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

Abstract

The present invention relates to GLP-1 / GIP / GCG triple receptor agonists and their use in the treatment or prevention of type 2 diabetes mellitus (T2DM), hyperlipidemia / dyslipidemia, metabolic syndromes, metabolic dysfunction-related fatty liver disease (MASLD), metabolic dysfunction-related steatohepatitis (MASH), neurodegenerative disorders, fibrosis, cardiovascular risk, and / or obesity.

Description

Cross-reference of related applications

[0001] This application claims the benefit of priority of Indian Patent Application No. 202321039646, filed 9 June 2023, which is incorporated herein by whole reference.

[0002] Reference to electronic sequence listings This application includes a sequence listing, which was filed electronically and is incorporated herein by reference in its entirety. The sequence listing was created on 7 June 2024, named "24-0750-WO_Sequence-Listing.xml", and has a size of 143,360 bytes. [Technical Field]

[0003] This disclosure relates to GLP-1 / GLP bireceptor agonists, GLP-1 / GCG bireceptor agonists, and GLP-1 / GIP / GCG triple receptor agonists. Specifically, this disclosure relates to GLP-1 / GIP / GCG triple receptor agonists comprising incretin analog polypeptides. The polypeptides described herein have structural features that provide balanced activity and long-lasting effects in each of these receptors. Polypeptides according to this disclosure may be useful in the treatment of type 2 diabetes mellitus (T2DM), hyperlipidemia / dyslipidemia, metabolic syndromes, metabolic dysfunction-related fatty liver disease (MASLD), metabolic dysfunction-related steatohepatitis (MASH), neurodegenerative disorders, fibrosis, obesity, and reduction of cardiovascular risk. [Background technology]

[0004] The prevalence of diabetes has been steadily increasing over the past few decades. T2DM is the most common form 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 syndromes, 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 injectable glucose-lowering agents, including oral medications and incretin therapies such as GLP-1 monoreceptor agonists and / or GLP-1 / GIP bireceptor agonists. As a new approach, novel therapies are being studied in which compounds are active not only at the GLP-1 monoreceptor or GLP-1 / GIP bireceptor, but also at the GCG receptor. Certain compounds have been described as having GLP-1 / GCG bireceptor and / or GLP-1 / GIP / GCG tripreceptor activity.

[0006] For example, cotadutide, MK-1462, and mazdutide are peptides that act as GLP-1 / GCG bireceptor agonists. Similarly, letatrutide (SEQ ID NO: 7) is another clinical candidate that acts as a GLP-1 / GIP / GCG triple receptor agonist. WIPO publications WO2019 / 193576, WO2006 / 097537, and WO1998 / 008871 disclose GLP-1 receptor 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. WIPO publications WO2015 / 067716, WO2016 / 198624, WO2014 / 049610, and WO2017 / 116204 disclose GLP-1 / GIP / GCG triple receptor agonist compounds.

[0007] Furthermore, recent research on GLP-1 / GCG dual receptor agonists and / or GLP-1 / GIP / GCG triple receptor agonists has emphasized the importance of understanding the contributions of individual hormone actions and branching effects by varying the GLP-1:GCG activity and ratio in GLP-1 / GCG dual receptor agonists and GLP-1 / GIP / GCG triple receptor agonists. Hope et al. Front. Endocrinol., 08 September 2021, Vol 12 - 2021.

[0008] In addition to being diabetogenic, glucagon receptor (GCGR) activation is known to increase heart rate and contractility, which can lead to adverse cardiovascular outcomes. Further chronic glucagon excess also results in amino acid and protein catabolism, which leads to loss of fat-free mass.

[0009] For example, despite showing increased weight loss in the treatment of obesity, retatrutide is associated with several side effects such as increased heart rate. These adverse effects may be due to the GCGR activation component of the drug action. Current research in Diabetes & Obesity Journal, July 26, 2023.

[0010] Therefore, although the broad metabolic benefits of GLP-1, GIP, and GCG receptor agonist compounds have been established in the treatment paradigm, there is still a need for treatments for type 2 diabetes mellitus (T2DM), as well as for related comorbidities such as cardiovascular disease and / or obesity, that can provide effective glucose control with weight loss benefits and reduction of adverse effect profiles such as increased heart rate, cardiac arrhythmias, and loss of fat mass. Also needed are therapeutic agents that can be used with a sufficiently long duration of action to allow dosing as infrequently as twice a week or as little as once a week.

[0011] Surprisingly, modification of the fatty acid side chain and / or substitution of the branched side chain containing an amino acid with a linear side chain amino acid in the sequence of the GLP-1 / GCG dual receptor agonist and / or the GLP-1 / GIP / GCG triple receptor agonist can substantially predominate GLP-1, and thereby, it has been found that weight loss without acute reduction of food consumption is enhanced along with reduction of side effects.

Summary of the Invention

[0012] In one aspect, the present disclosure is 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 (SEQ ID NO: 1) In the sequence, X1 is Y, X2 is Aib, X3 is Q or N, X10 is Y, X​​​​​​​​​​​​​​​​​​​​​​​​​​​​​​ During the ceremony, [ka] Here, R represents the binding site to Leu, and R is selected from C1-C6 alkyl, C3-C6 cycloalkylmethyl, and C3-C6 cycloalkyl. X21 is A, X23 is V or I, X24 is either Q or E, X25 is either W or Y, X27 is either I or L. X28 is A or E, X29 is G, X30 is G, X31 is P, X32 is S, X33 is S, X34 is G, X35 is A, X36 is P, X37 is P, X38 is P, X39 is S, The acid group of the C-terminal amino acid is either a free carboxylic acid group or is amidated at the C-terminal primary amide. However, at least one of X17 and X20 is K, and at least one of said K is C 16 -C 22 This relates to polypeptides or pharmaceutically acceptable salts thereof, provided they are conjugated with fatty acids.

[0013] In another aspect, the Disclosure relates to 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) In the array, X1 is Y, X2 is Aib, X3 is either Q or N, X10 is Y, X12 is I, X13 is αMe-L, X16 is K, X17 is either I or K. X18 is A, X19 is Q, X20 is K, Aib, or the L or D isomer of the following amino acid: [ka] During the ceremony, [ka] Here, R represents the binding site to Leu, and R is selected from C1-C6 alkyl, C3-C6 cycloalkylmethyl, and C3-C6 cycloalkyl. X21 is A, X23 is V or I, X24 is either Q or E, X25 is either W or Y, X27 is either I or L. X28 is A or E, X29 is G, X30 is G, X31 is P, X32 is S, X33 is S, X34 is G, X35 is A, X36 is P, X37 is P, X38 is P, X39 is S, The acid group of the C-terminal amino acid is either a free carboxylic acid group or amidated as a C-terminal primary amide. However, on the condition that at least one of X17 and X20 is K, and further, at least one of the Ks is aminoethoxyethoxyacetic acid - Aib - Glu - C 16 -C 22 fatty acid chain, aminoethoxyethoxyacetic acid - C(O) - diaminobutane - Glu - C 16 -C 22 fatty acid chain, Glu - C 16 -C 22 fatty acid chain, aminoethoxyethoxyacetic acid - aminoethoxyethoxyacetic acid - Glu - C 16 -C 22 fatty acid chain, and aminoethoxyethoxyacetic acid - Glu - C 16 -C 22 fatty acid chain, provided that it contains a side - chain amino (ε - amino) group acylated with a moiety of the formula selected from However, when X20 is Aib, the side - chain amino group is not acylated with either aminoethoxyethoxyacetic acid - aminoethoxyethoxyacetic acid - Glu - C 16 -C 22 fatty acid chain or aminoethoxyethoxyacetic acid - Glu - C 16 -C 22 fatty acid chain. It relates to a polypeptide or a pharmaceutically acceptable salt thereof.

[0014] In another aspect, the present disclosure is a polypeptide or a pharmaceutically acceptable salt thereof comprising the following amino acid sequence, 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 - X​​​​​​​​​​ X12 is I, X13 is Aib, Ser(OMe), nor-V, nor-L, or αMe-L. X16 is K, X17 is either I or K. X18 is A, X19 is Q, X20 is K, Aib, Ser(OMe), nor-V, or nor-L. X21 is A, X23 is V or I, X24 is either Q or E, X25 is either W or Y, X27 is E, I, or L, X28 is A or E, X29 is G, X30 is G, X31 is P, X32 is S, X33 is S, X34 is G, X35 is A, X36 is P, X37 is P, X38 is P, X39 is S, The acid group of the C-terminal amino acid is either a free carboxylic acid group or amidated as a C-terminal primary amide. However, provided that at least one of X17 and X20 is K, and further, that at least one of said K contains a side-chain amino (ε-amino) group acylated with a part of the formula selected from the following, [Table 1] TIFF2026524763000008.tif111155 relates to a polypeptide or a pharmaceutically acceptable salt thereof that is not Sequence ID No. 7, Sequence ID No. 23, or Sequence ID No. 30.

[0015] In another aspect, the Disclosure relates to 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 4) In the array, X1 is Y, X2 is Aib, X3 is either Q or N, X10 is Y, X12 is I, X13 is nor-V, nor-L, or αMe-L. X16 is K, X17 is either I or K. X18 is A, X19 is Q, X20 is K or Aib, X21 is A, X23 is V or I, X24 is either Q or E, X25 is either W or Y, X27 is either I or L. X28 is A or E, X29 is G, X30 is G, X31 is P, X32 is S, X33 is S, X34 is G, X35 is A, Each of X36, X37, and X38 is independently P. X39 is S, The present invention relates to a polypeptide or a pharmaceutically acceptable salt thereof, provided that at least one of X17 and X20 is K, and further, that K contains a side-chain amino (ε-amino) group acylated with a portion of a formula selected from the following. [Table 2]

[0016] In another aspect, the Disclosure relates to a polypeptide comprising the following amino acid sequence or a pharmaceutically acceptable salt thereof, Y-Aib-X3-GTFTSDYSI-X13-LDK-X17-AQ-X20-AF-X23-X24-X25-L-X27-X28-GGPSSGAPPPS(Sequence No. 5) In the array, X3 is either Q or N, X13 is nor-V, nor-L, or αMe-L. X17 is either I or K. X20 is K or Aib, X23 is V or I, X24 is either Q or E, X25 is either W or Y, X27 is either I or L. X28 is A or E, The acid group of the C-terminal amino acid is either a free carboxylic acid group or amidated as a C-terminal primary amide. However, provided that at least one of X17 and X20 is K, and further, that K contains a side-chain amino (ε-amino) group acylated with a part of the formula selected from the following, [Table 3] TIFF2026524763000011.tif71151 relates to polypeptides other than Sequence ID No. 7, or pharmaceutically acceptable salts thereof.

[0017] In another aspect, the Disclosure relates to a polypeptide comprising the following amino acid sequence or a pharmaceutically acceptable salt thereof, Y-Aib-X3-GTFTSDYSI-X13-LDK-X17-AQ-X20-AFIEYLLEGGPSSGAPPPS(Sequence No. 6) In the array, X3 is either Q or N, X13 is nor-V, nor-L, or αMe-L. X17 is K, X20 is Aib, nor-L, or nor-V. The acid group of the C-terminal amino acid is either a free carboxylic acid group or amidated as a C-terminal primary amide. The amino(ε-amino) group in the side chain of K at position X17 is acylated in the part of the formula selected from the following: [Table 4] The polypeptide in question is a polypeptide other than SEQ ID NO: 7, or a pharmaceutically acceptable salt thereof.

[0018] In another aspect, the present disclosure relates to an incretin analog polypeptide, A lysine residue containing a fatty acid extension group bonded to the lysine ε-nitrogen, A peptide residue containing the sequence Gly-Thr-Phe-Thr-Ser-Asp (SEQ ID NO: 31), indirectly bound to a lysine residue via the carboxyl terminus, A peptide residue having the sequence Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-CONH2 (SEQ ID NO: 32), wherein the peptide residue is indirectly bound to the carboxyl group of lysine via the amino terminus of the Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-CONH2 (SEQ ID NO: 32) residue, This invention relates to an incretin analog comprising a Gly-Thr-Phe-Thr-Ser-Asp (SEQ ID NO: 31) residue and a lysine residue, as well as a norvaline amino acid residue indirectly bound between them.

[0019] In another aspect, the present disclosure relates to an incretin analog, A lysine residue containing a group of formula (I) bonded to the ε-nitrogen of lysine, Equation (I) is, [ka] And, During the ceremony, U is either absent or represents -C(O)-CH2-O-(CH2)2-O-(CH2)2-NH-}, where} is the bond point to W. W -C(O)-CH2-O-(CH2)2-O-(CH2)2-NH--], --C(O)--NH--(CH2) 3--4 --NH--], --C(O)-C(CH3)2-NH-], or the following: [ka] ] is the connection point to Y, Y is either absent, 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 to Z. Z is -C(O)-(CH2) n -COOH or -C(O)-(CH2) n - Represents CH3, where n is an integer between 14 and 20, and consists of a lysine residue. A peptide residue containing the sequence Gly-Thr-Phe-Thr-Ser-Asp (SEQ ID NO: 31), indirectly bound to a lysine residue via the carboxyl terminus, A peptide residue having the sequence Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2 (SEQ ID NO: 32), wherein the peptide residue is indirectly bound to the carboxyl group of lysine via the amino terminus of the Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-CONH2 (SEQ ID NO: 32) residue, This invention relates to an incretin analog comprising a Gly-Thr-Phe-Thr-Ser-Asp (SEQ ID NO: 32) residue and a lysine residue, as well as a norvaline amino acid residue indirectly bound between them. [Modes for carrying out the invention]

[0020] 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

[0021]

[0022] definition The “pharmaceutically acceptable salts” according to the present invention include acid addition salts formed using either an organic or inorganic acid. Suitable pharmaceutically acceptable salts of the compounds of this disclosure include acid addition salts of inorganic acids such as hydrochloric acid, hydrobromic acid, and phosphoric acid, or of organic acids such as acetic acid, benzenesulfonic acid, methanesulfonic acid, benzoic acid, citric acid, lactic 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). Pharmaceutically acceptable acid addition salts of the compounds of this disclosure include salts formed by adding, for example, 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.)

[0023] As used herein, the term “effective dose” or “effective amount” refers to the amount of a compound that is sufficient, in a single or multiple dose, to cure, alleviate, reduce, or partially resolve a given disease or condition’s clinical manifestations and its complications beyond what would be expected in the absence of treatment. Therefore, the result may be a reduction and / or alleviation of the signs, symptoms, or causes of the disease, or any other desirable modification of 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.

[0024] The amino acid "Aib" used herein can be represented by the following structure. [ka] It can also be defined by its chemical name, "2-aminoisobutyric acid."

[0025] The amino acid "S(OMe)" or "Ser(OMe)" used herein can be represented by the following structure. [ka] It can also be defined by its chemical name, "serine methyl ether." The terms L-Ser(OMe) and D-Ser(OMe) refer to the "L" isomer and "D" isomer of Ser(OMe), respectively.

[0026] The amino acids "nor-V", "nor-Val", or "norvaline" as used herein can be represented by the following structures. [ka] It can also be defined by its chemical name, "2-aminopentanoic acid." The terms L-norvaline and D-norvaline refer to the "L" and "D" isomers of norvaline, respectively.

[0027] The amino acids "nor-L", "nor-Leu", or "norleucine" as used herein can be represented by the following structures. [ka] It can also be defined by its chemical name, "2-aminohexanoic acid." The terms L-norleucine and D-norleucine refer to the "L" and "D" isomers of norleucine, respectively.

[0028] The amino acids "αMe-L", "αMe-Leu", or "αMe-leucine" used herein can be represented by the following structures. [ka] It can also be defined by its chemical name, "2-amino-2,4-dimethylpentanoic acid." The terms L-α-Me-leucine and D-α-Me-leucine refer to the "L" isomer and "D" isomer of α-Me-leucine, respectively.

[0029] As described herein, this disclosure provides stable, long-acting GLP-1 monoreceptor agonists, GLP-1 / GIP dualreceptor agonists, GLP-1 / GCG dualreceptor agonists, and / or GLP-1 / GIP / GCG triplereceptor agonists that may be useful for the treatment of T2DM, hyperlipidemia / dyslipidemia, metabolic syndromes, metabolic dysfunction-related fatty liver disease (MASLD), metabolic dysfunction-related steatohepatitis (MASH), neurodegenerative disorders, fibrosis, and / or obesity, and for reducing cardiovascular risk.

[0030] In one embodiment, the present disclosure relates to 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) In the array, X1 is Y, X2 is Aib, X3 is either Q or N, X10 is Y, X12 is I, X13 is the L or D isomer of the amino acid in the following formula: [ka] During the ceremony, [ka] Here, R represents the binding site to Leu, and R is selected from C1-C6 alkyl, C3-C6 cycloalkylmethyl, and C3-C6 cycloalkyl. X16 is K, X17 is either I or K. X18 is A, X19 is Q, X20 is K, Aib, or the L or D isomer of the following amino acid: [ka] During the ceremony, [ka] Here, R represents the binding site to Leu, and R is selected from C1-C6 alkyl, C3-C6 cycloalkylmethyl, and C3-C6 cycloalkyl. X21 is A, X23 is V or I, X24 is either Q or E, X25 is either W or Y, X27 is either I or L. X28 is A or E, X29 is G, X30 is G, X31 is P, X32 is S, X33 is S, X34 is G, X35 is A, X36 is P, X37 is P, X38 is P, X39 is S, The acid group of the C-terminal amino acid is either a free carboxylic acid group or amidated as a C-terminal primary amide. However, at least one of X17 and X20 is K, and at least one of those K is C 16 -C 22 This relates to polypeptides or pharmaceutically acceptable salts thereof, provided they are conjugated with fatty acids.

[0031] In one embodiment, the polypeptide of SEQ ID NO: 1 is transmitted via a linker to C16 -C 22 It may have K conjugated to a fatty acid.

[0032] In certain embodiments, the linker is selected from the group consisting of aminoethoxyethoxyacetic acid, glutamic acid, diaminobutane, Aib, and any combination thereof.

[0033] In a preferred embodiment, glutamic acid is γ-glutamic acid.

[0034] In another aspect, the Disclosure relates to 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) In the array, X1 is Y, X2 is Aib, X3 is either Q or N, X10 is Y, X12 is I, X13 is αMe-L, X16 is K, X17 is either I or K. X18 is A, X19 is Q, X20 is K, Aib, or the L or D isomer of the following amino acid: [ka] During the ceremony, [ka] Here, R represents the binding site to Leu, and R is selected from C1-C6 alkyl, C3-C6 cycloalkylmethyl, and C3-C6 cycloalkyl. X21 is A, X23 is V or I, X24 is either Q or E, X25 is either W or Y, X27 is either I or L. X28 is A or E, X29 is G, X30 is G, X31 is P, X32 is S, X33 is S, X34 is G, X35 is A, X36 is P, X37 is P, X38 is P, X39 is S, The acid group of the C-terminal amino acid is either a free carboxylic acid group or amidated as a C-terminal primary amide. However, provided that at least one of X17 and X20 is K, and furthermore, at least one of those Ks is Aminoethoxyethoxyacetic acid-Aib-Glu-C 16 -C 22 fatty acid chain, Aminoethoxyethoxyacetic acid-C(O)-diaminobutane-Glu-C 16 -C 22 fatty acid chain, Glu-C 16 -C 22 fatty acid chain, Aminoethoxyethoxyacetic acid-aminoethoxyethoxyacetic acid-Glu-C 16 -C 22 Fatty acid chains, and Aminoethoxyethoxyacetic acid-Glu-C 16 -C 22 The condition is that the fatty acid chain contains an acylated side-chain amino(ε-amino) group in the selected part of the formula, However, when X20 is Aib, the side chain amino group is aminoethoxyethoxyacetic acid-aminoethoxyethoxyacetic acid-Glu-C 16 -C 22Fatty acid chains also include aminoethoxyethoxyacetic acid-Glu-C 16 -C 22 This relates to polypeptides or pharmaceutically acceptable salts thereof, provided that the fatty acid chains are not acylated.

[0035] In another aspect, the Disclosure relates to 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 3) In the array, X1 is Y, X2 is Aib, X3 is either Q or N, X10 is Y, X12 is I, X13 is Aib, Ser(OMe), nor-V, nor-L, or αMe-L. X16 is K, X17 is either I or K. X18 is A, X19 is Q, X20 is K, Aib, Ser(OMe), nor-V, or nor-L. X21 is A, X23 is V or I, X24 is either Q or E, X25 is either W or Y, X27 is E, I, or L, X28 is A or E, X29 is G, X30 is G, X31 is P, X32 is S, X33 is S, X34 is G, X35 is A, X36 is P, X37 is P, X38 is P, X39 is S, The acid group of the C-terminal amino acid is either a free carboxylic acid group or amidated as a C-terminal primary amide. However, provided that at least one of X17 and X20 is K, and furthermore, that at least one of the K groups has a side-chain amino (ε-amino) group acylated in a part of the formula selected from the following: [Table 5] TIFF2026524763000027.tif204153 relates to a polypeptide other than SEQ ID NO: 7, SEQ ID NO: 23, or SEQ ID NO: 30, or a pharmaceutically acceptable salt thereof.

[0036] In one embodiment, the polypeptide according to SEQ ID NO: 3 is X1 is Y, X2 is Aib, X3 is either Q or N, X10 is Y, X12 is I, X13 is nor-V, nor-L, or αMe-L. X16 is K, X17 is either I or K. X18 is A, X19 is Q, X20 is K, Aib, nor-V, or nor-L. X21 is A, X23 is V or I, X24 is either Q or E, X25 is either W or Y, X27 is either I or L. X28 is A or E, X29 is G, X30 is G, X31 is P, X32 is S, X33 is S, X34 is G, X35 is A, Each of X36, X37, and X38 is independently P. X39 is S, The amino(ε-amino) group in the side chain of K at position X17 is acylated in the part of the formula selected from the following: [Table 6] The polypeptide may also be one that is not sequence number 7.

[0037] In another embodiment, the polypeptide according to Sequence ID 3 is X1 is Y, X2 is Aib, X3 is either Q or N, X10 is Y, X12 is I, X13 is nor-V, nor-L, or αMe-L. X16 is K, X17 is K, X18 is A, X19 is Q, X20 is Aib, nor-V, or nor-L. X21 is A, X23 is I, X24 is E, X25 is Y, X27 is L, X28 is E, X29 is G, X30 is G, X31 is P, X32 is S, X33 is S, X34 is G, X35 is A, Each of X36, X37, and X38 is independently P. X39 is S, The amino(ε-amino) group in the side chain of K at position X17 is acylated in the part of the formula selected from the following: [Table 7] The polypeptide may also be one that is not sequence number 7.

[0038] In another aspect, the Disclosure relates to 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 4) In the array, X1 is Y, X2 is Aib, X3 is either Q or N, X10 is Y, X12 is I, X13 is nor-V, nor-L, or αMe-L. X16 is K, X17 is either I or K. X18 is A, X19 is Q, X20 is K or Aib, X21 is A, X23 is V or I, X24 is either Q or E, X25 is either W or Y, X27 is either I or L. X28 is A or E, X29 is G, X30 is G, X31 is P, X32 is S, X33 is S, X34 is G, X35 is A, Each of X36, X37, and X38 is independently P. X39 is S, However, this may be further provided that at least one of X17 and X20 is K, and that K contains a side-chain amino (ε-amino) group acylated with a part of the formula selected from the following. [Table 8]

[0039] In another aspect, the Disclosure relates to a polypeptide comprising the following amino acid sequence or a pharmaceutically acceptable salt thereof, Y-Aib-X3-GTFTSDYSI-X13-LDK-X17-AQ-X20-AF-X23-X24-X25-L-X27-X28-GGPSSGAPPPS(Sequence No. 5) In the array, X3 is either Q or N, X13 is nor-V, nor-L, or αMe-L. X17 is either I or K. X20 is K or Aib, X23 is V or I, X24 is either Q or E, X25 is either W or Y, X27 is either I or L. X28 is A or E, The acid group of the C-terminal amino acid is either a free carboxylic acid group or amidated as a C-terminal primary amide. However, provided that at least one of X17 and X20 is K, and further, that K contains a side-chain amino (ε-amino) group acylated with a part of the formula selected from the following, [Table 9] TIFF2026524763000032.tif108151 relates to polypeptides other than Sequence ID No. 7, or pharmaceutically acceptable salts thereof.

[0040] In one embodiment, the polypeptide according to SEQ ID NO: X3 is Q, X13 is αMe-L, X17 is K, X20 is Aib, X23 is I, X24 is E, X25 is Y, X27 is L, X28 is E, The amino(ε-amino) group of the K side chain at position X17 may be acylated in a part of the formula selected from the following: [Table 10]

[0041] In another embodiment, the polypeptide according to SEQ ID NO. 5 is X3 is Q, X13 is nor-V, X17 is K, X20 is Aib, X23 is I, X24 is E, X25 is Y, X27 is L, X28 is E, The amino(ε-amino) group of the K side chain at position X17 may be acylated in a part of the formula selected from the following: [Table 11]

[0042] In another embodiment, the polypeptide according to SEQ ID NO. 5 is X3 is Q, X13 is nor-L, X17 is K, X20 is Aib, X23 is I, X24 is E, X25 is Y, X27 is L, X28 is E, The amino(ε-amino) group of the K side chain at position X17 may be acylated in a part of the formula selected from the following: [Table 12]

[0043] In another embodiment, the polypeptide according to SEQ ID NO. 5 is X3 is Q, X13 is nor-V, X17 is I, X20 is K, X23 is V, X24 is Q, X25 is W, X27 is I, X28 is A, The side chain amino(ε-amino) group of K at position X20 may be acylated in a part of the formula selected from the following: [Table 13]

[0044] In another embodiment, the polypeptide according to SEQ ID NO. 5 is X3 is N, X13 is nor-V, X17 is I, X20 is K, X23 is V, X24 is Q, X25 is W, X27 is I, X28 is A, The side chain amino(ε-amino) group of K at position X20 may be acylated in a part of the formula selected from the following: [Table 14]

[0045] In another aspect, the Disclosure relates to a polypeptide comprising the following amino acid sequence or a pharmaceutically acceptable salt thereof, Y-Aib-X3-GTFTSDYSI-X13-LDK-X17-AQ-X20-AFIEYLLEGGPSSGAPPPS(Sequence No. 6) In the array, X3 is either Q or N, X13 is nor-V, nor-L, or αMe-L. X17 is K, X20 is Aib, nor-L, or nor-V. The acid group of the C-terminal amino acid is either a free carboxylic acid group or amidated as a C-terminal primary amide. The amino(ε-amino) group in the side chain of K at position X17 is acylated in the part of the formula selected from the following: [Table 15] This relates to polypeptides other than SEQ ID NO: 7, or pharmaceutically acceptable salts thereof.

[0046] In one embodiment, the polypeptide according to SEQ ID NO: 6 is X13 is either nor-V or nor-L. X20 is Aib, The amino(ε-amino) group of the K side chain at position X17 may be acylated in a part of the formula selected from the following: [Table 16]

[0047] In another embodiment, the polypeptide according to SEQ ID NO. 6 is X13 is αMe-L, X20 is either nor-L or nor-V. The amino(ε-amino) group of the K side chain at position X17 may be acylated in a part of the formula selected from the following: [Table 17]

[0048] In another aspect, the present disclosure relates to an incretin analog polypeptide, A lysine residue containing a fatty acid extension group bonded to the lysine ε-nitrogen, A peptide residue containing the sequence Gly-Thr-Phe-Thr-Ser-Asp (SEQ ID NO: 31), indirectly bound to a lysine residue via the carboxyl terminus, A peptide residue having the sequence Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-CONH2 (SEQ ID NO: 32), wherein the peptide residue is indirectly bound to the carboxyl group of lysine via the amino terminus of the Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-CONH2 (SEQ ID NO: 32) residue, This invention relates to an incretin analog comprising a Gly-Thr-Phe-Thr-Ser-Asp (SEQ ID NO: 31) residue and a lysine residue, as well as a norvaline amino acid residue indirectly bound between them.

[0049] In another aspect, the present disclosure relates to an incretin analog, A lysine residue containing a group of formula (I) bonded to the ε-nitrogen of lysine, wherein formula (I) is [ka] And, During the ceremony, U is either absent or represents -C(O)-CH2-O-(CH2)2-O-(CH2)2-NH-}, where} is the bond point to W. W -C(O)-CH2-O-(CH2)2-O-(CH2)2-NH--], --C(O)--NH--(CH2) 3--4 --NH--], --C(O)-C(CH3)2-NH-], or the following: [ka] ] is the connection point to Y, Y is either absent, 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 to Z. Z is -C(O)-(CH2) n -COOH or -C(O)-(CH2) n - Represents CH3, where n is an integer between 14 and 20, and consists of a lysine residue. A peptide residue containing the sequence Gly-Thr-Phe-Thr-Ser-Asp (SEQ ID NO: 31), indirectly bound to a lysine residue via the carboxyl terminus, A peptide residue having the sequence Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH2 (SEQ ID NO: 32), wherein the peptide residue is indirectly bound to the carboxyl group of lysine via the amino terminus of the Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-CONH2 (SEQ ID NO: 32) residue, This invention relates to an incretin analog comprising a Gly-Thr-Phe-Thr-Ser-Asp (SEQ ID NO: 31) residue and a lysine residue, as well as a norvaline amino acid residue indirectly bound between them.

[0050] In one embodiment, in the incretin analog, lysine is bound to the Gly-Thr-Phe-Thr-Ser-Asp (SEQ ID NO: 31) residue by a peptide residue containing 10 amino acids.

[0051] In another embodiment, in the incretin analog, lysine is bound to the Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-CONH2 (SEQ ID NO: 32) residue by a peptide residue containing 11 amino acids.

[0052] In another aspect, the disclosure relates to a pharmaceutical composition comprising a pharmaceutically acceptable carrier and a polypeptide or incretin analog described herein.

[0053] In another aspect, the present disclosure relates to a method for treating obesity, type 2 diabetes mellitus (T2DM), metabolic syndromes, metabolic dysfunction-related fatty liver disease (MASLD), metabolic dysfunction-related steatohepatitis (MASH), neurodegenerative disorders, fibrosis, hyperlipidemia / dyslipidemia, and reducing cardiovascular risk, comprising administering a polypeptide or incretin analog described herein to a patient in need of such treatment.

[0054] In another embodiment, the present disclosure relates to a polypeptide comprising an amino acid sequence selected from the group consisting of the following, or a pharmaceutically acceptable salt thereof, i.)Y-Aib-QGTFTSDYSI-(αMe-L)-LDKK*AQ-Aib-AFIEYLLEGGPSSGAPPPS-NH2(Sequence ID 8), ii.) Y-Aib-QGTFTSDYSI-(norvaline)-LDKK*AQ-Aib-AFIEYLLEGGPSSGAPPPS-NH2 (Sequence ID 9), iii.) Y-Aib-NGTFTSDYSI-(norvaline)-LDKK*AQ-Aib-AFIEYLLEGGPSSGAPPPS-NH2 (Sequence ID 10), iv.) Y-Aib-QGTFTSDYSI-(norleucine)-LDKK*AQ-Aib-AFIEYLLEGGPSSGAPPPS-NH2 (Sequence ID 11), v.)Y-Aib-QGTFTSDYSI-(αMe-L)-LDKK*AQ-(nor-V)-AFIEYLLEGGPSSGAPPPS-NH2(Sequence ID 12), vi.)Y-Aib-NGTFTSDYSI-(αMe-L)-LDKK*AQ-(nor-V)-AFIEYLLEGGPSSGAPPPS-NH2(Sequence ID 13), vii.)Y-Aib-QGTFTSDYSI-(αMe-L)-LDKK*AQ-(nor-L)-AFIEYLLEGGPSSGAPPPS-NH2(Sequence ID 14), viii.) Y-Aib-QGTFTSDYSI-(norvaline)-LDKIAQK*AFVQWLIAGGPSSGAPPPS-NH2 (SEQ ID NO: 15), and ix.)Y-Aib-NGTFTSDYSI-(norvaline)-LDKIAQK*AFVQWLIAGGPSSGAPPPS-NH2 (Sequence ID 16), The amino(ε-amino) group in the side chain of K* is acylated at the part of the formula selected from the following: [Table 18] TIFF2026524763000044.tif145151 relates to a polypeptide or a pharmaceutically acceptable salt thereof that is not Sequence ID No. 7, Sequence ID No. 23, or Sequence ID No. 30.

[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, all amino acids are in the "L" form unless otherwise indicated.

[0057] In another embodiment, the present invention relates to a polypeptide selected from one of the representative compounds in Table 1 or a pharmaceutically acceptable salt thereof. [Table 19] TIFF2026524763000046.tif201152

Table 20

[0058] In another aspect, the present disclosure relates to a method of treating or preventing type 2 diabetes (T2DM).

[0059] In another aspect, the present disclosure relates to a method of treating or preventing hyperlipidemia / dyslipidemia.

[0060] In another aspect, the present disclosure relates to a method of treating or preventing obesity.

[0061] In another aspect, the present disclosure relates to a method of treating or preventing metabolic syndrome, non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), neurodegenerative disorders, fibrosis, and / or cardiovascular risk.

[0062] In one embodiment, the treatment method comprises administering to a patient in need thereof an effective amount of the polypeptide described herein or a pharmaceutically acceptable salt thereof. <00,00921>

[0063] In another aspect, the present disclosure relates to a method of treating type 2 diabetes (T2DM) comprising administering to a patient in need of such treatment an effective amount of the polypeptide described herein or a pharmaceutically acceptable salt thereof.

[0064] In another aspect, the present disclosure relates to a method of treating obesity comprising administering to a patient in need of such treatment an effective amount of the polypeptide described herein or a pharmaceutically acceptable salt thereof.

[0065] In another aspect, the present disclosure relates to 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.

[0066] In another embodiment, the present invention relates to a pharmaceutical composition comprising a polypeptide described herein or a pharmaceutically acceptable salt thereof, together with one or more pharmaceutically acceptable carriers, diluents, or excipients.

[0067] 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 processes for preparing them are well known in the art. (See, for example, "Remington: The Science and 50 Practice of Pharmacy," edited by DB Troy, 21st Edition, Lippincott, Williams & Wilkins, 2006).

[0068] In another aspect, this disclosure relates to polypeptides described herein or pharmaceutically acceptable salts thereof for use as pharmaceuticals.

[0069] In another aspect, the disclosure relates to polypeptides described herein or pharmaceutically acceptable salts thereof for use in the treatment or prevention of type 2 diabetes mellitus (T2DM).

[0070] In another aspect, this disclosure relates to polypeptides described herein or pharmaceutically acceptable salts thereof for use in the treatment or prevention of hyperlipidemia / dyslipidemia.

[0071] In another aspect, this disclosure relates to polypeptides described herein or pharmaceutically acceptable salts thereof for use in the treatment or prevention of obesity.

[0072] In another aspect, the present disclosure relates to the 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 risk.

[0073] In another aspect, 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.

[0074] In another aspect, the pharmaceutical composition according to the present disclosure comprises a polypeptide described herein or a pharmaceutically acceptable salt thereof for use as a medicament.

[0075] In another aspect, the pharmaceutical composition according to the present disclosure comprises a polypeptide described herein or a pharmaceutically acceptable salt thereof for use in the treatment or prevention of type 2 diabetes (T2DM).

[0076] In another aspect, the pharmaceutical composition according to the present disclosure comprises a polypeptide described herein or a pharmaceutically acceptable salt thereof for use in the treatment or prevention of hyperlipidemia / dyslipidemia.

[0077] In another aspect, the pharmaceutical composition according to the present disclosure comprises a polypeptide described herein or a pharmaceutically acceptable salt thereof for use in the treatment or prevention of obesity.

[0078] In another aspect, the pharmaceutical composition according to the present disclosure comprises a polypeptide described herein or a pharmaceutically acceptable salt 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 risk.

[0079] 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 one or more additional therapeutic agents in an effective amount.

[0080] This disclosure may involve one or more embodiments described herein. It is understood that the embodiments described herein are illustrative of the invention and are not intended to limit the claims to any particular embodiment illustrated. It is also 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 intends to provide for all possible combinations and substitutions of various independently described embodiments.

[0081] 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. Therefore, any features, integers, characteristics, compounds, or chemical parts described in conjunction with a particular aspect, embodiment, or example of this disclosure will be understood to apply to any other aspect, embodiment, or example described herein, to the extent that they do not conflict.

[0082] Furthermore, unless otherwise specified, any feature disclosed herein may be substituted by an alternative feature that serves the same or similar purpose. [Examples]

[0083] Instruments and analytical methods. The instruments used for characterizing and analyzing the compounds described herein include a High Performance Liquid Chromatograph (HPLC) (Waters e2695 Alliance; Detector Waters (2489 UV / Visible)).

[0084] Mass instrument: HPLC: Waters e2695 Alliance, and detector: Acquity-QDa.

[0085] The compounds described herein were purified by the preparative HPLC procedure outlined below.

[0086] 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 μm, flow rate: 75 mL / min [Table 21]

[0087] The purity of the compounds described herein was analyzed by one of the RP-HPLC methods outlined below.

[0088] HPLC method A Column: Xbridge Peptide BEH C18 (4.6mm x 250mm, 3.5μ) 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°C Electrophoresis time: 90 minutes [Table 22]

[0089] 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°C Electrophoresis time: 38 minutes [Table 23]

[0090] 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 triethylamine 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°C Electrophoresis time: 90 minutes [Table 24]

[0091] 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 trimethylamine added and pH adjusted to 2.5 ± 0.1 with orthophosphate. Flow rate: 0.8mL / min Detection: UV detection at 210nm Column temperature: 60℃ Sample tray temperature: 5°C Electrophoresis time: 33 minutes [Table 25]

[0092] Preparation method Example A: Preparation of partial A-di-tert-butyl ester [ka]

[0093] Partially A-di-tert-butyl esters were prepared using solid-phase synthesis. 2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid was bonded 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 deblockage of the amino group using piperidine, and subsequently coupled with 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 deblockage using piperidine, and the free amino group was coupled with 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]acetic acid-2-chlorotrityl resin. The Fmoc group of the obtained compound was selectively deblocked using piperidine, and the free amino group was then coupled with 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, the 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 + ).

[0094] Preparation of Partial A-OSu [ka]

[0095] 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 then deprotected with trifluoroacetic acid to obtain the title compound, partial A-OSu.

[0096] Example B: Preparation of partial B-di-tert-butyl ester [ka]

[0097] Partially B-di-tert-butyl ester was prepared using a process similar to that described in Example A, but using 20-(tert-butoxy)-20-oxoicosanoic acid instead of octadecanediol monotert-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]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-[(20-tert-butoxy-20-oxo-icosanoyl)amino]-5-oxo-pentanoyl]amino]-2-methyl-propanoyl]amino]ethoxy]ethoxy]acetic acid (partially B-di-tert-butyl ester). LCMS=m / z:814.10(M+H + ).

[0098] Preparation of partial B-OSu [ka]

[0099] 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.

[0100] Example C: Preparation of partial C-di-tert-butyl ester [ka]

[0101] Partially C-di-tert-butyl esters were prepared using solid-phase synthesis. 2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid was bonded 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 deblockage 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 deblockage using piperidine, and the free amino group was then coupled 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]2-chlorotrityl acetate resin was selectively deblocked using piperidine and then coupled 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]2-chlorotrityl acetate resin. Next, the 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). + ).

[0102] Preparation of partial C-OSu [ka]

[0103] 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 then deprotected with trifluoroacetic acid to obtain the title compound, partial C-OSu.

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

[0105] Partially B-di-tert-butyl ester was prepared using a process similar to that described in Example C, but using 20-(tert-butoxy)-20-oxoeicosanoic acid instead of octadecanediic acid monotert-butyl 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]2-chlorotrityl acetate resin. Next, the 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 (partially D-di-tert-butyl ester). LCMS=m / z:843.14(M+H + ).

[0106] Preparation of partial D-OSu [ka]

[0107] 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 then deprotected with trifluoroacetic acid to obtain the title compound, partial D-OSu.

[0108] Example E: Preparation of partial E-OSu [ka]

[0109] 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 this is reacted with HOSu in the presence of IBCF and NMM to form CH3-(CH2) 14 -C(O)-Glu(OSu)-OtBu was obtained, and then deprotected with trifluoroacetic acid to obtain partial E-OSu.

[0110] Example F: Preparation of partial F-di-tert-butyl ester [ka]

[0111] Partially F-di-tert-butyl esters were prepared using solid-phase synthesis. 2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid was bonded 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 deblockage of the amino group using piperidine, and subsequently coupled with 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 deblockage using piperidine, and the free amino group was coupled with 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 coupled with octadecanediic acid monotertbutyl 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 + ).

[0112] Preparation of partial F-OSu [ka]

[0113] 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 then deprotected with trifluoroacetic acid to obtain the title compound, partial F-OSu.

[0114] Example G: Preparation of part G [ka]

[0115] Partially G-di-tert-butyl ester was prepared using a process similar to that described in Example F, but using 20-(tert-butoxy)-20-oxoeicosanoic acid 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]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-[(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 + ).

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

[0117] 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 title compound, partial G-OSu.

[0118] Example H: Preparation of partial H-di-tert-butyl ester [ka]

[0119] 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 bonded 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 deblockage of the amino group using piperidine, and then coupled 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 coupled with 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. The 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 + ).

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

[0121] 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 then deprotected with trifluoroacetic acid to obtain the title compound, partial H-OSu.

[0122] Example I: Partial Preparation [ka]

[0123] Partial I-di-tert-butyl ester was prepared using a process similar to that described in Example H, but using 20-(tert-butoxy)-20-oxoeicosanoic acid 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, the 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 + ).

[0124] Preparation of Partial I-OSu [ka]

[0125] 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 then deprotected with trifluoroacetic acid to obtain the title compound, partial I-OSu.

[0126] Example 12: Synthesis of Compound 12 The parent peptide 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 coupling of Fmoc-Ser(tBu)-OH with the Rink amide resin. Coupling was carried out using diisopropylcarbodiimide and N-hydroxybenzotriazole (DIPC-HOBt) as coupling reagents to obtain Ser(tBu)-Rink amide resin, thus completing the first cycle. Acetic anhydride and diisopropylethylamine were used to terminate / cap the uncoupled amino group in each amino acid coupling. Selective deblocking of the amino group of the Fmoc-Ser(tBu)-Rink amide resin using piperidine. Subsequently, Fmoc-Pro-Ser(tBu)-Rink amide resin was obtained by coupling with Fmoc-Pro-OH using HOBt and DIPC.

[0127] The three steps described above—selective capping, deblocking, and coupling of the Fmoc-protected amino acid residues bound to the resin with the Fmoc-protected amino group of an adjacent amino acid residue in the sequence—were repeated for the remaining 36 amino acid residues. The side chains of the Fmoc-protected amino acids were protected orthogonally (for example, 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-dioxocyclohex-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 coupling with the adjacent Fmoc-protecting amino acid—were performed to obtain the Fmoc-Aib-Gln(Trt)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-(αMethyl-Leu)-Leu-Asp(OtBu)-Lys(Boc)-Lys(IVDde)-Ala-Gln(Trt)-Aib-Ala-Phe-Ile-Glu(OtBu)-Tyr(tBu)-Leu-Leu-Glu(OtBu)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Resin.

[0128] Fmoc-Aib-Gln(Trt)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-(αMethyl-Leu)-Leu-Asp(OtBu)-Lys(Boc)-Lys(IVDde)-Ala-Gln(Trt)-Aib-Ala-Phe-Ile-Glu(OtBu)-Tyr(tBu)-Leu-Leu-Glu(OtBu)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Resin deblockage using piperidine, followed by diisopropylcarbodiimide and N-hydroxybenzotriazole (DIPC-HOB Boc-Tyr(tBu)-OH was coupled using t) as the coupling reagent to obtain Boc-Tyr(tBu)-Aib-Gln(Trt)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-(αMethyl-Leu)-Leu-Asp(OtBu)-Lys(Boc)-Lys(IVDde)-Ala-Gln(Trt)-Aib-Ala-Phe-Ile-Glu(OtBu)-Tyr(tBu)-Leu-Leu-Glu(OtBu)-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-resin. Deprotection of the IVDde group of the peptide resin using hydrazine hydrate, followed by coupling of the partial A-di-tert-butyl ester, was carried out in the presence of diisopropylcarbodiimide and N-hydroxybenzotriazole (DIPC-HOBt) as coupling reagents to obtain compound 12 on the resin. Compound 12 was obtained by cleavage and deprotection from the resin using trifluoroacetic acid together with ethane-1,2-dithiol and triisopropylsilane, followed by purification via preparative HPLC.

[0129] Mass (LCMS): m / z = 1197.92 (MH4 4+); Calculated mass = 4787.64; HPLC purity (Method C): 97.4%.

[0130] Example 13: Synthesis of Compound 13 Compound 13 was prepared by a solid-phase method following a process similar to that described in Example 12, but with partial B-di-tert-butyl ester coupling instead of partial A-di-tert-butyl ester coupling following IVDde deprotection.

[0131] Mass (LCMS): m / z = 1204.89 (MH4 4+); Mass calculated = 4815.52; HPLC purity (Method C): 97.2%.

[0132] Example 14: Synthesis of Compound 14 Compound 14 was prepared by solid-phase method according to a process similar to that described in Example 12, but with (i) Fmoc-norleucine-OH used instead of Fmoc-αMe-leucine-OH at the 13-position, and (ii) partial B-di-tert-butyl ester coupling instead of partial A-di-tert-butyl ester coupling following IVDde deprotection.

[0133] Mass (LCMS): m / z=1201.39 ((MH4 4+); Mass calculated: 4801.53; HPLC purity (Method C): 98.09%.

[0134] Example 15: Synthesis of Compound 15 Compound 15 was prepared by a solid-phase method following a process similar to that described in Example 14, but with partial A-di-tert-butyl ester coupling instead of partial B-di-tert-butyl ester coupling following IVDde deprotection.

[0135] Mass LCMS): m / z=1194.39 (MH4 4+); Mass calculated: 4773.53; HPLC purity (Method C): 96.4%.

[0136] Example 16: Synthesis of Compound 16 Compound 16 was prepared by solid-phase method according to a process similar to that described in Example 12, but using Fmoc-norvaline-OH instead of Fmoc-αMe-leucine-OH at position 13.

[0137] Mass (LCMS): m / z=1191.32 (MH4 4+) and calculated mass=4761.25.

[0138] Example 17: Synthesis of Compound 17 Compound 17 was prepared by a solid-phase method following a process similar to that described in Example 16, but with partial B-di-tert-butyl ester coupling instead of partial A-di-tert-butyl ester coupling following IVDde deprotection.

[0139] Example 18: Synthesis of Compound 18 Compound 18 was prepared by a solid-phase method following a process similar to that described in Example 16, but with partial G-di-tert-butyl ester coupling instead of partial A-di-tert-butyl ester coupling following IVDde deprotection.

[0140] Example 19: Synthesis of Compound 19 Compound 19 was prepared by a solid-phase method following a process similar to that described in Example 12, but with partial C-di-tert-butyl ester coupling instead of partial A-di-tert-butyl ester coupling following IVDde deprotection.

[0141] Mass (LCMS) m / z: 1205.64 (MH4 4+) and calculated mass: 4818.53.

[0142] Example 20: Synthesis of Compound 20 Compound 20 was prepared by a solid-phase method following a process similar to that described in Example 12, but with partial D-di-tert-butyl ester coupling instead of partial A-di-tert-butyl ester coupling following IVDde deprotection.

[0143] Mass (LCMS) m / z: 1212.64 (MH4 4+) and calculated mass: 4846.53.

[0144] Example 21: Synthesis of Compound 21 The parent peptide 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 coupling of Fmoc-Ser(tBu)-OH with the Rink amide resin. Coupling was carried out using diisopropylcarbodiimide and N-hydroxybenzotriazole (DIPC-HOBt) as coupling reagents to obtain Ser(tBu)-Rink amide resin, thus completing the first cycle. Uncoupled amino groups in each amino acid coupling were terminated / capped using acetic anhydride and diisopropylethylamine. Selective deblocking of the amino group of the Fmoc-Ser(tBu)-Rink amide resin using piperidine. Then, Fmoc-Pro-Ser(tBu)-Rink amide resin was obtained by coupling with Fmoc-Pro-OH using HOBt and DIPC. Uncoupled amino groups after each amino acid coupling were terminated using acetic anhydride and diisopropylethylamine.

[0145] The three steps described above—selective capping, deblocking, and coupling of the Fmoc-protected amino acid residues bound to the resin with the Fmoc-protected amino group of an adjacent amino acid residue in the sequence—were repeated for the remaining 36 amino acid residues. The side chains of the Fmoc-protected amino acids were protected orthogonally (for example, 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-dioxocyclohex-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 coupling with the adjacent Fmoc-protecting amino acid—were performed to obtain the Fmoc-Aib-Gln(Trt)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-norvaline-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(IVDde)-Ala-Phe-Val-Gln(Trt)-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Resin.

[0146] Fmoc-Aib-Gln(Trt)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-norvaline-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(IVDde)-Ala-Phe-Val-Gln(Trt)-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-Resin deblockage using piperidine, followed by diisopropylcarbodiimide, N-hydroxybenzotriazole (DIPC-HOB Boc-Tyr(tBu)-OH was coupled using t) as the coupling reagent to obtain Boc-Tyr(tBu)-Aib-Gln(Trt)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Tyr(tBu)-Ser(tBu)-Ile-norvaline-Leu-Asp(OtBu)-Lys(Boc)-Ile-Ala-Gln(Trt)-Lys(IVDde)-Ala-Phe-Val-Gln(Trt)-Trp-Leu-Ile-Ala-Gly-Gly-Pro-Ser(tBu)-Ser(tBu)-Gly-Ala-Pro-Pro-Pro-Ser(tBu)-resin. Compound 21 was obtained on the resin by deprotecting the IVDde group of the peptide resin using hydrazine hydrate, followed by coupling of the partial A-di-tert-butyl ester using diisopropylcarbodiimide and N-hydroxybenzotriazole (DIPC-HOBt) as coupling reagents. Compound 21 was obtained from the resin by cleavage and deprotection using trifluoroacetic acid together with ethane-1,2-dithiol and triisopropylsilane, followed by purification via preparative HPLC.

[0147] Mass (LCMS) m / z: 1185.39 (MH4 4+); Calculated mass: 4737.53; HPLC purity (Method C): 98.5%.

[0148] Example 22: Synthesis of Compound 22 Compound 22 was prepared by a solid-phase method following a process similar to that described in Example 21, but with partial B-di-tert-butyl ester coupling instead of partial A-di-tert-butyl ester coupling following IVDde deprotection.

[0149] Mass (LCMS): m / z=1192.41 (MH4 4+); Calculated mass: 4765.60; HPLC purity (Method C): 97.6%.

[0150] Example 23: Synthesis of Compound 23 Compound 23 was prepared by solid-phase method according to a process similar to that described in Example 22, but using Fmoc-Asn(Trt)-OH instead of Fmoc-Gln(Trt)-OH at the 3-position.

[0151] Mass (LCMS): m / z=1189.20 (MH4 4+) and calculated mass: 4752.77.

[0152] Example 24: Synthesis of Compound 24 Compound 24 was prepared by a solid-phase method following a process similar to that described in Example 21, but with Fmoc-Asn(Trt)-OH used instead of Fmoc-Gln(Trt)-OH at the 3-position.

[0153] Mass (LCMS): m / z=1182.22 (MH4 4+) and calculated mass: 4724.85.

[0154] Example 26: Synthesis of Compound 26 Compound 26 was prepared by a solid-phase method following a process similar to that described in Example 12, but with partial G-di-tert-butyl ester coupling instead of partial A-di-tert-butyl ester coupling following IVDde deprotection.

[0155] Mass (LCMS): m / z=1219.95 (MH4 4+) and calculated mass: 4875.76; HPLC purity (Method C): 96.3%.

[0156] biological research Example 1: Efficacy study in db / db mice at a dose of 10 nM / kg The effects of the compounds described herein on blood glucose levels, food intake, and body weight were studied in mice. This study was conducted in a type 2 diabetic mouse (db / db) model. The animals were divided into nine treatment groups (n=6): a diabetic control group and nine treatment groups: compound 12 (10 nM / kg), compound 13 (10 nM / kg), compound 26 (10 nM / kg), compound 14 (10 nM / kg), compound 15 (10 nM / kg), compound 21 (10 nM / kg), compound 22 (10 nM / kg), and tilzepatide (10 nM / kg). Baseline blood glucose levels were measured in all animals. All animals were administered the test products 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. Weight changes and cumulative food consumption were measured at 48 and 96 hours after the procedure. [Table 26] [Table 27] [Table 28]

[0157] The results presented above indicate that this compound may be effective in treating type 2 diabetes, diabetes with obesity, obesity, and hyperlipidemia.

[0158] Example 2: Oral glucose tolerance test (OGTT) in rats, single injection, 30 nM / kg dose Animals were divided into six groups (n=4 / group): a placebo control group, compound 17 (30 nM / kg), compound 18 (30 nM / kg), compound 19 (30 nM / kg), compound 20 (30 nM / kg), and letatrutide (30 nM / kg). Animals were fasted for 12 hours before the start of the OGTT. Blood glucose levels were measured using a blood glucose meter 24 hours after subcutaneous injection of the test drug or letatrutide (time 0 measurement). All animals were orally administered a 2 g / kg glucose solution. Blood glucose levels were measured at 10, 20, 40, 60, 90, and 120 minutes after the glucose challenge. Body weight and food consumption were recorded at 12, 48, and 72 hours. [Table 29] [Table 30] [Table 31]

[0159] Surprisingly, the inventors found that this compound showed weight reduction comparable to that of letatoltide, but without a dramatic reduction in food intake. This may be because the compound has lower potency in GCGR (vs. GLP-1R) compared to letatoltide.

[0160] Variations in the individual receptor agonisms of GLP-1 / GCG bireceptor agonists and GLP-1 / GIP / GCG triplereceptor agonists can lead to diverse outcomes in terms of therapeutic efficacy and adverse effects.

[0161] To achieve optimal efficacy and minimize side effects, a delicate balance must be achieved between GLP-1 / GIP / GCG receptor agonists.

[0162] The challenge is to devise molecules that enhance therapeutic effects and have fewer side effects than existing therapies. The weight reduction caused by compounds 17, 18, 19, and 20 without causing an acute reduction in food consumption may be related to the improved potency of these compounds against GLP-1R compared to GCGR, which can be derived from Example 7.

[0163] Example 3: In vitro assay The in vitro potency of the compounds described herein was determined 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 monitors the activation of GLP-1R, GIPR, or GCGR via Gi and Gs secondary messenger signaling using a technique developed by DiscoverX called enzyme fragment complementation (EFC) with β-galactosidase (β-Gal) as a functional reporter. The enzyme is split into two complementary parts: EA for the enzyme acceptor and ED for the enzyme donor. ED is fused to cAMP and, in the 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.

[0164] Three different assays were performed using cells expressing one of three receptors. The cAMP Hunter cell line was expanded from frozen stock according to standard procedure. Cells were seeded in a 384-well white-walled microplate with a total volume of 20 μL 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 conjugate antibody and 5 μL of the test compound. After appropriate compound incubation, the assay signal was generated via a 1-hour incubation with 20 μL of cAMP-ED cell lysis cocktail, followed by a 3-hour incubation at room temperature with 20 μL of cAMP-EA reagent. Free cAMP-ED available in the system complements free cAMP-EA to form active β-Gal, which reacts with the substrate to certify a chemiluminescent signal. The microplate was 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 as a result of the response. Logarithmic concentration-to-effect percentage curves were generated using different sample concentrations (different for different compounds). Four-parameter logistic curves were generated and the EC50 was determined. Appropriate assay references (Exendin-4 for GLP-1R, GIP for GIPR, and glucagon for GCGR) were used for each assay.

[0165] Cellular cAMP assays of letatoltide, compound 12, compound 13, compound 14, compound 17, compound 18, compound 19, and compound 20 showed the semi-effective concentrations on GLP-1R-expressing cells and GIPR-expressing cells as described in Table 9. [Table 32]

[0166] Due to its lower affinity for the GCG receptor compared to the GLP-1 receptor, some side effects caused by GCGR agonism may be reduced.

[0167] Glucagon activity is diabetic-inducing, causing amino acid and protein catabolism that leads to increased heart rate and loss of lean body mass. Therefore, the compounds of the present invention offer several benefits, along with a reduced side effect profile.

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) In the array, X1 is Y, X2 is Aib, X3 is Q or N, X10 is Y, X12 is I, X13 is the L or D isomer of the amino acid in the following formula: 【Chemistry 1】 During the ceremony, 【Chemistry 2】 This represents the connection point to Leu, and R is C １ -C ６ Alkyl, C ３ -C ６ Cycloalkylmethyl, and C ３ -C ６ Selected from cycloalkyl groups, X16 is K, X17 is I or K, X18 is A, X19 is Q, X20 is K, Aib, or the L or D isomer of an amino acid with the following formula: 【Transformation 3】 During the ceremony, 【Chemistry 4】 represents the binding point to Leu, and R is C １ -C ６ alkyl, C ３ -C ６ cycloalkylmethyl, and C ３ -C ６ selected from cycloalkyl, X21 is A, X23 is V or I, X24 is Q or E, X25 is either W or Y, X27 is I or L, X28 is A or E, X29 is G, X30 is G, X31 is P, X32 is S, X33 is S, X34 is G, X35 is A, X36 is P, X37 is P, X38 is P, X39 is S, The acid group of the C-terminal amino acid is either a free carboxylic acid group or amidated as a C-terminal primary amide. However, at least one of X17 and X20 is K, and at least one of the aforementioned K is C １６ -C ２２ A polypeptide that is conjugated to a fatty acid.

2. K, via the linker, C １６ -C ２２ The polypeptide according to claim 1, which is conjugated to a fatty acid.

3. The polypeptide according to claim 2, wherein the linker is selected from the group consisting of aminoethoxyethoxyacetic acid, glutamic acid, diaminobutane, Aib, and any combination thereof.

4. The polypeptide according to claim 3, wherein the glutamic acid is γ-glutamic acid.

5. 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) In the array, X1 is Y, X2 is Aib, X3 is Q or N, X10 is Y, X12 is I, X13 is αMe-L, X16 is K, X17 is I or K, X18 is A, X19 is Q, X20 is K, Aib, or the L or D isomer of an amino acid with the following formula: 【Transformation 5】 During the ceremony, 【Transformation 6】 This represents the connection point to Leu, and R is C １ -C ６ Alkyl, C ３ -C ６ Cycloalkylmethyl, and C ３ -C ６ Selected from cycloalkyl groups, X21 is A, X23 is V or I, X24 is Q or E, X25 is either W or Y, X27 is I or L, X28 is A or E, X29 is G, X30 is G, X31 is P, X32 is S, X33 is S, X34 is G, X35 is A, X36 is P, X37 is P, X38 is P, X39 is S, The acid group of the C-terminal amino acid is either a free carboxylic acid group or amidated as a C-terminal primary amide. However, provided that at least one of X17 and X20 is K, and furthermore, at least one of the K is Aminoethoxyethoxyacetic acid-Aib-Glu-C １６ -C ２２ fatty acid chain, Aminoethoxyethoxyacetic acid-C(O)-diaminobutane-Glu-C １６ -C ２２ fatty acid chain, Glu-C １６ -C ２２ Fatty acid lock Aminoethoxyethoxyacetic acid-aminoethoxyethoxyacetic acid-Glu-C １６ -C ２２ Fatty acid chains, and Aminoethoxyethoxyacetic acid-Glu-C １６ -C ２２ The condition is that the fatty acid chain contains an acylated side-chain amino (ε-amino) group in the selected part of the formula, However, when X20 is Aib, the side chain amino group is aminoethoxyethoxyacetic acid-aminoethoxyethoxyacetic acid-Glu-C １６ -C ２２ Fatty acid chains also include aminoethoxyethoxyacetic acid-Glu-C １６ -C ２２ A polypeptide or a pharmaceutically acceptable salt thereof, provided that the fatty acid chain is not acylated.

6. 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: 3) In the array, X1 is Y, X2 is Aib, X3 is Q or N, X10 is Y, X12 is I, X13 is Aib, Ser(OMe), nor-V, nor-L, or αMe-L. X16 is K, X17 is I or K, X18 is A, X19 is Q, X20 is K, Aib, Ser(OMe), nor-V, or nor-L. X21 is A, X23 is V or I, X24 is Q or E, X25 is either W or Y, X27 is E, I, or L, X28 is A or E, X29 is G, X30 is G, X31 is P, X32 is S, X33 is S, X34 is G, X35 is A, X36 is P, X37 is P, X38 is P, X39 is S, The acid group of the C-terminal amino acid is either a free carboxylic acid group or amidated as a C-terminal primary amide. However, provided that at least one of X17 and X20 is K, and further, that at least one of K contains a side-chain amino (ε-amino) group acylated with a part of the formula selected from the following, Table 1 The polypeptide is a polypeptide other than SEQ ID NO: 7, SEQ ID NO: 23, or SEQ ID NO: 30, or a pharmaceutically acceptable salt thereof.

7. X1 is Y, X2 is Aib, X3 is Q or N, X10 is Y, X12 is I, X13 is nor-V, nor-L, or αMe-L. X16 is K, X17 is I or K, X18 is A, X19 is Q, X20 is K, Aib, nor-V, or nor-L. X21 is A, X23 is V or I, X24 is Q or E, X25 is either W or Y, X27 is I or L, X28 is A or E, X29 is G, X30 is G, X31 is P, X32 is S, X33 is S, X34 is G, X35 is A, Each of X36, X37, and X38 is independently P, X39 is S, The amino (ε-amino) group of the K at position X17 is acylated with a portion of the formula selected from the following: Table 2 The polypeptide according to claim 6, wherein the polypeptide is not Sequence ID No.

7.

8. X1 is Y, X2 is Aib, X3 is Q or N, X10 is Y, X12 is I, X13 is nor-V, nor-L, or αMe-L. X16 is K, X17 is K, X18 is A, X19 is Q, X20 is Aib, nor-V, or nor-L. X21 is A, X23 is I, X24 is E, X25 is Y, X27 is L, X28 is E, X29 is G, X30 is G, X31 is P, X32 is S, X33 is S, X34 is G, X35 is A, Each of X36, X37, and X38 is independently P, X39 is S, The amino (ε-amino) group of the K at position X17 is acylated with a portion of the formula selected from the following: Table 3 The polypeptide according to claim 6, wherein the polypeptide is not Sequence ID No.

7.

9. 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: 4) In the array, X1 is Y, X2 is Aib, X3 is Q or N, X10 is Y, X12 is I, X13 is nor-V, nor-L, or αMe-L. X16 is K, X17 is I or K, X18 is A, X19 is Q, X20 is K or Aib, X21 is A, X23 is V or I, X24 is Q or E, X25 is either W or Y, X27 is I or L, X28 is A or E, X29 is G, X30 is G, X31 is P, X32 is S, X33 is S, X34 is G, X35 is A, Each of X36, X37, and X38 is independently P, X39 is S, A polypeptide or a pharmaceutically acceptable salt thereof, provided that at least one of X17 and X20 is K, and further, that K contains a side-chain amino (ε-amino) group acylated with a portion of a formula selected from the following. Table 4

10. A polypeptide comprising the following amino acid sequence or a pharmaceutically acceptable salt thereof, Y-Aib-X3-G-T-F-T-S-D-Y-S-I-X13-L-DK-X17-A-Q-X20-A-F-X23-X24-X25-L-X27-X28-G-G-P-S-S-G-A-P-P-P-S (SEQ ID NO: 5) In the array, X3 is Q or N, X13 is nor-V, nor-L, or αMe-L. X17 is I or K, X20 is K or Aib, X23 is V or I, X24 is Q or E, X25 is either W or Y, X27 is I or L, X28 is A or E, The acid group of the C-terminal amino acid is either a free carboxylic acid group or amidated as a C-terminal primary amide. However, provided that at least one of X17 and X20 is K, and further, that K contains a side-chain amino (ε-amino) group acylated with a portion of the formula selected from the following, Table 5 The polypeptide is a polypeptide other than SEQ ID NO: 7, or a pharmaceutically acceptable salt thereof.

11. X3 is Q, X13 is αMe-L, X17 is K, X20 is AIB, X23 is I, X24 is E, X25 is Y, X27 is L, X28 is E, The polypeptide according to claim 10, wherein the amino (ε-amino) group of the side chain at the X17 position of K is acylated with a portion of the formula selected from the following. Table 6

12. X3 is Q, X13 is no-V, X17 is K, X20 is AIB, X23 is I, X24 is E, X25 is Y, X27 is L, X28 is E, The polypeptide according to claim 10, wherein the amino (ε-amino) group of the side chain at the X17 position of K is acylated with a portion of the formula selected from the following. Table 7

13. X3 is Q, X13 is no-L, X17 is K, X20 is AIB, X23 is I, X24 is E, X25 is Y, X27 is L, X28 is E, The polypeptide according to claim 10, wherein the amino (ε-amino) group of the side chain at the X17 position of K is acylated with a portion of the formula selected from the following. Table 8

14. X3 is Q, X13 is no-V, X17 is I, X20 is K, X23 is V, X24 is Q, X25 is W, X27 is I, X28 is A, The polypeptide according to claim 10, wherein the amino (ε-amino) group of the side chain at the X20 position is acylated with a portion of the formula selected from the following. Table 9

15. X3 is N, X13 is no-V, X17 is I, X20 is K, X23 is V, X24 is Q, X25 is W, X27 is I, X28 is A, The polypeptide according to claim 10, wherein the amino (ε-amino) group of the side chain at the X20 position is acylated with a portion of the formula selected from the following. Table 10

16. A polypeptide comprising the following amino acid sequence or a pharmaceutically acceptable salt thereof, Y-Aib-X3-G-T-F-T-S-D-Y-S-I-X13-L-DK-X17-A-Q-X20-A-F-I-E-Y-L-L-E-G-G-P-S-G-A-P-P-P-S (SEQ ID NO: 6) In the array, X3 is Q or N, X13 is nor-V, nor-L, or αMe-L. X17 is K, X20 is Aib, nor-L, or nor-V. The acid group of the C-terminal amino acid is either a free carboxylic acid group or amidated as a C-terminal primary amide. The amino (ε-amino) group of the K at position X17 is acylated with a portion of the formula selected from the following: Table 11 The polypeptide is a polypeptide other than SEQ ID NO: 7, or a pharmaceutically acceptable salt thereof.

17. X13 is either no-V or no-L. X20 is AIB, The polypeptide according to claim 16, wherein the amino (ε-amino) group of the side chain of K at position X17 is acylated with a portion of the formula selected from the following. Table 12

18. X13 is αMe-L, X20 is either no-L or no-V. The polypeptide according to claim 16, wherein the amino (ε-amino) group of the side chain of K at position X17 is acylated with a portion of the formula selected from the following. Table 13

19. It is an incretin analog, A lysine residue containing a fatty acid extension group bonded to the lysine ε-nitrogen, A peptide residue containing the sequence Gly-Thr-Phe-Thr-Ser-Asp (SEQ ID NO: 31), indirectly bound to the lysine residue via the carboxyl terminus, Sequence Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-CONH ２ A peptide residue having (SEQ ID NO: 32), wherein the Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-CONH ２ A peptide residue indirectly bound to the carboxyl group of the lysine via the amino terminus of the residue (SEQ ID NO: 32), An incretin analog comprising the Gly-Thr-Phe-Thr-Ser-Asp (SEQ ID NO: 31) residue and the lysine residue, and norvaline amino acid residues indirectly bound between them.

20. It is an incretin analog, A lysine residue comprising a group of formula (I) bonded to the lysine ε-nitrogen, wherein formula (I) is 【Transformation 7】 And, During the ceremony, U is either absent or -C(O)-CH ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ This represents -NH-}, where} is the connection point 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 the following: 【Transformation 8】 ] is the connection point to 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 where x is 1, 2, or 3, and -- is the connection point to Z. Z is -C(O)-(CH ２ ) ｎ -COOH or -C(O)-(CH ２ ) ｎ -CH ３ This represents a lysine residue where n is an integer between 14 and 20, A peptide residue containing the sequence Gly-Thr-Phe-Thr-Ser-Asp (SEQ ID NO: 31), indirectly bound to the lysine residue via the carboxyl terminus, Sequence Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-NH ２ A peptide residue having (SEQ ID NO: 32), wherein the Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-CONH ２ A peptide residue indirectly bound to the carboxyl group of the lysine via the amino terminus of the residue (SEQ ID NO: 32), An incretin analog comprising the Gly-Thr-Phe-Thr-Ser-Asp (SEQ ID NO: 31) residue and the lysine residue, and norvaline amino acid residues indirectly bound between them.

21. A polypeptide or incretin analog selected from the following: Table 14

22. The incretin analog according to claim 19 or 20, wherein the lysine is bound to the Gly-Thr-Phe-Thr-Ser-Asp (SEQ ID NO: 31) residue by a peptide residue containing 10 amino acids.

23. The lysine is converted by a peptide residue containing 11 amino acids into the Gly-Gly-Pro-Ser-Ser-Gly-Ala-Pro-Pro-Pro-Ser-CONH ２ An incretin analog according to claim 19 or 20, which binds to a residue (SEQ ID NO: 32).

24. A pharmaceutical composition comprising a pharmaceutically acceptable carrier and a polypeptide or incretin analog according to any one of claims 1 to 22.

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

26. A method for treating type 2 diabetes mellitus (T2DM), comprising administering to a patient in need of such treatment a polypeptide or incretin analog according to any one of claims 1 to 22.

27. A method for treating a metabolic syndrome, comprising administering to a patient in need of such treatment a polypeptide or incretin analog described in any one of claims 1 to 22.

28. A method for treating metabolic disorder-related fatty liver disease (MASLD), comprising administering a polypeptide or incretin analog described in any one of claims 1 to 22 to a patient in need of such treatment.

29. A method for treating metabolic disorder-associated steatohepatitis (MASH), comprising administering a polypeptide or incretin analog described in any one of claims 1 to 22 to a patient in need of such treatment.

30. A method for treating a neurodegenerative disorder, comprising administering to a patient in need of such treatment a polypeptide or incretin analog according to any one of claims 1 to 22.

31. A method for treating fibrosis, comprising administering to a patient in need of such treatment a polypeptide or incretin analog according to any one of claims 1 to 22.

32. A method for reducing cardiovascular risk, comprising administering a polypeptide or incretin analog described in any one of claims 1 to 22 to a patient in need of such treatment.

33. A method for treating hyperlipidemia / dyslipidemia, comprising administering a polypeptide or incretin analog described in any one of claims 1 to 22 to a patient in need of such treatment.

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