecule did not produce sedative behavior. In conclusion, the sedative side effects of the multi-target molecule of this invention were significantly reduced compared to the CR845 group.

[0103] Example 8. Determination of depressive side effects of intraperitoneal injection of multi-target molecules

[0104] Forced swimming and tail suspension tests are classic behavioral experimental methods for evaluating depressive-like behaviors. Therefore, we used these two behavioral tests to evaluate the potential depressive-like behaviors in mice after intraperitoneal injection of the highest analgesic dose of a multi-target molecule.

[0105] Male Kunming mice weighing between 23±2g were selected for the experiment and were allowed free access to food. For the first two days of the experiment, the mice were moved from the temporary storage room to the experimental environment for 1 hour each day. On the morning of the experiment, the mice were placed in the experimental environment again for 30 minutes. Forced swimming was conducted 30 minutes after intraperitoneal administration of the drug. It is important to note that tap water was added to the cylindrical glass beaker, and the temperature must be controlled at approximately 24℃. The liquid level must be adjusted to avoid overfilling, which would allow the animals to escape, or underfilling, which would cause the animals' hind paws to touch the bottom. The experiment lasted 6 minutes and was divided into two phases: Phase 1 (first 2 minutes): training the animals to adapt to the experimental environment and eliminating those with poor swimming ability to prevent drowning. Phase 2 (last 4 minutes): testing phase, recording the time the animals remained still during the experiment. Observers were required to remain quiet throughout the experiment and observe and time the event from a distance of at least 1.5 meters from the beaker. After the 6-minute experiment, the animals were removed, dried, and placed on an electric blanket to restore body temperature. The water in the beaker was then poured out, and the beaker was wiped with 75% alcohol to prevent any residual odor from affecting the behavior of the next mouse. After preparation, the beaker was refilled with the required amount of tap water, and a new mouse was placed in to start the next batch of experiments.

[0106] The tail suspension test used a specially manufactured tail suspension box. This device was made from a cardboard box measuring (75×60×11.5cm). To prevent animals from observing or influencing each other, each mouse was suspended in its own three-walled rectangular compartment. Male Kunming mice weighing 23±2g were selected. Two days prior to the experiment, the mice were moved to the experimental environment to allow them to fully acclimatize. On the morning of the experiment, the animals were placed in the experimental environment for 30 minutes. Thirty minutes after intraperitoneal administration, the mice were suspended in the device, 75cm above the floor. The experiment required recording the immobility time of the mice in the last 4 minutes of the 6-minute total experimental period to reflect the degree of despair. Only the time when the mouse was completely limp and immobile was recorded. This experiment uses the time it takes for the mice to give up struggling in the extreme environment to determine whether the mice develop depressive symptoms. The depressive side effect in the mice was represented by recording the total immobility time in the last 4 minutes of the 6-minute total experimental phase.

[0107] Experimental data were expressed as the time of despair stillness in mice. Drug-induced depressive side effects were compared between the experimental and saline groups using the time of despair stillness. Data are expressed as mean ± standard error (Means ± SEM), and differences were statistically analyzed using one-way ANOVA (Bonferroni's test). *** P < 0.001 indicates a significant difference between the drug group and the saline group. ## P<0.01 indicates a significant difference between this multi-target molecular group and the CR845 group.

[0108] like Figure 7 As shown, intraperitoneal injection of the highest analgesic dose of CR845 significantly increased the immobility time in the forced swimming test (A) and tail suspension test (B) in mice compared to the saline group, indicating increased despair and significantly enhanced depressive-like behavior. However, intraperitoneal injection of the highest analgesic dose of the multi-target molecule did not result in a significant difference in immobility time between the mice in the forced swimming test (A) and tail suspension test (B) and the saline group. In conclusion, intraperitoneal injection of the multi-target molecule significantly reduced the immobility time in the forced swimming test (A) and tail suspension test (B) in mice compared to the CR845 group, indicating a significant reduction in the depressive side effects of the multi-target molecule in this invention.

[0109] Example 9. Determination of analgesic tolerance after intraperitoneal injection of multi-target molecules

[0110] The analgesic tolerance test evaluates the pharmacological activity of the multi-target molecule in this patent in terms of analgesia tolerance by observing the changes in the photothermal withdrawal latency of the drug in a photothermal withdrawal test after 8 consecutive days of drug administration. Male Kunming mice weighing 20–22 g were used in the experiment. Mice were intraperitoneally injected with the same concentration of drug once daily at the same time for 8 consecutive days, and the changes in withdrawal latency after each daily injection were measured.

[0111] Experimental data are expressed as claw withdrawal time. The analgesic tolerance of the drugs was compared using the claw withdrawal latency at the time point of maximum analgesic effect for different compounds. Claw withdrawal latency data are expressed as mean ± standard error (Means ± SEM). Differences in analgesic effect after 8 days of continuous intraperitoneal administration in mice were statistically analyzed using one-way ANOVA (Tukey's HSD test). ** P<0.01 and *** P<0.001 indicates a highly significant difference compared to the analgesic effect of the drug administered on the first day.

[0112] like Figure 8 As shown, compared with the saline group, the maximum analgesic percentage of the intraperitoneal injection multi-target molecule did not differ significantly between day 1 and day 8 after administration, indicating that it produced analgesia without tolerance. Similarly, the maximum analgesic percentage of the parent compound CR845 did not show a statistically significant difference between day 1 and day 8 after administration, meaning it did not exhibit analgesic tolerance. Therefore, consistent with CR845, the multi-target molecule in this invention produced analgesia without tolerance.

[0113] The above detailed description of the present invention does not limit the invention; those skilled in the art can make various modifications based on the present invention. Any changes or modifications made that do not depart from the spirit of this invention shall fall within the scope of the appended claims.

Claims

1. A peptide-based multi-target ligand molecule constructed with the kappa-opioid receptor agonist CR845 and the sigma 1 receptor antagonist S1RA as chemical template molecules, characterized in that, The sequence structure of the peptide multi-target ligand molecule is shown as formula I Ⅰ。 2. The method for preparing peptide multi-target ligand molecules according to claim 1, characterized in that, The synthesis is performed by combining Fmoc solid-phase synthesis method and liquid-phase synthesis method: (a) the side chain-protected N-terminal tetrapeptide Boc-D-Phe-D-Phe-D-Leu-D-Lys(Boc)-OH in the peptide multi-target ligand molecule of claim 1 is synthesized by Fmoc solid-phase synthesis method, (b) the condensation of the piperazinyl-modified S1RA analogue 1-(2-{[5-methyl-1-(naphthalen-2-yl)pyrazol-3-yl]oxy}ethyl)piperazine with the peptide segment is synthesized by liquid-phase synthesis method, thereby obtaining the peptide multi-target ligand molecule of claim 1 ⅠⅠ。 3. The method for preparing peptide multi-target ligand molecules according to claim 2, characterized in that, The Fmoc solid-phase polypeptide synthesis method comprises the following process steps: resin swelling, amino acid condensation, peptide chain extension, peptide chain compression, extraction, precipitation, purification and chemical identification.

4. The method for preparing peptide multi-target ligand molecules according to claim 3, characterized in that, The resin is 2-chlorotrityl chloride resin.

5. The method for preparing peptide multi-target ligand molecules according to claim 3, characterized in that, The condensing reagent used in the amino acid condensation is a combination of HOBt, HBTU and DIEA with a molar ratio of 1:1:

2.

6. The method for preparing peptide multi-target ligand molecules according to claim 2, characterized in that, The synthesis of the piperazinyl-modified S1RA analogue comprises the following steps: synthesis of N-(naphthalen-2-yl)acetohydrazide, synthesis of 5-methyl-1-(naphthalen-2-yl)pyrazol-3-ol and synthesis of 1-(2-{[5-methyl-1-(naphthalen-2-yl)pyrazol-3-yl]oxy}ethyl)piperazine.

7. The method for preparing peptide multi-target ligand molecules according to claim 2, characterized in that, The liquid-phase polypeptide synthesis method comprises the following process steps: condensation of the piperazinyl-modified S1RA analogue with the peptide segment, removal of the Boc protecting group, and extraction, precipitation, purification and chemical identification of the crude peptide.

8. A pharmaceutical composition containing the peptide multi-target ligand molecule constructed with kappa-opioid receptor agonist CR845 and sigma 1 receptor antagonist S1RA as chemical template molecules according to claim 1.

9. Use of the peptide multi-target ligand molecule of claim 1 in the preparation of an analgesic drug.

Images