Kappa opioid receptor agonist and its use.

Description

1 / 34 Opioid receptor κ agonist and its use.

[0001] This application claims priority over Chinese Patent Application No. 202310371594.6, filed with the China National Intellectual Property Administration on April 10, 2023, and entitled “LONG-ACTING K-OPIOID RECEPTOR AGONIST”, and Chinese Patent Application No. 202410112984.6, filed with the China National Intellectual Property Administration on January 26, 2024, and entitled “LONG-ACTING K-OPIOID RECEPTOR AGONIST”, which are incorporated herein by reference in their entirety. FIELD

[0002] The present disclosure relates to the field of biomedical technology and, in particular, to a highly selective κ opioid receptor agonist and its use. BACKGROUND

[0003] Opioid drugs exert physiological effects primarily through binding to three classic opioid receptors known as μ, δ, and κ. These three receptors are all members of the G protein-coupled receptor family, are primarily distributed in the central nervous system, and also exist in many peripheral tissues.

[0004] The most classic of these drugs is morphine, which exerts an analgesic effect primarily through the μ-opioid receptor. Clinically common analgesic medications also include other μ-opioid receptor drugs, such as traditional opioid medications as represented or dihydromorphone and fentanyl. However, long-term use of μ-opioid receptor drugs can lead to multiple side effects, such as tolerance, dependence, hypoventilation, effects on gastrointestinal motility, and the like, which not only increase treatment costs but also affect patient recovery periods. Some non-opioid injections, such as acetaminophen and nonsteroidal anti-inflammatory drugs, have ranges and Petition 870250091684, dated 07 / 10 / 2025, p. 53 / 199 2 / 34 application dosages are limited due to their weak analgesic effects; additionally, they also have certain side effects, for example, acetaminophen increases liver toxicity, and non-steroidal anti-inflammatory drugs can cause various gastrointestinal disorders.

[0005] The κ-opioid receptor exists in the brain, spinal cord, central and peripheral nerve endings, somatic and visceral sensory afferent nerve cell bodies, and immune cells. Research has found that, using κ-opioid receptor agonists, the κ-opioid receptor can be used as a therapeutic target to treat pain and prevent a wide variety of diseases and conditions, for example, the treatment of pain including hyperalgesia, use in eye disorders and eye pain, treatment of pruritus caused by uremia and opioids, and the like.

[0006] Difelicefalin (trade name KORSUVA) is a first-class, highly selective full κ-opioid receptor (KOR) agonist that can inhibit the activity of pruritus-producing peripheral neurons and has no significant activity at other receptors (including μ or δ opioid receptors), ion channels, or transporters. Furthermore, unlike small-molecule KOR agonists, Korsuva is a small synthetic peptide that primarily activates KORs expressed by peripheral neurons (PNS) and immune cells. The U.S. FDA has approved Difelicefalin for marketing for the treatment of moderate to severe pruritus related to chronic kidney disease in adults (CKD-aP) with underlying hemodialysis (HD). Difelicefalin has exhibited antipruritic and analgesic properties, as well as inhibition of acute postoperative pain (IV preparations in laparoscopic hysterectomy and ventral hernia and bunion resection), in several different Phase II or III studies.

[0007] The present disclosure is intended to provide a highly active κ opioid receptor agonist. SUMMARY

[0008] One objective of the present disclosure is to provide a class of peptide compounds that have high selectivity for the κ opioid receptor and are innovative, Petition 870250091684, dated 07 / 10 / 2025, page 54 / 199 3 / 34 highly active κ opioid receptor agonists. A further objective of the present disclosure is to provide a pharmaceutical composition. Another objective of the present disclosure is to provide a use of the compounds and the pharmaceutical composition.

[0009] To achieve the above objectives, the present disclosure provides the following technical solutions:

[0010] In a first aspect, the present disclosure provides a κ opioid receptor agonist having a structure as represented by formula I, a stereoisomer thereof and a pharmaceutically acceptable salt thereof: D-Phe-AA1-AA2-D-Lys-4-aminopiperidine-4-carboxamide-AA3-AA4-AA5 (formula I)

[0011] where AA1 is modified or unmodified D-Phe or modified or unmodified D-Tyr;

[0012] AA2 is D-Leu or D-cyclopropylalanine;

[0013] AA3 is -(PEGm1(CH2)m2CO)m3-, or (AA6)m4, or is absent, where:

[0014] m1 is an integer in the range of 1 to 10;

[0015] m2 is an integer in the range of 1 to 5;

[0016] m3 is an integer in the range of 1 to 5;

[0017] m4 is an integer in the range of 1 to 5;

[0018] AA6 is AEEA, or Gly, or D-Ala, or L-Ala, or D-Leu, or L-Leu, or D-Phe, or L-Phe, or D-Ser, or L-Ser, or D-Thr, or L-Thr, or D-Tyr, or L-Tyr, or D-Asp, or L-Asp, or D-Glu, or L-Glu, or D-Gln, or L-Gln, or D-Lys, or L-Lys, or D-Arg, or L-Arg, or D-His or L-His;

[0019] AA4 is (AA7)n, or is absent, where:

[0020] n is an integer in the range of 1 to 10;

[0021] AA7 is D-Lys, or L-Lys, or D-Dap, or L-Dap, or D-Dab, or L-Dab, or DOrn, or L-Orn, or D-Dah, or L-Dah, or D-Dao or L-Dao;

[0022] AA5 is OH or NH2.

[0023] Preferably, AA1 is modified D-Phe, and the modified D-Phe is selected from the group consisting of D-Phe(2-F), D-Phe(4-F), D-Phe(2Petition 870250091684, dated 07 / 10 / 2025, page 55 / 199 4 / 34 Cl), D-Phe(4-Cl), D-Phe(2-Br), D-Phe(4-Br), D-Phe(2-I), D-Phe(4-I), D-Phe(4-Me), D-Phe(4-Et), D-Phe(4-Ipr), D-Phe(4-NH2), D-Phe(4-NHCH3), D-Phe(4NHCH2CH3), D-Phe(4-NHCH(CH3)2), D-Phe(4-N(CH3)2) and D-Phe(4-N(CH3) CH2CH3).

[0024] Preferably, AA1 is modified D-Tyr, and the modified D-Tyr is selected from the group consisting of D-Tyr(Me), D-Tyr(Et), D-Tyr(CH2CF3) and D-Tyr(Ipr).

[0025] Additionally, the κ opioid receptor agonist is a compound having a structure as represented by formula II, a stereoisomer thereof, and a pharmaceutically acceptable salt thereof: D-Phe-AA1-AA2-D-Lys-4-aminopiperidine-4-carboxamide-R-(AA8)p-NH2(OH) (formula II)

[0026] Unless otherwise specified, in the structural formulas of the present disclosure, “-NH2(OH)” represents -NH2 or -OH.

[0027] Where, AA1 is modified D-Phe or modified D-Tyr, where specific modifications are as described above;

[0028] AA2 is D-Leu or D-cyclopropylalanine;

[0029] R is present or absent, and when R is present, R is selected from the group consisting of -PEGm1CH2CO-, -AEEAn1- and -Glyn2-; wherein:

[0030] m1 is an integer in the range of 1 to 5, and specifically, is selected from the group consisting of 1, 2, 3, 4 and 5;

[0031] πι is an integer in the range of 1 to 5, and specifically, it is selected from the group consisting of 1, 2, 3, 4 and 5;

[0032] Ϡ2 is an integer in the range of 1 to 5, and specifically, it is selected from the group consisting of 1, 2, 3, 4 and 5;

[0033] AA8 is Lys or D-Lys; p is an integer in the range of 0 to 6, and specifically, is selected from the group consisting of 0, 1, 2, 3, 4, 5 and 6; when p is 0, AA8 is absent.

[0034] In an optional embodiment of the present disclosure, the κ opioid receptor agonist is a compound having a structure as represented Petition 870250091684, dated 07 / 10 / 2025, p. 56 / 199 5 / 34 by formula III, a stereoisomer thereof and a pharmaceutically acceptable salt thereof: D-Phe-D-Tyr(X)-D-Leu-D-Lys-4-aminopiperidine-4-carboxamide-R1-(AA8)p1NH2(OH) (formula III)

[0035] where D-tyr(X) represents modified D-Tyr; the modified D-Tyr is selected from the group consisting of D-Tyr(Me), D-Tyr(Et), D-Tyr(CH2CF3) and D-Tyr(Ipr);

[0036] Ri is present or absent; when Ri is present, Ri is selected from the group consisting of -PEGk1CH2CO- and -Glyn2-; where:

[0037] k1 is an integer in the range of 1 to 5, and specifically, it is selected from the group consisting of 1, 2, 3, 4 and 5;

[0038] n2 is an integer in the range of 1 to 5, and specifically, it is selected from the group consisting of 1, 2, 3, 4 and 5;

[0039] AA8 is Lys or D-Lys; p1 is an integer in the range of 0 to 6, and specifically, is selected from the group consisting of 0, 1, 2, 3, 4, 5 and 6; when p2 is 0, AA8 is absent.

[0040] In an optional embodiment of the present disclosure, the κ opioid receptor agonist is a compound having a structure as represented by formula IV, a stereoisomer thereof and a pharmaceutically acceptable salt thereof: D-Phe-D-Tyr(X)-D-cyclopropylalanine-D-Lys-4-aminopiperidine-4-carboxamide -R2-(AA8)p2-NH2(OH) (formula IV)

[0041] where D-tyr(X) represents modified D-Tyr; the modified D-Tyr is selected from the group consisting of D-Tyr(Me), D-Tyr(Et) and D-Tyr(Ipr).

[0042] R2 is either present or absent; when R2 is present, R2 is PEGk2CH2CO-; where:

[0043] k2 is an integer in the range of 1 to 5, and specifically, it is selected from the group consisting of 1, 2, 3, 4 and 5; Petition 870250091684, dated 07 / 10 / 2025, p. 57 / 199 6 / 34

[0044] in the most preferred embodiments of the present disclosure, k2 is 1, 2 or 3;

[0045] AA8 is Lys or D-Lys; p2 is an integer in the range of 0 to 6, and specifically, it is selected from the group consisting of 0, 1, 2, 3, 4, 5 and 6; when p2 is 0, AA8 is absent;

[0046] in maximum preference modalities of the present disclosure, p2 is 0 or 1.

[0047] In an optional embodiment of the present disclosure, the κ opioid receptor agonist is a compound having a structure as represented by formula V, a stereoisomer thereof and a pharmaceutically acceptable salt thereof: D-Phe-D-Phe(X)-D-Leu-D-Lys-4-aminopiperidine-4-carboxamide-(AA8)p3NH2(OH) (formula V)

[0048] where, D-Phe(X) represents modified D-Phe; the modified D-Phe is selected from the group consisting of D-Phe(2-F), D-Phe(4-F), D-Phe(2Cl), D-Phe(4-Cl), D-Phe(2-Br), D-Phe(4-Br), D-Phe(2-I), D-Phe(4-I), D-Phe(4-Me), D-Phe(4-Et), D-Phe(4-Ipr), D-Phe(4-NH2).

[0049] AA8 is Lys or D-Lys; p3 is an integer in the range of 0 to 6, and specifically, it is selected from the group consisting of 0, 1, 2, 3, 4, 5 and 6; when p3 is 0, AA8 is absent;

[0050] in maximum preference modalities of the present disclosure, p3 is 0 or 1.

[0051] with additional preference, the κ opioid receptor agonist has one or more structural formulas selected from the group consisting of 1) to 10):

[0052] 1) D-Phe-D-Tyr(X)-D-Leu-D-Lys-4-aminopiperidine-4-carboxylic acidNH2(OH)

[0053] 2) D-Phe-D-Tyr(X)-D-Leu-D-Lys-4-aminopiperidine-4-carboxamideNH2(OH) Petition 870250091684, dated 07 / 10 / 2025, p. 58 / 199 7 / 34

[0054] 3) D-Phe-D-Tyr(X)-D-Leu-D-Lys-4-aminopiperidine-4-carboxamide(AA8)q-NH2(OH)

[0055] 4) D-Phe-D-Tyr(X)-D-Leu-D-Lys-4-aminopiperidine-4-carboxamidaPEGkiCH2CO-AA8-NH2(OH)

[0056] 5) D-Phe-D-Tyr(X)-D-Leu-D-Lys-4-aminopiperidine-4-carboxamidaGlyn2-AA8-NH2(OH)

[0057] 6) D-Phe-D-Tyr(X)-D-cyclopropylalanine-D-Lys-4-aminopiperidine-4-carboxamide-NH2(OH)

[0058] 7) D-Phe-D-Tyr(X)-D-cyclopropylalanine-D-Lys-4-aminopiperidine-4carboxamide-AA8-NH2(OH)

[0059] 8) D-Phe-D-Tyr(X)-D-cyclopropylalanine-D-Lys-4-aminopiperidine-4carboxamide-PEGk2CH2CO-AA8-NH2(OH)

[0060] 9) D-Phe-D-Phe(X)-D-Leu-D-Lys-4-aminopiperidine-4-carboxamideNH2(OH)

[0061] 10) D-Phe-D-Phe(X)-D-Leu-D-Lys-4-aminopiperidine-4-carboxamideAA8-NH2(OH),

[0062] In the structural formulas mentioned above, AA8 is Lys or D-Lys; where:

[0063] in formula 1), D-Tyr(X) is D-Tyr(Et);

[0064] in formulas 2) to 3), D-Tyr(X) is D-Tyr(Me) or D-Tyr(Et) or DTyr(CH2CF3) or D-Tyr(Ipr), and q is 1, or 2, or 3, or 4, or 5, or 6;

[0065] in formulas 4) to 8), D-Tyr(X) is D-Tyr(Et),

[0066] k1 is 1, or 2, or 3, or 4, or 5;

[0067] n2 is 1, or 2, or 3, or 4, or 5;

[0068] k2 is 1, or 2, or 3;

[0069] in formula 9), D-Phe(X) is D-Phe(4-F) or D-Phe(4-Cl) or D-Phe(4-Br) or D-Phe(4-I);

[0070] in formula 10), D-Phe(X) is D-Phe(4-F) or D-Phe(4-Cl) or D-Phe(4-Br) or D-Phe(4-I) or D-Phe(4-Me) or D-Phe(4-Et) or D-Phe(4-Ipr) or D-Phe(4-NH2);

[0071] In a second aspect, the present disclosure provides a Petition 870250091684, dated 07 / 10 / 2025, p. 59 / 199 8 / 34 pharmaceutical composition, wherein the pharmaceutical composition comprises the κ opioid receptor agonist provided in the present disclosure, the stereoisomer thereof and the pharmaceutically acceptable salt thereof.

[0072] Additionally, the pharmaceutical composition further comprises a pharmaceutically acceptable carrier.

[0073] In a third aspect, the present disclosure provides the use of the κ opioid receptor agonist, the stereoisomer, and the pharmaceutically acceptable salt thereof, and / or the pharmaceutical composition, in the manufacture of a medicament to prevent and / or relieve pruritus and / or an analgesic.

[0074] The κ-opioid receptor agonist provided in this disclosure has high selectivity for the κ-opioid receptor, with activity 3 to 15 times that of the commercially available drug Difelicefallina. The high drug activity makes it useful for treating, preventing and / or relieving pruritus and / or combating pain. DETAILED DESCRIPTION

[0075] As used in the present disclosure, the term stereoisomer refers to compounds that have the same chemical constitution but differ in the spatial arrangement of their atoms or groups. Stereoisomers include, but are not limited to, enantiomers, diastereomers, conformational isomers (rotamers), geometric isomers (cis / trans), and atropisomers.

[0076] The pharmaceutical composition described in this disclosure may refer to a composition for the treatment of a disease or for use in in vitro cell culture experiments. When intended for the treatment of a disease, the term pharmaceutical composition typically refers to a unit dosage form and may be prepared by any of the methods well known in the field of pharmacy. In general, all such methods include the step of combining the active ingredient with an excipient that constitutes one or more auxiliary components.

[0077] As used in this document, the term pharmaceutically acceptable refers to a substance or composition that is chemically and / or toxicologically compatible with the other ingredients of a formulation and / or Petition 870250091684, dated 07 / 10 / 2025, page 60 / 199 9 / 34 to the mammal to be treated with it. Preferably, pharmaceutically acceptable refers to what is approved by a regulatory agency of a federal or national government or listed in the U.S. Pharmacopoeia or other generally recognized pharmacopoeia for use in animals, and more particularly in humans.

[0078] Specifically, the term pharmaceutically acceptable carrier may include any and all solvents, solid excipients, diluents, liquid vehicles and the like, suitable for the particular dosage form desired. The use of any conventional carriers is also contemplated by this disclosure, except to the extent that they are incompatible with the κ opioid receptor agonist provided by this disclosure, for example, by producing any undesirable biological effect or otherwise interacting in a detrimental manner with any other component of the pharmaceutically acceptable composition.

[0079] The technical solutions in the embodiments of the present disclosure will be described clearly and completely below. It should be understood that the embodiments described are merely some, but not all, of the embodiments of the present disclosure. Based on the embodiments in the present disclosure, all other embodiments obtained by a person of common knowledge in the art without exercising creative effort shall fall within the scope of protection of the present disclosure.

[0080] As used in this document, the terms alleviate, improve, or mitigate may be used interchangeably. These terms refer to a method of achieving a beneficial or desired result, including, but not limited to, a therapeutic benefit. Therapeutic benefit means the eradication or improvement of the underlying disorder being treated. As used in this document, a therapeutic benefit is achieved by the eradication or improvement of one or more physiological symptoms associated with the underlying disorder, such that an improvement is observed in the individual, even though the individual is still afflicted with the underlying disorder. Petition 870250091684, dated 07 / 10 / 2025, page 61 / 199 10 / 34

[0081] The present disclosure provides a κ opioid receptor agonist and uses thereof. A person skilled in the art may, by reference to the contents of this disclosure, appropriately modify the relevant parameters to practice the present disclosure. Although the methods of the present disclosure have been described by preferred examples, it will be evident to those skilled in the art that appropriate modifications, or variations and combinations, can be made to the compounds and methods of preparation described herein without departing from the content, spirit and scope of the present disclosure to realize and apply the technology of the present disclosure.

[0082] The full names corresponding to the English abbreviations used in this disclosure are shown in the table below: Table 1 English Abbreviations Full Name English Abbreviations Full Name Fmoc 9-fluorenylmethyloxycarbonyl a OtBu tert-butoxy tBu tert-butyl Boc tert-butoxycarbonyl Trt trityl Pbf (2,3-dihydro-2,2,4,6,7-pentamethylbenzofuran-5-yl)sulfonyl Ala alanine Lys lysine Arg arginine Phe phenylalanine Asn asparagine Ser serine Gln glutamine Thr threonine Glu glutamic acid Trp tryptophan Petition 870250091684, dated 07 / 10 / 2025, page 62 / 199 11 / 34 English Abbreviations Full Name English Abbreviations Full Name Gly glycine Tyr tyrosine His histidine Val valine Leu leucine Dap 2,3-diaminopropionic acid 5-Ava 5-aminovaleric acid Dab 2,4-diaminobutyric acid Ada 2-aminoadipic acid Orn ornithine Apm 2-aminopimelic acid Dah 2,7-diaminoheptanoic acid Asu 2-aminosuberic acid Dao 2,8-diaminooctanoic acid

[0083] Example 1: preparation of compounds

[0084] The preparation method is solid-phase polypeptide synthesis, which comprises: preparing a peptide resin by solid-phase polypeptide synthesis, cleaving the peptide resin with an acid to obtain a crude product, and finally purifying the crude product to obtain a pure product. In which, the step of preparing the peptide resin by solid-phase polypeptide synthesis involves sequentially coupling the corresponding protected amino acids of the following sequence onto a carrier resin via a solid-phase coupling synthesis method to prepare the peptide resin:

[0085] In the preparation method mentioned above, the Fmoc-protected amino acid is used in an amount of 1.2 to 6 times the total molar amount of resin used; preferably 2.5 to 3.5 times.

[0086] In the preparation method mentioned above, the carrier resin has a substitution value of 0.3 to 1.5 mmol / g resin, preferably 0.6 to 1.0 Petition 870250091684, dated 07 / 10 / 2025, page 63 / 199 12 / 34 mmol / g resin.

[0087] In a preferred embodiment of the disclosure, the solid-phase coupling synthesis method is as follows: the protecting group Fmoc is removed from the protected amino acid resin obtained from the previous step, followed by a coupling reaction with the next protected amino acid. The deprotection step of removing the Fmoc group lasted from 10 to 60 min, preferably 15 to 25 min. The coupling reaction lasted from 60 to 300 min, preferably 100 to 140 min.

[0088] The coupling reaction requires the addition of a condensation reagent. The condensation reagent is selected from the group consisting of DIC (N,N-di-isopropylcarbodiimide), N,N-dicyclohexylcarbodiimide, benzotriazol-1-yl-oxytripyrrolidinophosphonium hexafluorophosphate, 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate, benzotriazol-N,N,N',N'-tetramethyluronium hexafluorophosphate and O-(benzotriazol)N,N,N',N'-tetramethyluronium tetrafluoroborate. N,N-di-isopropylcarbodiimide is preferred. The condensation reagent is used in a molar quantity of 1.2 to 6 times the total molar quantity of amino groups in the amino resin, preferably 2.5 to 3.5 times.

[0089] The coupling reaction requires the addition of an activating agent. The activating agent is selected from the group consisting of 1-hydroxybenzotriazole and N-hydroxy-7-azabenzotriazole, preferably 1-hydroxybenzotriazole. The activating agent is used in an amount of 1.2 to 6 times the total molar quantity of amino groups in the amino resin, preferably 2.5 to 3.5 times.

[0090] In a preferred embodiment of the present disclosure, the reagent for removing the Fmoc protecting group is a mixed solution of PIP / DMF (piperidine / N,N-dimethylformamide), wherein the mixed solution contains 10% to 30% (V) piperidine. The amount of Fmoc deprotecting reagent used is 5 to 15 ml per gram of amino resin, preferably 8 to 12 ml per gram of amino resin. Petition 870250091684, dated 07 / 10 / 2025, page 64 / 199 13 / 34

[0091] More preferably, the acidolysis agent used in the acidolysis step of the peptide resin is a mixed solvent of trifluoroacetic acid (TFA), 1,2-ethanedithiol (EDT) and water, wherein the mixed solvent has a volume ratio of: 80% to 95% TFA, 1% to 10% EDT, with the remainder being water.

[0092] Even more preferably, the mixed solvent has a volume ratio of: 89% to 91% TFA, 4% to 6% EDT, with the remainder being water. Most preferably, the mixed solvent has a volume ratio of: 90% TFA, 5% EDT, with the remainder being water.

[0093] The acidolysis agent is used in an amount of 4 to 15 ml per gram of peptide resin; preferably, 7 to 10 ml per gram of peptide resin.

[0094] The cleavage step using the acidolysis agent lasts 1 to 6 hours, preferably 3 to 4 hours, at room temperature.

[0095] Additionally, the crude product is purified by high-performance liquid chromatography and then lyophilized to obtain the pure product.

[0096] 1. Peptide resin synthesis

[0097] The peptide resin was prepared by taking a carrier resin and sequentially coupling the corresponding protected amino acids of the sequence via Fmoc coupling and deprotection reactions:

[0098] (1) Coupling of the first protected amino acid of the main chain

[0099] 0.03 mol of the first protected amino acid and 0.03 mol of HOBt were dissolved in an appropriate amount of DMF. Separately, 0.03 mol of DIC was slowly added to the DMF solution of the protected amino acid with stirring. The reaction was carried out at room temperature with stirring for 30 min to obtain an activated protected amino acid solution, which was reserved for later use.

[00100] 0.01 mol of carrier resin (substitution value was approximately 0.4 mmol / g) was taken and deprotected by treatment with Petition 870250091684, dated 07 / 10 / 2025, page 65 / 199 14 / 34 a 20% PIP / DMF solution for 25 min. The resin, unprotected by Fmoc, was obtained after washing and filtration.

[00101] The activated protected amino acid solution was added to the unprotected resin by Fmoc, and the coupling reaction was carried out for 60 to 300 min. The resin containing a protected amino acid was obtained after filtration and washing.

[00102] (2) Coupling of other protected amino acids

[00103] Using the same method as for coupling the first amino acid in the sequence, the other corresponding protected amino acids in the sequence were sequentially coupled to obtain a resin containing the peptides.

[00104] 2. Preparation of the raw product

[00105] The peptide resin obtained above was treated with a cleavage reagent (10 ml of cleavage reagent per gram of resin) with a TFA:water:EDT volume ratio of 95:5:5. The mixture was stirred uniformly and allowed to react at room temperature for 3 h. The reaction mixture was filtered through a frit funnel, and the filtrate was collected. The resin was washed three times with a small amount of TFA. The filtrates were combined and concentrated under reduced pressure. Anhydrous diethyl ether was added to precipitate the peptide. Then, the precipitate was washed three times with anhydrous diethyl ether and then dried under vacuum to produce a whitish powder.

[00106] 3. Preparation of the pure product

[00107] The crude product concentrate obtained above was purified by filtration through a mixed microporous filter membrane of 0.45 μm and reserved for later use.

[00108] Purification was performed using high-performance liquid chromatography. The chromatographic packing material for purification was 10 μm of reversed-phase C18 material, and the mobile phase system consisted of 0.1% TFA / water and 0.1% TFA / acetonitrile. For a 30 mm*250 mm chromatography column, the flow rate was 20 ml / min. An elution purification of Petition 870250091684, dated 07 / 10 / 2025, page 66 / 199 A 15 / 34 gradient and cyclic loading technique was used. The crude product solution was loaded onto the column, and the mobile phase was initiated for elution. The main peak was collected, and after removing acetonitrile by evaporation, a purified intermediate concentrate was obtained.

[00109] The purified intermediate concentrate was filtered through a 0.45 μm membrane and reserved for later use. Salt exchange was performed using high-performance liquid chromatography. The mobile phase system was 1% acetic acid / water-acetonitrile, and the chromatographic packing material for purification was 10 μm of C18 reversed-phase material. For a 30 mm*250 mm chromatography column, the flow rate was 20 ml / min (the flow rate can be adjusted according to different chromatography column specifications). A gradient elution and cyclic loading method was used. The sample was loaded onto the column, the mobile phase was initiated for elution, and chromatograms were collected. Changes in absorbance were observed, and the main peak of the salt exchange was collected. Purity was verified by analytical liquid chromatography.The fractions containing the main peak of salt exchange were combined, concentrated under reduced pressure to obtain an aqueous solution of purified product in acetic acid, and then lyophilized to obtain the pure product.

[00110] Using the method described above, the following compounds were synthesized: Table 2 Compound Number Sequence Diphenylkephalin D-Phe-D-Phe-D-Leu-D-Lys-4-aminopiperidine-4-carboxylic acid-OH Compound 1 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidine-4-carboxylic acid-NH2 Compound 2 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidine-4-carboxylic acid Petition 870250091684, dated 07 / 10 / 2025, p. 67 / 199 16 / 34 Compound Number Sequence carboxylic-OH Compound 3 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-Lys-NH2 Compound 4 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-Aminoperidine-Lyscarboxamide-4opost-OH 5 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-Lys-Lys-NH2 Compound 6 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-Lys-OH Compound Lys-7 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-Lys-Lys-Lys-NH2 Compound 8 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-Aminopiperidine-4carboxamide-Lys-Lys-OH-OH Compound 9 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-Lys-Lys-Lys- Lys-NH2 Compound 10 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-Aminopiperidine-4carboxamide-Lys-1-OH-Lys-O Compound D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-Aminopiperidine-4carboxamide-Lys-Lys-Lys- Lys- Lys- NH2 Compound 12 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-Aminopiperidine-4carboxamida-Lys-Lys-OH-Lys-OH Compound 13D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-Lys-Lys-Lys-Lys-Lys-Lys-NH2 Compound 14 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-Lys-Lys-Lys-Lys-Lys-Lys-OH Compound 15 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidine-4D-Lys-D-H2-Lys Petition 870250091684, of 07 / 10 / 2025, p. 68 / 199 17 / 34 Compound Number Sequence Compound 16 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-D-Lys-OH Compound 17 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-D-Lys-D-Lys-NH2 Compound 18 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-D-Lys-D-OH D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-Aminopiperidine-4carboxamide-D-Lys-D-Lys-D-Lys-NH2 Compound 20 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidine-4carboxamida-D-Lys-D-Lys-D-Lys-D-Lys-D-Lys-D-Lys-NH2 Compound 21 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-D-Lys-D-Lys-D-Lys-D-Lys -NH2 Compound 22 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-D-Lys-D-Lys-D-Lys-D-Lys -OH Compound 23 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-D-Lys-D-Lys-D-Lys-D-Lys -D-Lys-NH2 Compound 24 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-D-Lys-D-Lys-D-Lys-D-Lys -D-Lys-OH Compound 25 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidine-4-piperidine carboxamide-D-Lys-D-Lys-D-Lys-D-Lys -D-Lys-D-Lys-NH2 Compound26 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidine-4- carboxamide-D-Lys-D-Lys-D-Lys-D-Lys -D-Lys-D-Lys-OH Compound 27 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-PEG1CH2CO-Lys-NH2 Compound 28 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-PEG1CO-CH-OH-OH2 Compound 29 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidine-4- Petition 870250091684, of 07 / 10 / 2025, p. 69 / 199 18 / 34 Compound Number Sequence carboxamide-PEGiCH2CO-D-Lys-NH2 Compound 30 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-PEGiCH2CO-D-Lys-OH Compound 31 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide- PEG2CH2CO -Lys-NH2 Compound 32 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-Aminopiperidine-4carboxamide- PEG2CHCO D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide- PEG2CH2CO -D-Lys-NH2 Compound 34 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-Aminopiperidine-4carboxamide-PEG-CHD-CHD-4-CO35 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-PEG3CH2CO-Lys-NH2 Compound 36 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-PEG3CH-OH-OH32 Compound D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-PEG3CH2CO-D-Lys-NH2 Compound 38 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-Aminopiperidine-4carboxamide-PEG-CHD-3-CHD-3-CO D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-PEG4CH2CO-Lys-NH2 Compound 40D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-PEG4CH2CO-Lys-OH Compound 41 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-PEG4CH2CO-D-Lys-OH Petition 870250091684, dated 07 / 10 / 2025, p. 70 / 199 19 / 34 Compound Number Sequence Compound 43 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-PEG5CH2CO-Lys-NH2 Compound 44 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-PEG5CH2CO-Lys-OH Compound 45 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-PEG5CH2CO-D-Lys-NH2 Compound 46 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-PEG5CH2CO-D-Lys-OH Compound 47 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-Gly-Lys-NH2 Compound 48 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-Gly-Lys-OH Compound 49 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-Gly-D-Lys-NH2 Compound 50 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-Gly-Gly-Lys-NH2 Compound 52 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-Gly-Gly-D-Lys-NH2 Compound 54D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-Aminopiperidine-4carboxamide-Gly-Gly-D-Lys-OH Composto 55 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-Aminopiperidine-4carboxamida-Gly-Gly-Gly-Lys-NH52 Composto D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidine-4- Petition 870250091684, of 07 / 10 / 2025, p. 71 / 199 20 / 34 Número de Composto Sequência carboxamida-Gly-Gly-Gly-Lys-OH Composto 57 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidina-4carboxamida-Gly-Gly-Gly-D-Lys-NH2 Composto 58 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidina-4carboxamida-Gly-Gly-Gly-D-Lys-OH Composto 59 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidina-4carboxamida-Gly-Gly-Gly-Gly-Lys-NH2 Composto 60 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidina-4carboxam ida-Gly-Gly-Gly-Gly-Lys-OH Composto 61 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidina-4carboxamida-Gly-Gly-Gly-Gly-D-Lys-NH2 Composto 62 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidina-4carboxamida-Gly-Gly-Gly-Gly-D-Lys-OH Composto 63 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidina-4carboxamida-Gly-Gly-Gly-Gly-Gly-Lys-NH2 Composto 64 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidina-4carboxamida-Gly-Gly-Gly-Gly-Gly-Lys-OH Composto 65 D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidina-4carboxamida-Gly-Gly-Gly-Gly-Gly-D-Lys-NH2 Composto 66D-Phe-D-Tyr(Et)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-Gly-Gly-Gly-Gly-Gly-D-Lys-OH Compound 67 D-Phe-D-Tyr(Et)-D-cyclopropylalanine-D-Lys-4aminopiperidine-4-carboxamide-OH Compound 69 D-Phe-D-Tyr(Et)-D-cyclopropylalanine-D-Lys-4aminopiperidine-4-carboxamide-Lys-NH2 Petition 870250091684, dated 07 / 10 / 2025, p. 72 / 199 21 / 34 Compound Number Sequence Compound 70 D-Phe-D-Tyr(Et)-D-cyclopropylalanine-D-Lys-4-aminopiperidine-4-carboxamide-Lys-OH Compound 71 D-Phe-D-Tyr(Et)-D-cyclopropylalanine-D-Lys-4-aminopiperidine-4-carboxamide-D-Lys-NH2 Compound 72 D-Phe-D-Tyr(Et)-D-cyclopropylalanine-D-Lys-4-aminopiperidine-4-carboxamide-D-Lys-OH Compound 73 D-Phe-D-Tyr(Et)-D-cyclopropylalanine-D-Lys-4-aminopiperidine-4-carboxamide-PEGiCH2CO-Lys-NH2 Compound 74 D-Phe-D-Tyr(Et)-D-cyclopropylalanine-D-Lys-4- aminopiperidine-4-carboxamide- PEGiCH2CO-Lys-OH Compound 75 D-Phe-D-Tyr(Et)-D-cyclopropylalanine-D-Lys-4- aminopiperidine-4-carboxamide- PEGiCH2CO-D-Lys-NH2 Compound 76 D-Phe-D-Tyr(Et)-D-cyclopropylalanine-D-Lys-4- aminopiperidine-4-carboxamide- PEGiCH2CO-D-Lys-OH Compound 77 D-Phe-D-Tyr(Et)-D-cyclopropylalanine-D-Lys-4- aminopiperidine-4-carboxamide- PEG2CH2CO-Lys-NH2 Compound 78 D-Phe-D-Tyr(Et)-D-cyclopropylalanine-D-Lys-4aminopiperidine-4-carboxamide- PEG2CH2CO-Lys-OH Compound 79D-Phe-D-Tyr(Et)-D-cyclopropylalanine-D-Lys-4- aminopiperidine-4-carboxamide- PEG2CH2CO-D-Lys-NH2 Compound 80 D-Phe-D-Tyr(Et)-D-cyclopropylalanine-D-Lys-4- aminopiperidine-4-carboxamide- PEG2CH2CO-D-Lys-OH Compound 81 D-Phe-D-Tyr(Et)-D-cyclopropylalanine-D-Lys-4-aminopiperidine-4-carboxamide- PEG3CH2CO-Lys-NH2 Compound 82 D-Phe-D-Tyr(Et)-D-cyclopropylalanine-D-Lys-4aminopiperidine-4-carboxamide- PEG3CH2CO-Lys-OH Compound 83 D-Phe-D-Tyr(Et)-D-cyclopropylalanine-D-Lys-4- Petition 870250091684, dated 07 / 10 / 2025, p. 73 / 199 22 / 34 Compound Number | Sequence | aminopiperidine-4-carboxamide- PEG3CH2CO-D-Lys-NH2 | Compound 84 | D-Phe-D-Tyr(Et)-D-cyclopropylalanine-D-Lys-4-aminopiperidine-4-carboxamide- PEG3CH2CO-D-Lys-OH | Compound 85 | D-Phe-D-Tyr(Me)-D-Leu-D-Lys-4-aminopiperidine-4-carboxamide-NH2 | Compound 86 | D-Phe-D-Tyr(Me)-D-Leu-D-Lys-4-aminopiperidine-4-carboxamide-OH | Compound 87 | D-Phe-D-Tyr(Me)-D-Leu-D-Lys-4-aminopiperidine-4-carboxamide-Lys-NH2 | Compound 88 D-Phe-D-Tyr(Me)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-Lys-OH Compound 89 D-Phe-D-Tyr(Me)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-D-Lys-NH2 Compound 90 D-Phe-D-Tyr(Me)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-D-Lys-OH Compound 91 D-Phe-D-Tyr(CH2CF3)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-NH2 Compound 92 D-Phe-D-Tyr(CH2CF3)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-OH Compound 93 D-Phe-D-Tyr(CH2CF3)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-Lys-NH2 Compound 94 D-Phe-D-Tyr(CH2CF3)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-Lys-OH Compound 95D-Phe-D-Tyr(CH2CF3)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-D-Lys-NH2 Compound 96 D-Phe-D-Tyr(CH2CF3)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-D-Lys-OH Petition 870250091684, dated 07 / 10 / 2025, p. 74 / 199 23 / 34 Compound Number Sequence Compound 97 D-Phe-D-Tyr(Ipr)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-NH2 Compound 98 D-Phe-D-Tyr(Ipr)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-OH Compound 99 D-Phe-D-Tyr(Ipr)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-Lys-NH2 Compound 100 D-Phe-D-Tyr(Ipr)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-Lys-OH Compound 101 D-Phe-D-Tyr(Ipr)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-D-Lys-NH2 Compound 102 D-Phe-D-Tyr(Ipr)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-D-Lys-OH Compound 103 D-Phe-D-Phe(4-F)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-NH2 Compound 104 D-Phe-D-Phe(4-F)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-OH Compound 105 D-Phe-D-Phe(4-F)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-Lys-NH2 Compound 106 D-Phe-D-Phe(4-F)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-Lys-OH Compound 107 D-Phe-D-Phe(4-F)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-D-Lys-NH2 Compound 108 D-Phe-D-Phe(4-F)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-D-Lys-OH Compound 109D-Phe-D-Phe(4-Cl)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-NH2 Compound D-Phe-D-Phe(4-Cl)-D-Leu-D-Lys-4-aminopiperidine-4- Petition 870250091684, dated 07 / 10 / 2025, p. 75 / 199 24 / 34 Compound Number Sequence 110 carboxamide-OH Compound 111 D-Phe-D-Phe(4-Cl)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-Lys-NH2 Compound 112 D-Phe-D-Phe(4-Cl)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-Lys-OH Compound 113 D-Phe-D-Phe(4-Cl)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-D-Lys-NH2 Compound 114 D-Phe-D-Phe(4-Cl)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-D-Lys-OH Compound 115 D-Phe-D-Phe(4-Br)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-NH2 Compound 116 D-Phe-D-Phe(4-Br)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-OH Compound 117 D-Phe-D-Phe(4-Br)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-Lys-NH2 Compound 118 D-Phe-D-Phe(4-Br)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-Lys-OH Compound 119 D-Phe-D-Phe(4-Br)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-D-Lys-NH2 Compound 120 D-Phe-D-Phe(4-Br)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-D-Lys-OH Compound 121 D-Phe-D-Phe(4-I)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-NH2 Compound 122D-Phe-D-Phe(4-I)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-OH Compound 123 D-Phe-D-Phe(4-I)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-Lys-NH2 Petition 870250091684, dated 07 / 10 / 2025, p. 76 / 199 25 / 34 Compound Number Sequence Compound 124 D-Phe-D-Phe(4-I)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-Lys-OH Compound 125 D-Phe-D-Phe(4-I)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-D-Lys-NH2 Compound 126 D-Phe-D-Phe(4-I)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-D-Lys-OH Compound 127 D-Phe-D-Phe(4-Me)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-Lys-NH2 Compound 128 D-Phe-D-Phe(4-Me)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-Lys-OH Compound 129 D-Phe-D-Phe(4-Me)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-D-Lys-NH2 Compound 130 D-Phe-D-Phe(4-Me)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-D-Lys-OH Compound 131 D-Phe-D-Phe(4-Et)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-Lys-NH2 Compound 132 D-Phe-D-Phe(4-Et)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-Lys-OH Compound 133 D-Phe-D-Phe(4-Et)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-D-Lys-NH2 Compound 134 D-Phe-D-Phe(4-Et)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-D-Lys-OH Compound 135D-Phe-D-Phe(4-Ipr)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-Lys-NH2 Compound 136 D-Phe-D-Phe(4-Ipr)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-Lys-OH Compound D-Phe-D-Phe(4-Ipr)-D-Leu-D-Lys-4-aminopiperidine-4- Petition 870250091684, dated 07 / 10 / 2025, p. 77 / 199 26 / 34 Compound Number Sequence 137 carboxamide-D-Lys-NH2 Compound 138 D-Phe-D-Phe(4-Ipr)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-D-Lys-OH Compound 139 D-Phe-D-Phe(4-NH2)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-Lys-NH2 Compound 140 D-Phe-D-Phe(4-NH2)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-Lys-OH Compound 141 D-Phe-D-Phe(4-NH2)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-D-Lys-NH2 Compound 142 D-Phe-D-Phe(4-NH2)-D-Leu-D-Lys-4-aminopiperidine-4carboxamide-D-Lys-OH

[00111] Example 2: Determination of κ receptor agonist activities

[00112] 1. Determination Method

[00113] The human Gi-coupled opioid receptor kappa-1 (OPRK1), upon stimulation with its specific opioid receptor agonists, can inhibit the intracellular adenylate cyclase pathway and decrease cAMP levels. Forskolin can stimulate cAMP release in cell lines that highly express opioid receptors. By stimulating cell lines stably transfected with opioid receptors, the inhibitory effect of a test compound on forskolin-stimulated cAMP release can be measured to determine the compound's activity. The relative light units (RLU) of the cells after stimulation with different doses were measured via the homogeneous time-resolved fluorescence (HTRF) technique, and the EC50 of the agonist was subsequently calculated. This method of activity determination is a commonly used method currently used to determine the activities of opioid receptor agonists both domestically and internationally. Petition 870250091684, dated 07 / 10 / 2025, page 78 / 199 27 / 34

[00114] A CHO-K1 cell line that stably expresses opioid receptors was used. The stably transfected cells were stimulated with various concentrations of agonists (under stimulation with a certain concentration of forskolin). The relative light units of the cells after stimulation with different doses were measured, from which the EC50 value of the agonist was subsequently obtained by calculation.

[00115] 2. Determination results

[00116] The results of the determination are presented in the table below: Table 3 Compound Number EC50(pmol) % Relative Activity Diphelykephalin 607.2 100.0 Compound 1 115.7 524.8 Compound 2 117.0 519.0 Compound 3 39.4 1541.1 Compound 4 39.9 1521.8 Compound 5 41.0 1481.0 Compound 6 40.2 1510.4 Compound 7 42.2 1438.9 Compound 8 41.7 1456.1 Compound 9 47.7 1273.0 Compound 10 46.3 1311.4 Compound 11 53.4 1137.1 Compound 12 54.1 1122.4 Compound 13 66.9 907.6 Petition 870250091684, dated 07 / 10 / 2025, page 79 / 199 28 / 34 Compound Number EC (pmol) % Relative Activity Compound 14 65.5 927.0 Compound 15 42.9 1415.4 Compound 16 43.1 1408.8 Compound 17 45.3 1340.4 Compound 18 46.0 1320.0 Compound 19 47.9 1267.6 Compound 20 48.2 1259.8 Compound 21 50.1 1212.0 Compound 22 50.6 1200.0 Compound 23 55.4 1096.0 Compound 24 56.1 1082.4 Compound 25 68.4 887.7 Compound 26 67.1 904.9 Compound 27 46.2 1314.3 Compound 28 45.0 1349.3 Compound 29 45.6 1331.6 Compound 30 46.5 1305.8 Compound 31 53.2 1141.4 Compound 32 54.1 1122.4 Compound 33 53.6 1132.8 Compound 34 53.0 1145.7 Compound 35 54.9 1106.0 Petition 870250091684, dated 07 / 10 / 2025, page 80 / 199 29 / 34 Compound Number EC (pmol) % Relative Activity Compound 36 55.2 1100.0 Compound 37 56.1 1082.4 Compound 38 55.4 1096.0 Compound 39 60.2 1008.6 Compound 40 58.0 1046.9 Compound 41 59.6 1018.8 Compound 42 58.5 1037.9 Compound 43 88.3 687.7 Compound 44 86.7 700.3 Compound 45 85.9 706.9 Compound 46 87.0 697.9 Compound 47 43.9 1383.1 Compound 48 43.2 1405.6 Compound 49 44.5 1364.5 Compound 50 44.9 1352.3 Compound 51 47.7 1273.0 Compound 52 48.5 1252.0 Compound 53 46.3 1311.4 Compound 54 47.1 1289.2 Compound 55 52.1 1165.5 Compound 56 50.6 1200.0 Compound 57 51.4 1181.3 Petition 870250091684, dated 07 / 10 / 2025, page 81 / 199 30 / 34 Compound Number EC (pmol) % Relative Activity Compound 58 51.5 1179.0 Compound 59 69.4 874.9 Compound 60 71.3 851.6 Compound 61 70.6 860.1 Compound 62 70.1 866.2 Compound 63 90.5 670.9 Compound 64 89.9 675.4 Compound 65 88.4 686.9 Compound 66 89.5 678.4 Compound 67 127.1 477.7 Compound 68 125.6 483.4 Compound 69 42.4 1432.1 Compound 70 43.7 1389.5 Compound 71 41.0 1481.0 Compound 72 42.6 1425.4 Compound 73 46.8 1297.4 Compound 74 45.7 1328.7 Compound 75 47.3 1283.7 Compound 76 46.6 1303.0 Compound 77 55.1 1102.0 Compound 78 53.2 1141.4 Compound 79 54.9 1106.0 Petition 870250091684, dated 07 / 10 / 2025, page 82 / 199 31 / 34 Compound Number EC (pmol) % Relative Activity Compound 80 54.5 1114.1 Compound 81 56.2 1080.4 Compound 82 56.9 1067.1 Compound 83 55.7 1090.1 Compound 84 56.1 1082.4 Compound 85 125.5 483.8 Compound 86 121.2 501.0 Compound 87 42.2 1438.9 Compound 88 43.5 1395.9 Compound 89 43.6 1392.7 Compound 90 44.2 1373.8 Compound 91 110.4 550.0 Compound 92 108.7 558.6 Compound 93 51.2 1185.9 Compound 94 52.6 1154.4 Compound 95 50.5 1202.4 Compound 96 49.4 1229.1 Compound 97 147.6 411.4 Compound 98 145.2 418.2 Compound 99 72.6 836.4 Compound 100 71.6 848.0 Compound 101 72.8 834.1 Petition 870250091684, dated 07 / 10 / 2025, page 83 / 199 32 / 34 Compound Number EC (pmol) % Relative Activity Compound 102 73.0 831.8 Compound 103 220.8 275.0 Compound 104 215.4 281.9 Compound 105 79.6 762.8 Compound 106 78.0 778.5 Compound 107 77.3 785.5 Compound 108 78.5 773.5 Compound 109 151.0 402.1 Compound 110 148.6 408.6 Compound 111 52.1 1165.5 Compound 112 50.8 1195.3 Compound 113 49.4 1229.1 Compound 114 51.5 1179.0 Compound 115 137.4 441.9 Compound 116 135.5 448.1 Compound 117 65.0 934.2 Compound 118 63.8 951.7 Compound 119 61.5 987.3 Compound 120 62.7 968.4 Compound 121 122.4 496.1 Compound 122 120.1 505.6 Compound 123 53.5 1135.0 Petition 870250091684, dated 07 / 10 / 2025, page 84 / 199 33 / 34 Compound Number EC (pmol) % Relative Activity Compound 124 55.8 1088.2 Compound 125 55.0 1104.0 Compound 126 56.7 1070.9 Compound 127 157.4 385.8 Compound 128 159.7 380.2 Compound 129 151.5 400.8 Compound 130 152.6 397.9 Compound 131 133.6 454.5 Compound 132 130.4 465.6 Compound 133 131.5 461.7 Compound 134 132.7 457.6 Compound 135 146.3 415.0 Compound 136 149.2 407.0 Compound 137 143.5 423.1 Compound 138 145.9 416.2 Compound 139 112.4 540.2 Compound 140 115.0 528.0 Compound 141 111.8 543.1 Compound 142 109.7 553.5

[00117] The results of the experiments mentioned above show that the compounds provided in the present disclosure have a much higher activity than Difelicefalin. Compounds 1 to 142 provided in the Examples have a Petition 870250091684, dated 07 / 10 / 2025, page 85 / 199 34 / 34 activity 3 to 15 times that of Difelicefallina.

[00118] Finally, it should be noted that the embodiments above are provided for the purpose of illustrating the technical solutions of the present disclosure and are not intended to be limiting. Although the present disclosure has been described in detail with reference to the embodiments mentioned above, a person of ordinary skill in the art should understand that modifications to the described technical solutions, or equivalent substitutions for some or all of the technical features thereof, may still be made. Such modifications or substitutions, however, do not cause the essence of the respective technical solutions to depart from the scope of the technical solutions presented in the embodiments of the present disclosure. Petition 870250091684, dated 07 / 10 / 2025, page 86 / 199

Claims

1 / 6 CLAIMS 1. A κ opioid receptor agonist as represented by formula I, a stereoisomer thereof and a pharmaceutically acceptable salt thereof, characterized in that: D-Phe-AA1-AA2-D-Lys-4-aminopiperidine-4-carboxamide-AA3-AA4-AA5 (formula I) wherein AA1 is modified or unmodified D-Phe or modified or unmodified D-Tyr; AA2 is D-Leu or D-cyclopropylalanine; AA3 is -(PEGm1(CH2)m2CO)m3-, or (AA6)m4, or is absent, wherein: m1 is an integer in the range of 1 to 10; m2 is an integer in the range of 1 to 5; m3 is an integer in the range of 1 to 5; m4 is an integer in the range of 1 to 5; AA6 is AEEA, or Gly, or D-Ala, or L-Ala, or D-Leu, or L-Leu, or D-Phe, or L-Phe, or D-Ser, or L-Ser, or D-Thr, or L-Thr, or D-Tyr, or L-Tyr, or D-Asp, or L-Asp, or D-Glu, or L-Glu, or D-Gln, or L-Gln, or D-Lys, or L-Lys, or D-Arg, or L-Arg, or D-His or L-His; AA4 is (AA7)n, or is absent, where: n is an integer in the range 1 to 10;AA7 is D-Lys, or L-Lys, or D-Dap, or L-Dap, or D-Dab, or L-Dab, or D-Orn, or L-Orn, or D-Dah, or L-Dah, or D-Dao or L-Dao; AA5 is OH or NH2.

2. κ opioid receptor agonist, the stereoisomer thereof and the pharmaceutically acceptable salt thereof, according to claim 1, characterized in that AA1 is modified D-Phe or modified D-Tyr; the modified D-Phe is selected from the group consisting of D-Phe(2-F), D-Phe(4-F), D-Phe(2-Cl), D-Phe(4-Cl), D-Phe(2-Br), D-Phe(4-Br), D-Phe(2-I), DPhe(4-I), D-Phe(4-Me), D-Phe(4-Et), D-Phe(4-Ipr), D-Phe(4-NH2) CH2CH3);and the modified D-Tyr is selected from the group consisting of D-Tyr(Me), D-Tyr(Et) and D-Tyr(Ipr).

3. Opioid receptor κ agonist, the stereoisomer thereof and the pharmaceutically acceptable salt thereof, according to claim 2, characterized in that the opioid receptor κ agonist has a structure as represented by formula II: D-Phe-AA1-AA2-D-Lys-4-aminopiperidine-4-carboxamide-R-(AA8)pNH2(OH) (formula II) wherein AA1 is modified D-Phe or modified D-Tyr; AA2 is D-Leu or D-cyclopropylalanine; R is present or absent, and when R is present, R is selected from the group consisting of -PEGm1CH2CO-, -AEEAn1- and -Glyn2-; where: m1 is an integer in the range of 1 to 5, and specifically, is selected from the group consisting of 1, 2, 3, 4 and 5; n1 is an integer in the range of 1 to 5, and specifically, is selected from the group consisting of 1, 2, 3, 4 and 5;n2 is an integer in the range of 1 to 5, and specifically, it is selected from the group consisting of 1, 2, 3, 4, and 5; AA8 is Lys or D-Lys; p is an integer in the range of 0 to 6, and specifically, it is selected from the group consisting of 0, 1, 2, 3, 4, 5, and 6; when p is 0, AA8 is absent.

4. A κ opioid receptor agonist, its stereoisomer and its pharmaceutically acceptable salt, according to claim 3, characterized in that the κ opioid receptor agonist has a structure as represented by formula III: D-Phe-D-Tyr(X)-D-Leu-D-Lys-4-aminopiperidine-4-carboxamide-R1(AA8)p1-NH2(OH) (formula III) Petition 870250091684, dated 10 / 07 / 2025, p. 88 / 199 3 / 6 wherein, D-tyr(X) represents modified D-Tyr; the modified D-Tyr is selected from the group consisting of D-Tyr(Me), D-Tyr(Et), D-Tyr(CH2CF3) and D-Tyr(Ipr); Ri is either present or absent;When Ri is present, Ri is selected from the group consisting of -PEGk1CH2CO- and -Glyn2-; where: k1 is an integer in the range of 1 to 5, and specifically, is selected from the group consisting of 1, 2, 3, 4, and 5; n2 is an integer in the range of 1 to 5, and specifically, is selected from the group consisting of 1, 2, 3, 4, and 5; AA8 is Lys or D-Lys; p1 is an integer in the range of 0 to 6, and specifically, is selected from the group consisting of 0, 1, 2, 3, 4, 5, and 6; when p1 is 0, AA8 is absent.

5. A κ opioid receptor agonist, its stereoisomer, and its pharmaceutically acceptable salt, according to claim 3, characterized in that the κ opioid receptor agonist has a structure as represented by formula IV: D-Phe-D-Tyr(X)-D-cyclopropylalanine-D-Lys-4-aminopiperidine-4-carboxamide -R2-(AA8)p2-NH2(OH) (formula IV) where D-tyr(X) represents modified D-Tyr;The modified D-Tyr is selected from the group consisting of D-Tyr(Me), D-Tyr(Et), and D-Tyr(Ipr); R2 is either present or absent; when R2 is present, R2 is PEGk2CH2CO-; where: k2 is an integer in the range of 1 to 5, and specifically, is selected from the group consisting of 1, 2, 3, 4, and 5; in embodiments of the highest preference of the present disclosure, k2 is 1, 2, or 3; AA8 is Lys or D-Lys; p2 is an integer in the range of 0 to 6, and specifically, is selected from the group consisting of 0, 1, 2, 3, 4, 5, and 6;when p2 is 0, AA8 is absent.

6. Opioid receptor κ agonist, the stereoisomer thereof, and the pharmaceutically acceptable salt thereof, according to claim 3, characterized in that the opioid receptor κ agonist has a structure as represented by formula V: D-Phe-D-Phe(X)-D-Leu-D-Lys-4-aminopiperidine-4-carboxamide-(AA8)p3NH2(OH) (formula V) where D-Phe(X) represents modified D-Phe; The modified D-Phe is selected from the group consisting of D-Phe(2-F), D-Phe(4-F), D-Phe(2Cl), D-Phe(4-Cl), D-Phe(2-Br), D-Phe(4-Br), D-Phe(2-I), D-Phe(4-I), D-Phe(4Me), D-Phe(4-Et), D-Phe(4-Ipr), D-Phe(4-NH2); AA8 is Lys or D-Lys; p3 is an integer in the range of 0 to 6, and specifically, is selected from the group consisting of 0, 1, 2, 3, 4, 5, and 6;when p3 is 0, AA8 is absent.

7. κ opioid receptor agonist, the stereoisomer thereof, and the pharmaceutically acceptable salt thereof, according to any one of claims 1 to 6, characterized in that the κ opioid receptor agonist has one or more structural formulas selected from the group consisting of 1) to 10): 1) D-Phe-D-Tyr(X)-D-Leu-D-Lys-4-aminopiperidine-4-carboxylic acidNH2(OH); 2) D-Phe-D-Tyr(X)-D-Leu-D-Lys-4-aminopiperidine-4-carboxamide- NH2(OH) 3) D-Phe-D-Tyr(X)-D-Leu-D-Lys-4-aminopiperidine-4-carboxamide-(AA8)qNH2(OH) 4) D-Phe-D-Tyr(X)-D-Leu-D-Lys-4-aminopiperidine-4-carboxamide- PEGkiCH2CO-AA8-NH2(OH) 5) D-Phe-D-Tyr(X)-D-Leu-D-Lys-4-aminopiperidine-4-carboxamide-Glyn2-AA8-NH2(OH) Petition 870250091684, dated 07 / 10 / 2025, page 90 / 199 5 / 6 6) D-Phe-D-Tyr(X)-D-cyclopropylalanine-D-Lys-4-aminopiperidine-4-carboxamide-NH2(OH) 7) D-Phe-D-Tyr(X)-D-cyclopropylalanine-D-Lys-4-aminopiperidine-4-carboxamide-AA8-NH2(OH) 8) D-Phe-D-Tyr(X)-D-cyclopropylalanine-D-Lys-4-aminopiperidine-4-carboxamide-PEGk2CH2CO-AA8-NH2(OH) 9) D-Phe-D-Phe(X)-D-Leu-D-Lys-4-aminopiperidine-4-carboxamide- NH2(OH) 10) D-Phe-D-Phe(X)-D-Leu-D-Lys-4-aminopiperidine-4-carboxamide-AA8-NH2(OH), in the structural formulas mentioned above, AA8 is Lys or D-Lys; where: in formula 1), D-Tyr(X) is D-Tyr(Et); in formulas 2) to 3), D-Tyr(X) is D-Tyr(Me) or D-Tyr(Et) or D-Tyr(CH2CFa) or D-Tyr(Ipr), eq is 1, or 2, or 3, or 4, or 5, or 6; in formulas 4) to 8), D-Tyr(X) is D-Tyr(Et), k1 is 1, or 2, or 3, or 4, or 5; n2 is 1, or 2, or 3, or 4, or 5; k2 is 1, or 2, or 3; in formula 9), D-Phe(X) is D-Phe(4-F) or D-Phe(4-Cl) or D-Phe(4-Br) or D-Phe(4-I); in formula 10), D-Phe(X) is D-Phe(4-F) or D-Phe(4-Cl) or D-Phe(4-Br) or D-Phe(4-I) or D-Phe(4-Me) or D-Phe(4-Et) or D-Phe(4-Ipr) or D-Phe(4-NH2).

8. Pharmaceutical composition characterized by comprising the κ opioid receptor agonist, the stereoisomer thereof and the pharmaceutically acceptable salt thereof, as defined in any one of claims 1 to 7. 9.Pharmaceutical composition, according to claim 8, wherein the pharmaceutical composition is characterized by further comprising a pharmaceutically acceptable carrier.

10. Use of the κ opioid receptor agonist, the stereoisomer thereof and the pharmaceutically acceptable salt thereof, as defined in any one of claims 1 to 7, and / or the pharmaceutical composition as defined in any one of claims 8 to 9, characterized in the manufacture of a medicament for preventing and / or relieving pruritus and / or an analgesic. Petition 870250091684, dated 07 / 10 / 2025, p. 92 / 199.