Novel long-acting amylin receptor agonists and uses thereof

Abstract

The present application provides novel long-acting Amylin receptor agonists and uses thereof. The Amylin receptor agonists are prepared by solid phase synthesis and / or solution phase synthesis, cleavage of the peptide resin, formation of a disulfide bond between the thiol groups of Cys2 and Cys7, RCM ring closure of the side chains of AA2 and AA7 double bond, hydrogenation, and optional isolation and purification. The compounds disclosed herein can be used in combination with one or more additional therapeutic agents for the treatment of diabetes and its complications, obesity and its complications, dyslipidemia, hypertension, OSA, and / or NASH, either simultaneously, separately or sequentially.

Description

Technical Field

[0001] This invention belongs to the field of pharmaceutical technology and relates to amylin receptor agonists, particularly to novel long-acting amylin receptor agonists and their application in the preparation of drugs for the treatment of diabetes, obesity, long-term weight management, dyslipidemia, non-alcoholic steatohepatitis (NASH), and OSA. Background Technology

[0002] The global obesity situation is serious and has become a major public health problem.

[0003] Most acquired obesity is caused by unhealthy lifestyles. Excessive obesity is often accompanied by many complications such as dyslipidemia, hypertension, coronary heart disease, obstructive sleep apnea syndrome, obesity-related cardiomyopathy, and fatty liver. Furthermore, obesity is closely related to type 2 diabetes and can cause insulin resistance.

[0004] There are generally three ways to treat obesity. One is to adopt a healthy lifestyle, control your diet, reduce fat intake, and exercise regularly.

[0005] Secondly, surgical treatment involves reducing the size of the stomach or the length of the intestines through external interventions such as gastric bypass surgery, sleeve gastrectomy, and bile-pancreatic diversion, thereby reducing food intake and absorption and achieving weight loss. Obesity surgery is suitable for individuals with a BMI exceeding 35 kg / m². 2 Patients with obesity-related complications.

[0006] Thirdly, there is drug therapy. For example, orlistat can reduce fat absorption in the gastrointestinal tract by inhibiting the activity of gastric and pancreatic lipases. GLP-1 receptor agonists (such as liraglutide, semaglutide, and dulaglutide) reduce weight by delaying gastric emptying, increasing satiety, suppressing appetite, and increasing energy expenditure. GIP / GLP-1 dual-target receptor agonists (such as tirzepatide) achieve better weight loss through synergistic effects of multiple targets. The clinically tested GIP / GCG / GLP-1 tri-target receptor agonist retatrutide (phase III clinical trial) has shown better weight loss effects than tirzepatide.

[0007] In addition, Novo Nordisk's GLP-1 and amylin receptor agonist combination therapy (Cagrisema) also showed excellent weight loss results in Clinical III trials (68 weeks, weight loss >20%).

[0008] Amylin, also known as isletamyloid polypeptide (IAPP), is an endogenous polypeptide hormone composed of 37 amino acids, secreted by pancreatic β cells. Amylin is an appetite suppressant that increases satiety and reduces food intake by activating the functions of the central nervous system, including the nucleus tractus solitarius (NTS) and lateral hypothalamus (LH). Additionally, amylin can act on pancreatic α cells to reduce glucagon secretion, thereby helping to maintain blood glucose homeostasis.

[0009] The amino acid sequence of endogenous human amylin is as follows:

[0010] H-KC * NTATC * ATQRLANFLVHSSNNFGAILSSTNVGSNTY-NH2, the H- at the nitrogen end represents a hydrogen atom, and the -NH2 at the carbon end represents a tyrosine residue at the carbon end. It is an amidated structure, with cysteine ​​at position 2. * And cysteine ​​C at position 7 * It forms an intramolecular disulfide bond.

[0011] The amino acid sequence from position 20 to 29 of human amylin readily forms a β-sheet, making it particularly prone to aggregation into insoluble amyloid proteins. Furthermore, human amylin has a plasma half-life of only 13 minutes. These two properties make human amylin unsuitable for direct development into therapeutic drugs. Inspired by the fact that mouse amylin is more stable than human amylin and does not tend to aggregate, Amylin Pharmaceuticals developed Pramlintide (trade name Symlin), the first amylin receptor agonist, for adjunctive treatment of type 1 and type 2 diabetes. Structurally, Pramlintide mutates the 25th (Ala), 28th (Ser), and 29th (Ser) positions of human amylin to Pro, reducing the tendency to aggregate into protein fibrils and increasing the plasma half-life to 60 minutes.

[0012] The modification of the fatty chain of peptides can enhance the binding rate of peptide molecules to plasma albumin, thereby significantly prolonging the half-life of peptide molecules in plasma. Examples include liraglutide (palmitoylation of Lys at position 20, with a plasma half-life of about 13 hours) and smegglutide (Lys at position 20 is linked to a carbooctadecyl diacid via a linker, with a plasma half-life of about 168 hours).

[0013] NovoNordisk's patent WO2012168430A2 discloses Cagrilintide, a long-acting amylin derivative modified with a nitrogen-terminated carbon eicosyl diacid. Its plasma half-life is about 160 hours. Currently, Cagrilintide, used in combination with semaglutide, is in Phase III clinical trials, with an average weight loss of 22.7% over 68 weeks.

[0014] Patents WO2006105527A2 / WO2012168431A2 / CN103596972A / WO2013156594A1 disclose amyloid analogs with improved pharmacokinetic properties.

[0015] Patents CN118108832A / WO2022187305A1 / WO2023227133A1 / WO2024022465A1 disclose a series of amylin analogs modified with long-chain fatty acid diacids. However, the currently disclosed amylin analogs suffer from drawbacks such as similar long-acting modification strategies, poor solubility, and no significant improvement in efficacy.

[0016] The literature J.Med.Chem. 2021, 64, 11183-11194 points out that the N-terminus of Cagrilintide forms a stable circuit through the disulfide bond between cysteine ​​residues at positions 2 and 7, pointing the N-terminus outward from the transmembrane region of the Amylin receptor, thus allowing for modification with long-chain fatty acids at the N-terminus. Furthermore, this stable circuit at the N-terminus is crucial for its agonistic activity. Disulfide bonds in peptides are easily broken, reconstructed, and polymerized by disulfide reductases in vivo. Additionally, intermolecular reactions can easily occur during peptide synthesis to form dimer impurities and disulfide bond mismatch impurities.

[0017] Patent WO2019207427A2 reports an Amylin analog of a nitrogen-terminated cyclic thioacetamide that retains receptor agonist activity.

[0018] In patent WO2022187305A1, the Cys at the second and seventh positions are cyclized using a thioacetal, which greatly enhances the stability of the nitrogen-terminal cyclic peptide compared to a disulfide bond. However, acetals have poor tolerance to acidic environments and are easily degraded in gastric acid when administered orally.

[0019] Patent WO2018046719A1 reports an Amylin analog with higher chemical stability, specifically by constructing a nitrogen-terminated cyclic peptide structure through amidation of the carboxyl and amino groups of the second and seventh amino acid residues, thereby maintaining agonistic activity. Summary of the Invention

[0020] To address the aforementioned problems, the present invention provides novel long-acting amylin receptor agonists or pharmaceutically acceptable salts or solvates thereof, wherein the amylin receptor agonists have the following general structural formula:

[0021]

[0022] R1 is a fatty chain with an acidic group at the end; R2 is a linker; R1 is linked to the α-amino group of the N-terminal amino acid of the 37 peptide through linker R2; R1 and R2 are linked by an amide bond, and R2 is linked to the α-amino group of the first amino acid X1 by an amide bond; AA2 and AA7 of the main peptide chain form a cyclic peptide structure through the R4 group.

[0023] X1 is selected from des (missing), Lys, homoLys, Dab, Orn, Nva, or Nle;

[0024] X5 is selected from Ala or Ser;

[0025] Where X 10 Selected from Gln or Ala;

[0026] Where X 19 Selected from Ser or Lys;

[0027] Where X 27 Selected from Leu or Trp;

[0028] Where X 29 Choose from Pro or Lys;

[0029] Where X 34 Selected from Ser or Pro;

[0030] Where X 37 Selected from Pro, αMePro, homoPro, Aze, or transHyp;

[0031] R1 is selected from the following structures, where n1 = 8 or 9; n2 = 17, 19 or 21;

[0032] ;

[0033] R2 is selected from the following structures, where n3 = 1 or 2; n4 = 0, 1 or 2;

[0034] ;

[0035] Where R3 is NH2;

[0036] R4 is selected from the following structure, where n5 = 1 or 2;

[0037] .

[0038] To facilitate understanding of this invention, the terminology used herein is first defined and explained. Unless otherwise stated, those skilled in the art should have the same understanding of the terminology used herein. In the general structural formula shown below, R1 / R2 / R3 / R4 represent structural segments with a certain chemical structure, the specific structure of which will be explained below; the main chain of the general structural formula described in this invention is a 37-amino acid peptide formed by 37 amino acids, wherein X1 indicates that the first amino acid can be selected from one or more amino acids, X5 indicates that the fifth amino acid can be selected from one or more amino acids, X... 10 / X 19 / X 27 / X 29 / X 34 / X 37 This indicates that one or more amino acids can be selected at the corresponding position; the second and seventh amino acids in the general formula are represented by structural formulas; AA is an abbreviation for aminoacid; "—" in the general formula represents a single bond; "=" in the general formula represents a double bond; "CH2" represents a methylene group; amino acids at other positions in the general formula are represented by single-letter abbreviations (as shown in Table 1 below):

[0039] Table 1. Abbreviation Table

[0040]

[0041] The above description mentions some non-natural amino acids, which are amino acids not encoded by the existing 64 genetic codons; for example, X1 is selected from des (deleted), Lys, homoLys, Dab, Orn, Nva, or Nle; X 37 Selected from Pro, αMePro, homoPro, Aze, or transHyp; where des indicates the deletion of an amino acid at this position; homoLys indicates high lysine; Dab indicates L-2,4-diaminobutyric acid; Orn indicates ornithine; Nva indicates L-valine; Nle indicates L-leucine; αMePro indicates L-2-methylpyrrolidine-2-carboxylic acid; homoPro indicates L-piperidine-2-carboxylic acid; Aze indicates L-azacyclobutane-2-carboxylic acid; transHyp indicates L-hydroxyproline; the structural formulas of all non-natural amino acids involved in this invention are as follows:

[0042] .

[0043] The "human amylin" used in this invention is a human endogenous polypeptide hormone composed of 37 amino acids, with an intramolecular disulfide bond between Cys at positions 2 and 7, as shown below:

[0044] .

[0045] The "Cagrilintide" used in this invention is a long-acting human amylin analogue modified with fatty acid chains, developed by Novo Nordisk and currently in clinical Phase III trials. Its structure is shown below:

[0046] .

[0047] The "Pramlintide" used in this invention is an analogue of human amylin, a commercially available drug used as adjunctive therapy for type 1 and type 2 diabetes. Its structure is shown below:

[0048] .

[0049] The novel, long-acting Amylin receptor agonist according to the present invention is preferably derived from the polypeptide sequence shown in the following structure.

[0050] The preferred sequence uses standard single-letter amino acid codes for most positions, but at positions 1, 2, 7, 37, the linker, and the aliphatic chain, the chemical structural formulas are used as needed for the representation.

[0051] The preferred embodiment of the present invention comprises the following sequence: R1-R2-KC*NTATC*ATQRLAEFLRHSSNNFGPILPPTNVGSNTP-NH2 (No. 003), wherein an intramolecular disulfide bond is formed between Cys at positions 2 and 7, and the α-amino group of the N-terminal Lys of the main peptide chain is linked by γ-Glu (R2) and the terminal iminodiacetic acid-modified octadecyl diacid (R1). The structure of sequence No. 003 is shown below, where most positions use standard single-letter amino acid codes, but the Lys at position 1, the Cys at position 2, the Cys at position 7, the disulfide bond between residues at positions 2 and 7, the linker γ-Glu, and the R1 portion of the aliphatic chain are extended using chemical structural formulas:

[0052] No. 003 .

[0053] The preferred embodiment of the present invention comprises the following sequence: R1-R2-KC*NTATC*ATQRLAEFLRHSSNNFGPILPPTNVGSNTP-NH2 (No. 005), wherein an intramolecular disulfide bond is formed between Cys at positions 2 and 7, and the α-amino group of the N-terminal Lys of the main peptide chain is linked by γ-Glu (R2) and an eicosyl diacid modified with iminodiacetic acid (R1). The structure of sequence No. 005 is shown below, wherein most positions use standard single-letter amino acid codes, but the Lys at position 1, the Cys at position 2, the Cys at position 7, the disulfide bond between residues at positions 2 and 7, the linker γ-Glu, and the R1 portion of the aliphatic chain are extended through chemical structural formulas:

[0054] No. 005 .

[0055] The preferred embodiment of the present invention comprises the following sequence: R1-R2-KC*NTATC*ATQRLAEFLRHSSNNFGPILPPTNVGSNTP-NH2 (No. 006), wherein an intramolecular disulfide bond is formed between Cys at positions 2 and 7, and the α-amino group of the N-terminal Lys of the main peptide chain is linked by γ-Glu (R2) and 18-phosphono-octadecylcarboxylic acid (R1). The structure of sequence No. 006 is shown below, where most positions use standard single-letter amino acid codes, but the Lys at position 1, the Cys at position 2, the Cys at position 7, the disulfide bond between residues at positions 2 and 7, the linker γ-Glu, and the R1 portion of the aliphatic chain are extended using chemical structural formulas:

[0056] No. 006 .

[0057] The preferred embodiment of the present invention comprises the following sequence: R1-R2-KC*NTATC*ATQRLAEFLRHSSNNFGPILPPTNVGSNTP-NH2 (No. 007), wherein an intramolecular disulfide bond is formed between Cys at positions 2 and 7, and the α-amino group of the N-terminal Lys of the main peptide chain is linked by γ-Glu (R2) and 22-phosphono-dodecylcarboxylic acid (R1). The structure of sequence No. 007 is shown below, where most positions use standard single-letter amino acid codes, but the Lys at position 1, the Cys at position 2, the Cys at position 7, the disulfide bond between residues at positions 2 and 7, the linker γ-Glu, and the R1 portion of the aliphatic chain are extended using chemical structural formulas:

[0058] No. 007 .

[0059] The preferred embodiment of the present invention comprises the following sequence: R1-R2-KC*NTATC*ATQRLAEFLRHSSNNFGPILPPTNVGSNTP-NH2 (No. 008), wherein an intramolecular disulfide bond is formed between Cys at positions 2 and 7, and the α-amino group of the N-terminal Lys of the main peptide chain is linked by γ-Glu (R2) and 20-phosphono-eicosylcarboxylic acid (R1). The structure of sequence No. 008 is shown below, where most positions use standard single-letter amino acid codes, but the Lys at position 1, the Cys at position 2, the Cys at position 7, the disulfide bond between residues at positions 2 and 7, the linker γ-Glu, and the R1 portion of the aliphatic chain are extended using chemical structural formulas:

[0060] No. 008 .

[0061] The preferred embodiment of the present invention comprises the following sequence: R1-R2-homoLys-C*NTATC*ATQRLAEFLRHSSNNFGPILPPTNVGSNTP-NH2 (No. 009), wherein an intramolecular disulfide bond is formed between Cys at positions 2 and 7, and the α-amino group of the N-terminus homoLys of the main peptide chain is linked by γ-Glu (R2) and 20-phosphono-eicosylcarboxylic acid (R1). The structure of sequence No. 009 is shown below, where most positions use standard single-letter amino acid codes, but the homoLys at position 1, the Cys at position 2, the Cys at position 7, the disulfide bond between residues at positions 2 and 7, the linker γ-Glu, and the R1 portion of the aliphatic chain are extended using chemical structural formulas:

[0062] No. 009 .

[0063] The preferred embodiment of the present invention comprises the following sequence: R1-R2-Dab-C*NTATC*ATQRLAEFLRHSSNNFGPILPPTNVGSNTP-NH2 (No. 011), wherein an intramolecular disulfide bond is formed between Cys at positions 2 and 7, and the α-amino group of the nitrogen-terminal Dab of the main peptide chain is linked by γ-Glu (R2) and 20-phosphono-eicosylcarboxylic acid (R1). The structure of sequence No. 011 is shown below, where most positions use standard single-letter amino acid codes, but Dab at position 1, Cys at position 2, Cys at position 7, the disulfide bond between residues at positions 2 and 7, the linker γ-Glu, and the R1 portion of the aliphatic chain are extended using chemical structural formulas:

[0064] No. 011 .

[0065] The preferred embodiment of the present invention comprises the following sequence: R1-R2-Orn-C*NTATC*ATQRLAEFLRHSSNNFGPILPPTNVGSNTP-NH2 (No. 013), wherein an intramolecular disulfide bond is formed between Cys at positions 2 and 7, and the α-amino group of the N-terminus Orn of the main peptide chain is linked by γ-Glu (R2) and 20-phosphono-eicosylcarboxylic acid (R1). The structure of sequence No. 013 is shown below, where most positions use standard single-letter amino acid codes, but the Orn at position 1, the Cys at position 2, the Cys at position 7, the disulfide bond between residues at positions 2 and 7, the linker γ-Glu, and the R1 portion of the aliphatic chain are extended using chemical structural formulas:

[0066] No. 013 .

[0067] The preferred embodiment of the present invention comprises the following sequence: R1-R2-Nva-C*NTATC*ATQRLAEFLRHSSNNFGPILPPTNVGSNTP-NH2 (No. 015), wherein an intramolecular disulfide bond is formed between Cys at positions 2 and 7, and the α-amino group of the N-terminus Nva of the main peptide chain is linked by γ-Glu (R2) and 20-phosphono-eicosylcarboxylic acid (R1). The structure of sequence No. 015 is shown below, where most positions use standard single-letter amino acid codes, but Nva at position 1, Cys at position 2, Cys at position 7, the disulfide bond between residues at positions 2 and 7, the linker γ-Glu, and the R1 portion of the aliphatic chain are extended using chemical structural formulas:

[0068] No. 015 .

[0069] The preferred embodiment of the present invention comprises the following sequence: R1-R2-Nle-C*NTATC*ATQRLAEFLRHSSNNFGPILPPTNVGSNTP-NH2 (No. 017), wherein an intramolecular disulfide bond is formed between Cys at positions 2 and 7, and the α-amino group of the N-terminus Nle of the main peptide chain is linked by γ-Glu (R2) and 20-phosphono-eicosylcarboxylic acid (R1). The structure of sequence No. 017 is shown below, where most positions use standard single-letter amino acid codes, but Nle at position 1, Cys at position 2, Cys at position 7, the disulfide bond between residues at positions 2 and 7, the linker γ-Glu, and the R1 portion of the aliphatic chain are extended using chemical structural formulas:

[0070] No. 017 .

[0071] The preferred embodiment of the present invention comprises the following sequence: R1-R2-KC*NTATC*ATQRLAEFLRHSSNNFGPILPPTNVGSNT-transHyp-NH2 (No. 020), wherein an intramolecular disulfide bond is formed between Cys at positions 2 and 7, and the α-amino group of the N-terminal Lys of the main peptide chain is linked by γ-Glu (R2) and 20-phosphono-eicosylcarboxylic acid (R1). The structure of sequence No. 020 is shown below, where most positions use standard single-letter amino acid codes, but the Lys at position 1, the Cys at position 2, the Cys at position 7, the disulfide bond between residues at positions 2 and 7, the transHyp at position 37, the linker γ-Glu, and the R1 portion of the aliphatic chain are extended using chemical structural formulas:

[0072] No. 020 .

[0073] The preferred embodiment of the present invention comprises the following sequence: R1-R2-KC*NTATC*ATQRLAEFLRHSSNNFGPILPPTNVGSNT-αMePro-NH2 (No. 022), wherein an intramolecular disulfide bond is formed between Cys at positions 2 and 7, and the α-amino group of the N-terminal Lys of the main peptide chain is linked by γ-Glu (R2) and 20-phosphono-eicosylcarboxylic acid (R1). The structure of sequence No. 022 is shown below, where most positions use standard single-letter amino acid codes, but the Lys at position 1, the Cys at position 2, the Cys at position 7, the disulfide bond between residues at positions 2 and 7, the αMePro at position 37, the linker γ-Glu, and the R1 portion of the aliphatic chain are extended using chemical structural formulas:

[0074] No. 022 .

[0075] The preferred embodiment of the present invention comprises the following sequence: R1-R2-KC*NTATC*ATQRLAEFLRHSSNNFGPILPPTNVGSNT-homoPro-NH2 (No. 024), wherein an intramolecular disulfide bond is formed between Cys at positions 2 and 7, and the α-amino group of the N-terminal Lys of the main peptide chain is linked by γ-Glu (R2) and 20-phosphono-eicosylcarboxylic acid (R1). The structure of sequence No. 024 is shown below, where most positions use standard single-letter amino acid codes, but Lys at position 1, Cys at position 2, Cys at position 7, the disulfide bond between residues at positions 2 and 7, homoPro at position 37, the linker γ-Glu, and the R1 portion of the aliphatic chain are extended using chemical structural formulas:

[0076] No. 024 .

[0077] The preferred embodiment of the present invention comprises the following sequence: R1-R2-KC*NTATC*ATQRLAEFLRHSSNNFGPILPPTNVGSNTP-NH2 (No. 025), wherein an intramolecular disulfide bond is formed between Cys at positions 2 and 7, and the α-amino group of the N-terminal Lys of the main peptide chain is linked by -γ-Glu-γ-Glu- (R2) and 20-phosphono-eicosylcarboxylic acid (R1). The structure of sequence No. 025 is shown below, where most positions use standard single-letter amino acid codes, but the Lys at position 1, the Cys at position 2, the Cys at position 7, the disulfide bond between residues at positions 2 and 7, the linker -γ-Glu-γ-Glu-, and the R1 portion of the aliphatic chain are extended using chemical structural formulas:

[0078] No. 025 .

[0079] The preferred embodiment of the present invention comprises the following sequence: R1-R2-Des-C*NTATC*ATQRLAEFLRHSSNNFGPILPPTNVGSNTP-NH2 (No. 026), wherein an intramolecular disulfide bond is formed between Cys at positions 2 and 7, and the α-amino group of the nitrogen-terminal Cys of the main peptide chain is linked by -γ-Glu-γ-Glu- (R2) and 20-phosphono-eicosylcarboxylic acid (R1). The structure of sequence No. 026 is shown below, where most positions use standard single-letter amino acid codes, but the Cys at positions 2 and 7, the disulfide bond between residues at positions 2 and 7, the linker -γ-Glu-γ-Glu-, and the R1 portion of the aliphatic chain are extended using chemical structural formulas:

[0080] No. 026 .

[0081] The preferred embodiment of the present invention comprises the following sequence: R1-R2-homoLys-C*NTATC*ATQRLAEFLRHSSNNFGPILPPTNVGSNTP-NH2 (No. 027), wherein an intramolecular disulfide bond is formed between Cys at positions 2 and 7, and the α-amino group of the N-terminus homoLys of the main peptide chain is linked by -γ-Glu-γ-Glu- (R2) and 20-phosphono-eicosylcarboxylic acid (R1). The structure of sequence No. 027 is shown below, where most positions use standard single-letter amino acid codes, but the homoLys at position 1, Cys at position 2, Cys at position 7, the disulfide bond between residues at positions 2 and 7, the linker -γ-Glu-γ-Glu-, and the R1 portion of the aliphatic chain are extended using chemical structural formulas:

[0082] No. 027 .

[0083] The preferred embodiment of the present invention comprises the following sequence: R1-R2-KC*NTATC*ATQRLAEFLRHSSNNFGPILPPTNVGSNTP-NH2 (No. 029), wherein an intramolecular disulfide bond is formed between Cys at positions 2 and 7, and the α-amino group of the N-terminal Lys of the main peptide chain is linked by -AEEA-AEEA-γ-Glu- (R2) and 20-phosphono-eicosylcarboxylic acid (R1). The structure of sequence No. 029 is shown below, where most positions use standard single-letter amino acid codes, but the Lys at position 1, the Cys at position 2, the Cys at position 7, the disulfide bond between residues at positions 2 and 7, the linker -AEEA-AEEA-γ-Glu-, and the R1 portion of the aliphatic chain are extended using chemical structural formulas:

[0084] No. 029 .

[0085] The preferred embodiment of the present invention comprises the following sequence: R1-R2-KC*NTATC*ATQRLAEFLRHKSNNFGPILPKTNVGSNTP-NH2 (No. 035), wherein an intramolecular disulfide bond is formed between Cys at positions 2 and 7, and the α-amino group of the N-terminal Lys of the main peptide chain is linked by -γ-Glu- (R2) and 20-phosphono-eicosylcarboxylic acid (R1). The structure of sequence No. 035 is shown below, where most positions use standard single-letter amino acid codes, but the Lys at position 1, the Cys at position 2, the Cys at position 7, the disulfide bond between residues at positions 2 and 7, the linker -γ-Glu-, and the R1 portion of the aliphatic chain are extended using chemical structural formulas:

[0086] No. 035 .

[0087] The preferred embodiment of the present invention comprises the following sequence: R1-R2-KC*NTSTC*ATARLAEFLRHSSNNFGPILPPTNVGSNTP-NH2 (No. 036), wherein an intramolecular disulfide bond is formed between Cys at positions 2 and 7, and the α-amino group of the N-terminal Lys of the main peptide chain is linked by -γ-Glu- (R2) and 20-phosphono-eicosylcarboxylic acid (R1). The structure of sequence No. 036 is shown below, where most positions use standard single-letter amino acid codes, but the Lys at position 1, the Cys at position 2, the Cys at position 7, the disulfide bond between residues at positions 2 and 7, the linker -γ-Glu-, and the R1 portion of the aliphatic chain are extended using chemical structural formulas:

[0088] No. 036 .

[0089] The literature J.Med.Chem. 2021, 64, 11183-11194 points out that the N-terminus of Cagrilintide forms a stable circuit through the disulfide bond between cysteine ​​residues at positions 2 and 7, pointing the N-terminus outward from the transmembrane region of the Amylin receptor, thus allowing for modification with long-chain fatty acids at the N-terminus. Furthermore, this stable circuit at the N-terminus is crucial for its agonistic activity. Disulfide bonds in peptides are easily broken, reconstructed, and polymerized by disulfide reductases in vivo. Additionally, intermolecular reactions can easily occur during peptide synthesis to form dimer impurities and disulfide bond mismatch impurities. This invention mutates the 2nd and 7th amino acids of the canagliflozin main peptide chain into non-natural amino acids (such as (S)-2-amino-4-pentenoic acid and (S)-2-amino-5-pentenoic acid) containing terminal double bonds. After the main peptide chain is constructed, a stable carbon-carbon cyclization strategy is constructed on a solid-phase resin under the catalysis of a second-generation Grubbs catalyst via a Ring-Closing Metathesis (RCM) reaction and a hydrogenation reaction (as shown below).

[0090] .

[0091] In an embodiment of the RCM cyclization strategy according to the present invention, a preferred embodiment comprises the following sequence: R1-R2-KX2NTATX7ATQRLAEFLRHSSNNFGPILPPTNVGSNTP-NH2 (No. 037), wherein an intramolecular stapled peptide ring is formed between the α-carbons of the amino acids at positions 2 and 7 via -(CH2)4-, and the α-amino group of the N-terminus Lys of the main peptide chain is linked via -γ-Glu- (R2) and 20-phosphono-eicosylcarboxylic acid (R1). The structure of sequence No. 037 is shown below, wherein most positions use standard single-letter amino acid codes, but the Lys at position 1, X2 at position 2, X7 at position 7, the stapled peptide ring between residues at positions 2 and 7, the linker -γ-Glu-, and the R1 portion of the adipose chain are extended using chemical structural formulas:

[0092] No. 037 .

[0093] In an embodiment of the RCM cyclization strategy according to the present invention, a preferred embodiment comprises the following sequence: R1-R2-KX2NTATX7ATQRLAEFLRHSSNNFGPILPPTNVGSNTP-NH2 (No. 038), wherein an intramolecular stapled peptide ring is formed between the α-carbons of the amino acids at positions 2 and 7 via -(CH2)6-, and the α-amino group of the N-terminus Lys of the main peptide chain is linked via -γ-Glu- (R2) and 20-phosphono-eicosylcarboxylic acid (R1). The structure of sequence No. 038 is shown below, wherein most positions use standard single-letter amino acid codes, but the Lys at position 1, X2 at position 2, X7 at position 7, the stapled peptide ring between residues at positions 2 and 7, the linker -γ-Glu-, and the R1 portion of the adipose chain are extended using chemical structural formulas:

[0094] No. 038 .

[0095] In an embodiment of the RCM cyclization strategy according to the present invention, a preferred embodiment comprises the following sequence: R1-R2-KX2NTATX7ATQRLAEFLRHSSNNFGPILPPTNVGSNT-transHyp-NH2 (No. 039), wherein an intramolecular stapled peptide ring is formed between the α-carbons of the amino acids at positions 2 and 7 via -(CH2)4-, and the α-amino group of the N-terminus Lys of the main peptide chain is linked via -γ-Glu- (R2) and 20-phosphono-eicosylcarboxylic acid (R1). The structure of sequence No. 039 is shown below, wherein most positions use standard single-letter amino acid codes, but Lys at position 1, X2 at position 2, X7 at position 7, the stapled peptide ring between residues at positions 2 and 7, transHyp at position 37, the linker -γ-Glu-, and the R1 portion of the adipose chain are extended using chemical structural formulas:

[0096] No. 039 .

[0097] In an embodiment of the RCM cyclization strategy according to the present invention, a preferred embodiment comprises the following sequence: R1-R2-KX2NTATX7ATQRLAEFLRHKSNNFGPILPKTNVGSNTP-NH2 (No. 040), wherein an intramolecular stapled peptide ring is formed between the α-carbons of the amino acids at positions 2 and 7 via -(CH2)4-, and the α-amino group of the N-terminus Lys of the main peptide chain is linked via -γ-Glu- (R2) and 20-phosphono-eicosylcarboxylic acid (R1). The structure of sequence No. 040 is shown below, wherein most positions use standard single-letter amino acid codes, but the Lys at position 1, X2 at position 2, X7 at position 7, the stapled peptide ring between residues at positions 2 and 7, the linker -γ-Glu-, and the R1 portion of the adipose chain are extended using chemical structural formulas:

[0098] No. 040 .

[0099] In an embodiment of the RCM cyclization strategy according to the present invention, a preferred embodiment comprises the following sequence: R1-R2-KX2NTSTX7ATARLAEFLRHSSNNFGPILPPTNVGSNTP-NH2 (No. 041), wherein an intramolecular stapled peptide ring is formed between the α-carbons of the amino acids at positions 2 and 7 via -(CH2)4-, and the α-amino group of the N-terminus Lys of the main peptide chain is linked via -γ-Glu- (R2) and 20-phosphono-eicosylcarboxylic acid (R1). The structure of sequence No. 041 is shown below, wherein most positions use standard single-letter amino acid codes, but the Lys at position 1, X2 at position 2, X7 at position 7, the stapled peptide ring between residues at positions 2 and 7, the linker -γ-Glu-, and the R1 portion of the adipose chain are extended by chemical structural formulas:

[0100] No. 041 .

[0101] In an embodiment of the RCM cyclization strategy according to the present invention, a preferred embodiment comprises the following sequence: R1-R2-KX2NTATX7ATQRLAEFLRHKSNNFGPILPKTNVGSNT-transHyp-NH2 (No. 050), wherein an intramolecular stapled peptide ring is formed between the α-carbons of the amino acids at positions 2 and 7 via -(CH2)4-, and the α-amino group of the N-terminus Lys of the main peptide chain is linked via -γ-Glu- (R2) and 20-phosphono-eicosylcarboxylic acid (R1). The structure of sequence No. 050 is shown below, wherein most positions use standard single-letter amino acid codes, but Lys at position 1, X2 at position 2, X7 at position 7, the stapled peptide ring between residues at positions 2 and 7, transHyp at position 37, the linker -γ-Glu-, and the R1 portion of the adipose chain are extended by chemical structural formulas:

[0102] No. 050 .

[0103] The present invention also relates to the amylin receptor agonist No.003, No.005, No.006, No.007, No.008, No.009, No.011, No.013, No.015, No.017, No.020, No.022, No.024, No.025, No.026, No.027, No.029, No.035, No.036, No.037, No.03 8. Methods for the synthesis and purification of compounds, pharmaceutically acceptable salts, or solvates comprising No. 039, No. 040, No. 041, or No. 050, wherein the methods include solid-phase synthesis (assembly one by one or via fragment peptide assembly) and / or liquid-phase synthesis, methods for cleaving peptide resins, methods for forming disulfide bonds between the thiol groups at positions 2 and 7 of Cys, methods for RCM cyclization and hydrogenation of the side chain double bonds at positions 2 and 7 of AA, and optional separation and purification methods. The solid-phase resin used in the solid-phase synthesis of the present invention is an amino resin. The above-described synthesis methods also include a method for synthesizing a terminally phosphonylated long-chain fatty acid.

[0104] This invention provides compounds, pharmaceutically acceptable salts, or solvates composed of No. 003, No. 005, No. 006, No. 007, No. 008, No. 009, No. 011, No. 013, No. 015, No. 017, No. 020, No. 022, No. 024, No. 025, No. 026, No. 027, No. 029, No. 035, No. 036, No. 037, No. 038, No. 039, No. 040, No. 041, or No. 050; this invention provides... A pharmaceutical composition comprising No. 003, No. 005, No. 006, No. 007, No. 008, No. 009, No. 011, No. 013, No. 015, No. 017, No. 020, No. 022, No. 024, No. 025, No. 026, No. 027, No. 029, No. 035, No. 036, No. 037, No. 038, No. 039, No. 040, No. 041, or No. 050, and one or more pharmaceutically acceptable carriers, diluents, or excipients. The pharmaceutical composition must be manufactured under aseptic conditions and stored stably. The pharmaceutical composition may be in dosage forms such as solutions, microemulsions, liposomes, oral tablets, or lyophilized powder for injection. The unit dose of the pharmaceutical composition is 0.05 mg to 50 mg, more preferably 0.5 mg to 5 mg. The routes of administration of the pharmaceutical composition include intravenous, intramuscular, subcutaneous, spinal / spinal cord, or other parenteral routes.

[0105] This invention provides a pharmaceutical composition for treating diabetes, obesity, dyslipidemia, and / or NASH, comprising an effective dose of a compound, pharmaceutically acceptable salt or solvate, or a pharmaceutical composition comprising No. 003, No. 005, No. 006, No. 007, No. 008, No. 009, No. 011, No. 013, No. 015, No. 017, No. 020, No. 022, No. 024, No. 025, No. 026, No. 027, No. 029, No. 035, No. 036, No. 037, No. 038, No. 039, No. 040, No. 041, or No. 050; this invention also provides a method for lowering blood lipids. Pharmaceutical compositions that reduce food intake, reduce weight, reduce blood glucose, reduce HbA1c and / or reduce triglycerides, including compounds, pharmaceutically acceptable salts or solvates, or pharmaceutical compositions containing No. 003, No. 005, No. 006, No. 007, No. 008, No. 009, No. 011, No. 013, No. 015, No. 017, No. 020, No. 022, No. 024, No. 025, No. 026, No. 027, No. 029, No. 035, No. 036, No. 037, No. 038, No. 039, No. 040, No. 041 or No. 050, or pharmaceutically acceptable salts or solvates, or pharmaceutical compositions containing them, in a dose effective for use in a patient.

[0106] This invention provides the use of compounds, pharmaceutically acceptable salts or solvates comprising No. 003, No. 005, No. 006, No. 007, No. 008, No. 009, No. 011, No. 013, No. 015, No. 017, No. 020, No. 022, No. 024, No. 025, No. 026, No. 027, No. 029, No. 035, No. 036, No. 037, No. 038, No. 039, No. 040, No. 041 or No. 050 in the treatment of, inhibition of or reduction of weight gain, promotion of weight loss, reduction of food intake and / or reduction of overweight, treatment of diabetes, treatment of non-alcoholic steatohepatitis, treatment of obstructive sleep apnea syndrome, treatment of atherosclerotic dyslipidemia, and treatment of diabetes and obesity-related complications.

[0107] The compounds disclosed in this invention can be used simultaneously, separately, or sequentially in combination with one or more additional therapeutic agents for the treatment of diabetes and its complications, obesity and its complications, dyslipidemia, hypertension, OSA, and / or NASH.

[0108] The additional therapeutic agents mentioned include antidiabetic agents such as metformin, sulfonylureas, meglitinides, DPP-IV inhibitors, thiazolidinediones, GLP-1 receptor agonists, SGLT2 inhibitors, GPR40 agonists, insulin, or insulin analogs.

[0109] The additional therapeutic agents mentioned include anti-obesity agents such as peptide YY or its analogues, neuropeptide Y (NPY) or its analogues, cannabinoid receptor 1 antagonists, lipase inhibitors, human pro-insulin peptide (HIP), melanocortin receptor 4 agonists, GLP-1 receptor agonists, GIP receptor agonists, GCG receptor agonists, orlistat, sibutramine, phentermine, melanin-concentrating hormone receptor 1 antagonists, CCK, or leptin.

[0110] The additional therapeutic agents mentioned include antihypertensive agents such as angiotensin-converting enzyme inhibitors, angiotensin II receptor blockers, diuretics, beta-blockers, or calcium channel blockers.

[0111] The additional therapeutic agents mentioned include anti-dyslipidemia agents such as statins, fibrates, niacin, PSCK9 (proteinogen type 9 converting enzyme subtilisin / kexin) inhibitors, or cholesterol absorption inhibitors. Attached Figure Description

[0112] To more clearly illustrate the technical solutions in the embodiments of this application or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of this application. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0113] Figure 1 This is the hydrogen spectrum of the 20-phosphono-eicosylcarboxylic acid product obtained in Example 3.

[0114] Figure 2 This is the HPLC chromatogram of No. 003 obtained in Example 5.

[0115] Figure 3 This is the mass spectrum of No. 003 obtained in Example 5.

[0116] Figure 4 This is a comparison chart of the half-lives of No. 008 and No. 036. Detailed Implementation

[0117] The technical solutions of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0118] The definitions of the abbreviations used in this invention are shown in Table 2.

[0119] Table 2. Abbreviations

[0120]

[0121] Example 1

[0122] This embodiment provides the synthesis of aliphatic chain 1:

[0123]

[0124] Octadecanedioic acid (100 mmol, 31.45 g) was weighed into a 500 mL round-bottom flask at room temperature. 100 mL of DMF was added and stirred to dissolve. Then, DIPEA (150 mmol, 19.39 g) was added, followed by the condensing agent DMTMM (100 mmol, 29.47 g) in portions. The mixture was stirred at room temperature for 5 minutes, followed by the slow addition of di-tert-butyl iminodiacetic acid (100 mmol, 24.53 g) in portions. The reaction mixture was then reacted at room temperature for 5 hours. The reaction mixture was diluted with 500 mL of dichloromethane, washed twice with 0.2 M hydrochloric acid solution (150 mL x 2), and washed twice with water (150 mL x 2). The organic phase was collected, dried, filtered, and concentrated under reduced pressure to obtain crude aliphatic chain 1. This crude aliphatic chain 1 was then purified by silica gel column chromatography (mobile phase: n-heptane:ethyl acetate = 10:1~5:1) to obtain 16.36 g (30.2 mmol, yield 30.2%) of pure aliphatic chain 1.

[0125] Fat chain 1: 1 H-NMR (400M, DMSO-d6) 1.11~1.25(m, 24H), 1.32(s, 9H), 1.38(s,9H), 1.41~1.49(m, 4H), 2.12~2.21(m, 4H), 3.78(s, 2H), 4.11(s, 2H).

[0126] Example 2

[0127] This embodiment provides the synthesis of aliphatic chain 2:

[0128] .

[0129] The preparation process of fatty acid chain 2 is similar to that of fatty acid chain 1.

[0130] Fat chain 2: 1 H-NMR (400M, DMSO-d6) 1.11~1.25(m, 32H), 1.35(s, 9H), 1.39(s,9H), 2.12~2.22(m, 4H), 3.77(s, 2H), 4.10(s, 2H).

[0131] Example 3

[0132] This embodiment provides a method for synthesizing 20-phosphono-eicosylcarboxylic acid:

[0133] Starting with monomethyl eicosanoate, the carboxyl group was selectively reduced to a primary alcohol by borane at low temperature, the primary alcohol was brominated under carbon tetrachloride and triphenylphosphine conditions, then reacted with dibenzyl phosphite under alkaline conditions to form a phosphonyl group, and finally the carboxyl methyl ester was selectively hydrolyzed to a carboxylic acid under sodium hydroxide conditions. The target product was obtained through four chemical transformations.

[0134] .

[0135] Step 1: Selectively reduce carboxyl groups using borane

[0136] .

[0137] Eicosanoic acid monomethyl ester (35.65 g, 1.0 equiv, 100.0 mmol) was weighed into a three-necked flask at room temperature. Tetrahydrofuran (300 mL) was added and stirred until homogeneous. The mixture was cooled to -20 °C, and then 1 M BH3-THF (150 mL, 1.5 equiv, 150.0 mmol) was slowly added dropwise. After the addition was complete, the mixture was stirred at -20 °C for 1 h, and then naturally warmed to room temperature and stirred for 3-4 h. TLC plate showed that the starting material had reacted completely. The reaction flask was cooled to below 10 °C in an ice bath, and the reaction was quenched by slowly adding ice water (200 mL). The tetrahydrofuran was then concentrated under reduced pressure. The aqueous phase was extracted with ethyl acetate (300 mL each time, 3 extractions). The organic phases were combined and washed with saturated sodium chloride aqueous solution (100 mL each time, 2 washes). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain 33.86 g of crude product, a white solid.

[0138] 1H-NMR (400M, DMSO-d6) 1.13~1.25(m, 30H), 1.30~1.40(m, 2H), 1.40~1.50(m, 2H), 2.25(t, J=7.4Hz, 2H), 3.35(q, J=5.8Hz, 2H), 3.55(s, 3H).

[0139] Step 2: Bromination of primary alcohols under carbon tetrachloride and triphenylphosphine conditions .

[0140] At room temperature, methyl 20-hydroxy-eicosanoate (17.12 g, 1.0 equiv, 50.0 mmol) was weighed into a three-necked flask and dissolved in dichloromethane (200 mL). The mixture was cooled in an ice bath. Then, triphenylphosphine (19.67 g, 1.5 equiv, 75.0 mmol) was added to the reaction flask and stirred until dissolved. Carbon tetrabromide (24.87 g, 1.5 equiv, 75.0 mmol) was then slowly added in portions. After the addition was complete, the mixture was allowed to heat naturally for 3–4 hours. TLC plate readings showed complete reaction of the starting material. The reaction was quenched by slowly adding ice water (200 mL) in an ice bath. The aqueous phase was extracted with dichloromethane (200 mL each time, 3 extractions). The organic phases were combined and washed with saturated sodium chloride aqueous solution (50 mL each time, 2 washes). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. The product was then purified using a 200-300 mesh silica gel column (n-hexane:ethyl acetate = 20:1) to obtain 16.77 g of the pure brominated product, with a yield of 83%.

[0141] Step 3: Adding phosphonyl groups

[0142] .

[0143] Dibenzyl phosphite (11.80 g, 1.5 equiv, 45.0 mmol) was weighed into a three-necked flask at room temperature and dissolved in DMF (200 mL). Cesium carbonate (29.32 g, 3.0 equiv, 90.0 mmol) and tetrabutylammonium iodide (16.62 g, 1.5 equiv, 45.0 mmol) were weighed and added to the three-necked flask, and stirred at room temperature for 1 h. Methyl 20-bromo-eicosanoate (12.13 g, 1.0 equiv, 30.0 mmol) was weighed and added to the three-necked flask in portions, and the reaction was allowed to proceed at room temperature for 24 h. The reaction was confirmed by TLC plate analysis to be complete. The reaction was then quenched by adding water (300 mL) at room temperature. The aqueous phase was extracted with dichloromethane (300 mL each time, 3 extractions). The organic phases were combined and washed with saturated sodium chloride aqueous solution (50 mL each time, 2 washes). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. It was then purified using a 200-300 mesh silica gel column (n-hexane:ethyl acetate = 3:1) to give 11.62 g of the pure phosphonylated product, with a yield of 66%.

[0144] Step 4: Hydrolysis of methyl ester

[0145] .

[0146] At room temperature, methyl 20-phosphono n-eicosylcarboxylate (11.62 g, 1.0 equiv, 19.8 mmol) was weighed into a three-necked flask and dissolved in tetrahydrofuran (150 mL). The mixture was then cooled in an ice bath. Sodium hydroxide (0.74 g, 1.5 equiv, 29.7 mmol) was weighed into an Erlenmeyer flask and dissolved in MeOH / water (1 / 1, V / V, 10 mL). The solution was then slowly added dropwise to the reaction system in an ice bath. After the addition was complete, the temperature was raised to 40 °C and the reaction was carried out for 2–3 h. The mixture was cooled in an ice bath, and the pH was adjusted to 3–5 with 1 M hydrochloric acid solution. The tetrahydrofuran was concentrated under reduced pressure, and the mixture was diluted with 100 mL of ice water. The aqueous phase was then extracted with dichloromethane (200 mL each time, 3 extractions). The organic phases were combined and washed with saturated sodium chloride solution (50 mL each time, 2 washes). The organic phase was dried over anhydrous sodium sulfate, filtered, and concentrated under reduced pressure to obtain the crude product. It was then purified using a 200-300 mesh silica gel column (hexane:ethyl acetate = 1:1) to give 9.87 g of pure 20-phosphono-eicosylcarboxylic acid product, with a yield of 87%.

[0147] 1¹H-NMR (400M, CDCl₃) 1.19–1.39 (m, 30H), 1.47–1.69 (m, 4H), 1.70–1.82 (m, 2H), 2.33 (t, J=7.4Hz, 2H), 4.91–5.11 (m, 4H), 7.31–7.41 (m, 10H) (See also) Figure 1 ).

[0148] Example 4

[0149] This embodiment provides the synthesis of 22-phosphono-n-dococarboxylic acid:

[0150] .

[0151] The preparation process of 22-phosphono-n-eicosylcarboxylic acid is similar to that of 20-phosphono-n-eicosylcarboxylic acid.

[0152] Example 5

[0153] This embodiment provides the preparation of polypeptide sequence No. 003.

[0154] The peptide sequence was synthesized using the Fmoc (alkaline method) solid-phase synthesis, specifically through monomer-by-monomer coupling. Rink Amide-AM Resin was used as the solid-phase support, with DIC+HOBt or HATU+DIPEA as the coupling reagents, and 10%–30% piperidine DMF solution as the Fmoc removal reagent. Reaction monitoring was performed using the ninhydrin method or the tetrachlorobenzoquinone method (to detect the amino group of Pro). After synthesis, the resin was washed with DMF and DCM and dried. The peptide resin was lysed using a TFA / TIPS / EDT / water (90 / 4 / 4 / 2, V / V) lysis buffer, followed by precipitation with 5–10 volumes of methyl tert-butyl ether or isopropyl ether. After centrifugation, washing, and drying, crude linear peptides were obtained. The linear peptides were oxidized with iodine to form intramolecular disulfide bonds, and then excess iodine was neutralized with ascorbic acid. After purification, pure peptide No. 003 was obtained, with an HPLC purity of 98.84% (see [link to relevant documentation]). Figure 2 (See Table 3). The molecular weight of the peptides was confirmed by MS; see [reference]. Figure 3 (MS, ESI) + 1124.8177[M+4H] 4+ .

[0155] Table 3. HPLC Purity

[0156]

[0157] Purification method: The crude peptide was purified by high-pressure DAC preparative chromatography. For specific preparation and purification methods, please refer to Table 4.

[0158] Table 4. Purification Methods

[0159]

[0160] The main parameters of the preparation and purification method are shown in Table 4: the mobile phase A is 0.1% trifluoroacetic acid aqueous solution, and the mobile phase B is acetonitrile; UniSil 10-120 C18 produced by Changshu Nano Microbial Technology Co., Ltd. is used as the packing material; the target fraction is collected in segments, then concentrated under reduced pressure, and then freeze-dried to obtain pure lyophilized peptide No. 003 powder.

[0161] The preparation processes for peptides No. 005, No. 006, No. 007, No. 008, No. 009, No. 011, No. 013, No. 015, No. 017, No. 020, No. 022, No. 024, No. 025, No. 026, No. 027, No. 029, No. 035, and No. 036 are similar to those for peptide No. 003. Their detection and identification results are shown in Table 4. The preparation processes for peptides No. 037, No. 038, No. 039, No. 040, No. 041, and No. 050 are similar to those for peptide No. 003, except for an additional RCM ring-closing on the resin and palladium carbon hydrogenation after deprotection. Their detection and identification results are shown in Table 5.

[0162] Table 5. Purity and Molecular Weight

[0163]

[0164] Example 6

[0165] Freeze-thaw stability of the polypeptide compounds described in this invention:

[0166] The prepared peptide compounds were dissolved in phosphate-buffered saline (PBS) at pH 7.4 to prepare solutions with a concentration of 0.2 mg / mL. The samples were stored at -20°C for 48 hours, then naturally warmed to room temperature for reconstitution. The phenomena and changes in properties during the dissolution and reconstitution processes were observed, and the concentrations of the peptide compounds before and after freeze-thaw cycles were determined using high-performance liquid chromatography (HPLC).

[0167] Sample dissolution: Weigh a certain amount of the peptide compound and dissolve it in 20 nM pH 7.4 PBS buffer to prepare a 0.2 mg / mL peptide PBS solution. Centrifuge the sample at 10000 rpm, 4℃, and 5 min. Take the supernatant from the centrifugation and perform HPLC detection.

[0168] Sample redissolving: Remove the frozen sample from the refrigerator and place it at room temperature (25℃). After the ice has completely melted, centrifuge the sample at 10,000 rpm, 4℃, and 5 min. Take the supernatant from the centrifugation and perform HPLC analysis.

[0169] HPLC detection method: The detection method is shown in Table 6.

[0170] Table 6. HPLC Detection Methods

[0171]

[0172] Freeze-thaw yield = concentration of peptide after freeze-thaw / concentration of peptide before freeze-thaw. The dissolution and freeze-thaw properties of the peptide compounds are shown in Table 7. Most of the peptide compounds of this invention exhibit good freeze-thaw stability and freeze-thaw yield.

[0173] Table 7. Statistics on melting and freeze-thaw data

[0174]

[0175] Example 7

[0176] This embodiment provides an in vitro hCT-R and hAmy-R3 agonist activity assay for peptide compounds.

[0177] The main reagents, consumables, and instruments used for in vitro hCT-R and hAmy-R3 agonist activity assays are shown in Table 8.

[0178] Table 8. Main Reagents, Consumables and Instruments

[0179]

[0180] To evaluate the in vitro activity of the peptide compounds, stable cell lines expressing either recombinant human amylin 3 receptor (hAmyR3) or recombinant human calcitonin receptor (hCT-R) were used in a COS-7 cell line background. hAmyR3 is a heterooligomer of the two genes formed when calcitonin receptor (gene ID 799) and RAMP3 (gene ID 10268) are expressed in the same cell. The hCT-R cell line expressed only the recombinant human calcitonin receptor gene (gene ID 799). Activation of hCT-R or hAmyR3 with the tested peptide compounds induced cAMP formation, which was measured using a cAMP assay kit (cAMP-Gs Dynamic Kit) from Cisbio.

[0181] After removing the cultured cells from the culture medium, wash them with 5 ml of DPBS, then add 2.5 ml of 0.25% trypsin and digest for 1-2 min. Carefully transfer the cells to sterile 15 / 50 mL test tubes, add 10-50 mL of preheated 37°C complete culture medium, incubate the cells for 5 min, then centrifuge at 900 rpm for 5 min. Resuspend the cells in experimental buffer. To ensure experimental stability, the optimized cell density is 1000 cells / well. Seed 10 μL of COS-7 cells expressing hCT-R or hAmyR3 per well (containing 1000 cells / well) into a 384-well microtiter plate, then add 50 nmol of the test peptide compound at different concentrations to each well; rotate at 1000 rpm for 10 seconds and incubate at 37°C, 5% CO2 for 30 min.

[0182] Dilute cAMP and Eu-Anti-cAMP reagents according to Table 9 using the lysis and detection buffers provided in the kit.

[0183] Table 9. Reagent Kits

[0184]

[0185] Add 25 μL of cAMP-d and 5 μL of Anti-cAMP-Cryptate at the concentrations listed in the table above to each well of a 384-well plate containing the test peptide compound and cells. Incubate at room temperature for 60 minutes, and read the signal at 665 / 615 nm using a microplate reader. The data were processed using XLfit 5.5.0.5.

[0186] Results of in vitro activity (EC) 50 The values ​​are summarized in Table 10.

[0187] Table 10. Results of in vitro activity (EC) 50 value)

[0188]

[0189] Example 8

[0190] This example illustrates the effects of a polypeptide compound on the food intake and body weight of normal Sprague Dawley rats.

[0191] SD (Sprague Dawley) rats were purchased from Shanghai Silex Laboratory Animal Co., Ltd. Rats weighing approximately 250 grams were selected for this animal experiment. Before the experiment, the rats were housed in a reversed light / dark environment (light was off during the day and on at night) for two weeks to acclimatize to the experimental environment. Throughout the acclimatization period and the subsequent drug administration period, the rats had free access to food and water. Five rats were used in each test group, and each test included parallel setups for a solvent group and a positive control group (AM833, cagrilintide). Each rat was administered the drug at a dosage of 10 nmol / kg. An automated feeding system was used to record feeding online for four days, and food intake was manually recorded and body weight measured at 24h, 48h, 72h, and 96h after drug administration. Specific data are shown in Table 11. After a single subcutaneous injection, the food intake and body weight of the SD rats were significantly reduced. Compared with the positive control AM833, compounds No. 020 / No. 035 / No. 036 of this invention showed significant advantages.

[0192] Table 11. Effects of peptide compounds on food intake and body weight in normal Sprague Dawley rats

[0193] Example 9

[0194] This embodiment provides a weight loss experiment of DIO mice using a combination of peptide compounds and semaglutide.

[0195] DIO mice were purchased from Beijing Vital River Laboratory Animal Technology Co., Ltd., and were subsequently acclimatized in the animal facility for more than 10 days. During the entire acclimatization and drug administration experiment, they were fed a high-fat diet and had free access to food and water. DIO mice weighing between 38 and 42 grams were selected for the experiment. The DIO mouse weight loss experiment was set up as a Vehicle group, a combined drug positive control group (AM833 10 nmol / kg + Semaglutide 10 nmol / kg), and a combined drug group (peptide compound 10 nmol / kg + Semaglutide 10 nmol / kg). The drugs were administered once a day for 2 consecutive weeks. The results are shown in Table 12.

[0196] Table 12. Results of weight loss test in DIO mice using peptide compounds combined with semaglutide.

[0197] The weight loss test results of DIO mice showed that the weight loss effects of the polypeptide compounds No. 008, No. 009, No. 020, No. 025, No. 035, No. 036, No. 039 and No. 050 of the present invention were better than or comparable to those of the positive control group AM833+sema, among which No. 020 and No. 036 were significantly better than the positive control group.

[0198] Example 10

[0199] This example is a pharmacokinetic analysis in rats.

[0200] The pharmacokinetic properties of compounds No. 008 and No. 036 were investigated in SD rats. Ten rats were used for each compound, and a single dose of 10 nmol / kg was administered. Blood samples were collected from the tail vein at 0.25 h, 0.5 h, 1.0 h, 2.0 h, 4.0 h, 8.0 h, 24.0 h, 48.0 h, and 72.0 h after administration. Serum was separated, and blood drug concentrations were detected by LC-MS.

[0201] The main pharmacokinetic parameters are shown in Table 13.

[0202] Table 13. Pharmacokinetic Parameters

[0203]

[0204] In Table 13, Lambda_z is a parameter representing the slope of the terminal elimination phase of the drug, reflecting the rate of drug elimination from the body; T 1 / 2 Half-life; T max Peak time; C max Peak concentration; AUC 0-t Area under the drug-time curve from 0 to t; AUC 0-inf_obs The area under the observation curve from 0 to infinity; MRT 0-inf_obs The average residence time from 0 to infinity; Vz _obs Eliminate apparent phase distribution volume; Cl _obs Apparent clearance rate.

[0205] Conclusion: After subcutaneous administration, the half-life (T008) of polypeptide compound No. 008 in SD rats was... 1 / 2 The half-life (T036) of No. 036 in SD rats is 26.5 hours. 1 / 2 ) is 32.1 hours (see Figure 4 ), comparing the half-life (T) of AM833 in SD rats. 1 / 2 ) of 25 (WO2024022465A1), compound No. 036 of T 1 / 2 Significant improvements have been made.

[0206] The above description is merely a preferred embodiment of the present invention and is not intended to limit the present invention in any form or substance. It should be noted that those skilled in the art can make various improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention. Any modifications, alterations, and equivalent changes made by those skilled in the art based on the above-disclosed technical content without departing from the spirit and scope of the present invention are equivalent embodiments of the present invention. Furthermore, any modifications, alterations, and evolutions made to the above embodiments based on the essential technology of the present invention still fall within the scope of the technical solution of the present invention.

Claims

1. An amylin receptor agonist or a pharmaceutically acceptable salt or solvate thereof, characterized in that, The amylin receptor agonist mentioned above is selected from the following compounds: No. 039: ； No. 050: 。 2. A pharmaceutical composition, characterized in that, It includes the amylin receptor agonist of claim 1 or a pharmaceutically acceptable salt or solvate thereof, as well as a pharmaceutically acceptable excipient.

3. A medicine box, characterized in that, It comprises the amylin receptor agonist of claim 1 or a pharmaceutically acceptable salt or solvate thereof, and also comprises packaging and / or instructions for use.

4. The medicine box according to claim 3, wherein It also includes antidiabetic agents, antiobesity agents, or anti-dyslipidemia agents.

5. The medicine box according to claim 3, wherein It also includes medications for treating metabolic syndrome.

6. A method for synthesizing the amylin receptor agonist according to claim 1, characterized in that, The method includes synthesizing the amylin receptor agonist by solid-phase or liquid-phase methods, isolating and purifying the final product, and further includes the step of forming a disulfide bond between the thiol groups of the cysteine ​​side chains at positions 2 and 7, and further includes a method of constructing a cyclic peptide by means of an RCM reaction of the olefin bond at the ends of the amino acid side chains at positions 2 and 7, and further includes a method of synthesizing a terminally phosphorylated 20-carbon fatty acid side chain.

7. Use of the amylin receptor agonist of claim 1 or a pharmaceutically acceptable salt or solvate thereof in the preparation of a medicament for the treatment of obesity, diabetes, non-alcoholic steatohepatitis, or atherosclerotic dyslipidemia.

8. The use according to claim 7, characterized in that, The medication includes additional therapeutic agents, such as antidiabetic agents, antiobesity agents, anti-dyslipidemia agents, antihypertensive agents, proton pump inhibitors, or anti-inflammatory agents.

9. The use according to claim 7, characterized in that, The medications also include agents for the treatment of metabolic syndrome.

10. The use according to claim 8, characterized in that, The antidiabetic agents mentioned are metformin, sulfonylureas, meglitinides, DPP-IV inhibitors, thiazolidinediones, GLP-1 receptor agonists, SGLT2 inhibitors, GPR40 agonists, insulin or insulin analogs.

11. The use according to claim 8, characterized in that, The anti-obesity agents mentioned are peptide YY, neuropeptide Y, cannabinoid receptor 1 antagonist, lipase inhibitor, human pro-insulin peptide, melanocortin receptor 4 agonist, GLP-1 receptor agonist, GIP receptor agonist, GCG receptor agonist, phentermine, melanin-concentrating hormone receptor 1 antagonist, CCK or leptin.

12. The use according to claim 11, wherein The lipase inhibitor mentioned is orlistat.

13. The use according to claim 8, wherein The antihypertensive agents mentioned are angiotensin-converting enzyme inhibitors, angiotensin II receptor blockers, diuretics, β-blockers, or calcium channel blockers.

14. The use according to claim 8, wherein The aforementioned anti-dyslipidemia agents are statins, fibrates, niacin, PSCK9 inhibitors, or cholesterol absorption inhibitors.

15. The use according to claim 8, characterized in that, The proton pump inhibitor is a benzimidazole derivative or an imidazopyridine derivative.

16. The use according to claim 8, wherein The anti-inflammatory agent mentioned is a steroid, a nonsteroidal anti-inflammatory agent, 5-aminosalicylic acid, or metformin.

17. The use according to claim 8, characterized in that, The anti-inflammatory agents mentioned are corticosteroids, COX II inhibitors, 5-aminosalicylic acid, or metformin.

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