GLP-1 / GIP bireceptor agonists, GLP-1 / GCG bireceptor agonists, and GLP-1 / GIP / GCG triplereceptor agonists

Abstract

This disclosure provides GLP-1 / GIP and GLP-1 / GCG dual receptor agonists comprising incretin analog polypeptides, as well as their use in the treatment or prevention of type 2 diabetes mellitus (T2DM), hyperlipidemia / dyslipidemia, metabolic syndrome, metabolic dysfunction-related fatty liver disease (MASLD), metabolic dysfunction-related steatohepatitis (MASH), neurodegenerative disorders, fibrosis, cardiovascular risk, and / or obesity.

Description

[Technical Field]

[0001] Cross-reference of related applications This application claims priority to Indian Patent Application No. 202321039646, filed on 9 June 2023, which is incorporated herein by reference in its entirety. Reference to electronic sequence listings

[0002] This application includes an electronically filed sequence listing, which is incorporated herein by reference in its entirety. The sequence listing was created on 7 June 2024, named "24-0805-WO_Sequence-Listing.xml", and has a size of 110,592 bytes. field

[0003] This disclosure relates to glucagon-like peptide-1 (GLP-1) monoreceptor agonists, GLP-1 / glucose-dependent insulin secretion-stimulating polypeptides or gastrointestinal peptide (GIP) dualreceptor agonists, and GLP-1 / glucagon (GCG) dualreceptor agonists. In particular, this disclosure relates to GLP-1 / GIP and GLP-1 / GCG dualreceptor agonists comprising incretin analog polypeptides. The polypeptides described herein have structural features that provide balanced activity and long-lasting action in each of these receptors. Polypeptides according to the present invention may be useful in the treatment of type 2 diabetes mellitus (T2DM), hyperlipidemia / dyslipidemia, metabolic syndrome, metabolic dysfunction-related fatty liver disease (MASLD), metabolic dysfunction-related steatohepatitis (MASH), neurodegenerative disorders, fibrosis, and / or obesity, as well as in reducing cardiovascular risk. [Background technology]

[0004] Over the past several decades, the prevalence of diabetes has continued to rise. T2DM is the most common type of diabetes and is characterized by high blood glucose levels caused by insulin resistance. People with T2DM are more likely to develop comorbidities such as hyperlipidemia / dyslipidemia, metabolic syndrome, metabolic dysfunction-related fatty liver disease (MASLD), metabolic dysfunction-related steatohepatitis (MASH), neurodegenerative disorders, fibrosis, cardiovascular risk, and / or obesity.

[0005] Current therapies for T2DM include diet and exercise, as well as oral medications and injectable glucose-lowering agents, including incretin therapies such as GLP-1 monoreceptor agonists and / or GLP-1 / GIP dual receptor agonists. For example, cotadutide (SEQ ID NO: 6), MK-1462 (SEQ ID NO: 7), and mazdotide (SEQ ID NO: 8) are peptides that act as GLP-1 / GCG dual receptor agonists.

[0006] WIPO Publications WO2019 / 193576, WO2006 / 097537, and WO1998 / 008871 disclose GLP-1 monoreceptor agonist compounds. WIPO Publications WO2022 / 079639, WO2021 / 260530, WO2017 / 74714A1, WO2020 / 23386, WO2020 / 023388, WO2015 / 067715, WO2016 / 111971, WO2014 / 192284, WO2011 / 119657, and WO2013 / 164483 disclose GLP-1 / GIP dual receptor agonist compounds. WIPO Publications WO2011 / 075393, WO2012 / 177444, WO2014 / 091316, and WO2017 / 153575 disclose GLP-1 / GCG bireceptor agonist compounds.

[0007] Recent studies on GLP-1 / GCG bireceptor agonists and / or GLP-1 / GIP / GCG triple receptor agonists have also highlighted the importance of understanding the contributions of individual hormonal actions and branching effects by altering GLP-1:GCG activity and ratio in GLP-1 / GCG bireceptor agonists and GLP-1 / GIP / GCG triple receptor agonists.

[0008] In addition to being diabetic, glucagon receptor (GCGR) agonists are known to increase heart rate and contractility, which can lead to adverse cardiovascular consequences. Furthermore, chronic excess glucagon also leads to catabolism of amino acids and proteins, resulting in loss of lean body mass. These side effects offset the weight-loss effect provided by GCG agonism.

[0009] A delicate balance between GLP-1 receptor agonists and GCG receptor agonists is necessary to achieve optimal results with minimal side effects.

[0010] Cotadutide, which is currently discontinued, also shows higher efficacy against GCGR compared to GLP-1R.

[0011] While the broad metabolic benefits of GLP-1 monoreceptor agonists or GLP-1 / GIP bireceptor agonists are well-established in the therapeutic paradigm, there remains a need for therapies that offer effective glucose control and weight loss, along with a favorable side effect profile, particularly for the treatment of T2DM. There is also a need for therapeutic agents with sufficiently long durations of action to allow for less frequent administration, such as twice or once per week. [Overview of the project]

[0012] In one embodiment, the present disclosure provides a polypeptide comprising the following amino acid sequence or a pharmaceutically acceptable salt thereof. X1-X2-X3-G-T-F-T-S-D-X10-S-X12-X13-L-D-X16-X17-X18-X19-X20-X21-F-X23-X24-X25-L-X27-X28-X29-X30-X31-X32-X33-X34-X35-X36-X37-X38-X39(Array No. 1) In the formula, X1 is H, X2 is D-Ser(OMe), Aib or D-S, X3 is Q, X10 is K or Y, X12 is E, K, or I, X13 is Y, S(OMe), nor-V, nor-L, or αMe-L, X16 is S, E, or A, X17 is E, R, or K, X18 is R, K, or A, X19 is A, X20 is R, Q, or K, X21 is D or E, X23 is V or I, X24 is A, Q, or E, X25 is W, X27 is E or L, X28 is A, D or E, X29 is G or T, X30 does not exist or is G, X31 does not exist or is P, X32 does not exist or is S, X33 does not exist or is S, X34 does not exist or is G, X35 does not exist, X36 does not exist, X37 does not exist, X38 does not exist, X39 does not exist, In the formula, the acid group of the C-terminal amino acid is either a free carboxylic acid group or is amidated as a C-terminal primary amide. However, at least one of X10 and X20 is K, and at least one of K is [ka] [ka] The selected formula contains an acylated side-chain amino(ε-amino) group.

[0013] In another embodiment, the disclosure provides a polypeptide comprising the following amino acid sequence or a pharmaceutically acceptable salt thereof. X1-X2-X3-GTFTSD-X10-S-X12-X13-LD-X16-X17-X18-X19-X20-X21-F-X23-X2 4-X25-L-X27-X28-X29-X30-X31-X32-X33-X34-X35-X36-X37-X38-X39 (Sequence number 2) During the ceremony, X1 is H, X2 is S, D-Ser(OMe), Aib, or DS. X3 is Q, X10 is either K or Y. X12 is E, K, or I. X13 is Y, S(OMe), nor-V, nor-L, or αMe-L. X16 is S, E, or A. X17 is E, R, or K. X18 is R, K, or A. X19 is A, X20 is R, Q, or K. X21 is either D or E. X23 is V, X24 is A, Q, or E. X25 is W, X27 is either E or L. X28 is A, D, or E. X29 is either G or T. X30 does not exist, or it is G. X31 does not exist, or it is P. X32 does not exist, or it is S. X33 does not exist, or it is S. X34 does not exist, or it is G. The X35 does not exist. X36 does not exist. X37 does not exist. X38 does not exist. X39 does not exist. In the formula, the acid group of the C-terminal amino acid is either a free carboxylic acid group or is amidated as a C-terminal primary amide. However, at least one of X10 and X20 is K, and at least one of K is [ka] The selected formula contains an acylated side-chain amino(ε-amino) group.

[0014] In another embodiment, the disclosure provides a polypeptide comprising the following amino acid sequence or a pharmaceutically acceptable salt thereof. H-X2-QGTFTSD-X10-SEYLDSERARDFVAWLEAGG (Sequence ID 3) During the ceremony, X2 is S, DS(OMe) or Aib, X10 is K, In the formula, the acid group of the C-terminal amino acid is either a free carboxylic acid group or is amidated as a C-terminal primary amide. The amino(ε-amino) side chain of K at position X10 is [ka] The part of the expression selected from is acylated, However, the polypeptide in question is not Sequence ID No. 6.

[0015] In another embodiment, the disclosure provides a polypeptide comprising the following amino acid sequence or a pharmaceutically acceptable salt thereof. H-Aib-QGTFTSDYS-X12-X13-LDEKKA-X20-EFVEWLLEGGPSSG(Sequence No. 4) During the ceremony, X12 is either K or I. X13 is Y or Nor-V, X20 is K, In the formula, the acid group of the C-terminal amino acid is either a free carboxylic acid group or is amidated as a C-terminal primary amide. The amino(ε-amino) side chain of K at position X20 is [ka] The part of the expression selected from is acylated.

[0016] In another embodiment, the disclosure provides a polypeptide comprising the following amino acid sequence or a pharmaceutically acceptable salt thereof. H-(DSer)-QGTFTSD-X10-SKYLDARAAQDFVQWLLDT(Sequence ID 5) X10 is K, In the formula, the acid group of the C-terminal amino acid is either a free carboxylic acid group or is amidated as a C-terminal primary amide. The amino(ε-amino) side chain of K at position X10 is [ka] The part of the expression selected from is acylated.

[0017] In one aspect, this disclosure is, [ka] The present invention provides polypeptides selected from the group consisting of the following.

[0018] In another aspect, this disclosure is: X 1 A peptide residue having the sequence -Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys (SEQ ID NO: 29), X 1 The peptide residue includes a peptide residue where Aib or Ser(OMe) represents Aib or Ser(OMe), and lysine is bonded to the lysine ε-nitrogen, This invention relates to an incretin analog containing a glycerin-glycerin-OH peptide residue indirectly bound to the carboxyl group of lysine.

[0019] In another aspect, this disclosure is: X 1 A peptide residue having the sequence -Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys (SEQ ID NO: 29), X 1 A peptide residue containing a group of formula (I) in which represents Aib or Ser(OMe) and lysine is bonded to the lysine ε-nitrogen, [ka] (In the formula, U is either nonexistent or -C(O)-CH2-O-(CH2)2-O-(CH2)2-NH-}, where} is a bond point with W. W is -C(O)-CH2-O-(CH2)2-O-(CH2)2-NH-], -C(O)-NH-(CH2) 3‐4 -NH-], -C(O)-C(CH3)2-NH-], or [ka] This represents the connection point with Y, where ] is the point of connection with Y. Y is either nonexistent, or -C(O)-(CH2)2-CH(CO2H)NH-- or -C(O)CH((CH2) xrepresents --NH--CO₂H, where x is 1, 2, or 3, and where -- represents the point of attachment to Z, Z is -C(O)-(CH₂) n -COOH or -C(O)-(CH₂) n -CH₃, where n is an integer from 14 to 20), Provided is an incretin analog comprising a Gly-Gly-OH peptide residue indirectly linked to the carboxy of lysine.

[0020] In another aspect, the present disclosure a peptide residue having the Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp sequence (SEQ ID NO: 30), and a lysine residue indirectly linked to the carboxy of the Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp (SEQ ID NO: 30) residue, wherein the lysine comprises a group of formula (I) attached to the lysine ε-nitrogen, [Chemical formula] (wherein U is absent or is -C(O)-CH₂-O-(CH₂)₂-O-(CH₂)₂-NH-}, where} is the point of attachment to W, W is -C(O)-CH₂-O-(CH₂)₂-O-(CH₂)₂-NH-], or -C(O)-NH-(CH₂) 3‐4 -NH-], -C(O)-C(CH₃)₂-NH-], where ] is the point of attachment to Y, Y is absent or is -C(O)-(CH₂)₂-CH(CO₂H)NH-- or -C(O)CH((CH₂) x CO₂H)NH--, where x is 1, 2, or 3, and -- is the point of attachment to Z, Z is -C(O)-(CH₂) n -COOH or -C(O)-(CH₂) n -CH₃, where n is an integer from 14 to 20), The present invention provides an incretin analog containing a Gly-Gly-Pro-Ser-Ser-Gly-CONH2 peptide residue indirectly bound to the carboxyl group of lysine.

[0021] In another aspect, the disclosure relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an incretin analog or polypeptide as described herein.

[0022] In another aspect, the Disclosure relates to a method for treating obesity, type 2 diabetes mellitus (T2DM), metabolic syndrome, metabolic dysfunction-related fatty liver disease (MASLD), metabolic dysfunction-related steatohepatitis (MASH), neurodegenerative disorders, fibrosis, cardiovascular risk, and / or hyperlipidemia / dyslipidemia, comprising administering an incretin analog or polypeptide described herein to a patient requiring such treatment. [Modes for carrying out the invention]

[0023] Abbreviation A1: 2-aminoisobutyric acid DIPEA: N,N′-di-isopropylethylamine HOBt: 1-hydroxybenzotriazole DIPC:N,N′-di-isopropylcarbodiimide THF: Tetrahydrofuran DCM: Dichloromethane Fmoc: Fluorenylmethyloxycarbonyl HOSu:N-hydroxysuccinimide DCC: Dicyclohexylcarbodiimide DMAc: Dimethylacetamide IBCF: Isobutylchloroformate NMM: N-methylmorpholine DIC: Diisopropylcarbodiimide

[0024] definition

[0025] The “pharmaceutically acceptable salts” as defined in this disclosure include acid addition salts formed with either organic or inorganic acids. Suitable pharmaceutically acceptable salts of the compounds of this disclosure include, for example, salts of inorganic acids such as hydrochloric acid, hydrobromic acid, and phosphoric acid, or acid addition salts that may be salts of organic acids such as acetic acid, fumaric acid, succinic acid, adipic acid, pimelic acid, suberic acid, azelaic acid, malic acid, tartaric acid, and amino acids (e.g., glutamic acid or aspartic acid). Examples of pharmaceutically acceptable acid addition salts of the compounds of this disclosure include salts formed by the addition of one or more acid equivalents, such as monohydrochloride or dihydrochloride. Salts can be prepared by any process at the responsibility of those skilled in the art. See Berge et al., J. Pharm. Sci., 1977, 66, 1-19, and “Handbook of Pharmaceutical Salts: Properties, Selection, and Use,” edited by Stahl et al., Verlag Helv. Chim. Acta, Zurich, Switzerland, and Wiley-VCH, Weinheim, Germany, 2002.

[0026] As used herein, the term “effective dose” or “effective amount” refers to an amount of a compound sufficient to cure, reduce, alleviate, or partially address the clinical symptoms and complications of a particular disease or condition, beyond the effects expected without such treatment, through a single or multiple administration to a subject. Therefore, the result may be a reduction and / or alleviation of the signs, symptoms, or causes of the disease, or any other desirable change in the biological system. It is understood that the “therapeutic effective dose” may vary from subject to subject, depending on age, weight, the subject’s overall health, the condition being treated, the severity of the condition being treated, and the judgment of the prescribing physician.

[0027] As used herein, the amino acid "Aib" may also be represented by the following structure: [ka] It may also be defined as "2-aminoisobutyric acid" by its chemical name.

[0028] As used herein, the amino acid "S(OMe)" or "Ser(OMe)" may also be represented by the following structure: [ka] It may also be defined as "serine methyl ether" by its chemical name. The terms L-Ser(OMe) and D-Ser(OMe) refer to the "L" isomer and "D" isomer of Ser(OMe), respectively.

[0029] As used herein, the amino acids “nor-V”, “nor-Val”, or “norvaline” may also be represented by the following structures: [ka] It may also be defined as "2-aminopentanoic acid" by its chemical name. The terms L-norvaline and D-norvaline refer to the "L" and "D" isomers of norvaline, respectively.

[0030] As used herein, the amino acids "nor-L", "nor-Leu", or "norleucine" may also be represented by the following structures: [ka] It may also be defined as "2-aminohexanoic acid" by its chemical name. The terms D-norleucine and D-norleucine refer to the "L" and "D" isomers of norleucine, respectively.

[0031] As used herein, the amino acid "αMe-L", "αMe-Leu", or "αMe-leucine" may also be represented by the following structures: [ka] It may also be defined as "2-amino-2,4-dimethylpentanoic acid" by its chemical name. The terms L-α-Me-leucine and D-α-Me-leucine refer to the "L" isomer and "D" isomer of α-Me-leucine, respectively.

[0032] As described herein, the disclosure provides stable, long-acting GLP-1 mono, GLP-1 / GIP dual, and / or GLP-1 / GCG dual receptor agonists that may be useful for the treatment of T2DM, hyperlipidemia / dyslipidemia, metabolic syndrome, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), neurodegenerative disorders, fibrosis, and / or obesity, as well as for reducing cardiovascular risk.

[0033] In one embodiment, the present disclosure provides a polypeptide comprising the following amino acid sequence or a pharmaceutically acceptable salt thereof. X1-X2-X3-GTFTSD-X10-S-X12-X13-LD-X16-X17-X18-X19-X20-X21-F-X23-X2 4-X25-L-X27-X28-X29-X30-X31-X32-X33-X34-X35-X36-X37-X38-X39 (Sequence number 1) During the ceremony, X1 is H, X2 is D-Ser(OMe), Aib, or DS. X3 is Q, X10 is either K or Y. X12 is E, K, or I. X13 is Y, S(OMe), nor-V, nor-L, or αMe-L. X16 is S, E, or A. X17 is E, R, or K. X18 is R, K, or A. X19 is A, X20 is R, Q, or K. X21 is either D or E. X23 is V or I, X24 is A, Q, or E. X25 is W, X27 is either E or L. X28 is A, D, or E. X29 is either G or T. X30 does not exist, or it is G. X31 does not exist, or it is P. X32 does not exist, or it is S. X33 does not exist, or it is S. X34 does not exist, or it is G. The X35 does not exist. X36 does not exist. X37 does not exist. X38 does not exist. X39 does not exist. In the formula, the acid group of the C-terminal amino acid is either a free carboxylic acid group or is amidated as a C-terminal primary amide. However, at least one of X10 and X20 is K, and at least one of K is [ka] [ka] The selected formula contains an acylated side-chain amino(ε-amino) group.

[0034] In one embodiment, the polypeptide of SEQ ID NO: 1 comprises the following sequence: X2 is either D-Ser(OMe) or Aib. X10 is K, X12 is E, X13 is Y, X16 is S, X17 is E, X18 is R, X20 is R, X21 is D, X24 is A, X27 is E, X28 is A, X29 is G, X30 is G, X31, X32, X33, and X34 do not exist. The amino(ε-amino) side chain of K at position X10 is [ka] The part of the expression selected from is acylated.

[0035] In another embodiment, the disclosure provides a polypeptide comprising the following amino acid sequence or a pharmaceutically acceptable salt thereof. X1-X2-X3-GTFTSD-X10-S-X12-X13-LD-X16-X17-X18-X19-X20-X21-F-X23-X2 4-X25-L-X27-X28-X29-X30-X31-X32-X33-X34-X35-X36-X37-X38-X39 (Sequence number 2) During the ceremony, X1 is H, X2 is S, D-Ser(OMe), Aib, or DS. X3 is Q, X10 is either K or Y. X12 is E, K, or I. X13 is Y, S(OMe), nor-V, nor-L, or αMe-L. X16 is S, E, or A. X17 is E, R, or K. X18 is R, K, or A. X19 is A, X20 is R, Q, or K. X21 is either D or E. X23 is V, X24 is A, Q, or E. X25 is W, X27 is either E or L. X28 is A, D, or E. X29 is either G or T. X30 does not exist, or it is G. X31 does not exist, or it is P. X32 does not exist, or it is S. X33 does not exist, or it is S. X34 does not exist, or it is G. The X35 does not exist. X36 does not exist. X37 does not exist. X38 does not exist. X39 does not exist. In the formula, the acid group of the C-terminal amino acid is either a free carboxylic acid group or is amidated as a C-terminal primary amide. However, at least one of X10 and X20 is K, and at least one of K is [ka] The selected formula contains an acylated side-chain amino(ε-amino) group.

[0036] In one embodiment, the polypeptide of SEQ ID NO: 1 or SEQ ID NO: 2 includes the following sequence: X2 is a DS, X10 is K, X12 is K, X13 is Y, X16 is A, X17 is R, X18 is A, X20 is Q, X21 is D, X24 is Q, X27 is L, X28 is D, X29 is T, X31, X32, X33, and X34 do not exist. The amino(ε-amino) side chain of K at position X10 is [ka] The part of the expression selected from is acylated.

[0037] In another embodiment, the polypeptide of SEQ ID NO. 1 or SEQ ID NO. 2 includes the following sequence: X2 is Aib, X10 is Y, X12 is either K or I. X13 is Y, Nor-V, Nor-L, or αMe-L. X16 is E, X17 is K, X18 is K, X20 is K, X21 is E, X24 is E, X27 is L, X28 is E, X29 is G, X30 is G, X31 is P, X32 is S, X33 is S, X34 is G, In the formula, the amino(ε-amino) group of the K side chain at position X20 is, [ka] The part of the expression selected from is acylated.

[0038] In another embodiment, the disclosure provides a polypeptide comprising the following amino acid sequence or a pharmaceutically acceptable salt thereof. H-X2-QGTFTSD-X10-SEYLDSERARDFVAWLEAGG (Sequence ID 3) During the ceremony, X2 is S, DS(OMe) or Aib, X10 is K, In the formula, the acid group of the C-terminal amino acid is either a free carboxylic acid group or is amidated as a C-terminal primary amide. The amino(ε-amino) side chain of K at position X10 is [ka] The part of the expression selected from is acylated, However, the polypeptide is not sequence number 6.

[0039] In one embodiment of the polypeptide of SEQ ID NO: 3, X2 is S, The amino(ε-amino) side chain of K at position X10 is [ka] The part of the expression selected from is acylated.

[0040] In another embodiment of the polypeptide of SEQ ID NO: 3, X2 is D-Ser(OMe), The amino(ε-amino) side chain of K at position X10 is, [ka] It is acylated in that part.

[0041] In another embodiment of the polypeptide of SEQ ID NO: 3, X2 is Aib, The amino(ε-amino) group of the K at position X10 is, [ka] The part of the expression selected from is acylated.

[0042] In another embodiment, the disclosure provides a polypeptide comprising the following amino acid sequence or a pharmaceutically acceptable salt thereof. H-Aib-QGTFTSDYS-X12-X13-LDEKKA-X20-EFVEWLLEGGPSSG(Sequence No. 4) During the ceremony, X12 is either K or I. X13 is Y or Nor-V, X20 is K, In the formula, the acid group of the C-terminal amino acid is either a free carboxylic acid group or is amidated as a C-terminal primary amide. The amino(ε-amino) side chain of K at position X20 is [ka] The part of the expression selected from is acylated.

[0043] In one embodiment of the polypeptide of Sequence ID No. 4, X12 is K, X13 is Y, The amino(ε-amino) side chain of K at position X20 is [ka] The part of the expression selected from is acylated.

[0044] In another embodiment of the polypeptide of Sequence ID No. 4, X12 is I, X13 is Nor-V, The amino(ε-amino) side chain of K at position X20 is, [ka] It is acylated in that part.

[0045] In another embodiment of the polypeptide of Sequence ID No. 4, X12 is K, X13 is Nor-V, The amino(ε-amino) side chain of K at position X20 is, [ka] It is acylated in that part.

[0046] In another embodiment, the disclosure provides a polypeptide comprising the following amino acid sequence or a pharmaceutically acceptable salt thereof. H-(DSer)-QGTFTSD-X10-SKYLDARAAQDFVQWLLDT(Sequence ID 5) In the formula, X10 is K, In the formula, the acid group of the C-terminal amino acid is either a free carboxylic acid group or is amidated as a C-terminal primary amide. The amino(ε-amino) side chain of K at position X10 is [ka] The part of the expression selected from is acylated.

[0047] In another aspect, this disclosure is: X 1 A peptide residue having the sequence -Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys (SEQ ID NO: 29), X 1 The peptide residue includes a peptide residue where Aib or Ser(OMe) represents Aib or Ser(OMe), and lysine is bonded to the lysine ε-nitrogen, The present invention provides an incretin analog comprising a glycerin-glycerin-OH peptide residue indirectly bound to the carboxyl group of lysine.

[0048] In one embodiment, the incretin analog has lysine bound to a glycy-glycy-OH residue by a peptide residue containing 18 amino acids.

[0049] In another aspect, this disclosure is: X 1 A peptide residue having the sequence -Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys (SEQ ID NO: 29), X 1 A peptide residue containing a group of formula (I) in which represents Aib or Ser(OMe) and lysine is bonded to the lysine ε-nitrogen, [ka] (In the formula, U is either nonexistent or -C(O)-CH2-O-(CH2)2-O-(CH2)2-NH-}, where} is a bond point with W. W is -C(O)-CH2-O-(CH2)2-O-(CH2)2-NH-], -C(O)-NH-(CH2) 3‐4 -NH-], -C(O)-C(CH3)2-NH-], or [ka] In the equation, ] is the point of connection with Y, Y is either nonexistent, or -C(O)-(CH2)2-CH(CO2H)NH-- or -C(O)CH((CH2) x This represents CO2H)NH--, where x is 1, 2, or 3, and -- is the bond point with Z. Z is -C(O)-(CH2). n -COOH or -C(O)-(CH2) n -CH3, where n is an integer between 14 and 20) The present invention provides an incretin analog comprising a glycerin-glycerin-OH peptide residue indirectly bound to the carboxyl group of lysine.

[0050] In one embodiment, the incretin analog has lysine bound to a glycy-glycy-OH residue by a peptide residue containing 18 amino acids.

[0051] In another aspect, this disclosure is: A peptide residue having the sequence Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp (SEQ ID NO: 30), A lysine residue indirectly bound to the carboxyl group of the Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp (SEQ ID NO: 30) residue, wherein the lysine residue contains a group of formula (I) bonded to the lysine ε-nitrogen, [ka] (In the formula, U is either nonexistent or -C(O)-CH2-O-(CH2)2-O-(CH2)2-NH-}, where} is a bond point with W. W is -C(O)-CH2-O-(CH2)2-O-(CH2)2-NH], or -C(O)-NH-(CH2) 3‐4 [-NH-], [-C(O)-C(CH3)2-NH-], In the equation, ] is the point of connection with Y, Y does not exist, or -C(O)-(CH2)2-CH(CO2H)NH-- or -C(O)CH((CH2) x This represents CO2H)NH--, where x is 1, 2, or 3, and -- is the bond point with Z. Z is -C(O)-(CH2). n -COOH or -C(O)-(CH2) n -CH3, where n is an integer between 14 and 20) The present invention provides an incretin analog containing a Gly-Gly-Pro-Ser-Ser-Gly-CONH2 peptide residue indirectly bound to the carboxyl group of lysine.

[0052] In one embodiment, the incretin analog has lysine bound to the Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp (SEQ ID NO: 30) residue by a peptide residue containing 10 amino acids.

[0053] In another embodiment, the polypeptides described herein exclude the polypeptides of SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, and SEQ ID NO: 27.

[0054] In another embodiment, the disclosure provides a polypeptide comprising an amino acid sequence selected from the group consisting of the following: a pharmaceutically acceptable salt thereof. i.) HSQGTFTSDK * SEYLDSERARDFVAWLEAGG-OH (SEQ ID NO: 9) ii.) H-(DS(OMe))-QGTFTSDK *SEYLDSERARDFVAWLEAGG-OH(SEQ ID NO: 10); iii.) H-Aib-SQGTFTSDK * SEYLDSERARDFVAWLEAGG-OH(SEQ ID NO: 11); iv.) H-(DS)-QGTFTSDK * SKYLDARAAQDFVQWLLDT-NH2 (Sequence ID 12), v.) H-Aib-QGTFTSDYSKYLDEKKAK * EFVEWLLEGGPSSG-NH2 (Sequence ID 13), vi.) H-Aib-QGTFTSDYSK-(nor-V)-LDEKKAK * EFVEWLLEGGPSSG-NH2 (Sequence ID 14); and vii.) H-Aib-QGTFTSDYSI-(nor-V)-LDEKKAK * EFVEWLLEGGPSSG-NH2 (Sequence ID 15), In the formula, K * The side chain amino(ε-amino) group is [ka] [ka] The part of the expression selected from is acylated, The polypeptide is not selected from SEQ ID NO: 6, SEQ ID NO: 7, SEQ ID NO: 8, or SEQ ID NO: 27.

[0055] The polypeptide sequences described herein are represented by either single-letter or three-letter amino acid codes approved by the International Union of Pure and Applied Chemistry (IUPAC).

[0056] Unless otherwise specified, this disclosure is intended to cover both the L and D isomers of the amino acids in the sequences described herein. However, in certain preferred embodiments, unless otherwise suggested, all amino acids are of the "L" form.

[0057] In another embodiment, the disclosure provides polypeptides selected from representative compounds disclosed in Table 1 or pharmaceutically acceptable salts thereof. [Table 1-1] [Table 1-2] [Table 2-1] [Table 2-2]

[0058] In another aspect, this disclosure is: [ka] Provides polypeptides selected from.

[0059] In another aspect, the present disclosure provides a pharmaceutical composition comprising a pharmaceutically acceptable carrier and an incretin analog or polypeptide as described herein.

[0060] In another aspect, the Disclosure provides a method for treating obesity, type 2 diabetes mellitus (T2DM), metabolic syndrome, metabolic dysfunction-related fatty liver disease (MASLD), metabolic dysfunction-related steatohepatitis (MASH), fibrosis, cardiovascular disease, and / or hyperlipidemia / dyslipidemia, the method comprising administering an incretin analog or polypeptide described herein to a patient requiring such treatment.

[0061] In another aspect, the disclosure provides a method for treating or preventing type 2 diabetes mellitus (T2DM).

[0062] In another aspect, the disclosure provides a method for treating or preventing hyperlipidemia / dyslipidemia.

[0063] In another aspect, the disclosure provides a method for treating or preventing obesity.

[0064] In another aspect, the disclosure provides methods for treating or preventing metabolic syndrome, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), neurodegenerative disorders, fibrosis, and / or cardiovascular risks.

[0065] In one embodiment, the treatment method includes administering an effective amount of the polypeptide described herein or a pharmaceutically acceptable salt thereof to a patient in need of treatment.

[0066] In another aspect, the present disclosure provides a method for treating type 2 diabetes mellitus (T2DM), comprising administering an effective amount of a polypeptide described herein or a pharmaceutically acceptable salt thereof to a patient in need of such treatment.

[0067] In another aspect, the present disclosure provides a method for treating obesity, comprising administering to a patient in need of such treatment an effective amount of a polypeptide described herein or a pharmaceutically acceptable salt thereof.

[0068] In another aspect, the Disclosure provides a method for treating hyperlipidemia / dyslipidemia, comprising administering an effective amount of a polypeptide described herein or a pharmaceutically acceptable salt thereof to a patient in need of such treatment.

[0069] In another embodiment, the Disclosure provides a pharmaceutical composition comprising a polypeptide described herein or a pharmaceutically acceptable salt thereof, and one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0070] The compounds of the present invention are preferably formulated as pharmaceutical compositions administered via parenteral routes (e.g., subcutaneous, intravenous, intraperitoneal, intramuscular, or transdermal). Such pharmaceutical compositions and methods for preparing them are well known in the art. See, for example, "Remington: The Science and 50 Practices of Pharmacy," edited by DB Troy, 21st Edition, Lippincott, Williams & Wilkins, 2006.

[0071] In another aspect, the present disclosure provides polypeptides described herein or pharmaceutically acceptable salts thereof for use as pharmaceuticals.

[0072] In another aspect, the Disclosure provides polypeptides described herein or pharmaceutically acceptable salts thereof for use in the treatment or prevention of type 2 diabetes mellitus (T2DM).

[0073] In another aspect, the Disclosure provides polypeptides described herein or pharmaceutically acceptable salts thereof for use in the treatment or prevention of hyperlipidemia / dyslipidemia.

[0074] In another aspect, the present disclosure provides polypeptides described herein or pharmaceutically acceptable salts thereof for use in the treatment or prevention of obesity.

[0075] In another aspect, the Disclosure provides polypeptides described herein or pharmaceutically acceptable salts thereof for use in the treatment or prevention of diseases selected from the group consisting of metabolic syndrome, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), neurodegenerative disorders, fibrosis, and cardiovascular risks.

[0076] In another embodiment, the polypeptides described herein or pharmaceutically acceptable salts thereof may be administered simultaneously, separately, or sequentially in combination with an effective amount of one or more additional therapeutic agents.

[0077] In another embodiment, the pharmaceutical compositions according to this disclosure comprise a polypeptide described herein or a pharmaceutically acceptable salt thereof for use as a pharmaceutical.

[0078] In another embodiment, the pharmaceutical compositions according to this disclosure comprise a polypeptide described herein or a pharmaceutically acceptable salt thereof for use in the treatment or prevention of type 2 diabetes mellitus (T2DM).

[0079] In another embodiment, the pharmaceutical compositions according to this disclosure comprise a polypeptide described herein or a pharmaceutically acceptable salt thereof for use in the treatment or prevention of hyperlipidemia / dyslipidemia.

[0080] In another embodiment, the pharmaceutical compositions according to this disclosure comprise a polypeptide described herein or a pharmaceutically acceptable salt thereof for use in the treatment or prevention of obesity.

[0081] In another embodiment, the pharmaceutical compositions according to the present disclosure include polypeptides described herein or pharmaceutically acceptable salts thereof for use in the treatment or prevention of diseases selected from the group consisting of metabolic syndrome, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), neurodegenerative disorders, fibrosis, and cardiovascular risks.

[0082] In another embodiment, the pharmaceutical composition according to the Disclosure comprises a polypeptide described herein or a pharmaceutically acceptable salt thereof, provided simultaneously, separately, or sequentially, in combination with an effective amount of one or more additional therapeutic agents.

[0083] This disclosure may include one or more embodiments described herein. It should be understood that the embodiments described herein are illustrative of the disclosure and are not intended to limit the claims to any particular embodiment illustrated herein. It should also be understood that the embodiments defined herein may be used independently or in conjunction with any definition or any other embodiment defined herein. Therefore, this disclosure may attempt to accommodate all possible combinations and substitutions of various independently described embodiments.

[0084] Other features of this disclosure will become apparent to those skilled in the art based on the following examples. In general, this disclosure may extend to any novel features described herein, including the appended claims and drawings. Accordingly, any features, integers, features, compounds, or chemical parts described in conjunction with a particular aspect, embodiment, or example of this disclosure should be understood to apply to any other aspect, embodiment, or example described herein, to the extent that they do not conflict.

[0085] Furthermore, unless otherwise stated, any feature disclosed herein may be replaced by an alternative feature that serves the same or similar purpose. [Examples]

[0086] Instruments and analytical methods: The instruments used for characterizing and analyzing the compounds described herein include high-performance liquid chromatography (HPLC) (Waters e2695 Alliance; Detector Waters (2489 UV / Visible)).

[0087] Mass instrument: HPLC: Waters e2695 Alliance; and Detector: Acquity-QDa.

[0088] The compounds described herein were purified by the preparative HPLC procedure outlined below.

[0089] Preparative HPLC: WATERS 2555 Quaternary gradient module (maximum total flow rate: 300 mL / min, maximum pressure: 3000 psi) or Shimadzu LC-8A (maximum total flow rate: 150 mL, maximum pressure: 30 MPa), column: phenyl, 10 μF flow rate: 75 mL / min

[0090] Mobile phase: [Table 3]

[0091] The purity of the compounds described herein was analyzed by one of the RP-HPLC methods outlined below.

[0092] HPLC method A: Column: Xbridge Peptide BEH C18 (4.6mm x 250mm, 3.5u) Eluent: Mobile phase A: Buffer:Acetonitrile = 900:100 Mobile phase B: Buffer: Acetonitrile = 300:700 Buffer solution: Potassium dihydrogen orthophosphate in water, pH adjusted to 3.0 ± 0.1 with orthophosphate. Flow rate: 0.8mL / min Detection: UV detection at 210nm Column temperature: 65℃ Sample tray temperature: 5℃ Execution time: 90 minutes [Table 4]

[0093] HPLC method B: Column: YMC-Pack Pro C18 (4.6mm x 250mm, 3.0μ) Eluent: Mobile phase A: Buffer:Acetonitrile = 900:100 Mobile phase B: Buffer: Acetonitrile = 300:700 Buffer solution: Potassium dihydrogen orthophosphate in water, pH adjusted to 3.0 ± 0.1 with orthophosphate. Flow rate: 1.0mL / min Detection: UV detection at 210nm Column temperature: 50℃ Sample tray temperature: 5℃ Execution time: 38 minutes [Table 5]

[0094] HPLC method C: Column: X-Select CSH C18, 130A°, 2.5μm, (4.6×150)mm Eluent: Mobile phase A: Buffer:Acetonitrile = 900:100 Mobile phase B: Buffer: Acetonitrile = 300:700 Buffer solution: Potassium dihydrogen orthophosphate in water, with trimethylamine added and pH adjusted to 2.5 ± 0.1 with orthophosphate. Flow rate: 0.5mL / min Detection: UV detection at 214nm Column temperature: 60℃ Sample tray temperature: 5℃ Execution time: 90 minutes [Table 6]

[0095] HPLC method D: Column: X-Select CSH C18, 130A°, 2.5μm, (4.6×150)mm Eluent: Mobile phase A: Buffer:Acetonitrile = 900:100 Mobile phase B: Buffer: Acetonitrile = 300:700 Buffer solution: Potassium dihydrogen orthophosphate in water, with triethylamine added and pH adjusted to 2.5±0.1 with orthophosphate. Flow rate: 0.8mL / min Detection: UV detection at 210nm Column temperature: 60 °C Sample tray temperature: 5 °C Running time: 33 minutes

Table 7

[0096] Preparation of Example A part A - di - tert - butyl ester

Chemical formula

[0097] Partially A-di-tert-butyl esters were prepared using solid-phase synthesis. 2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid was attached to a 2-chlorotrityl chloride resin in the presence of DIPEA to obtain 2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid-2-chlorotrityl resin. The Fmoc protecting group was removed by selective deblocking of the amino group using piperidine, and subsequently bonded to Fmoc-Aib-OH in THF using DIPC and HOBt to obtain 2-[2-[2-[(2-Fmoc-amino-2-methyl-propanoyl)amino]ethoxy]ethoxy]acetic acid-2-chlorotrityl resin. The Fmoc group was removed by selective deblocking using piperidine, and the free amino group was bonded to Fmoc-Glu-OtBu using HOBt and DIPC to obtain 2-[2-[2-[[2-[[(4S)-4-Fmoc-amino-5-tert-butoxy-5-oxo-pentanoyl]amino]-2-methyl-propanoyl]amino]ethoxy]ethoxy]2-chlorotrityl acetate resin. The Fmoc group of the obtained compound was selectively deblocked using piperidine, and the free amino group was then bonded to octadecanediic acid monotertbutyl ester to obtain 2-[2-[2-[[2-[[(4S)-5-tert-butoxy-4-[(18-tert-butoxy-18-oxo-octadecanoyl)amino]-5-oxo-pentanoyl]amino]-2-methyl-propanoyl]-amino]ethoxy]ethoxy]2-chlorotrityl acetate resin. Next, this intermediate was cleaved from the 2-chlorotrityl resin using trifluoroethanol:DCM (1:1) to obtain 2-[2-[2-[[2-[[(4S)-5-tert-butoxy-4-[(18-tert-butoxy-18-oxo-octadecanoyl)amino]-5-oxo-pentanoyl]amino]-2-methyl-propanoyl]amino]ethoxy]ethoxy]acetic acid (partially A-di-tert-butyl ester). LCMS=m / z:786.39(M+H + ).

[0098] Preparation of Partial A-OSu [ka]

[0099] Next, the obtained partial A - di - tert - butyl ester was reacted with HOSu in the presence of dicyclohexylcarbodiimide (DCC) to obtain a succinimide - protected intermediate, which was deprotected using trifluoroacetic acid to obtain the title compound, partial A - OSu.

[0100] Example B Preparation of partial B - di - tert - butyl ester

Chemical formula

[0101] The partial B - di - tert - butyl ester was prepared using a similar process as described in Example A, using 20 - (tert - butoxy) - 20 - oxoicosanoic acid instead of octadecanedioic acid monoter - t - butyl ester, to obtain 2 - [2 - [2 - [[2 - [[(4S) - 5 - tert - butoxy - 4 - [(20 - tert - butoxy - 20 - oxo - icosanoyl)amino] - 5 - oxo - pentanoyl]amino] - 2 - methyl - propanoyl]amino]ethoxy]ethoxy]acetic acid - 2 - chlorotrityl - resin. Then, this intermediate was cleaved from the 2 - chlorotrityl - resin using trifluoroethanol:DCM (1:1) to obtain 2 - [2 - [2 - [[2 - [[(4S) - 5 - tert - butoxy - 4 - [(20 - tert - butoxy - 20 - oxo - icosanoyl)amino] - 5 - oxo - pentanoyl]amino] - 2 - methyl - propanoyl]amino]ethoxy]ethoxy]acetic acid (partial B - di - tert - butyl ester). LCMS = m / z: 814.10 (M + H + )

[0102] Preparation of partial B - OSu

Chemical formula

[0103] Next, the obtained partial B-di-tert-butyl ester was reacted with HOSu in the presence of dicyclohexylcarbodiimide (DCC) to obtain a succinimide-protected intermediate, which was then deprotected with trifluoroacetic acid to obtain the title compound, partial B-OSu.

[0104] Example C: Preparation of partial C-di-tert-butyl ester [ka]

[0105] Partially C-di-tert-butyl esters were prepared using solid-phase synthesis. 2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid was attached to a 2-chlorotrityl chloride resin in the presence of DIPEA to obtain 2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid-2-chlorotrityl resin. The Fmoc protecting group was removed by selective deblocking of the amino group using piperidine, and the free amino group was then activated with p-nitrophenyl chloroformate in THF and DIPEA, followed by reaction with Fmoc-aminobutylamine hydrochloride in THF:DMAc and DIPEA to obtain 2-[2-[2-(4-Fmoc-aminobutylcarbamoylamino)ethoxy]ethoxy]acetic acid-2-chlorotrityl resin. The Fmoc group was removed by selective deblocking using piperidine, and the free amino group was then bonded to Fmoc-Glu-OtBu using HOBt and DIPC to obtain 2-[2-[2-[4-[[(4S)-4-Fmoc-amino-5-tert-butoxy-5-oxo-pentanoyl]amino]butylcarbamoylamino]ethoxy]ethoxy]2-chlorotrityl acetate resin. The obtained 2-[2-[2-[4-[[(4S)-4-Fmoc-amino-5-tert-butoxy-5-oxo-pentanoyl]amino]-butylcarbamoylamino]ethoxy]ethoxy]acetic acid-2-chlorotrityl resin was selectively deblocked using piperidine and then bonded with octadecanediic acid monotert-butyl ester to obtain the intermediate 2-[2-[2-[4-[[(4S)-5-tert-butoxy-4-[(18-tert-butoxy-18-oxo-octadecanoyl)amino]-5-oxo-pentanoyl]amino]butylcarbamoylamino]ethoxy]ethoxy]acetic acid]-2-chlorotrityl resin. Next, this intermediate was cleaved from the 2-chlorotrityl resin using trifluoroethanol:DCM (1:1) to obtain 2-[2-[2-[4-[[(4S)-5-tert-butoxy-4-[(18-tert-butoxy-18-oxo-octadecanoyl)amino]-5-oxo-pentanoyl]amino]butylcarbamoylamino]ethoxy]ethoxy]acetic acid (partially C-di-tert-butyl ester).LCMS = m / z: 814.56 (M+H。 + )。

[0106] Preparation of partial C-OSu

Chemical formula

[0107] Next, the obtained partial C-di-tert-butyl ester was reacted with HOSu in the presence of dicyclohexylcarbodiimide (DCC) to obtain a succinimide-protected intermediate, which was deprotected using trifluoroacetic acid to obtain the title compound, partial C-OSu.

[0108] Example D Preparation of partial D-di-tert-butyl ester

Chemical formula

[0109] The partial B-di-tert-butyl ester was prepared using a similar procedure as described in Example C, using 20-(tert-butoxy)-20-oxoicosanoic acid instead of octadecanedioic acid monoterbutyl ester, to obtain the intermediate 2-[2-[2-[4-[[5-tert-butoxy-4-[(20-tert-butoxy-20-oxo-icosanoyl)amino]-5-oxo-pentanoyl]amino]butylcarbamoylamino]ethoxy]ethoxy]acetic acid-2-chlorotrityl-resin. Subsequently, this intermediate was cleaved from the 2-chlorotrityl-resin using trifluoroethanol:DCM (1:1) to obtain 2-[2-[2-[4-[[(5-tert-butoxy-4-[(20-tert-butoxy-20-oxo-icosanoyl)amino]-5-oxo-pentanoyl]amino]butylcarbamoylamino]ethoxy]ethoxy]acetic acid (partial D-di-tert-butyl ester). LCMS = m / z: 843.14 (M+H + )。

[0110] Preparation of partial D-OSu

Chemical formula

[0111] Next, the obtained partial D-di-tert-butyl ester was reacted with HOSu in the presence of dicyclohexylcarbodiimide (DCC) to obtain a succinimide-protected intermediate, which was deprotected with trifluoroacetic acid to obtain the title compound, partial D-OSu.

[0112] Example E: Preparation of partial E-OSu [ka]

[0113] L-glutamic acid alpha-tert-butyl ester (H-Glu-OtBu) is reacted with palmitic acid in the presence of IBCF and NMM to form CH3-(CH2) 14 -C(O)-Glu-OtBu is obtained, and then it is reacted with HOSu in the presence of IBCF and NMM to obtain CH3-(CH2) 14 -C(O)-Glu(OSu)-OtBu was obtained, and then deprotected with trifluoroacetic acid to obtain partial E-OSu.

[0114] Example F: Preparation of partial F-di-tert-butyl ester [ka]

[0115] Partially F-di-tert-butyl esters were prepared using solid-phase synthesis. 2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid was attached to a 2-chlorotrityl chloride resin in the presence of DIPEA to obtain 2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid-2-chlorotrityl resin. The Fmoc protecting group was removed by selective deblocking of the amino group using piperidine, and subsequently bonded to 2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid in THF using DIPC and HOBt to obtain {(Fmoc-aminoethoxy)-ethoxy}acetyl-{(-aminoethoxy)-ethoxy}acetic acid-2-chlorotrityl resin. The Fmoc group was removed by selective deblocking using piperidine, and the free amino group was bonded to Fmoc-Glu-OtBu using HOBt and DIPC to obtain Fmoc-Glu({(amino-ethoxy)-ethoxy}-acetyl-{(-amino-ethoxy)-ethoxy}-acetic acid-2-chlorotrityl-resin)-OtBu. The Fmoc group of the obtained compound was selectively deblocked using piperidine, and the free amino group was then bonded to octadecanediic acid mono-tert-butyl ester to obtain 2-[2-[2-[[2-[2-[2-[[5-tert-butoxy-4-[(18-tert-butoxy-18-oxo-octadecanoyl)amino]-5-oxo-pentanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetic acid-2-chlorotrityl-resin. Next, the intermediate was cleaved from the 2-chlorotrityl resin using trifluoroethanol:DCM (1:1) to obtain 2-[2-[2-[[2-[2-[2-[[5-tert-butoxy-4-[(18-tert-butoxy-18-oxo-octadecanoyl)amino]-5-oxo-pentanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetic acid (partially F-di-tert-butyl ester). LCMS=m / z:846.10(M+H + ).

[0116] Preparation of partial F-OSu [ka]

[0117] Next, the obtained partial F-di-tert-butyl ester was reacted with HOSu in the presence of dicyclohexylcarbodiimide (DCC) to obtain a succinimide-protected intermediate, which was deprotected with trifluoroacetic acid to obtain the title compound, partial F-OSu.

[0118] Example G: Preparation of Part G [ka]

[0119] Partially G-di-tert-butyl esters were prepared using a similar process described in Example F, and 20-(tert-butoxy)-20-oxoicosanoic acid was used instead of octadecanediic acid monotert-butyl ester to obtain the intermediate 2-[2-[2-[[2-[2-[2-[[5-tert-butoxy-4-[(20-tert-butoxy-20-oxo-icosanoyl)amino]-5-oxo-pentanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]2-chlorotrityl acetate resin. Next, the intermediate was cleaved from the 2-chlorotrityl resin using trifluoroethanol:DCM (1:1) to obtain 2-[2-[2-[[2-[2-[2-[[5-tert-butoxy-4-[(20-tert-butoxy-20-oxo-icosanoyl)amino]-5-oxo-pentanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetic acid (partially G-di-tert-butyl ester). LCMS=m / z:874.15(M+H + ).

[0120] Preparation of partial G-OSu [ka]

[0121] Next, the obtained partial G-di-tert-butyl ester was reacted with HOSu in the presence of dicyclohexylcarbodiimide (DCC) to obtain a succinimide-protected intermediate, which was then deprotected with trifluoroacetic acid to obtain the partial G-OSu of the title compound.

[0122] Example H: Preparation of partial H-di-tert-butyl ester [ka]

[0123] Partially H-di-tert-butyl esters were prepared using solid-phase synthesis with 2-chlorotrityl chloride resin. 2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid was attached to 2-chlorotrityl chloride resin in the presence of DIPEA to obtain 2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid-2-chlorotrityl resin. The Fmoc protecting group was removed by selective deblocking of the amino group using piperidine, followed by coupling with Fmoc-Glu-OtBu using HOBt and DIPC to obtain 2-[2-[2-[[(4S)-5-tert-butoxy-4-(9H-fluoren-9-ylmethoxycarbonylamino)-5-oxo-pentanoyl]amino]ethoxy]ethoxy]acetic acid-2-chlorotrityl resin. The Fmoc group of the obtained compound was selectively deblocked using piperidine, and the free amino group was then bonded to octadecanediic acid monotert-butyl ester to obtain 2-[2-[2-[[(4S)-5-tert-butoxy-4-[(18-tert-butoxy-18-oxo-octadecanoyl)amino]-5-oxo-pentanoyl]amino]ethoxy]ethoxy]acetic acid-2-chlorotrityl resin. This intermediate was then cleaved from the 2-chlorotrityl resin using trifluoroethanol:DCM (1:1) to obtain 2-[2-[2-[[(4S)-5-tert-butoxy-4-[(18-tert-butoxy-18-oxo-octadecanoyl)amino]-5-oxo-pentanoyl]amino]ethoxy]ethoxy]acetic acid (partially H-di-tert-butyl ester). LCMS = m / z: 700.94(M+H + ).

[0124] Preparation of partial H-OSu [ka]

[0125] Next, the obtained partial H-di-tert-butyl ester was reacted with HOSu in the presence of dicyclohexylcarbodiimide (DCC) to obtain a succinimide-protected intermediate, which was deprotected with trifluoroacetic acid to obtain the title compound, partial H-OSu.

[0126] Example I: Preparation of Part I [ka]

[0127] Partial I-di-tert-butyl esters were prepared using a similar process described in Example H, and 20-(tert-butoxy)-20-oxoicosanoic acid was used instead of octadecanediic acid monotert-butyl ester to obtain the intermediate 2-[2-[2-[[(4S)-5-tert-butoxy-4-[(20-tert-butoxy-20-oxo-icosanoyl)amino]-5-oxo-pentanoyl]amino]ethoxy]ethoxy]2-chlorotrityl acetate resin. Next, this intermediate was cleaved from the 2-chlorotrityl resin using trifluoroethanol:DCM (1:1) to obtain 2-[2-[2-[[(4S)-5-tert-butoxy-4-[(20-tert-butoxy-20-oxo-icosanoyl)amino]-5-oxo-pentanoyl]amino]ethoxy]ethoxy]acetic acid (partially I-di-tert-butyl ester). LCMS=m / z:728.99(M+H + ).

[0128] Preparation of Partial I-OSu [ka]

[0129] Next, the obtained partial I-di-tert-butyl ester was reacted with HOSu in the presence of dicyclohexylcarbodiimide (DCC) to obtain a succinimide-protected intermediate, which was deprotected with trifluoroacetic acid to obtain the title compound, partial I-OSu.

[0130] Example 1: Synthesis of Compound 1:

[0131] Part A: Synthesis of Linear Peptide Skeletons

[0132] The peptide skeleton was synthesized by solid-phase method. The starting resin used for synthesis was Wang resin. Fmoc-protected Gly-OH was used for bonding with the Wang resin. Bonding was carried out in the presence of 4-dimethylaminopyridine (DMAP) using diisopropylcarbodiimide and N-hydroxybenzotriazole (DIC-HOBt) as the bonding agent to obtain Fmoc-Gly-Wang resin. Unbound amino groups at each amino acid bond were terminated using acetic anhydride and diisopropylethylamine / pyridine. Following selective deblocking of the amino groups of the Fmoc-Gly-Wang resin using piperidine, Fmoc-Gly-Gly-Wang resin was obtained by bonding with Fmoc-Gly-OH using HOBt and DIPC. This completed one cycle.

[0133] The three steps described above—selective capping of the Fmoc protection of amino acids attached to the resin, selective deblocking, and binding to the Fmoc-protected amino group of an adjacent amino acid residue in the sequence—were repeated for the remaining 28 amino acid residues. Selective deblocking (deprotection of the Fmoc group) was performed using piperidine, and binding to the adjacent Fmoc-protected amino acid was performed using HOBt / DIPC. The side chains of Fmoc-protected amino acids were orthogonally protected (hydroxyl groups of serine, tyrosine, or threonine were protected with tert-butyl (-tBu) groups; amino and guanide groups of lysine and arginine were protected with tert-butyloxycarbonyl (-Boc) and 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (-Pbf) groups, respectively; the imidazole of histidine was protected with tert-butyloxycarbonyl (-Boc); the carboxylic acid group of aspartic acid or glutamic acid was protected with (-tBu) groups; and the amide group of glutamine was protected with trityl (-Trt) groups). The two steps described above, namely selective deblocking and subsequent binding with the adjacent Fmoc-protecting amino acid, were carried out to obtain Fmoc-His(Boc)-Ser(tBu)-Gln(Trt)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Lys(Boc)-Ser(tBu)-Glu(OtBu)-Tyr(tBu)-Leu-Asp(OtBu)-Ser(tBu)-Glu(OtBu)-Arg(Pbf)-Ala-Arg(Pbf)-Asp(OtBu)-Phe-Val-Ala-Trp-Leu-Glu(OtBu)-Ala-Gly-Gly-resin.

[0134] Fmoc-His(Boc)-Ser(tBu)-Gln(Trt)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Lys(Boc)-Ser(tBu)-Glu(OtBu)-Tyr(tBu)-Leu-Asp(OtBu)-Ser(tBu)-Glu(OtBu)-Arg(Pbf)-Ala-Arg(Pbf)-Asp(OtBu)-Phe-Val-Ala-Trp-Leu-Glu(OtBu)-Ala-Gly- Deblocking of the Gly-resin, followed by cleavage and deprotection using trifluoroacetic acid together with ethane-1,2-dithiol and triisopropylsilane, yielded unpurified H-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(ε-NH2)-Ser-Glu-Tyr-Leu-Asp-Ser-Glu-Arg-Ala-Arg-Asp-Phe-Val-Ala-Trp-Leu-Glu-Ala-Gly-Gly-OH, which was purified by preparative HPLC.

[0135] Part B: Grafting of activated fatty acid chains onto linear peptides

[0136] The activated fatty acid chain portion C-Osu was grafted onto a purified linear peptide obtained in part A of water:acetonitrile at approximately pH 11 to obtain unpurified title peptide compound 1, which was then purified by preparative HPLC.

[0137] Mass (LCMS): m / z = 10¹².36 (MH₄⁴⁺), calculated mass = 40⁴⁵.40, HPLC purity: (Method B) 94.48%.

[0138] Example 2: Synthesis of Compound 2:

[0139] Part A: Synthesis of Linear Peptide Skeletons

[0140] The linear peptide skeleton of compound 2 was prepared by solid-phase method according to a similar process described in Part A of Example 1, with Fmoc-D-Ser(OMe)-OH used at position 2 instead of Fmoc-Ser(tBu)-OH.

[0141] Part B: Grafting of activated fatty acid chains onto linear peptides

[0142] The activated fatty acid chain portion E-OSu was grafted onto a purified linear peptide: H-His-D-Ser(OMe)-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(ε-NH2)-Ser-Glu-Tyr-Leu-Asp-Ser-Glu-Arg-Ala-Arg-Asp-Phe-Val-Ala-Trp-Leu-Glu-Ala-Gly-Gly-OH obtained in water:acetonitrile at pH approximately 11 to obtain unpurified title peptide compound 2, which was purified by preparative HPLC.

[0143] Mass (LCMS): m / z = 936.10 (MH44+), calculated mass = 3739.96, HPLC purity: (Method B) 95.05%.

[0144] Example 3 Synthesis of Compound 3:

[0145] Part A: Synthesis of Linear Peptide Skeletons

[0146] The linear peptide skeleton of compound 3 was prepared by solid-phase method according to a similar process described in Part A of Example 1, with Fmoc-Aib-OH used at position 2 instead of Fmoc-Ser(tBu)-OH.

[0147] Part B: Grafting of activated fatty acid chains onto linear peptides

[0148] The activated fatty acid chain portion E-OSu was grafted onto the purified linear peptide obtained in Part A: H-His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(ε-NH2)-Ser-Glu-Tyr-Leu-Asp-Ser-Glu-Arg-Ala-Arg-Asp-Phe-Val-Ala-Trp-Leu-Glu-Ala-Gly-Gly-OH in water:acetonitrile at pH approximately 11 to obtain the unpurified title peptide compound 3, which was purified by preparative HPLC.

[0149] Mass (LCMS): m / z = 932.13 (MH44+), calculated mass = 3724.48, HPLC purity: (Method B) 96.1%.

[0150] Example 4 Synthesis of Compound 4:

[0151] Part A: Synthesis of Linear Peptide Skeletons

[0152] The linear peptide skeleton of compound 4 was prepared by a solid-phase method according to a similar process shown in Part A of Example 3.

[0153] Part B: Grafting of activated fatty acid chains onto linear peptides

[0154] The activated fatty acid chain portion A-OSu was grafted onto the purified linear peptide H-His-Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(ε-NH2)-Ser-Glu-Tyr-Leu-Asp-Ser-Glu-Arg-Ala-Arg-Asp-Phe-Val-Ala-Trp-Leu-Glu-Ala-Gly-Gly-OH obtained in water:acetonitrile at pH approximately 11 to obtain the unpurified title peptide compound 4, which was purified by preparative HPLC.

[0155] Mass (LCMS): m / z = 1004.34 (MH4 4+), calculated mass = 4013.33, HPLC purity: (Method D) 96.93%.

[0156] Example 5 Synthesis of Compound 5:

[0157] Part A: Synthesis of Linear Peptide Skeletons

[0158] The linear peptide skeleton of compound 4 was prepared by a solid-phase method according to a similar process shown in Part A of Example 1.

[0159] Part B: Grafting of activated fatty acid chains onto linear peptides

[0160] The activated fatty acid chain portion A-OSu was grafted onto the purified linear peptide obtained in Part A: H-His-Ser-Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys(ε-NH2)-Ser-Glu-Tyr-Leu-Asp-Ser-Glu-Arg-Ala-Arg-Asp-Phe-Val-Ala-Trp-Leu-Glu-Ala-Gly-Gly-OH in water:acetonitrile at pH approximately 11 to obtain the unpurified title peptide compound 5, which was purified by preparative HPLC.

[0161] Mass (LCMS): m / z = 1004.96 (MH44+), calculated mass = 4015.81, HPLC purity: (Method B) 97.33%.

[0162] Example 6: Synthesis of Compound 6:

[0163] Part A: Synthesis of Linear Peptide Skeletons

[0164] Compound 6 was synthesized by solid-phase method. The starting resin used for synthesis was Fmoc-Rink amide resin. The Fmoc-protecting amino group of the Rink amide resin was selectively deblocked using piperidine, followed by the bonding of Fmoc-Gly-OH to the Rink amide resin. Coupling was performed using diisopropylcarbodiimide and N-hydroxybenzotriazole (DIPC-HOBt) as coupling reagents to obtain Fmoc-Gly-Rink amide resin, completing the first cycle. Unbound amino groups at each amino acid bond were terminated / capped using acetic anhydride and diisopropylethylamine. The amino groups of the Fmoc-Gly-Rink amide resin were selectively deblocked using piperidine. Then, bonding with Fmoc-Ser(tBu)-OH using HOBt and DIPC was performed to obtain Fmoc-Ser(tBu)-Gly-Rink amide resin, completing the second cycle.

[0165] The three steps described above—selective capping, deblocking of the Fmoc protection of the amino acids bonded to the resin, and sequential bonding of the next amino acid residue with the Fmoc-protected amino group—were repeated for the remaining 32 amino acid residues. The side chains of the Fmoc-protected amino acids were orthogonally protected (the hydroxyl groups of serine, tyrosine, or threonine were protected with tert-butyl (-tBu) groups; the amino groups of lysine were protected with tert-butyloxycarbonyl (-Boc) and (4,4-dimethyl-2,6-dioxocyclohexa-1-ylidene)-3-methylbutyl (IVDde) groups, respectively; the carboxylic acid groups of aspartic acid or glutamic acid were protected with (-tBu) groups; and the amide group of glutamine was protected with trityl (-Trt) groups). The three steps described above—selective capping, deblocking, and subsequent binding with the adjacent Fmoc-protecting amino acid—were performed to obtain the Fmoc-His(Boc)-Aib-Gln(Trt)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Lys(Boc)-Tyr(tBu)-Leu-Asp(OtBu)-Glu(OtBu)-Lys(Boc)-Lys(Boc)-Ala-Lys(IVDde)-Glu(OtBu)-Phe-Val-Glu(OtBu)-Trp-Leu-Leu-Glu(OtBu)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-resin.

[0166] Deblocking of Fmoc-His(Boc)-Aib-Gln(Trt)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Lys(Boc)-Tyr(tBu)-Leu-Asp(OtBu)-Glu(OtBu)-Lys(Boc)-Lys(Boc)-Ala-Lys(IVDde)-Glu(OtBu)-Phe-Val-Glu(OtBu)-Trp-Leu-Leu-Glu(OtBu)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-resin using piperidine. Boc protection was performed on the peptide-resin obtained using Boc anhydrous to obtain Boc-His(Boc)-Aib-Gln(Trt)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Lys(Boc)-Tyr(tBu)-Leu-Asp(OtBu)-Glu(OtBu)-Lys(Boc)-Lys(Boc)-Ala-Lys(IVDde)-Glu(OtBu)-Phe-Val-Glu(OtBu)-Trp-Leu-Leu-Glu(OtBu)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-resin By deprotecting the IVDde group of the peptide resin obtained using hydrazine hydrate, a linear peptide resin, Boc-His(Boc)-Aib-Gln(Trt)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Lys(Boc)-Tyr(tBu)-Leu-Asp(OtBu)-Glu(OtBu)-Lys(Boc)-Lys(Boc)-Ala-Lys(ε-NH2)-Glu(OtBu)-Phe-Val-Glu(OtBu)-Trp-Leu-Leu-Glu(OtBu)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-resin, was obtained.

[0167] Part B: Grafting of activated fatty acid chains onto linear peptides

[0168] The partial β-di-tert-butyl ester was coupled to a linear peptide-resin as obtained in Part A, using diisopropylcarbodiimide and N-hydroxybenzotriazole (DIPC-HOBt) as coupling reagents, to obtain compound 6-resin. After cleavage from the resin, deprotection with trifluoroacetic acid using ethane-1,2-dithiol and triisopropylsilane, purification by preparative HPLC was performed to obtain pure compound 6.

[0169] Mass (LCMS): m / z = 1126.36 (MH4 4+), calculated mass = 4501.41, HPLC purity: (Method A) 97.16%.

[0170] Example 7: Synthesis of Compound 7:

[0171] Compound 7 was prepared by a solid-phase method according to a similar process described in Example 6, and after IVDde deprotection, partial A-di-tert-butyl ester coupling was performed instead of partial B-di-tert-butyl ester coupling.

[0172] Mass (LCMS): m / z = 1120.09 (MH4 4+), calculated mass = 4476.33, HPLC purity: (Method A) 98.95%.

[0173] Example 8: Synthesis of Compound 8:

[0174] Compound 8 was prepared by a solid-phase method according to a similar process described in Example 6, followed by IVDde deprotection and then coupling with a partial D-di-tert-butyl ester instead of a partial B-di-tert-butyl ester coupling.

[0175] Mass (LCMS): m / z = 1134.05 (MH44+), calculated mass = 4532.17.

[0176] Example 9: Synthesis of Compound 9:

[0177] Compound 9 was prepared by a solid-phase method according to a similar process described in Example 6, followed by IVDde deprotection and then coupling with a partial C-di-tert-butyl ester instead of a partial B-di-tert-butyl ester coupling.

[0178] Mass (LCMS): m / z = 1127.22 (MH44+), calculated mass = 4504.85.

[0179] Example 10: Synthesis of Compound 10:

[0180] Compound 9 was prepared by a solid-phase method according to a similar process described in Example 6, with Fmoc-norvaline-OH used at the 13-position instead of Fmoc-Tyr(tBu)-OH, and partial A-di-tert-butyl ester coupling instead of partial B-di-tert-butyl ester coupling following IVDde deprotection.

[0181] Mass (LCMS): m / z = 1103.56 (MH44+), calculated mass = 4410.21.

[0182] Example 11: Synthesis of Compound 11:

[0183] Compound 11 was prepared by a solid-phase method according to a similar process described in Example 6, (i) using Fmoc-Ile-OH at position 12 instead of Fmoc-Lys(Boc)-OH, (ii) using Fmoc-norvaline-OH at position 13 instead of Fmoc-Tyr(tBu)-OH, and (iii) coupling with a partial A-di-tert-butyl ester instead of a partial B-di-tert-butyl ester coupling after IVDde deprotection.

[0184] Mass (LCMS): m / z = 1100.11 (MH44+), calculated mass = 4396.41.

[0185] Example 25 Synthesis of Compound 25:

[0186] Compound 25 was prepared by a solid-phase method according to a similar process described in Example 6, followed by IVDde deprotection and then coupling with a partial F-di-tert-butyl ester instead of a partial B-di-tert-butyl ester coupling.

[0187] Mass (LCMS): m / z = 1135.11 (MH4 4+), calculated mass = 4536.41, HPLC purity (Method A): 98.69%. Biological tests

[0188] Example 1: Oral glucose tolerance test (OGTT) in rats, single injection, 1 mg / kg

[0189] Study 1: Animals were divided into four groups (n=4 / group): a normal control group, cotadutide (1 mg / kg), compound 1 (1 mg / kg), and semaglutide (1 mg / kg). Animals were fasted for 12 hours before the start of the OGTT. Blood glucose levels were measured using a blood glucose meter 22 and 166 hours after subcutaneous injection of the test drug, cotadutide, and semaglutide (time 0 measurement). All animals were orally administered a 2 g / kg glucose solution. Blood glucose levels were measured 20, 40, 60, 90, and 120 minutes after the glucose test. Body weight and food consumption were recorded at 48 and 154 hours.

[0190] Study 2: Animals were divided into four groups (n=4 / group): a normal control group, compound 2 (1 mg / kg), compound 3 (1 mg / kg), and semaglutide (1 mg / kg). Animals were fasted for 12 hours before the start of the OGTT. Blood glucose levels were measured using a blood glucose meter 22 and 166 hours after subcutaneous injection of the test drug and semaglutide (time 0 measurement). All animals were orally administered a 2 g / kg glucose solution. Blood glucose levels were measured 20, 40, 60, 90, and 120 minutes after the glucose test. Body weight and food consumption were recorded at 48 and 154 hours. [Table 8] [Table 9] [Table 10] [Table 11]

[0191] Example 2: Efficacy study in db / db mice at a dose of 12 nM / kg

[0192] The effects of the compounds described herein on blood glucose levels, food intake, and body weight were tested in mice. This study was conducted using a type 2 diabetic mouse (db / db) model. Animals were divided into five treatment groups (n=4 / group): a diabetic control group, cotadutide (12 nM / kg), compound 3 (12 nM / kg), compound 4 (12 nM / kg), and semaglutide (12 nM / kg). Baseline blood glucose levels were measured in all animals. All animals were administered the test compound subcutaneously. Blood glucose levels were measured at 4, 8, 24, 48, 72, and 96 hours post-treatment. Delta blood glucose levels (mM) were calculated. [Table 12]

[0193] Example 3: Efficacy study in db / db mice at a dose of 10 nM / kg

[0194] The effects of the compounds described herein on blood glucose levels, food intake, and body weight were tested in mice. This study was conducted using a type 2 diabetic mouse (db / db) model. Animals were divided into two treatment groups: a diabetic control group and a group receiving compound 5 (10 nM / kg) (n=5 / group). Baseline blood glucose levels were measured in all animals. All animals were administered the test compound subcutaneously. Blood glucose levels were measured at 4, 8, 12, 24, 48, 72, and 96 hours post-treatment. Delta blood glucose levels (mM) were calculated. Body weight change and cumulative food consumption were measured at 48 and 96 hours post-treatment. [Table 13] [Table 14] [Table 15]

[0195] Example 4: Efficacy study in db / db mice at a dose of 10 nM / kg

[0196] The effects of the compounds described herein on blood glucose levels, food intake, and body weight were tested in mice. This study was conducted using a type 2 diabetic mouse (db / db) model. Animals were divided into 12 treatment groups (n=4 / group): a diabetic control group and 12 treatment groups (n=4 / group) receiving mazdotide (10 nM / kg), compound 6 (10 nM / kg), compound 7 (10 nM / kg), compound 25 (10 nM / kg), and tilzepatide (10 nM / kg). Baseline blood glucose levels were measured in all animals. All animals were administered the test compound subcutaneously. Blood glucose levels were measured at 4, 8, 12, 24, 48, 72, and 96 hours post-treatment. Delta blood glucose levels (mM) were calculated. Body weight change and cumulative food consumption were measured at 48 and 96 hours post-treatment. [Table 16] [Table 17] [Table 18]

[0197] Example 5: In vitro assay

[0198] The in vitro potency of the compounds described herein was measured using stably expressing GLP-1R, GIPR, or GCGR cell lines. Signaling by GLP-1R, GIP-R, and GCG-R involves activation of adenylyl cyclase and cAMP production. The Hit Hunter® cAMP assay uses the Enzyme Fragment Complementation (EFC) technique developed by DiscoverX to monitor the activation of GLP-1R, GIPR, or GCGR via Gi and Gs secondary messenger signaling, with β-galactosidase (β-Gal) as a functional reporter. The enzyme is split into two complementary parts: EA for the enzyme receptor and ED for the enzyme donor. ED is fused with cAMP and, in this assay, competes with cell-produced cAMP for binding to cAMP-specific antibodies. Active β-Gal is formed by the complementation of exogenous EA to any unbound ED cAMP. Subsequently, the active enzyme can transform the chemiluminescent substrate, generating an output signal detectable on a standard microplate reader.

[0199] Three different assays were performed using cells expressing one of three receptors. cAMP hunter cell lines were grown from frozen stock according to standard procedures. Cells were seeded in 20 μL total volume in a 384-well white-walled microplate and incubated at 37°C for an appropriate time before testing. The medium was aspirated, and the cells were then treated with 15 μL of cAMP-binding antibody and 5 μL of the test compound. Assay signals were generated by incubation with the appropriate compound, followed by incubation with 20 μL of cAMP-ED cell lysis cocktail for 1 hour, and then incubation with 20 μL of cAMP-EA reagent at room temperature for 3 hours. Free cAMP-ED available in the system, in combination with free cAMP-EA, reacts with the substrate to form active β-Gal, which gives a chemiluminescent signal. Microplates were read after signal generation using a PerkinElmer Envision® instrument for chemiluminescent signal detection. The amount of signal is directly proportional to the concentration of cAMP generated by the response. Logarithmic concentration-to-effect percentage curves were generated using samples of various concentrations (different for different compounds). Four parametric logistic curves were generated, and the EC50 was determined. Appropriate assay criteria (Exendin-4 for GLP-1R, GIP for GIPR, and glucagon for GCGR) were used for each assay.

[0200] Cellular cAMP assays were performed for mazdotide, compound 6, compound 7, and compound 8, and the semi-effective concentrations on GLP-1R-expressing cells and GIPR-expressing cells are listed in Table 14 below. [Table 19]

Claims

1. A polypeptide comprising the following amino acid sequence or a pharmaceutically acceptable salt thereof, X1-X2-X3-G-T-F-T-SD-X10-S-X12-X13-LD-X16-X17-X18-X19-X20-X21-F-X23 -X24-X25-L-X27-X28-X29-X30-X31-X32-X33-X34-X35-X36-X37-X38-X39 (SEQ ID NO: 1) During the ceremony, X1 is H, X2 is D-Ser(OMe), Aib, or D-S. X3 is Q, X10 is either K or Y. X12 is E, K, or I. X13 is Y, S(OMe), Nor-V, Nor-L, or αMe-L. X16 is S, E, or A. X17 is E, R, or K. X18 is R, K, or A. X19 is A, X20 is R, Q, or K. X21 is either D or E, X23 is V or I, X24 is A, Q, or E. X25 is W, X27 is E or L, X28 is A, D, or E. X29 is G or T, X30 does not exist, or it is G. X31 does not exist, or it is P. X32 does not exist, or it is S. X33 does not exist, or it is S. X34 does not exist, or it is G. X35 does not exist. X36 does not exist. X37 does not exist. X38 does not exist. X39 does not exist. In the formula, the acid group of the C-terminal amino acid is either a free carboxylic acid group or is amidated as a C-terminal primary amide. However, at least one of X10 and X20 is K, and furthermore, at least one of the K is 【Chemistry 1-1】 【Chemistry 1-2】 A polypeptide or a pharmaceutically acceptable salt thereof, comprising a side-chain amino (ε-amino) group acylated in a portion of the formula selected from the above.

2. A polypeptide comprising the following amino acid sequence or a pharmaceutically acceptable salt thereof, X1-X2-X3-G-T-F-T-SD-X10-S-X12-X13-LD-X16-X17-X18-X19-X20-X21-F-X23 -X24-X25-L-X27-X28-X29-X30-X31-X32-X33-X34-X35-X36-X37-X38-X39 (SEQ ID NO: 2) During the ceremony, X1 is H, X2 is S, D-Ser(OMe), Aib, or D-S. X3 is Q, X10 is either K or Y. X12 is E, K, or I. X13 is Y, S(OMe), Nor-V, Nor-L, or αMe-L. X16 is S, E, or A. X17 is E, R, or K. X18 is R, K, or A. X19 is A, X20 is R, Q, or K. X21 is either D or E, X23 is V, X24 is A, Q, or E. X25 is W, X27 is E or L, X28 is A, D, or E. X29 is G or T, X30 does not exist, or it is G. X31 does not exist, or it is P. X32 does not exist, or it is S. X33 does not exist, or it is S. X34 does not exist, or it is G. X35 does not exist. X36 does not exist. X37 does not exist. X38 does not exist. X39 does not exist. In the formula, the acid group of the C-terminal amino acid is either a free carboxylic acid group or is amidated as a C-terminal primary amide. However, at least one of X10 and X20 is K, and furthermore, at least one of the K is 【Chemistry 2】 A polypeptide or a pharmaceutically acceptable salt thereof, comprising a side-chain amino (ε-amino) group acylated in a portion of the formula selected from the above.

3. X2 is D-Ser(OMe) or Aib, X10 is K, X12 is E, X13 is Y, X16 is S, X17 is E, X18 is R, X20 is R, X21 is D, X24 is A, X27 is E, X28 is A, X29 is G, X30 is G, X31, X32, X33, and X34 do not exist. The amino (ε-amino) group of the side chain of K at position X10, 【Transformation 3】 The part of the expression selected from is acylated. The polypeptide according to claim 1.

4. X2 is D-S, X10 is K, X12 is K, X13 is Y, X16 is A, X17 is R, X18 is A, X20 is Q, X21 is D, X24 is Q, X27 is L, X28 is D, X29 is T, X31, X32, X33, and X34 do not exist. The amino (ε-amino) group of the side chain of K at position X10, 【Chemistry 4】 The part of the expression selected from is acylated. The polypeptide according to claim 1 or 2.

5. X2 is Aib, X10 is Y, X12 is K or I, X13 is Y, or Nor-V, Nor-L, or αMe-L. X16 is E, X17 is K, X18 is K, X20 is K, X21 is E, X24 is E, X27 is L, X28 is E, X29 is G, X30 is G, X31 is P, X32 is S, X33 is S, X34 is G, The amino (ε-amino) group of the side chain of K at position X20, 【Transformation 5】 The part of the expression selected from is acylated. The polypeptide according to claim 1 or 2.

6. A polypeptide comprising the following amino acid sequence or a pharmaceutically acceptable salt thereof, H-X2-Q-G-T-F-T-S-D-X10-S-E-Y-L-D-S-E-R-A-R-D-F-V-A-W-LE-A-G-G (SEQ ID NO: 3) During the ceremony, X2 is S, D-S (OMe) or Aib, X10 is K, In the formula, the acid group of the C-terminal amino acid is either a free carboxylic acid group or is amidated as a C-terminal primary amide. The amino (ε-amino) group in the side chain of K at position X10 is 【Transformation 6】 The part of the expression selected from is acylated, However, the polypeptide is not Sequence ID No. 6, but a polypeptide or a pharmaceutically acceptable salt thereof.

7. X2 is S, The amino (ε-amino) group of the side chain of K at position X10, 【Transformation 7】 The part of the expression selected from is acylated. The polypeptide according to claim 6.

8. X2 is D-Ser(OMe), The amino (ε-amino) group of the K at position X10 is, formula: 【Transformation 8】 It is acylated in that part. The polypeptide according to claim 6.

9. X2 is Aib, The amino (ε-amino) group of the side chain of K at position X10, 【Chemistry 9】 The part of the expression selected from is acylated. The polypeptide according to claim 6.

10. A polypeptide comprising the following amino acid sequence or a pharmaceutically acceptable salt thereof, H-Aib-Q-G-T-F-T-S-D-Y-S-X12-X13-L-D-E-K-K-A-X20-E-F-V-E-W-L-L-E-G-G-P-S-SG (SEQ ID NO: 4) During the ceremony, X12 is K or I, X13 is Y or Nor-V, X20 is K, In the formula, the acid group of the C-terminal amino acid is either a free carboxylic acid group or is amidated as a C-terminal primary amide. The amino (ε-amino) group in the side chain of K at position X20, 【Chemistry 10】 A polypeptide or a pharmaceutically acceptable salt thereof that is acylated in a portion of the formula selected from the above.

11. X12 is K, X13 is Y, The amino (ε-amino) group of the side chain of K at position X20, 【Chemistry 11】 The part of the expression selected from is acylated. The polypeptide according to claim 10.

12. X12 is I, X13 is Nor-V, The amino (ε-amino) group of the side chain of K at position X20 is, formula: 【Chemistry 12】 It is acylated in that part. The polypeptide according to claim 10.

13. X12 is K, X13 is Nor-V, The amino (ε-amino) group of the side chain of K at position X20 is, formula: 【Chemistry 13】 It is acylated in that part. The polypeptide according to claim 10.

14. A polypeptide comprising the following amino acid sequence or a pharmaceutically acceptable salt thereof, H-(DSer)-Q-G-T-F-T-S-D-X10-S-K-Y-L-D-A-R-A-A-Q-D-F-V-Q-W-L-L-D-T (Sequence No. 5) In the formula, X10 is K, In the formula, the acid group of the C-terminal amino acid is either a free carboxylic acid group or is amidated as a C-terminal primary amide. The amino (ε-amino) group in the side chain of K at position X10, 【Chemistry 14】 A polypeptide or a pharmaceutically acceptable salt thereof that is acylated in a portion of the formula selected from the above. 【Request Item 15】 【Chemistry 15】 A polypeptide selected from the following.

16. X １ A peptide residue having the sequence -Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys (SEQ ID NO: 29), X １ However, it represents Aib or Ser(OMe), and lysine is a peptide residue containing a fatty acid extension group bonded to the lysine ε-nitrogen, It contains a glycy-glycy-OH peptide residue indirectly bound to the carboxyl group of lysine. Incretin analog.

17. X １ A peptide residue having the sequence -Gln-Gly-Thr-Phe-Thr-Ser-Asp-Lys (SEQ ID NO: 29), X １ A peptide residue containing a group of formula (I) in which lysine is bonded to lysine ε-nitrogen, where represents Aib or Ser(OMe), 【Chemistry 16】 (In the formula, U is absent or -C(O)-CH ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -NH-}, where} is the point of attachment to W, W is -C(O)-CH ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -NHH-]、 ‐C(O)‐NH‐(CH ２ ) ３‐４ ‐NH‐]、‐C(O)-C(CH ３ ) ２ -NH-]、or 【Chemistry 17】 This represents the relationship, where ] is the point of connection with Y. Y does not exist, or -C(O)-(CH ２ ) ２ -CH(CO) ２ H)NH-- or -C(O)CH((CH ２ ) ｘ CO ２ H) NH-- represents the junction with Z, where x is 1, 2, or 3, and -- is the point of connection with Z. Z is -C(O)-(CH ２ ) ｎ -COOH or -C(O)-(CH ２ ) ｎ -CH ３ (where n is an integer between 14 and 20) It contains a glycy-glycy-OH peptide residue indirectly bound to the carboxyl group of lysine. Incretin analog.

18. The incretin analog according to claim 16 or claim 17, wherein the lysine is bound to the Gly-Gly-OH residue by a peptide residue containing 18 amino acids.

19. A peptide residue having the sequence Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp (SEQ ID NO: 30), A lysine residue indirectly bonded to the carboxyl group of the Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp (SEQ ID NO: 30) residue, wherein the lysine residue contains a group of formula (I) bonded to the lysine ε-nitrogen, [Chemistry 18] (In the formula, U does not exist, or -C(O)-CH ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -NH-}, where} is the bonding point with W, W is -C(O)-CH ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -NH-], or -C(O)-NH-(CH ２ ) ３‐４ -NH-], -C(O)-C(CH ３ ) ２ -NH- represents, In the formula, ] is the point of connection with Y, Y does not exist, or -C(O)-(CH ２ ) ２ -CH(CO) ２ H)NH-- or -C(O)CH((CH ２ ) ｘ CO ２ H) NH-- represents the junction with Z, where x is 1, 2, or 3, and -- is the point of connection with Z. Z is -C(O)-(CH ２ ) ｎ -COOH or -C(O)-(CH ２ ) ｎ -CH ３ (where n is an integer between 14 and 20) Gly-Gly-Pro-Ser-Ser-Gly-CONH indirectly bonded to the carboxyl group of lysine. ２ Including peptide residues, Incretin analog.

20. The incretin analog according to claim 19, wherein the lysine is bound to the Aib-Gln-Gly-Thr-Phe-Thr-Ser-Asp (SEQ ID NO: 30) residue by a peptide residue containing 10 amino acids.

21. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and an incretin analog according to any one of claims 1 to 20.

22. A method for treating obesity, comprising administering an incretin analog according to any one of claims 1 to 20 to a patient in need of such treatment.

23. A method for treating type 2 diabetes mellitus (T2DM), comprising administering an incretin analog according to any one of claims 1 to 20 to a patient in need of such treatment.

24. A method for treating metabolic syndrome, comprising administering an incretin analog according to any one of claims 1 to 20 to a patient in need of such treatment.

25. A method for treating metabolic dysfunction-related fatty liver disease (MASLD), comprising administering an incretin analog according to any one of claims 1 to 20 to a patient in need of such treatment.

26. A method for treating metabolic dysfunction-related steatohepatitis (MASH), comprising administering an incretin analog according to any one of claims 1 to 20 to a patient in need of such treatment.

27. A method for treating a neurodegenerative disorder, comprising administering an incretin analog according to any one of claims 1 to 20 to a patient in need of such treatment.

28. A method for treating fibrosis, comprising administering an incretin analog according to any one of claims 1 to 20 to a patient in need of such treatment.

29. A method for reducing cardiovascular risk, comprising administering an incretin analog according to any one of claims 1 to 20 to a patient in need of such treatment.

30. A method for treating hyperlipidemia / dyslipidemia, comprising administering an incretin analog according to any one of claims 1 to 20 to a patient in need of such treatment.

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