New GLP-1 analogs

Abstract

The present disclosure relates to novel glucagon-like peptide-1 (GLP-1) (7-37) analogs having an amino acid sequence with Leu or Ile at the C-terminus. The novel analogs are potent GLP-1 agonists with reduced side effects and improved duration of action. The present disclosure also relates to acylated derivatives of the novel analogs, which have further improved potency and duration of action and are suitable for oral administration. The analogs of the present disclosure can be used to treat diabetes and obesity.

Description

[0001] Related applications

[0002] This application claims the benefit of three Indian provisional applications having application numbers IN201821013109 (filed on April 5, 2018); IN 201821040468 (filed on October 26, 2018) and IN201821040474 (filed on October 26, 2018), which are incorporated herein by reference.

[0003] Areas of public content

[0004] The present disclosure relates to novel glucagon-like peptide-1 (GLP-1)(7-38) analogs having an amino acid sequence with Leu or Ile at the C-terminus. The novel analogs are potent GLP-1 agonists with reduced side effects and improved duration of action. The present disclosure also relates to acylated derivatives of the novel analogs having further improved potency and duration of action and being suitable for oral administration. The analogs disclosed herein are acylated with an extended moiety, which increases the duration of activity of the compound. The analogs disclosed herein can be used to treat diabetes and obesity.

[0005] Background of the Public Content

[0006] Glucagon-like peptide-1 (GLP-1) is a hormone that is produced primarily in the enteroendocrine L cells of the intestine and is secreted into the bloodstream when a meal containing fat, protein hydrolysates and / or glucose enters the duodenum. GLP-1 is derived from cell-specific post-translational processing of the preproglucagon gene. Initially, the peptide GLP-1(1-37) was identified from this process, but it was two N-terminally truncated products, GLP-1(7-37) (SEQ ID NO: 1) and GLP-1(7-36)amide, that were found to recognize pancreatic receptors and were identified as the active species in vivo. GLP-1 has been found to stimulate insulin secretion, thereby causing glucose uptake by cells and reduced serum glucose levels. GLP-1 agonists may be advantageous drugs for the treatment of type 2 diabetes mellitus (T2DM) as they do not cause hypoglycemia and have the positive benefit of weight loss. The endogenous substances GLP-1 (7-37) and GLP-1 (7-36) amide are cleaved by peptidases and therefore have very short half-lives. Efforts have been made to improve performance by developing GLP-1 analogs with improved half-lives. The first drug approved in 2005 was Exenatide, which was administered at a dose level of 10mcg twice a day, and was found to show significant improvements in HbA1c (a marker of glucose control). In addition, Novo Nordisk developed Liraglutide (Liraglutide) (U.S. Patent No. 6,268,343) (SEQ ID NO: 2) with a once-daily dose of 1.8mg sc / day and approved it in 2010. Further research and development have produced once-weekly products such as Albiglutide, developed by GSK, and Dulaglutide, developed by Eli Lilly. Recently, the GLP-1 analogue Semaglutide (International Publication No. WO 2006 / 097537 A2) was approved by the USFDA. Semaglutide (SEQ ID NO: 3) is marketed under the trade name It is administered as a once-weekly subcutaneous injection.

[0007] Many attempts to prepare GLP-1 analogs with improved potency and duration of action have been reported in the literature. U.S. Patent No. 7,291,594 B2 (US'594 patent) discloses GLP-1 (7-35) derivatives having several arginine and / or lysine residues added to their C-termini to provide high bioavailability through the mucosa. The US'594 patent also discloses that these derivatives can be conferred resistance to dipeptidyl peptidase IV (DPP-IV) by replacing amino acid 8 in their GLP-1 amino acid sequence with Ser, or by conferring resistance to trypsin by replacing amino acids 26 and 34 with Gln and Asn, respectively.

[0008] U.S. Patent No. 7,893,017 B2 (the US'017 patent) discloses acylated GLP-1 analogs, wherein the GLP-1 analogs are stabilized against DPP-IV by modifying at least one of the amino acid residues at positions 7 and 8 relative to the sequence GLP-1 (7-37), and wherein the acylation is directly linked to the diacid of the C-terminal amino acid residue of the GLP-1 analog.

[0009] U.S. Patent No. 8,951,959 B2 (the US'959 patent) discloses DPP-IV-resistant GLP-1(7-37) analogs having a non-proteinogenic amino acid residue containing a trifluoromethyl group in position 8 relative to the sequence of GLP-1 and acylation of the lysine residue in position 26 by a moiety comprising two acidic groups.

[0010] US Patent No. 7,084,243 B2 (US'243 patent) discloses GLP-1(7-37) analogs having Val or Gly at position 8 relative to the sequence GLP-1(7-37) as DPP-IV resistant peptides.

[0011] International Publication No. WO 2017 / 149070 A1 (WO'070) discloses GLP-1 analogs having Trp at the position corresponding to position 8 of GLP-1(7-37), and these Trp8 compounds were shown to be very stable against degradation by DPP-IV.

[0012] International Publication No. WO 2004 / 103390 A2 (WO '390) discloses that modifications at the P'1 position (corresponding to position 9 in the case of GLP-1(7-37)) can produce GLP-1 analogs that have greatly reduced susceptibility to enzyme-mediated cleavage (e.g., DPP-IV) relative to the natural substrate, yet retain the biological activity of the natural substrate. WO '390 also discloses GLP-1(7-37) analogs having amino acids with tetrasubstituted Cβ carbons at position 9 (e.g., tert-leucine), providing GLP analogs that are resistant to degradation by DPP-IV.

[0013] International Publication No. WO 2015 / 086686 A2 (the WO '686 publication) discloses the incorporation of α-methyl functionalized amino acids directly into the backbone of GLP-1 analogs, which has been determined to produce protease-resistant (including DPP-IV-resistant) peptides.

[0014] A variety of other DPP-IV-resistant GLP-1 agonists are disclosed in patent publications, such as International Publication Nos. WO 2007 / 030519 A2, WO 2004 / 078777 A2, WO 2007 / 039140 A1, WO 2014 / 209886 A1, WO 2012 / 016419 A1, WO 2017 / 211922 A2, WO 2016 / 198544 A1, and WO 2013 / 051938 A2.

[0015] Various patent applications disclose C-terminally extended GLP-1 analogs with increased stability and longer duration of action, for example, U.S. Patent Nos. 7,482,321 B2, 9,498,534 B2, and 7,897,566 B2.

[0016] Various patent applications disclose acylated GLP-1 analogs, wherein the GLP-1 analog is optionally linked to a lipophilic substituent via a linker to provide a longer duration of action.

[0017] US Patent No. 8,603,972 B2 (US'972) discloses monoacylated derivatives of GLP-1 analogs, wherein the Lys residue at position 37 or 38 of the GLP-1 analog is acylated.

[0018] U.S. Patent Nos. 8,648,041 B2, 9,758,560 B2, 9,006,178 B2, 9,266,940 B2, 9,708,383 B2 and U.S. Patent Application Publication Nos. US 2015 / 0152157 A1 and US 2015 / 0133374 A1 disclose diacylated derivatives of GLP-1 analogs.

[0019] U.S. Patent Application Publication No. US 2016 / 0200791 A1 discloses triacylated derivatives of GLP-1 analogs.

[0020] International Publication Nos. WO 2016 / 083499 A1, WO 2016 / 097108 A1, and WO 2014 / 202727 A1 disclose acylated GLP-1 analogs, wherein a Lys residue of the GLP-1 analog is linked to two prolonging moieties via a branched linker.

[0021] International Publication Nos. WO 2009 / 030771 A1 and WO 2018 / 083335 A1 disclose various acylating agents (side chains) that can be attached to the Lys residue of GLP-1 analogs to provide a longer duration of action.

[0022] International Publication No. WO 2013 / 186240 A2 discloses exendin-4 peptide analogs having Gly, Ser, or functionalized Ser, such as Ser(OCH3), D-Ser, or functionalized D-Ser, such as D-Ser(OCH3), Aib, Ala, or D-Ala, at position 2 of the exendin-4 amino acid sequence.

[0023] A variety of other GLP-1 analogs are disclosed in patent applications, such as International Publication Nos. WO 2005 / 027978 A2, WO 1998 / 008871 A1, WO 1999 / 043705 A1, WO 1999 / 043706 A1, WO 1999 / 043707 A1, WO 1999 / 043708 A1, WO 2000 / 034331 A2, WO 2009 / 030771 A1, WO 2011 / 080103 A1, WO 2012 / 140117 A1, WO 2012 / 062803 A1, WO 2012 / 062804 A1, WO 2013 / 037690 A1, WO 2014 / 202727 A1, WO 2015 / 000942 A1, WO 2015 / 022400 A1, WO 2016 / 083499 A1, WO 2016 / 097108 A1 and WO 2017 / 149070 A1.

[0024] There remains a need to develop GLP-1 analogues with optimally desired properties in terms of stability and duration of action.

[0025] Overview of public content

[0026] One aspect of the present disclosure provides a polypeptide comprising the following amino acid sequence:

[0027] H-X2-X3-X4-GTFTSDVSSYL-X16-GQAA-X21-EF-X24-AWLVRGRG-X33-X34

[0028] wherein X2 is Ser, Ser(OMe), D-Ser, D-Ser(OMe), Ala or Aib;

[0029] X3 does not exist or is Gln;

[0030] X4 is Glu;

[0031] X16 is Glu;

[0032] X24 is Ile;

[0033] X33 is Leu, D-Leu, D-Ile, or Ile;

[0034] X34 does not exist, and

[0035] X21 is Lys, wherein the side chain amino group (ε amino group) of Lys is partially acylated as follows:

[0036] {-QTUWYZ

[0037] Where Q and T do not exist;

[0038] U is absent or is -C(O)-CH2-O-(CH2)2-O-(CH2)2-NH-}, where} is the point of attachment to the group W;

[0039] W is absent or selected from -C(O)-CH2-O-(CH2)2-O-(CH2)2-NH-], -C(O)-NH-(CH2) 3-4 -NH-], -C(O)-C(CH3)2-NH-] and wherein] is the point of attachment to group Y;

[0040] Y is -C(O)-(CH2)2-CH(COOH)NH--, where -- is the point of attachment to group Z;

[0041] Z is -C(O)-(CH2) n -COOH or -C(O)-(CH2) n -CH3, wherein n is an integer from 14 to 20.

[0042] The polypeptides of the present disclosure are effective GLP-1 agonists with fewer side effects. In addition, the polypeptides of the present disclosure are stable, have a long duration of action and are suitable for oral administration.

[0043] Description of the drawings

[0044] Figure 1A The preparation of Part A-Osu (Intermediate 3) is shown. Figure 1B The preparation of part A-OSu (Intermediate 3) is shown.

[0045] Figure 2 The preparation of part of C-OSu is shown.

[0046] Figure 3 The preparation of a portion of D-OSu is shown.

[0047] Figure 4 The preparation of a portion of E-OSu is shown.

[0048] Figure 5 Part of the preparation of F-OSu is shown.

[0049] Figure 6A and 6B show the results of oral glucose tolerance test (OGTT) of Compound 1 in rats; single injection; 1 mg / kg glucose AUC 0-120 min ( Figure 6A =22 hours later, Figure 6B = after 46 hours).

[0050] Figure 7 It is shown that blood glucose levels are reduced in db / db type 2 diabetic mice after long-term treatment with compound 1.

[0051] Figure 8 Shown is a decrease in food intake in db / db mice following treatment with Compound 1.

[0052] Figure 9 The efficacy of Compound 1 in reducing body weight in db / db mice is shown.

[0053] Figure 10 Shown is a decrease in Hb1Ac in db / db mice following treatment with Compound 1.

[0054] abbreviation

[0055] Aib: 2-aminoisobutyric acid

[0056] ADO: 8-amino-3,6-dioxo-octanoic acid

[0057] OGTT: Oral Glucose Tolerance Test

[0058] DIPEA: N,N'-diisopropylethylamine

[0059] HOBt: 1-hydroxybenzotriazole

[0060] DIPC: N,N'-diisopropylcarbodiimide

[0061] HOSu: N-hydroxysuccinimide

[0062] IBCF: Isobutyl Chloroformate

[0063] NMM: N-methylmorpholine

[0064] THF: Tetrahydrofuran

[0065] DCM: dichloromethane

[0066] DMAP: 4-dimethylaminopyridine

[0067] DCC: dicyclohexylcarbodiimide

[0068] DMAc: dimethylacetamide

[0069] Description of public content

[0070] The present disclosure provides stable, long-acting GLP-1 analogs that do not require frequent subcutaneous administration and are also suitable for oral administration. Surprisingly, it was found that the addition of an additional Leu at the C-terminus of the sequence produced peptides with significantly improved potency and duration of action when compared to the parent peptide. Peptides with additional Ile also showed similar effects of improved potency and duration of action when compared to the parent peptide. In addition, the disclosure herein demonstrates that it is possible to attach to a portion of a peptide that is an analog of GLP-1 (7-37) via an acylation reaction to produce compounds with significantly improved potency and longer duration of action. The extended portion of the disclosed compound has a more stable bond that is less sensitive to cleavage by biological enzymes. Therefore, the compounds disclosed herein are more stable and require less frequent administration, increasing patient compliance. Therefore, in some embodiments, the present disclosure provides a polypeptide comprising the following amino acid sequence:

[0071] H-X2-X3-X4-GTFTSDVSSYL-X16-GQAA-X21-EF-X24-AWLVRGRG-X33-X34(SEQ ID NO:4)

[0072] wherein X2 is Ser, Ser(OMe), D-Ser, D-Ser(OMe), Ala or Aib;

[0073] X3 does not exist or is Gln;

[0074] X4 is Glu;

[0075] X16 is Glu;

[0076] X24 is Ile;

[0077] X33 is Leu, D-Leu, D-Ile, or Ile;

[0078] X34 does not exist, and

[0079] X21 is Lys, wherein the side chain amino group (ε amino group) of Lys is acylated.

[0080] In some embodiments, X21 can be acylated with the extending moieties reported in U.S. Patent Nos. 6,268,343, 8,951,959 B2, 8,603,972 B2, 8,648,041 B2, 9,758,560 B2, 9,006,178 B2, 9,266,940 B2, 9,708,383 B2, and U.S. Patent Application Publication Nos. US 2015 / 0152157 A1 and US 2015 / 0133374 A1; International Publication Nos. WO 2009 / 030771 A1, WO 2006 / 097537 A2, and WO 2018 / 083335 A1.

[0081] In some embodiments, X21 Lys is acylated at its side chain amino group (ε amino group) by a moiety comprising a fatty acid group. The fatty acid group can be linked to X21 Lys via a linker. Thus, in some embodiments, the present disclosure provides a polypeptide comprising the following amino acid sequence:

[0082] H-X2-X3-X4-GTFTSDVSSYL-X16-GQAA-X21-EF-X24-AWLVRGRG-X33-X34

[0083] wherein X2 is Ser, Ser(OMe), D-Ser, D-Ser(OMe), Ala or Aib;

[0084] X3 does not exist or is Gln;

[0085] X4 is Glu;

[0086] X16 is Glu;

[0087] X24 is Ile;

[0088] X33 is Leu, D-Leu, D-Ile, or Ile;

[0089] X34 does not exist, and

[0090] X21 is Lys, wherein the side chain amino group (ε amino group) of Lys is partially acylated as follows:

[0091] {-QTUWYZ

[0092] Where Q and T do not exist;

[0093] U is absent or is -C(O)-CH2-O-(CH2)2-O-(CH2)2-NH-}, where} is the point of attachment to the group W;

[0094] W is absent or selected from -C(O)-CH2-O-(CH2)2-O-(CH2)2-NH-], -C(O)-NH-(CH2) 3-4 -NH-], -C(O)-C(CH3)2-NH-] and wherein] is the point of attachment to group Y;

[0095] Y is -C(O)-(CH2)2-CH(COOH)NH--, where -- is the point of attachment to group Z;

[0096] Z is -C(O)-(CH2) n -COOH or -C(O)-(CH2) n -CH3, wherein n is an integer from 14 to 20.

[0097] In some embodiments, the amino acid at X2 is selected from Ser, Ser(OMe), D-Ser, D-Ser(OMe), Ala, or Aib.

[0098] In some embodiments, X2 is Aib.

[0099] In some embodiments, X3 is absent.

[0100] In some embodiments, X33 is Leu.

[0101] In some embodiments, X33 is He.

[0102] In some embodiments, X21 is Lys, wherein the side chain amino group (epsilon amino group) of Lys is acylated with the following moiety:

[0103] {-QTUWYZ,

[0104] Wherein W is selected from -C(O)-NH-(CH2) 3-4 -NH-], -C(O)-C(CH3)2-NH-] and

[0105] In some embodiments, X21 is Lys, wherein the side chain amino group (epsilon amino group) of Lys is acylated with the following moiety:

[0106] {-QTUWYZ,

[0107] Wherein U and W are absent, and Z is -C(O)-(CH2) n -CH3, wherein n is an integer of 14.

[0108] In some embodiments, X21 is Lys, wherein the side chain amino group (epsilon amino group) of Lys is acylated with the following moiety:

[0109] {-QTUWYZ,

[0110] wherein W is -C(O)-CH2-O-(CH2)2-O-(CH2)2-NH-].

[0111] In some embodiments, X21 is Lys, wherein the side chain amino group (epsilon amino group) of Lys is acylated with the following moiety:

[0112] {-QTUWYZ,

[0113] wherein W is -C(O)-C(CH3)2-NH-].

[0114] In some embodiments, X21 is Lys, wherein the side chain amino group (epsilon amino group) of Lys is acylated with the following moiety:

[0115] {-QTUWYZ,

[0116] wherein W is -C(O)-NH-(CH2)4-NH-].

[0117] In some embodiments, X21 is Lys, wherein the side chain amino group (epsilon amino group) of Lys is acylated with the following moiety:

[0118] {-QTUWYZ,

[0119] wherein W is -C(O)-NH-(CH2)3-NH-].

[0120] In some embodiments, X21 is Lys, wherein the side chain amino group (epsilon amino group) of Lys is acylated with the following moiety:

[0121] {-QTUWYZ,

[0122] Where W is

[0123] In some embodiments, X21 is Lys, wherein the side chain amino group (epsilon amino group) of Lys is acylated with the following moiety:

[0124] {-QTUWYZ,

[0125] Where Z is -C(O)-(CH2) n -COOH, wherein n is the integer 16.

[0126] In some embodiments, X21 is Lys, wherein the side chain amino group (epsilon amino group) of Lys is acylated with the following moiety:

[0127] {-QTUWYZ,

[0128] Where Z is -C(O)-(CH2) n -CH3, wherein n is an integer of 14.

[0129] In some embodiments, X2 is Ala or Aib;

[0130] X3 does not exist;

[0131] X33 is Leu;

[0132] U does not exist;

[0133] W does not exist;

[0134] Y is -C(O)-(CH2)2-CH(COOH)NH--, where -- is the point of attachment to group Z;

[0135] Z is -C(O)-(CH2) n -CH3, wherein n is an integer of 14.

[0136] In some embodiments, the present disclosure provides a polypeptide comprising the following amino acid sequence:

[0137] H-X2-X3-X4-GTFTSDVSSYL-X16-GQAA-X21-EF-X24-AWLVRGRG-X33-X34

[0138] Where X2 is Aib;

[0139] X3 does not exist;

[0140] X4 is Glu;

[0141] X16 is Glu;

[0142] X24 is Ile;

[0143] X33 is Leu;

[0144] X34 does not exist, and

[0145] X21 is Lys, wherein the side chain amino group (ε amino group) of Lys is partially acylated as follows:

[0146] {-QTUWYZ

[0147] Where Q and T do not exist;

[0148] U is -C(O)-CH2-O-(CH2)2-O-(CH2)2-NH-}, where} is the point of attachment to the group W;

[0149] W is -C(O)-CH2-O-(CH2)2-O-(CH2)2-NH-], wherein] is the point of attachment of the group Y;

[0150] Y is -C(O)-(CH2)2-CH(COOH)NH--, where -- is the point of attachment to group Z;

[0151] Z is -C(O)-(CH2) n -COOH, wherein n is the integer 16.

[0152] In some embodiments, the present disclosure provides a polypeptide comprising the following amino acid sequence:

[0153] H-X2-X3-X4-GTFTSDVSSYL-X16-GQAA-X21-EF-X24-AWLVRGRG-X33-X34

[0154] Where X2 is Aib;

[0155] X3 does not exist;

[0156] X4 is Glu;

[0157] X16 is Glu;

[0158] X24 is Ile;

[0159] X33 is Leu;

[0160] X34 does not exist, and

[0161] X21 is Lys, wherein the side chain amino group (ε amino group) of Lys is partially acylated as follows:

[0162] {-QTUWYZ

[0163] Where Q and T do not exist;

[0164] U is -C(O)-CH2-O-(CH2)2-O-(CH2)2-NH-}, where} is the point of attachment to the group W;

[0165] W is -C(O)-C(CH3)2-NH-], wherein] is the point of attachment to group Y;

[0166] Y is -C(O)-(CH2)2-CH(COOH)NH--, where -- is the point of attachment to group Z;

[0167] Z is -C(O)-(CH2) n -COOH, wherein n is the integer 16.

[0168] In some embodiments, the present disclosure provides a polypeptide comprising the following amino acid sequence:

[0169] H-X2-X3-X4-GTFTSDVSSYL-X16-GQAA-X21-EF-X24-AWLVRGRG-X33-X34

[0170] Where X2 is Aib;

[0171] X3 does not exist;

[0172] X4 is Glu;

[0173] X16 is Glu;

[0174] X24 is Ile;

[0175] X33 is Leu;

[0176] X34 does not exist, and

[0177] X21 is Lys, wherein the side chain amino group (ε amino group) of Lys is partially acylated as follows:

[0178] {-QTUWYZ

[0179] Where Q and T do not exist;

[0180] U is -C(O)-CH2-O-(CH2)2-O-(CH2)2-NH-}, where} is the point of attachment to the group W;

[0181] W is -C(O)-NH-(CH2) 3-4 -NH-], wherein] is the point of attachment to the group Y;

[0182] Y is -C(O)-(CH2)2-CH(COOH)NH--, where -- is the point of attachment to group Z;

[0183] Z is -C(O)-(CH2) n -COOH, wherein n is the integer 16.

[0184] In some embodiments, X21 is Lys, wherein the side chain amino group (epsilon amino group) of Lys is acylated with the following moiety:

[0185] {-QTUWYZ,

[0186] wherein W is -C(O)-NH-(CH2)4-NH-].

[0187] In some embodiments, X21 is Lys, wherein the side chain amino group (epsilon amino group) of Lys is acylated with the following moiety:

[0188] {-QTUWYZ,

[0189] wherein W is -C(O)-NH-(CH2)3-NH-].

[0190] In some embodiments, the present disclosure provides a polypeptide comprising the following amino acid sequence:

[0191] H-X2-X3-X4-GTFTSDVSSYL-X16-GQAA-X21-EF-X24-AWLVRGRG-X33-X34

[0192] Where X2 is Aib;

[0193] X3 does not exist;

[0194] X4 is Glu;

[0195] X16 is Glu;

[0196] X24 is Ile;

[0197] X33 is Leu;

[0198] X34 does not exist, and

[0199] X21 is Lys, wherein the side chain amino group (ε amino group) of Lys is partially acylated as follows:

[0200] {-QTUWYZ

[0201] Where Q and T do not exist;

[0202] U is -C(O)-CH2-O-(CH2)2-O-(CH2)2-NH-}, where} is the point of attachment to the group W;

[0203] W is -C(O)-NH-(CH2)4-NH-], wherein] is the point of attachment of the group Y;

[0204] Y is -C(O)-(CH2)2-CH(COOH)NH--, where -- is the point of attachment to group Z;

[0205] Z is -C(O)-(CH2) n -COOH or -C(O)-(CH2) n -CH3, wherein n is an integer from 14 to 20.

[0206] In some embodiments, X21 is Lys, wherein the side chain amino group (epsilon amino group) of Lys is acylated with the following moiety:

[0207] {-QTUWYZ,

[0208] Where Z is -C(O)-(CH2) n -COOH, wherein n is the integer 16.

[0209] In some embodiments, X21 is Lys, wherein the side chain amino group (epsilon amino group) of Lys is acylated with the following moiety:

[0210] {-QTUWYZ,

[0211] Where Z is -C(O)-(CH2) n -CH3, wherein n is an integer of 14.

[0212] In some embodiments, the present disclosure provides a polypeptide comprising the following amino acid sequence:

[0213] H-X2-X3-X4-GTFTSDVSSYL-X16-GQAA-X21-EF-X24-AWLVRGRG-X33-X34

[0214] Where X2 is Aib;

[0215] X3 does not exist;

[0216] X4 is Glu;

[0217] X16 is Glu;

[0218] X24 is Ile;

[0219] X33 is Leu;

[0220] X34 does not exist, and

[0221] X21 is Lys, wherein the side chain amino group (ε amino group) of Lys is partially acylated as follows:

[0222] {-QTUWYZ

[0223] Where Q and T do not exist;

[0224] U is -C(O)-CH2-O-(CH2)2-O-(CH2)2-NH-}, where} is the point of attachment to the group W;

[0225] W is wherein] is the point of attachment to group Y;

[0226] Y is -C(O)-(CH2)2-CH(COOH)NH--, where -- is the point of attachment to group Z; and

[0227] Z is -C(O)-(CH2) n -COOH, wherein n is the integer 16.

[0228] In some embodiments, the present disclosure provides a polypeptide comprising the following amino acid sequence:

[0229] H-X2-X3-X4-GTFTSDVSSYL-X16-GQAA-X21-EF-X24-AWLVRGRG-X33-X34

[0230] wherein X2 is Ser, Ser(OMe), D-Ser, or D-Ser(OMe);

[0231] X3 does not exist;

[0232] X4 is Glu;

[0233] X16 is Glu;

[0234] X24 is Ile;

[0235] X33 is Leu;

[0236] X34 does not exist, and

[0237] X21 is Lys, wherein the side chain amino group (ε amino group) of Lys is partially acylated as follows:

[0238] {-QTUWYZ

[0239] Where Q and T do not exist;

[0240] U is -C(O)-CH2-O-(CH2)2-O-(CH2)2-NH-}, where} is the point of attachment to the group W;

[0241] 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-], wherein] is the point of attachment to the group Y;

[0242] Y is -C(O)-(CH2)2-CH(COOH)NH--, where -- is the point of attachment to group Z; and

[0243] Z is -C(O)-(CH2) n -COOH or -C(O)-(CH2) n -CH3, wherein n is an integer from 14 to 20.

[0244] In some embodiments, X21 is Lys, wherein the side chain amino group (epsilon amino group) of Lys is acylated with the following moiety:

[0245] {-QTUWYZ,

[0246] Where W is -C(O)-NH-(CH2) 3-4 -NH-].

[0247] In some embodiments, X21 is Lys, wherein the side chain amino group (epsilon amino group) of Lys is acylated with the following moiety:

[0248] {-QTUWYZ,

[0249] wherein W is -C(O)-CH2-O-(CH2)2-O-(CH2)2-NH-;

[0250] In some embodiments, X21 is Lys, wherein the side chain amino group (epsilon amino group) of Lys is acylated with the following moiety:

[0251] {-QTUWYZ,

[0252] wherein W is -C(O)-C(CH3)2-NH-].

[0253] In some embodiments, X21 is a lipid-modified Lys, wherein the side chain amino group (ε amino group) of Lys is acylated by

[0254] {-QTUWYZ

[0255] This is represented by the fractions provided in Table 1.

[0256] Table 1: Representative moieties of the group {-QTUWYZ

[0257]

[0258]

[0259] In another embodiment, the present disclosure provides a polypeptide according to any one of the preceding embodiments, which is selected from the peptides provided in Table 2:

[0260] Table 2: Representative polypeptide compounds of the present disclosure

[0261]

[0262]

[0263]

[0264] *Unless otherwise noted, all amino acids in the structure have the L-configuration at the α-position. D, when used as a prefix for an amino acid in a sequence, indicates the D-configuration of the amino acid. For example, (DSer) indicates that the serine amino acid in the sequence has the D-configuration.

[0265] Unless otherwise indicated, the present disclosure is intended to encompass both L and D isomers of the amino acids in the sequence.

[0266] As described in the disclosure herein, Ser(OMe) is the amino acid serine which is hydroxymethylated and has the structure

[0267]

[0268] Polypeptide sequences referred to in this disclosure are represented by the single-letter code for amino acids as approved by IUPAC.

[0269] As used herein, Q, T, U, W, Y, and Z, which are used to define acylated moieties according to embodiments of the present disclosure, are distinct from the single letter codes used to designate amino acids of polypeptide sequences.

[0270] When oral glucose tolerance test (OGTT) is carried out in SD rats, the polypeptide of the present disclosure surprisingly shows significant reduction of blood glucose.When challenged with oral glucose, the percentage reduction of blood glucose in SD rats is significantly lower than the corresponding polypeptide lacking additional Leu or Ile at position X33.

[0271] The present invention is further described in detail with reference to the following examples.It is intended that the examples be considered illustrative in all aspects and not intended to limit the scope of the present invention. Example:

[0272] General preparation method:

[0273] The polypeptide compounds of the present disclosure can be prepared by the method described herein below. The method involves two steps, involving the preparation of a parent linear peptide and the subsequent attachment of a fatty acid chain to the parent peptide.

[0274] The peptides described herein can be prepared by chemical synthesis using solid-phase techniques, such as those described in G. Barany and RB Merrifield, "The Peptides: Analysis, Synthesis, Biology"; Vol. 2, "Special Methods in Peptide Synthesis, Part A", pp. 3-284, E. Gross and J. Meienhofer, eds., Academic Press, New York, 1980; and JM Stewart and JD Young, "Solid-Phase Peptide Synthesis", 2nd ed., Pierce Chemical Co., Rockford, 111., 1984. The desired strategy is based on the Fmoc (9-fluorenylmethyl-oxycarbonyl) group for temporary protection of the α-amino group in combination with protecting groups for temporary protection of amino acid side chains, such as tert-butyl (-tBu), tert-butoxycarbonyl (-Boc), trityl (-Trt) (see, e.g., E. Atherton and RC Sheppard, "The Fluorenylmethoxycarbonyl Amino Protecting Group", in "The Peptides: Analysis, Synthesis, Biology"; Vol. 9 - "Special Methods in Peptide Synthesis, Part C", pp. 1-38, S. Undenfriend and J. Meienhofer, eds., Academic Press, San Diego, 1987).

[0275] Peptides can be synthesized in a stepwise manner starting from the C-terminus of the peptide on an insoluble polymer support (also referred to as a "resin"). Synthesis begins by attaching the C-terminal amino acid of the peptide to the resin via the formation of an amide or ester linkage. This allows the resulting peptide to be ultimately released as a C-terminal amide or carboxylic acid, respectively.

[0276] The C-terminal amino acid and all other amino acids used in the synthesis are required to have differentially protected α-amino groups and side chain functional groups (if present) so that the α-amino protecting groups can be selectively removed during the synthesis. The coupling of the amino acid is carried out by activation of its carboxyl group to an active ester and its reaction with the unblocked α-amino group of the N-terminal amino acid attached to the resin. The sequence of α-amino deprotection and coupling is repeated until the entire peptide sequence is assembled. The peptide is then released from the resin, usually in the presence of an appropriate scavenger to limit side reactions, with concomitant deprotection of the side chain functional groups. The resulting peptide is finally purified by reverse phase HPLC.

[0277] The parent peptide can then be coupled to the fatty acid chain by coupling the activated fatty acid chain to the parent peptide. The fatty acid chain can be prepared by methods well known in organic chemistry. For example, a solid phase synthesis method capable of preparing linear fatty acid chains can be used to prepare the fatty acid chain.

[0278] The synthesized linear peptide was purified by preparative HPLC method as outlined below:

[0279] Preparative HPLC: WATERS 2555 Quaternary Gradient Module (maximum total flow: 300 mL / min, maximum pressure: 3000 psi) or

[0280] Shimadzu LC-8A (maximum total flow rate: 150mL; maximum pressure: 20Mpa)

[0281] Column: C18, 10μ

[0282] Flow rate: 75mL / min

[0283] Mobile phase: For the first purification

[0284] Mobile phase A: pH 7.5 phosphate buffer

[0285] Mobile phase B: acetonitrile

[0286] Gradient: 10% to 40% mobile phase-B in 300 min.

[0287] For the second purification:

[0288] Mobile phase A: 1% acetic acid in water

[0289] Mobile phase B: 1% acetic acid in acetonitrile: n-propanol (50:50)

[0290] Gradient: 15% to 45% mobile phase-B in 300 min

[0291] The final compounds of the present disclosure were purified by preparative HPLC methods as outlined below:

[0292] Preparative HPLC: WATERS 2555 Quaternary Gradient Module (maximum total flow: 300 mL / min, maximum pressure: 3000 psi) or

[0293] Shimadzu LC-8A (maximum total flow rate: 150mL; maximum pressure: 20Mpa)

[0294] Column: C18, 10μ

[0295] Flow rate: 75mL / min

[0296] Mobile phase:

[0297] For the first purification For the second purification Mobile phase A pH 7.5 phosphate buffer 1% acetic acid in water Mobile phase B Acetonitrile 1% acetic acid in acetonitrile:n-propanol (50:50) gradient 10% to 40% mobile phase-B in 300 minutes 15% to 45% mobile phase-B in 300 minutes

[0298] The purity of the compounds of the present disclosure was analyzed by RP-HPLC method as outlined below:

[0299] HPLC Method B1:

[0300] Column: YMC Pack-Ph (4.6mm×150mm 3μ)

[0301] Eluent: Mobile phase A: 0.1% trifluoroacetic acid in water

[0302] Mobile phase B: 0.1% trifluoroacetic acid in acetonitrile

[0303] Flow rate: 1.5 mL / min

[0304] Detection: UV detection at 210 nm

[0305] Column temperature: 50°C

[0306] Running time: 50 minutes.

[0307] gradient:

[0308] time Mobile phase A% Mobile phase B% 0.01 90 10 35.0 20 80 40.0 20 80 41.0 90 10 50.0 90 10

[0309] HPLC Method B2:

[0310] Column: YMC-Pack Pro C18 (4mm×250mm, 3μ)

[0311] Eluent: Mobile phase A: buffer: acetonitrile (900:100)

[0312] Mobile phase B: buffer: acetonitrile (300:700)

[0313] Buffer: Potassium dihydrogen orthophosphate in water, pH adjusted to 3.0 ± 0.1 with orthophosphoric acid

[0314] Flow rate: 1.0 mL / min

[0315] Detection: UV detection at 210 nm

[0316] Column temperature: 50°C

[0317] Sample tray temperature: 8°C

[0318] Running time: 38 minutes.

[0319] time Mobile phase A% Mobile phase B% 0 100 0 5 100 0 30 0 100 32 0 100 32.1 100 0 38 100 0

[0320] HPLC Method B3:

[0321] Column: Waters X-Select CSH-C18 (150mm×4.6mm; 2.5μ)

[0322] Eluent: Mobile phase A: buffer: acetonitrile (900:100)

[0323] Mobile phase B: buffer: acetonitrile (300:700)

[0324] Buffer: Potassium dihydrogen orthophosphate in water, pH adjusted to 1.5 ± 0.1 with orthophosphoric acid

[0325] Flow rate: 0.9 mL / min

[0326] Detection: UV detection at 210 nm

[0327] Column temperature: 40°C

[0328] Sample tray temperature: 5°C

[0329] Running time: 100 minutes.

[0330]

[0331]

[0332] Compounds of the present disclosure were analyzed by LCMS as outlined below:

[0333] Using Waters Waters Micromass Quattro Micro API or Thermoscientific LCQ Fleet TM, mass spectra were recorded on an LCMS. Test solutions were prepared by dissolving the appropriate amount of analyte in a diluent to a final concentration of 1 μg / mL to 50 μg / mL, depending on the ionization of the analyte. The test solution was infused into the LCMS at a rate of approximately 10 μL to 50 μL / min for 1 minute, and mass spectra were recorded in electrospray ionization (ESI) positive or negative mode and within the appropriate mass range.

[0334] Example 1: Preparation of activated fatty acid side chains:

[0335] 1. Preparation of 18-[[(1S)-1-carboxy-4-[2-[2-[2-[2-[2-[2-(2,5-dioxopyrrolidin-1-yl)oxy-2-oxo-ethoxy]ethoxy]ethylamino]-2-oxo-ethoxy]ethoxy]ethylamino]-4-oxo-butyl]amino]-18-oxo-octadecanoic acid (Part A-OSu, Intermediate 3)

[0336] like Figure 1ASchematically represented in , the activated fatty acid side chain portion A-OSu is prepared by solid phase synthesis using 2-chlorotrityl chloride resin. In the presence of N, N'-diisopropylethylamine (DIPEA), 2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid (intermediate 1) is connected to 2-chlorotrityl chloride resin, which produces 2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid-2-Cl-Trt-resin. Intermediate 1 can be prepared by coupling 2-[2-(2-aminoethoxy)ethoxy]acetic acid with Fmoc N-hydroxysuccinimide ester. Alternatively, intermediate 1 is commercially available and can be obtained as such. The Fmoc protecting group was removed by selectively deblocking the amino group of 2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid-2-Cl-Trt-resin using piperidine, and then the free amino group was coupled to 2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid using 1-hydroxybenzotriazole (HOBt) and N,N'-diisopropylcarbodiimide (DIPC), which produced 2-[2-[2-[[2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetyl]amino]ethoxy]ethoxy]acetic acid-2-Cl-Trt-resin. The Fmoc group was then removed by selectively deblocking the amino group of 2-[2-[2-[[2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetyl]amino]ethoxy]ethoxy]acetic acid-2-Cl-Trt-resin using piperidine, and the free amino group was then coupled to Fmoc-Glu-OtBu using HOBt and DIPC to obtain 2-[2-[2-[[2-[2-[[(4S)-4-Fmoc-amino-5-tert-butoxy-5-oxo-pentanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetic acid-2-Cl-Trt-resin. The resulting 2-[2-[2-[[2-[2-[2-[[(4S)-4-Fmoc-amino-5-tert-butoxy-5-oxo-pentanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetic acid-2-Cl-Trt-resin was selectively deblocked using piperidine and then coupled with mono-tert-butyl octadecanoate to afford intermediate 2, i.e., [2-[2-[[2-[2-[2-[[(4S)-5-tert-butoxy-4-[(18-tert-butoxy-18-oxo-octadecanoyl)amino]-5-oxo-pentanoyl]amino]ethoxy]ethoxy]acetyl]amino]ethoxy]ethoxy]acetic acid]-2-Cl-Trt-resin. Intermediate 2 was then cleaved from the 2-Cl-Trt-resin using trifluoroethanol:DCM (1:1).The resulting compound is then reacted with N-hydroxysuccinimide (HOSu) in the presence of isobutyl chloroformate (IBCF) and N-methylmorpholine (NMM), followed by deprotection with trifluoroacetic acid to produce the title compound (part A-OSu, intermediate 3). The entire process can also be as follows. Figure 1B Schematically represented in the depiction.

[0337] 2. Preparation of N-palmitoyl-L-γ-glutamyl succinimide ester (Part B-OSu)

[0338]

[0339] L-glutamic acid α-tert-butyl ester (H-Glu-OtBu) was reacted with palmitic acid in the presence of IBCF and NMM to produce CH3-(CH2) 14 -C(O)-Glu-OtBu, which is then reacted with HOSu in the presence of IBCF and NMM to produce CH3-(CH2) 14 -C(O)-Glu(OSu)-OtBu, which is then deprotected with trifluoroacetic acid to yield moiety B-OSu.

[0340] 3. Preparation of 18-[[(1S)-1-carboxy-4-[4-[2-[2-[2-(2,5-dioxopyrrolidin-1-yl)oxy-2-oxo-ethoxy]ethoxy]ethylcarbamoylamino]butylamino]-4-oxo-butyl]amino]-18-oxo-octadecanoic acid (part C-OSu)

[0341] like Figure 2Schematically represented in, using 2-chlorotrityl chloride resin, solid phase synthesis is used to prepare the activated fatty acid side chain portion C-OSu. In the presence of DIPEA, 2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid is connected to 2-chlorotrityl chloride resin to produce 2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid-2-Cl-Trt-resin. The Fmoc protecting group is removed by selectively blocking the amino group using piperidine, and the free amino group is then activated using p-nitrophenyl chloroformate in THF and DIPEA, followed by reaction with Fmoc-aminobutylamine hydrochloride in THF:DMAc and DIPEA, which produces 2-[2-[2-(4-Fmoc-aminobutylcarbamoylamino)ethoxy]ethoxy]acetic acid-2-Cl-Trt-resin. The Fmoc group was removed by selective deblocking with piperidine, and the free amino group was then coupled to Fmoc-Glu-OtBu using HOBt and DIPC, which yielded 2-[2-[2-[4-[[(4S)-4-Fmoc-amino-5-tert-butoxy-5-oxo-pentanoyl]amino]butylcarbamoylamino]ethoxy]ethoxy]acetic acid-2-Cl-Trt-resin. The resulting 2-[2-[2-[4-[[(4S)-4-Fmoc-amino-5-tert-butoxy-5-oxo-pentanoyl]amino]-butylcarbamoylamino]ethoxy]ethoxy]acetic acid-2-Cl-Trt-resin was selectively deblocked using piperidine and then coupled with mono-tert-butyl octadecandioate to give 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-Cl-Trt-resin. The intermediate was then cleaved from the 2-Cl-Trt-resin using trifluoroethanol:DCM (1:1) to afford 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 (LCMS=m / z: 814.56 (M+H) + )). The resulting compound is then reacted with HOSu in the presence of dicyclohexylcarbodiimide (DCC) to produce a succinimide protected intermediate, which is deprotected with trifluoroacetic acid to produce the title compound (partial C-OSu).

[0342] 4. Preparation of 18-[[(1S)-1-carboxy-4-[[2-[2-[2-[2-(2,5-dioxopyrrolidin-1-yl)oxy-2-oxo-ethoxy]ethoxy]ethylamino]-1,1-dimethyl-2-oxo-ethyl]amino]-4-oxo-butyl]amino]-18-oxo-octadecanoic acid (partial D-OSu)

[0343] like Figure 3 Schematically represented in , the fatty acid side chains are prepared using solid phase synthesis using 2-chlorotrityl chloride resin. In the presence of DIPEA, 2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid is connected to 2-chlorotrityl chloride resin to produce 2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid-2-Cl-Trt-resin. The Fmoc protecting group is removed by selectively blocking the amino group using piperidine, followed by coupling with Fmoc-Aib-OH in THF:DMAc using DIPC and HOBt, which produces 2-[2-[2-[(2-Fmoc-amino-2-methyl-propionyl)amino]ethoxy]ethoxy]acetic acid-2-Cl-Trt-resin. The Fmoc group was removed by selective deblocking using piperidine, and the free amino group was coupled with Fmoc-Glu-OtBu using HOBt and DIPC to produce 2-[2-[2-[[2-[[(4S)-4-Fmoc-amino-5-tert-butoxy-5-oxo-pentanoyl]amino]-2-methyl-propionyl]amino]ethoxy]ethoxy]acetic acid-2-Cl-Trt-resin. The Fmoc group of the resulting compound was selectively deblocked using piperidine, and the free amino group was coupled with mono-tert-butyl octadecanoate to give 2-[2-[2-[[(4S)-5-tert-butoxy-4-[(18-tert-butoxy-18-oxo-octadecanoyl)amino]-5-oxo-pentanoyl]amino]-2-methyl-propionyl]-amino]ethoxy]ethoxy]acetic acid-2-Cl-Trt-resin. The intermediate was then cleaved from the 2-Cl-Trt-resin using trifluoroethanol:DCM (1:1) to afford 2-[2-[2-[[2-[[(4S)-5-tert-butoxy-4-[(18-tert-butoxy-18-oxo-octadecanoyl)amino]-5-oxo-pentanoyl]amino]-2-methyl-propionyl]amino]ethoxy]ethoxy]acetic acid (LCMS=m / z: 786.39 (M+H + )). The resulting compound is then reacted with HOSu in the presence of DCC to produce a succinimide protected intermediate, which is deprotected with trifluoroacetic acid to yield the title compound (partial D-OSu).

[0344] 5. Preparation of 18-[[(1S)-1-carboxy-4-[3-[2-[2-[2-(2,5-dioxopyrrolidin-1-yl)oxy-2-oxo-ethoxy]ethoxy]ethylcarbamoylamino]propylamino]-4-oxo-butyl]amino]-18-oxo-octadecanoic acid (Part E-OSu)

[0345] like Figure 4 As schematically shown in Figure 2, the fatty acid side chains were prepared using solid-phase synthesis using 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 produce 2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid-2-Cl-Trt-resin. The Fmoc protecting group was removed by selectively deblocking the amino group with piperidine, and the free amino group was activated using p-nitrophenyl chloroformate in THF and DIPEA. The reaction was then reacted with 1,3-diaminopropane in THF:DMAc using HOBt in the presence of DIPEA to form NH2-(CH2)3-NH-C(O)-{(2-(2-amino-ethoxy)-ethoxy}-acetic acid-2-Cl-Trt-resin. The free amino group was then coupled to Fmoc-Glu-OtBu using HOBt and DIPC, which yielded 2-[2-[2-[3-[[(4S)-4-Fmoc-amino-5-tert-butoxy-5-oxo-pentanoyl]amino]propylcarbamoylamino]ethoxy]ethoxy]acetic acid-2-Cl-Trt-resin. The resulting 2-[2-[2-[3-[[(4S)-4-Fmoc-amino-5-tert-butoxy-5-oxo-pentanoyl]amino]propylcarbamoylamino]ethoxy]ethoxy]acetic acid-2-Cl-Trt-resin was reacted with piperidine. The resin was selectively deblocked and then coupled with octadecanoic acid mono-tert-butyl ester to give 2-[2-[2-[3-[[(4S)-5-tert-butoxy-4-[(18-tert-butoxy-18-oxo-octadecanoyl)amino]-5-oxo-pentanoyl]amino]-propylcarbamoylamino]ethoxy]-ethoxy]acetic acid-2-Cl-Trt-resin to give 2-[2-[2-[3-[[(4S)-5-tert-butoxy-4-[(18-tert-butoxy-18-oxo-octadecanoyl)amino]-5-oxo-pentanoyl]amino]-propylcarbamoylamino]ethoxy ... The intermediate was then cleaved from the 2-Cl-Trt-resin using trifluoroethanol:DCM (1:1) to afford 2-[2-[2-[3-[[(4S)-5-tert-butoxy-4-[(18-tert-butoxy-18-oxo-octadecanoyl)amino]-5-oxo-pentanoyl]amino]-propylcarbamoylamino]ethoxy]ethoxy]acetic acid (LCMS=m / z: 801.41 (M+H +)). The resulting compound is then reacted with HOSu in the presence of dicyclohexylcarbodiimide (DCC) to produce a succinimide protected intermediate, which is deprotected with trifluoroacetic acid to produce the title compound (partial E-OSu).

[0346] 6. Preparation of 18-[[(1S)-1-carboxy-4-[4-[2-[2-[2-(2,5-dioxopyrrolidin-1-yl)oxy-2-oxo-ethoxy]ethoxy]ethylcarbamoylamino]-1-piperidinyl]-4-oxo-butyl]amino]-18-oxo-octadecanoic acid (Part F-OSu)

[0347] like Figure 5As schematically shown in FIG, a solid phase synthesis is used to prepare fatty acid side chains using 2-chlorotrityl chloride resin. 2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid is attached to 2-chlorotrityl chloride resin in the presence of DIPEA to produce 2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid-2-Cl-Trt-resin. The Fmoc protecting group is removed by selectively deblocking the amino group using piperidine, and the free amino group is then activated using p-nitrophenyl chloroformate in THF and DIPEA, followed by reaction with 4-amino-Boc-piperidine in THF:DMAc using HOBt in the presence of DIPEA to produce (2-[2-[2-(4-Boc-piperidinylcarbamoylamino)ethoxy]ethoxy]acetic acid-2-Cl-Trt-resin. The resulting compound is cleaved using trifluoroacetic acid to produce 2-[2-[2-(4-piperidinylcarbamoylamino)ethoxy]acetic acid-2-Cl-Trt-resin. [2-[2-(4-Fmoc-piperidinylcarbamoylamino)ethoxy]ethoxy]acetic acid, which is further reacted with Fmoc-OSu in the presence of triethylamine (TEA) to produce 2-[2-[2-(4-Fmoc-piperidinylcarbamoylamino)ethoxy]ethoxy]acetic acid. The obtained compound is then further connected to 2-chlorotrityl chloride resin in the presence of DIPEA to produce 2-[2-[2-(4-Fmoc-piperidinylcarbamoylamino)ethoxy]ethoxy]acetic acid-2-Cl-Trt-resin. The Fmoc group is removed by selective deblocking using piperidine, and then HOBt and DIPC, the free amino group is coupled with Fmoc-Glu-OtBu, which produces 2-[2-[2-[[1-[(4S)-4-amino-5-tert-butoxy-5-oxo-pentanoyl]-4-piperidinyl]carbamoylamino]ethoxy]ethoxy]acetic acid-2-Cl-Trt-resin. The resulting compound is selectively deblocked using piperidine and then coupled with mono-tert-butyl octadecanoate to give 2-[2-[2-[[1-[(4S)-5-tert-butoxy-4-[(18-tert-butoxy-18-oxo-octadecanoyl) [amino]-5-oxo-pentanoyl]-4-piperidinyl]carbamoylamino]ethoxy]ethoxy]acetic acid-2-Cl-Trt-resin. The intermediate was then cleaved from the 2-Cl-Trt-resin using trifluoroethanol:DCM (1:1) to give 2-[2-[2-[[1-[(4S)-5-tert-butoxy-4-[(18-tert-butoxy-18-oxo-octadecanoyl)amino]-5-oxo-pentanoyl]-4-piperidinyl]carbamoylamino]ethoxy]ethoxy]acetic acid (LCMS=m / z: 827.40 (M+H + )). The resulting compound is then reacted with HOSu in the presence of DCC to produce a succinimide protected intermediate, which is deprotected using trifluoroacetic acid to produce the title compound moiety F-OSu.

[0348] Example 2: Synthesis of Compound 1:

[0349] N-ε 26 -[2-(2-[2-(2-[2-(2-[4-(17-carboxyheptadecanoylamino)-4(S)carboxybutanoylamino]ethoxy)ethoxy]acetylamino)ethoxy]ethoxy)acetyl][(Aib 8 ,Arg 34 ,Leu 38 GLP-1(7-38) peptide

[0350] Part A. Synthesis of parent linear peptide Aib 8 ,Arg 34 ,Leu 38 GLP-1 (7-38)

[0351] The parent peptide is synthesized by a solid phase method. The starting resin used for synthesis is Wang resin. Fmoc-protected leucine is used for coupling with Wang resin. In the presence of 4-dimethylaminopyridine (DMAP), diisopropylcarbodiimide and N-hydroxybenzotriazole (DIC-HOBt) are used as coupling reagents to couple to produce Fmoc-Leu-Wang resin. Piperidine is used to selectively block the amino group of Fmoc-Leu-Wang resin, followed by HOBt and DIPC coupling with Fmoc-Gly-OH to produce Fmoc-Gly-Leu-Wang resin. This completes a cycle. Acetic anhydride and diisopropylethylamine / pyridine are used to block the uncoupled amino group at each amino acid coupling site.

[0352] For remaining 30 amino acid residues, repeat above-mentioned 2 steps, i.e. the Fmoc-protected selectivity of the amino acid being connected to resin goes to block, and the amino coupling of next amino acid residue in sequence and Fmoc protection.Use piperidine to complete selectivity to block, i.e. the deprotection of Fmoc group, and use HOBt / DIPC to complete the coupling with the amino acid of next Fmoc protection.The side chain of the amino acid of Fmoc protection is orthogonally protected, and for example the hydroxyl group of serine, tyrosine or threonine is protected by tert-butyl (-tBu), and the amino and guanidino of lysine and arginine are protected by tert-butyloxycarbonyl (-Boc) and 2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (-Pbf) respectively, and the imidazole of histidine is protected by trityl (-Trt), and the carboxylic acid group of aspartic acid or glutamic acid is protected by tBu group. The two steps mentioned above, i.e., selective deblocking and subsequent coupling with the next Fmoc-protected amino acid, were performed to obtain Fmoc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-Glu(OtBu)-Gly-Gln-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-Ile-Ala-Trp-Leu-Val-Arg(Pbf)-Gly-Arg(Pbf)-Gly-Leu-resin.

[0353] Fmoc-His(Trt)-Aib-Glu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-T yr(tBu)-Leu-Glu(OtBu)-Gly-Gln-Ala-Ala-Lys(Boc)-Glu(OtBu)-Phe-Ile-Ala-Trp-Leu-Val-Arg(Pbf)-Gly-Ar The g(Pbf)-Gly-Leu-resin was deblocked with piperidine, followed by cleavage and deprotection using trifluoroacetic acid and ethane-1,2-dithiol to afford crude H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-Leu-OH(Aib 8 ,Arg 34 ,Leu 38GLP-1 (7-38) peptide), which was purified by HPLC.

[0354] Part B:

[0355] The activated fatty acid chain portion A-OSu was grafted onto the purified (linear peptide) H-His-Aib-Glu-Gly-Thr-Phe-Thr-Ser-Asp-Val-Ser-Ser-Tyr-Leu-Glu-Gly-Gln-Ala-Ala-Lys-Glu-Phe-Ile-Ala-Trp-Leu-Val-Arg-Gly-Arg-Gly-Leu-OH obtained in part A in acetonitrile at pH about 10 to give the crude title peptide, which was purified by preparative HPLC. The characterization of the compounds is provided in Table 3.

[0356] Example 3: Preparation of Compounds 2, 3, 5, 9, 10 and 12

[0357] Linear peptides of compounds 2, 3, 5, 9, 10, and 12 were prepared by solid phase methods similar to those given for Example 1, part A. Compounds 2, 3, 5, 9, 10, and 12 were provided by grafting the activated fatty acid chain moiety A-OSu onto the corresponding linear peptides according to the method of Example 1, part B.

[0358] Example 4: Preparation of compounds 4 and 11:

[0359] Linear peptides of compounds 4 and 11 were prepared by solid-phase methods similar to those described for Example 2, Part A, except that Fmoc-protected D-leucine was coupled to Wang resin first, followed by the sequential coupling of the other amino acids. Compounds 4 and 11 were provided by grafting the activated fatty acid chain moiety, A-OSu, onto the corresponding linear peptides according to the method of Example 2, Part B.

[0360] Example 5: Preparation of Compound 8

[0361] The linear peptide was prepared by solid phase methods similar to those given for Example 2, part A, except that here Fmoc-protected isoleucine was coupled to Wang resin first, followed by the sequential coupling of the other amino acids. Compound 8 was provided by grafting the activated fatty acid chain moiety A-OSu onto the linear peptide according to the method of Example 2, part B.

[0362] Example 6: Preparation of Compound 6

[0363] The linear peptide was prepared by solid phase method following a similar procedure as given for Example 2, part A. Compound 6 was provided by grafting the activated fatty acid chain moiety B-OSu onto the linear peptide by following a similar procedure as given for Example 2, part B.

[0364] Example 7: Preparation of Compound 7

[0365] Compound 7 was provided by grafting the activated fatty acid chain moiety B-OSu onto the linear peptide of Example 2, Part A, following a procedure similar to that of Example 2, Part B.

[0366] Example 8: Preparation of Compound 13

[0367] Compound 13 was provided by grafting the activated fatty acid chain moiety C-OSu onto the linear peptide of Example 1, Part A, by following a procedure similar to that of Example 1, Part B.

[0368] Example 9: Preparation of Compound 14

[0369] The linear peptide was prepared by solid phase method following a similar procedure as given for Example 2, part A. Compound 14 was provided by grafting the activated fatty acid chain moiety C-OSu onto the linear peptide by following a similar procedure as given for Example 2, part B.

[0370] Example 10: Preparation of Compound 15

[0371] The linear peptide was prepared by solid phase methods, starting with coupling Fmoc-protected isoleucine to Wang resin, followed by sequential coupling of the other amino acids, following a procedure analogous to that given for Example 2, Part A. Compound 15 was provided by grafting the activated fatty acid chain moiety, C-OSu, onto the linear peptide following a procedure analogous to that described for Example 2, Part B.

[0372] Example 11: Preparation of Compound 16

[0373] Compound 16 was provided by grafting the activated fatty acid chain moiety D-OSu onto the linear peptide of Example 2, Part A, by following a procedure analogous to that of Example 2, Part B.

[0374] Example 12: Preparation of Compound 17

[0375] Compound 17 was provided by grafting the activated fatty acid chain moiety E-OSu onto the linear peptide of Example 2, Part A, by following a procedure similar to that of Example 2, Part B.

[0376] Example 13: Preparation of Compound 18

[0377] Compound 18 was provided by grafting the activated fatty acid chain moiety F-OSu onto the linear peptide of Example 2, Part A, by following a procedure similar to that of Example 2, Part B.

[0378] Characterization data for the synthesized compounds of the present disclosure are provided below in Table 3 below.

[0379] Table 3: Characterization data of representative compounds of the present disclosure

[0380] Compound# LCMS data HPLC purity 1 <![CDATA[m / z=1057.52(MH4 4+ ), calculated mass = 4226.05]]> 98.32% (Method B2), RT = 24.85 min. 2 <![CDATA[m / z=1061.74(MH4 4+ ), calculated mass = 4242.93]]> 99.02% (Method B1), RT = 18.53 min. 3 <![CDATA[m / z=1087.65(M-4H) -4 , calculated mass = 4354.63]]> 98.12% (Method B1), RT = 18.48 min. 4 <![CDATA[m / z=1055.68(M-4H) -4 , calculated mass = 4226.75]]> 98.97% (Method B1), RT = 18.32 min. 5 <![CDATA[m / z=1057.88(MH4 4+ ), calculated mass = 4227.49]]> 96.75% (Method B1), RT = 17.09 min. 6 <![CDATA[m / z=967.26(MH4 4+ ), calculated mass: 3865.01]]> 98.68% (Method B3), RT = 44.04 min. 7 <![CDATA[m / z=968.53(M-4H) -4 , calculated mass = 3878.15]]> 97.39% (Method B3), RT = 27.79 min. 8 <![CDATA[m / z=1057.72(MH4 4+ ), calculated mass = 4226.85]]> 95.70% (Method B1), RT = 16.62 min. 9 <![CDATA[m / z=1061.67(MH4 4+ ), calculated mass = 4242.65]]> 95.15% (Method B1), RT = 16.48 min. 10 <![CDATA[m / z=1058.18(MH4 4+ ), calculated mass = 4228.69]]> 93.66% (Method B1), RT = 16.13 min. 11 <![CDATA[m / z=1056.95(MH4 4+ ), calculated mass: 4223.77]]> 95.70% (Method B2), RT = 24.46 min 12 <![CDATA[m / z=1405.12(MH3 3+ ), calculated mass: 4212.34]]> 97.51% (Method B1), RT = 19.06 min. 13 <![CDATA[m / z=1049.59(MH4 4+ ), calculated mass = 4194.33]]> 96.01% (Method B2), RT = 25.16 min. 14 <![CDATA[m / z=1050.13(MH4 4+ ), calculated mass = 4196.49]]> 92.06% (Method B2), RT = 24.55 min. 15 <![CDATA[m / z=1049.61(MH4 4+ ), calculated mass = 4194.41]]> 94.41% (Method B2), RT = 24.82 min. 16 <![CDATA[m / z=1042.34(MH4 4+ ), calculated mass = 4165.32]]> 94.56% (Method B2), RT = 25.26 min. 17 <![CDATA[m / z=1046.18(MH4 4+ ), calculated mass = 4180.72]]> 94.33% (Method B2), RT = 25.17 min. 18 <![CDATA[m / z=1052.77(MH4 4+ ), calculated mass = 4207.08]]> 93.12% (Method B2), RT = 24.92 min.

[0381] Example 14: Oral glucose tolerance test (OGTT) in rats; single injection; 1 mg / kg

[0382] The animals were divided into three groups - a normal control group, a test group and a third semaglutide group, with 4 animals in each group. Before the start of the OGTT, the animals were fasted for 12 hours. For the test group animals, compound 1 was injected subcutaneously at a dose of 1 mg / kg. For the semaglutide group, a dose of 1 mg / kg was injected subcutaneously. Blood glucose was measured with a glucometer 22 hours, 166 hours and 334 hours after the subcutaneous injection of the test drug or semaglutide (time 0 measurement). All animals were then orally administered a 2g / kg glucose solution. Blood glucose was measured at 20, 40, 60, 90 and 120 minutes after the glucose attack. Body weight and food intake were recorded. Blood glucose data were analyzed using a two-way ANOVA followed by a Bonferroni post hoc test using PRISM (Graph Pad version 5.03). Blood glucose AUC was analyzed using a t-test. 0-120分钟 data.

[0383] When studied in an oral glucose tolerance test (OGTT) in rats, the polypeptides of the present disclosure have shown a significant glucose lowering effect compared to the control group. For example, Figure 6 provides the changes in blood glucose levels from time 0 to 120 minutes after 22 hours and 46 hours in the test group and semaglutide treatment group administered with Compound 1. At 22 hours after a single dose, Compound 1 showed a statistically significant reduction in blood glucose levels relative to normal controls in an ANOVA followed by a Bonferroni post hoc test with p < 0.001. The glucose lowering effect of Compound 1 was superior to the glucose lowering effect observed with semaglutide (see Figure 6A Even after 46 hours of subcutaneous administration, the superiority of the glucose lowering effect of Compound 1 was observed ( Figure 6B). In addition, when observed on day 2 and day 4, both Compound 1 and semaglutide showed statistically significant reductions in food intake compared to the control (see Tables 4 and 5). Compound 1 showed a greater reduction in food intake than semaglutide on day 4 (see Table 5). In terms of weight loss, only the test compound showed a significant reduction in body weight on day 4.

[0384] Table 4: Effects of treatment on food intake and body weight at day 2

[0385]

[0386] *p<0.05, **p<0.01, ***p<0.001 relative to normal control; one-way ANOVA followed by Bonferroni post hoc test

[0387] Table 5: Effects of treatment on food intake and body weight on day 4

[0388]

[0389] *p<0.05, **p<0.01, ***p<0.001 relative to normal control; one-way ANOVA followed by Bonferroni post hoc test

[0390] #p<0.05, ##p<0.01, ###p<0.001 vs. semaglutide; one-way ANOVA followed by Bonferroni post hoc test

[0391] Surprisingly, it was found that in a given study, compounds with X33 being Leu and Ile showed a significant reduction in blood glucose, while compounds with amino acids other than Leu and Ile had a significantly smaller effect in reducing blood glucose. Compared to the control group, the polypeptides of the present disclosure have shown a significant reduction in blood glucose. Compounds with amino acids other than Leu or Ile at position X33 were also tested. For example, Leu at position 32 in compound 1 (SEQ ID NO: 05) was replaced with Lys and Ser to obtain compounds Std-1 and Std-2, respectively. Std-1 and Std-2 only showed a blood glucose AUC of 0.04. 0-120分钟 Approximately 35% and 15% reduction (Table 6).

[0392] Table 6: Blood glucose AUC at 1 mg / Kg dose after 24 hours in OGTT test 0-120分钟 percentage of reduction.

[0393]

[0394] Similarly, compound 6, which differs from liraglutide by having an additional Leu at position 32, and compound 7, which differs from liraglutide by replacing the second amino acid Ala with Aib and having Leu as the additional 32nd amino acid, showed a blood glucose lowering effect significantly greater than that of liraglutide at 24 hours (Table 6).

[0395] Once it was determined that Compound 1 was significantly better in terms of glucose reduction, food intake, and weight loss, experiments were conducted to determine the duration of action of the compounds of the invention. The effects of representative compounds of the invention (Compounds 1, 13, and 16) were studied after 166 hours (7 days) and 334 hours (14 days) and compared with the effects of semaglutide. The compounds were tested according to the method provided below:

[0396] The animals were divided into three groups - a normal control group, a test group and a third semaglutide group, with four animals in each group. Before the start of the OGTT, the animals were fasted for 12 hours. For the test group animals, compound 1, compound 13 and compound 16 were injected subcutaneously at a dose of 1 mg / kg. For the semaglutide group, a dose of 1 mg / kg was injected subcutaneously. Blood glucose was measured with a glucometer 22 hours, 166 hours and 334 hours after subcutaneous injection of the test compound or semaglutide (time 0 measurement). All animals were then orally administered a 2g / kg glucose solution. Blood glucose was measured at 20, 40, 60, 90 and 120 minutes after the glucose attack. Body weight and food intake were recorded. Blood glucose data were analyzed using a two-way ANOVA followed by a Bonferroni post hoc test using PRISM (Graph Pad version 5.03). Blood glucose AUC was analyzed using a t-test. 0-120分钟 data.

[0397] Table 7 provides the reduction in blood glucose AUC for representative compounds of the invention (Compounds 1, 13, and 16) compared to the control group after 1 day, 7 days, and 14 days of administration.

[0398] Table 7: Blood glucose AUC at 1 mg / Kg dose in OGTT test 0-120分钟 percentage of reduction.

[0399]

[0400]

[0401] Compounds 1 and 13 were studied in one trial (Trial 1) and compared with semaglutide, and compound 16 was studied in a separate trial (Trial 2) and compared with semaglutide. At 168 hours after injection, compounds 1, 13, and 16 of the present invention showed a reduction in blood glucose AUC of approximately 60% when compared to time zero blood glucose levels. On the other hand, semaglutide only showed a reduction in blood glucose levels of approximately 25% relative to time zero blood glucose levels.

[0402] Similar observations were made regarding the amount of food consumed and changes in body weight. As shown in Table 8 below, animals administered with representative compounds (Compounds 1, 13, and 16) consumed significantly less food when compared to animals administered with semaglutide. Compound 16 showed a significant decrease in body weight, demonstrating its potential utility for the treatment of obesity.

[0403] Table 8: Effects of 1 mg / Kg dose on food consumption and body weight in OGTT test

[0404]

[0405] Example 15: HbA1c reduction in db / db type 2 diabetic mice after long-term treatment

[0406] This study was done in a diabetic mouse model. The animals were divided into three treatment groups - a diabetic control group, a test group and a semaglutide treatment group. Compound 1 of the present disclosure was administered subcutaneously once daily at a dose of 0.3 mg / kg for 3 days (qd×3), followed by a dose of 0.1 mg / kg every other day for a total of 7 doses (q2d×7), followed by a dose of 0.1 mg / kg once every four days for a total of 2 dose cycles (q4d×2). The semaglutide treatment group was administered the same dosing regimen. Blood glucose levels and body weight were measured daily. %HbA1c was measured by column chromatography on days 0, 7, 14 and 27. Cumulative food intake was calculated on day 27. %HbA1C data were analyzed by two-way ANOVA followed by Bonferroni post hoc test using PRISM (Graph Pad version 5.03).

[0407] The test group animals administered with Compound 1 showed a statistically significant decrease in blood glucose levels compared to the diabetic control group (see Figure 7 ), and the effect was superior to that of the semaglutide treatment group in the later stages of the study. Figure 8 As can be seen from the results provided in , the animals in the test group administered with Compound 1 showed a significant decrease in food intake. Figure 8Cumulative food intake from day 0 to day 27 for control and test compound treated db / db mice is provided. Both the test compound and semaglutide showed a statistically significant reduction in food intake compared to the diabetic control group. In addition, the test compound showed significantly lower food intake compared to semaglutide. In the same study, compound 1 also showed a significant reduction in body weight when compared to the diabetic control group. Figure 9 Results are provided for the % change in body weight of the control and test groups from day 0 to day 27. Test compound 1 showed a significant decrease of -16% compared to -8% as observed in the semaglutide treated group (see Figure 09).

[0408] In diabetes, higher amounts of HbA1c, which indicate poorer control of blood sugar levels, have been associated with cardiovascular disease, nephropathy, neuropathy, and retinopathy. In a 27-day study, Compound 1 showed a statistically significant reduction in HbA1c levels in db / db type 2 diabetic mice after long-term treatment. Table 9 and Figure 10 Presented are HbA1c levels at 0 and 27 days in the diabetic control group and in the group after long-term treatment with Compound 1. The effect was statistically significant even when compared with semaglutide.

[0409] Table 9: Effect of treatment on %HbA1c levels in db / db mice

[0410]

[0411] *p<0.05, **p<0.01, ***p<0.001 relative to diabetic controls; two-way ANOVA followed by Bonferroni post hoc test

[0412] #p<0.05, ##p<0.01, ###p<0.001 vs. semaglutide; one-way ANOVA followed by Bonferroni post hoc test

[0413] In a separate study, test compounds 1, 13, and 16 were investigated and compared with semaglutide for their effects on HbA1c and insulin levels, as well as cumulative food consumption, body weight change, and blood glucose AUC. This study was conducted in a diabetic mouse model in a similar manner as described above. Animals were divided into three treatment groups: a diabetic control group, a test group, and a semaglutide-treated group. Representative compounds of the present disclosure, Compound 1, Compound 13, and Compound 16, were subcutaneously injected at a dose of 3.04 or 6.078 nM (every other day until day 28 (q2d×15). The same dosing regimen was administered to the semaglutide treatment group. Blood glucose levels and body weight were measured daily. % HbA1c and insulin were measured on days 0, 14, and 29. Cumulative food intake and body weight changes were calculated on days 14 and 29. % HbA1c and insulin data were analyzed by two-way ANOVA followed by Bonferroni post hoc test using PRISM (Graph Pad version 5.03). % HbA1c and insulin data were analyzed by one-way ANOVA followed by PRISM (Graph Pad version 5.03). Blood glucose AUC, body weight change, and cumulative food intake data were analyzed using a Bonferroni post hoc test using Pad version 5.03. From day 29 to day 45, animals were maintained in a recovery period, during which no drug treatment was given. Blood glucose and body weight were measured during this period. On day 45, body weight change, %HbA1c, and insulin were measured.

[0414] The results are provided in Tables 10, 11 and 12 below.

[0415] Table 10: HbA1C (%): Compound 1 (6.078 nM), Compound 13 (3.04 & 6.078 nM), Compound 16 (3.04 & 6.078 nM); (q2d×15) (n=7)

[0416]

[0417] *=p<0.05, **=p<0.01, ***=p<0.001 vs. diabetic controls, #p<0.05, ##p<0.01, ###p<0.001 vs. semaglutide; one-way ANOVA followed by Bonferroni post hoc test

[0418] Table 11: Insulin (ng / mL): Compound 1 (6.078 nM), Compound 13 (3.04 & 6.078 nM), Compound 16 (3.04 & 6.078 nM); (q2d×15) (n=7)

[0419]

[0420] *=p<0.05, **=p<0.01, ***=p<0.001 vs. diabetic controls, #p<0.05, ##p<0.01, ###p<0.001 vs. semaglutide; one-way ANOVA followed by Bonferroni post hoc test

[0421] Table 12: Cumulative food consumption, blood glucose AUC (mg / dL×天) and body weight changes: Compound 1 (6.078 nM), Compound 13 (3.04 & 6.078 nM), Compound 16 (3.04 & 6.078 nM); (q2d×15)

[0422]

[0423]

[0424] *=p<0.05, **=p<0.01, ***=p<0.001 vs. diabetic controls, #p<0.05, ##p<0.01, ###p<0.001 vs. semaglutide; one-way ANOVA followed by Bonferroni post hoc test

[0425] When compared to the control, representative compounds of the present disclosure (Compounds 1, 13, and 16) at doses of about 3 nM and 6 nM showed significant reductions in HbA1c, blood glucose, food consumption, and body weight (Table 12). This reduction was comparable to the reduction shown by semaglutide at a dose of about 12 nM. Furthermore, this effect was seen even after 29 days (Tables 13 and 14), demonstrating the potential of the compounds of the present invention for the development of long-acting drugs that do not require frequent administration and thus increase patient compliance.

[0426] Table 13: Recovery Study - Blood Glucose AUC (mg / dL×天)

[0427]

[0428] *p<0.05, **p<0.01, ***p<0.001 relative to diabetic controls and #p<0.05, ##p<0.01, ###p<0.001 relative to semaglutide; one-way ANOVA followed by Bonferroni post hoc test

[0429] Table 14: Recovery Study - % HbA1C and Insulin (ng / mL)

[0430]

[0431] *p<0.05, **p<0.01, ***p<0.001 relative to diabetic controls and #p<0.05, ##p<0.01, ###p<0.001 relative to semaglutide; one-way ANOVA followed by Bonferroni post hoc test

[0432] These results demonstrate that the compounds of the present invention may find potential use in the treatment of diabetes and obesity.

Claims

1. A polypeptide having the following amino acid sequence: Part A's structure is as follows:

2. A polypeptide having the following amino acid sequence: Among them, Ser(OMe) is And part A has the following structure:

3. A polypeptide having the following amino acid sequence: Part A's structure is as follows:

4. A polypeptide having the following amino acid sequence: wherein DLeu is a leucine residue in the D-configuration; and Part A's structure is as follows:

5. A polypeptide having the following amino acid sequence: Part A's structure is as follows:

6. A polypeptide having the following amino acid sequence: The structure of part B is as follows:

7. A polypeptide having the following amino acid sequence: The structure of part B is as follows:

8. A polypeptide having the following amino acid sequence: Part A's structure is as follows:

9. A polypeptide having the following amino acid sequence: Wherein DSer is a serine residue in the D-configuration; Among them, Ser(OMe) is And part A has the following structure:

10. A polypeptide having the following amino acid sequence: wherein DSer is a serine residue in the D-configuration; and Part A's structure is as follows:

11. A polypeptide having the following amino acid sequence: wherein DLeu is a leucine residue in the D-configuration; and Part A's structure is as follows:

12. A polypeptide having the following amino acid sequence: Part A's structure is as follows:

13. A polypeptide having the following amino acid sequence: Some of the C structures are as follows:

14. A polypeptide having the following amino acid sequence: Some of the C structures are as follows:

15. A polypeptide having the following amino acid sequence: Some of the C structures are as follows:

16. A polypeptide having the following amino acid sequence: Some of the D structures are as follows:

17. A polypeptide having the following amino acid sequence: Some of the E structures are as follows:

18. A polypeptide having the following amino acid sequence: Some of the F structures are as follows:

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