Novel GLP-1 analog

Abstract

To provide a GLP-1 analog that has optimally desirable properties in terms of stability and duration of action. [Solution] This disclosure relates to a novel glucagon-like peptide-1 (GLP-1)(7-37) analog having an amino acid sequence with Leu or Ile at the C-terminus. This novel analog is a potent GLP-1 agonist with reduced adverse effects and improved duration of action. This disclosure further relates to a novel acylated derivative of the analog that has further improved potency and duration of action and is suitable for oral administration. The analog of this disclosure may be useful in the treatment of diabetes and obesity.

Description

[Technical Field]

[0001] Cross-reference of related applications This application is application number IN2018 / 21013109 (filed April 5, 2018), I N2018 / 21040468 (filed October 26, 2018), and IN2018 / The benefits of three Indian provisional applications, including 21040474 (filed October 26, 2018) These are assertions, and they are incorporated herein by reference.

[0002] This disclosure relates to a novel glucago having an amino acid sequence with Leu or Ile at the C-terminus. This relates to an analog of GLP-1 (GLP-1) (7-38). This new analog is This is a potent GLP-1 agonist with reduced harmful effects and improved duration of action. The disclosure further reveals a new formulation with improved efficacy and duration of action, and suitable for oral administration. This relates to acylated derivatives of analogs. The analogs disclosed herein are acylated in the extended portion. This is converted, which increases the duration of activity of the compound. The analogs disclosed herein are sugars It may be useful in the treatment of urinary tract diseases and obesity. [Background technology]

[0003] Glucagon-like peptide-1 (GLP-1) is mainly produced in enteroendocrine L cells of the intestine. Foods containing fat, protein hydrolysates, and / or glucose enter the duodenum. It is a hormone that is secreted into the bloodstream when it enters the body. GLP-1 is a preproglucagon gene. It originates from cell-specific post-translational processing. Initially, the peptide GLP-1(1-37) Identified from this processing, it was found to recognize pancreatic receptors, and Two N-terminal cleavage products, GLP-1(7-37), were determined to be active species in vivo. The drugs were (SEQ ID NO: 1) and GLP-1(7-36) amide. GLP-1 is an insulin It stimulates glucose secretion, thereby increasing glucose uptake by cells and reducing serum blood glucose levels. It has been found that it causes. GLP-1 agonists do not cause hypoglycemia and weight loss. Because it offers significant benefits, it can be used as a preferred drug for the treatment of type 2 diabetes (T2DM). These are endogenous substances: GLP-1(7-37) and GLP-1(7-36) amides. It is cleaved by peptidase and therefore has a very short half-life. The half-life is improved. Efforts were made to improve performance by developing GLP-1 analogs. The first drug approved in 2005 was exocrine, administered twice daily at a dose level of 10 mcg. Exenatide is a glucose control marker that affects HbA1c. It was found to show a significant improvement. Furthermore, Novo Nordisk showed a daily rate of Liraglutide is administered by subcutaneous injection at a dose of 1.8 mg once daily. They developed U.S. Patent No. 6,268,343 (Sequence ID 2), which was approved in 2010. Further research and development led to the development of albiglutide (Albig) by GSK. Dulaglutide, developed by Eli Lilly, is also known as dulaglutide. Products administered once a week, such as aglutide, have been manufactured. More recently, GLP- 1. An analogue: semaglutide (International Publication WO2006 / 09) Semaglutide (SEQ ID NO: 7537(A2)) was approved by the USFDA. It is marketed under the brand name Ozempic®. This is administered by subcutaneous injection weekly. is administered once.

[0004] Many attempts have been made to produce GLP-1 analogs with improved efficacy and duration of action reported in the literature. U.S. Patent No. 7,291,594 (B2) (US'594 patent) discloses GLP-1(7-35) derivatives that add some residues of arginine and / or lysine to its C-terminus to provide high bioavailability via the mucosa. The US'594 patent further discloses that these derivatives can confer resistance to dipeptidyl peptidase IV (DPP-IV) by substituting amino acid 8 with Ser in its GLP-1 amino acid sequence, or can confer resistance to trypsin by substituting amino acids 26 and 34 with Gln and Asn, respectively. U.S. Patent No. 7,893,017 (B2) (US'017 patent) states that a GLP-1 analog is stable to DPP-IV by modifying at least one amino acid residue at positions 7 and 8 relative to GLP-1(7-37), and the acylation is a diacid directly attached to the C-terminal amino acid residue of the GLP-1 analog.

[0005] U.S. Patent No. 8,951,959 (B2) (US'959 patent) discloses a non-proteinogenic amino acid residue containing a trifluoromethyl group at position 8 relative to the sequence GLP-1, and a DPP-IV-resistant GL P-1(7-37) analog acylated with a moiety containing two acidic groups at the lysine residue at position 26.

[0006] U.S. Patent No. 7,084,243(B2) (US'243 patent) is for DPP-IV resistance. As a peptide, it has Val or Gly at position 8 relative to the sequence GLP-1(7-37). The document discloses a GLP-1(7-37) analog.

[0007] International Publication No. WO2017 / 149070(A1) (WO'070) is GLP-1( The disclosure includes a GLP-1 analog having Trp at the position corresponding to position 8 in 7-37). These Trp8 compounds are highly stable against degradation by DPP-IV. This was shown.

[0008] International publication no. WO2004 / 103390(A2) (WO'390) is located at P'1 position ( Modification at position 9 (corresponding to position 9 in the case of GLP-1(7-37)) affects the natural substrate. Although its sensitivity to enzyme-mediated cleavage (e.g., DPP-IV) is significantly reduced, it is still natural. The present invention discloses that it is possible to generate GLP-1 analogs that maintain the biological activity of the substrate. WO'390 has a tetrasubstituted Cβ carbon (e.g., tert-leucine) at position 9. Further disclosures include GLP-1(7-37) analogs having amino acids, and DPP-IV This provides a GLP analog that is resistant to degradation by [unspecified factor].

[0009] International publication no. WO2015 / 086686(A2) (published as WO'686) is GLP- By directly incorporating alpha-methyl-functionalized amino acids into the main chain of one analog, It has been determined that thease-resistant peptides (including DPP-IV resistance) are produced. They have disclosed the following.

[0010] Various other DPP-IV resistant GLP-1 agonists were published internationally in WO2007 / 03. No. 0519(A2), No. WO2004 / 078777(A2), No. WO2007 / Issues 039140(A1), WO2014 / 209886(A1), and WO201 2 / 016419(A1), WO2017 / 211922(A2), WO2 Issues 016 / 198544(A1) and WO2013 / 051938(A2) In which patent publication is it disclosed?

[0011] Various patent applications describe C-terminal extensions that have increased stability and a longer duration of action. GLP-1 analogs are disclosed. For example, U.S. Patent No. 7,482,321(B2), These are registration numbers 9,498,534(B2) and 7,897,566(B2).

[0012] Various patent applications describe how GLP-1 analogs can be selectively linked to lipophilic substituents via a linker. This document discloses an acylated GLP-1 analog that adheres to surfaces and provides a longer duration of action. .

[0013] U.S. Patent No. 8,603,972(B2) (US'972) is for the position of a GLP-1 analog. In the monoa of the GLP-1 analog, the Lys residue is acylated at position 37 or 38. A sylated derivative is disclosed.

[0014] U.S. Patent No. 8,648,041(B2), U.S. Patent No. 9,758,560(B2), U.S. Patent No. No. 9,006,178(B2), No. 9,266,940(B2), No. 9,708, Patent No. 383(B2) and U.S. Patent Application Publication No. US2015 / 0152157(A1) US2015 / 0133374(A1) describes the diacylation induction of GLP-1 analogs. She is exposing her body.

[0015] U.S. Patent Application Publication US2016 / 0200791(A1) relates to a GLP-1 analog. Triacylated derivatives are disclosed.

[0016] International publication no. WO2016 / 083499(A1), international publication no. WO2016 / 097108 (A1), and WO2014 / 202727(A1), are GLP-1 analogs. Lys residues are attached to two extended portions via a branched linker, acylated GLP- 1. An analogue is disclosed.

[0017] International publications WO2009 / 030771(A1) and WO2018 / 0833 Substance 35(A1) attaches to the Lys residue of the GLP-1 analog, resulting in a longer duration of action. Various acylating agents (side chains) that can be used are disclosed.

[0018] International Publication No. WO2013 / 186240(A2) is an exendin-4 amino acid compound At position 2 of the column, Gly, Ser, or functionalized Ser, for example, Ser(OCH3 ), D-Ser or functionalized D-Ser, for example, D-Ser(OCH3), Aib, The present invention discloses exendin-4 peptide analogs having Ala or D-Ala. .

[0019] Various other GLP-1 analogues are described in International Publication No. WO2005 / 027978(A2), Same as WO1998 / 008871(A1), same as WO1999 / 043705(A1) Issues WO1999 / 043706(A1) and WO1999 / 043707(A 1) No. WO1999 / 043708(A1), No. WO2000 / 034331 (A2), WO2009 / 030771(A1), WO2011 / 0801 No. 03(A1), No. WO2012 / 140117(A1), No. WO2012 / 06 2803(A1), WO2012 / 062804(A1), WO2013 / Issues 037690(A1), WO2014 / 202727(A1), and WO201 5 / 000942(A1), WO2015 / 022400(A1), WO2 Issues 016 / 083499(A1), WO2016 / 097108(A1), and This is disclosed in patent applications such as WO2017 / 149070(A1).

[0020] GLP- 1. There is a need to develop an analog. [Overview of the project]

[0021] One aspect of this disclosure is a polypeptide comprising the following amino acid sequence, H-X2-X3-X4-GTFTSDVSSYL-X16-GQ -AA-X21-EF-X24-AWLVRGRG-X33-X34 In the formula, X2 is Ser, Ser(OMe), D-Ser, D-Ser(OMe), Ala, or Aib, X3 does not exist, or it is Gln. X4 is Glu, X16 is Glu, X24 is Ile, X33 is Leu, D-Leu, D-Ile, or Ile. X34 does not exist. X21 is Lys, and the amino(ε-amino) group in the side chain of Lys is acylated at the following site. And, [ka] In the formula, Q and T are absent. U is either absent or -C(O)-CH2-O-(CH2)2-O-(CH2)2- NH-}, where} is the attachment point with the base W, W is either absent, or -C(O)-CH2-O-(CH2)2-O-(CH2)2- NH-, -C(O)-NH-(CH2) 3-4 -NH-], -C(O)-C(CH3) 2-NH-, and [ka] Selected from the group consisting of, where ] is the attachment point with base Y, Y is -C(O)-(CH2)2-CH(COOH)NH--, and in the formula -- is the group Z and It is the attachment point, Z is -C(O)-(CH2) n -COOH or -C(O)-(CH2) n -CH3 The formula provides polypeptides where n is an integer between 14 and 20.

[0022] The polypeptides disclosed herein are potent GLP-1 agonists with fewer adverse effects. Furthermore, the polypeptides of this disclosure are stable, have a long duration of action, and can be administered orally. Suitable for giving. [Brief explanation of the drawing]

[0023] [Figure 1A] The preparation of partial A-OSu (intermediate 3) is illustrated. [Figure 1B] The preparation of partial A-OSu (intermediate 3) is illustrated. [Figure 2] The preparation of partial C-OSu is illustrated. [Figure 3] The preparation of partial D-OSu is illustrated. [Figure 4] The preparation of partial E-OSu is illustrated. [Figure 5] The preparation of partial F-OSu is illustrated. [Figure 6A] The results of the oral glucose tolerance test (OGTT) of compound 1 in rats with a single injection, at a glucose AUC of 1 mg / kg from 0 to 120 minutes (Figure 6A = 22 hours later, Figure 6B = 46 hours later), are shown. [Figure 6B] The results of the oral glucose tolerance test (OGTT) of compound 1 in rats with a single injection, at a glucose AUC of 1 mg / kg from 0 to 120 minutes (Figure 6A = 22 hours later, Figure 6B = 46 hours later), are shown. [Figure 7] The reduction in blood glucose levels in db / db2 diabetic mice after chronic treatment with compound 1 is illustrated. [Figure 8] The figure illustrates the decrease in food intake in db / db mice after treatment with compound 1. [Figure 9] The efficacy of compound 1 in weight loss in db / db mice is illustrated. [Figure 10] The figure illustrates the decrease in Hb1Ac in db / db mice after treatment with compound 1.

[0024] Abbreviation A1: 2-aminoisobutyric acid ADO: 8-amino-3,6-dioxo-octanoic acid OGTT: Oral glucose tolerance test DIPEA: N,N'-di-isopropylethylamine HOBt: 1-hydroxybenztriazole DIPC: N,N'-di-isopropylcarbodiimide HOSu:N-hydroxysuccinimide IBCF: Isobutylchloroformate NMM: N-methylmorpholine THF: Tetrahydrofuran DCM: Dichloromethane DMAP: 4-dimethylaminopyridine DCC: Dicyclohexylcarbodiimide DMAc: Dimethylacetamide [Modes for carrying out the invention]

[0025] This disclosure provides a stable, long-lasting effect that does not require frequent subcutaneous administration and is also suitable for oral administration. It provides a GLP-1 analogue. Surprisingly, it adds an extra Leu to the C-terminus of the sequence. This resulted in significantly improved potency and duration of action compared to the parent peptide. It was found that it produces polypeptides containing extra Ile. Peptides containing extra Ile also Compared to the parent peptide, it showed similar effects in improving potency and duration of action. Furthermore, this disclosure uses the following terms to describe PEP, an analog of GLP-1(7-37). The addition of this compound to the cytoside via an acylation reaction resulted in significantly improved potency and longer-lasting action. The present invention demonstrates that it is possible to produce a compound that has time. The portion has a more stable bond, which makes it more susceptible to cleavage by biological enzymes. Low. Therefore, the compounds disclosed herein are more stable and less frequent. This requires administration of medication and increases patient adherence to medication. Therefore, in some embodiments, this opening The diagram shows a polypeptide containing the following amino acid sequence, H-X2-X3-X4-GTFTSDVSSYL-X16-GQ -AA-X21-EF-X24-AWLVRGRG-X33-X34 (Sequence No. 4) In the formula, X2 is Ser, Ser(OMe), D-Ser, D-Ser(OMe), Ala, or Aib, X3 does not exist, or it is Gln. X4 is Glu, X16 is Glu, X24 is Ile, X33 is Leu, D-Leu, D-Ile, or Ile. X34 does not exist. X21 is Lys, and the amino (ε-amino) group in the side chain of Lys is acylated, poly We provide peptides.

[0026] In some embodiments, X21 is U.S. Patent No. 6,268,343, and No. 8,95 No. 1,959(B2), No. 8,603,972(B2), No. 8,648,041( B2), No. 9,758,560 (B2), No. 9,006,178 (B2), 9 Patent Nos. 266,940(B2), 9,708,383(B2), and U.S. Patent Publication No. Application Publication No. US2015 / 0152157(A1) and No. US2015 / 0133 Publication No. 374(A1); International Publication No. WO2009 / 030771(A1), and WO200 Reported in issue 6 / 097537(A2) and issue WO2018 / 083335(A1). The extended portion can be acylated.

[0027] In some embodiments, X21Lys has a side-chain amino(ε-amino) group, The fatty acid group is acylated at the moiety containing the fatty acid group. The fatty acid group is linked to X21Lys via a linker. It can adhere to. Therefore, in some embodiments, the present disclosure uses the following amino acid sequence Polypeptides containing, H-X2-X3-X4-GTFTSDVSSYL-X16-GQ -AA-X21-EF-X24-AWLVRGRG-X33-X34 In the formula, X2 is Ser, Ser(OMe), D-Ser, D-Ser(OMe), Ala, or Aib, X3 does not exist, or it is Gln. X4 is Glu, X16 is Glu, X24 is Ile, X33 is Leu, D-Leu, D-Ile, or Ile. X34 does not exist. X21 is Lys, and the amino(ε-amino) group in the side chain of Lys is acylated at the following site. And, [ka] In the formula, Q and T are absent. U is either absent or -C(O)-CH2-O-(CH2)2-O-(CH2)2- NH-}, where} is the attachment point with the base W, W is either absent, or -C(O)-CH2-O-(CH2)2-O-(CH2)2- NH-, -C(O)-NH-(CH2) 3-4 -NH-], -C(O)-C(CH3) 2-NH-, and [ka] Selected from the group consisting of, where ] is the attachment point with base Y, Y is -C(O)-(CH2)2-CH(COOH)NH--, and in the formula -- is the group Z and It is the attachment point, Z is -C(O)-(CH2) n -COOH or -C(O)-(CH2) n -CH3 The formula provides polypeptides where n is an integer between 14 and 20.

[0028] In some embodiments, the amino acids of X2 are Ser, Ser(OMe), D-Ser Selected from D-Ser(OMe), Ala, or Aib.

[0029] In some embodiments, X2 is Aib.

[0030] In some embodiments, X3 is absent.

[0031] In some embodiments, X33 is Leu.

[0032] In some embodiments, X33 is Ile.

[0033] In some embodiments, X21 is Lys, and the side chain amino(ε-amino) group of Lys The following part is acylated: [ka] In the formula, W is -C(O)-NH-(CH2) 3-4 -NH-], -C(O)-C(CH3 )2-NH-], and [ka] It is selected from the group consisting of the following.

[0034] In some embodiments, X21 is Lys, and the side chain amino(ε-amino) group of Lys The following part is acylated: [ka] In the formula, neither U nor W exists, and Z is -C(O)-(CH2) n -CH3, In the formula, n is an integer 14.

[0035] In some embodiments, X21 is Lys, and the side chain amino(ε-amino) group of Lys The following part is acylated: [ka] In the formula, W is -C(O)-CH2-O-(CH2)2-O-(CH2)2-NH- .

[0036] In some embodiments, X21 is Lys, and the side chain amino(ε-amino) group of Lys The following part is acylated: [ka] In the formula, W is C(O)-C(CH3)2-NH-.

[0037] In some embodiments, X21 is Lys, and the side chain amino(ε-amino) group of Lys The following part is acylated: [ka] In the formula, W is -C(O)-NH-(CH2)4-NH-].

[0038] In some embodiments, X21 is Lys, and the side chain amino(ε-amino) group of Lys The following part is acylated: [ka] In the formula, W is -C(O)-NH-(CH2)3-NH-].

[0039] In some embodiments, X21 is Lys, and the side chain amino(ε-amino) group of Lys The following part is acylated: [ka] In the formula, W is [ka] That is the case.

[0040] In some embodiments, X21 is Lys, and the side chain amino(ε-amino) group of Lys is acylated at the following portion,

Chemical formula

[0041] In some embodiments, X21 is Lys, and the side-chain amino (ε-amino) group of Lys is acylated at the following portion,

Chemical formula

[0042] In some embodiments, X2 is Ala or Aib, X3 does not exist, X33 is Leu, U does not exist, W does not exist, Y is -C(O)-(CH2)2-CH(COOH)NH--, where -- is the attachment point to group Z and Z is -C(O)-(CH2)n-CH3, where n is the integer 14.

[0043] [[ID=4৬]] In some embodiments, the present disclosure is a polypeptide comprising the following amino acid sequence , H-X2-X3-X4-G-T-F-T-S-D-V-S-S-Y-L-X16-G-Q -A-A-X21-E-F-X24-A-W-L-V-R-G-R-G-X33-X34 where X2 is Aib, X3 does not exist, X4 is Glu, X16 is Glu, X24 is Ile, X33 is Leu, X34 does not exist. X21 is Lys, and the amino(ε-amino) group in the side chain of Lys is acylated at the following site. And, [ka] In the formula, Q and T are absent. U is -C(O)-CH2-O-(CH2)2-O-(CH2)2-NH-, and in the formula ,} is the attachment point with the base W, W is -C(O)-CH2-O-(CH2)2-O-(CH2)2-NH-, and in the formula ,] is the attachment point of base Y, Y is -C(O)-(CH2)2-CH(COOH)NH--, and in the formula -- is the group Z and It is the attachment point, Z is -C(O)-(CH2) n -COOH, where n is an integer 16, polypeptide We will provide Chido.

[0044] In some embodiments, the present disclosure relates to a polypeptide comprising the following amino acid sequence. , H-X2-X3-X4-GTFTSDVSSYL-X16-GQ -AA-X21-EF-X24-AWLVRGRG-X33-X34 In the equation, X2 is Aib, X3 does not exist. X4 is Glu, X16 is Glu, X24 is Ile, X33 is Leu, X34 does not exist. X21 is Lys, and the amino(ε-amino) group in the side chain of Lys is acylated at the following site. And, [ka] In the formula, Q and T are absent. U is -C(O)-CH2-O-(CH2)2-O-(CH2)2-NH-, and in the formula ,} is the attachment point with the base W, W is -C(O)-(CH3)2-NH-], where ] is the attachment point with group Y. Y is -C(O)-(CH2)2-CH(COOH)NH--, and in the formula -- is the group Z and It is the attachment point, Z is -C(O)-(CH2) n -COOH, where n is an integer 16, polypeptide We will provide Chido.

[0045] In some embodiments, the present disclosure relates to a polypeptide comprising the following amino acid sequence. , H-X2-X3-X4-GTFTSDVSSYL-X16-GQ -AA-X21-EF-X24-AWLVRGRG-X33-X34 In the equation, X2 is Aib, X3 does not exist. X4 is Glu, X16 is Glu, X24 is Ile, X33 is Leu, X34 does not exist. X21 is Lys, and the amino(ε-amino) group in the side chain of Lys is acylated at the following site. And, [ka] In the formula, Q and T are absent. U is -C(O)-CH2-O-(CH2)2-O-(CH2)2-NH-, and in the formula ,} is the attachment point with the base W, W is -C(O)-NH-(CH2) 3-4 -NH-], where ] is the attachment point with base Y. And, Y is -C(O)-(CH2)2-CH(COOH)NH--, and in the formula -- is the group Z and It is the attachment point, Z is -C(O)-(CH2) n -COOH, where n is an integer 16, polypeptide We will provide Chido.

[0046] In some embodiments, X21 is Lys, and the side chain amino(ε-amino) group of Lys The following part is acylated: [ka] In the formula, W is -C(O)-NH-(CH2)4-NH-].

[0047] In some embodiments, X21 is Lys, and the side chain amino(ε-amino) group of Lys The following part is acylated: [ka] In the formula, W is -C(O)-NH-(CH2)3-NH-].

[0048] In some embodiments, the present disclosure relates to a polypeptide comprising the following amino acid sequence. , H-X2-X3-X4-GTFTSDVSSYL-X16-GQ -AA-X21-EF-X24-AWLVRGRG-X33-X34 In the equation, X2 is Aib, X3 does not exist. X4 is Glu, X16 is Glu, X24 is Ile, X33 is Leu, X34 does not exist. X21 is Lys, and the amino(ε-amino) group in the side chain of Lys is acylated at the following site. And, [ka] In the formula, Q and T are absent. U is -C(O)-CH2-O-(CH2)2-O-(CH2)2-NH-, and in the formula ,} is the attachment point with the base W, W is -C(O)-NH-(CH2)4-NH-], where ] is the attachment point of group Y. , Y is -C(O)-(CH2)2-CH(COOH)NH--, and in the formula -- is the group Z and It is the attachment point, Z is -C(O)-(CH2) n -COOH or -C(O)-(CH2) n -CH3 The formula provides polypeptides where n is an integer between 14 and 20.

[0049] In some embodiments, X21 is Lys, and the side chain amino(ε-amino) group of Lys The following part is acylated: [ka] In the formula, Z is -C(O)-(CH2) n The expression is -COOH, where n is the integer 16.

[0050] In some embodiments, X21 is Lys, and the side chain amino(ε-amino) group of Lys The following part is acylated: [ka] In the formula, Z is -C(O)-(CH2) n -CH3, where n is an integer 14.

[0051] In some embodiments, the present disclosure relates to a polypeptide comprising the following amino acid sequence. , H-X2-X3-X4-GTFTSDVSSYL-X16-GQ -AA-X21-EF-X24-AWLVRGRG-X33-X34 In the equation, X2 is Aib, X3 does not exist. X4 is Glu, X16 is Glu, X24 is Ile, X33 is Leu, X34 does not exist. X21 is Lys, and the amino(ε-amino) group in the side chain of Lys is acylated at the following site. And, [ka] In the formula, Q and T are absent. U is -C(O)-CH2-O-(CH2)2-O-(CH2)2-NH-, and in the formula ,} is the attachment point with the base W, W [ka] In the formula, ] is the attachment point with base Y, Y is -C(O)-(CH2)2-CH(COOH)NH--, and in the formula -- is the group Z and It is the attachment point, Z is -C(O)-(CH2)n-COOH, where n is an integer 16. We will provide Chido.

[0052] In some embodiments, the present disclosure relates to a polypeptide comprising the following amino acid sequence. , H-X2-X3-X4-GTFTSDVSSYL-X16-GQ -AA-X21-EF-X24-AWLVRGRG-X33-X34 In the equation, X2 is Ser, Ser(OMe), D-Ser, D-Ser(OMe), X3 does not exist. X4 is Glu, X16 is Glu, X24 is Ile, X33 is Leu, X34 does not exist. X21 is Lys, and the amino(ε-amino) group in the side chain of Lys is acylated at the following site. And, [ka] In the formula, Q and T are absent. U is -C(O)-CH2-O-(CH2)2-O-(CH2)2-NH-, and in the formula ,} is the attachment point with the base W, W is -C(O)-CH2-O-(CH2)2-O-(CH2)2-NH-, -C(O)- NH-(CH2) 3-4 Select from -NH-, -C(O)-C(CH3)2-NH- In the formula, ] is the attachment point with base Y, Y is -C(O)-(CH2)2-CH(COOH)NH--, and in the formula -- is the group Z and It is the attachment point, Z is -C(O)-(CH2) n -COOH or -C(O)-(CH2) n -CH3 The formula provides polypeptides where n is an integer between 14 and 20.

[0053] In some embodiments, X21 is Lys, and the side chain amino(ε-amino) group of Lys The following part is acylated: [ka] In the formula, W is -C(O)-NH-(CH2) 3-4 -NH-]

[0054] In some embodiments, X21 is Lys, and the side chain amino(ε-amino) group of Lys The following part is acylated: [ka] In the formula, W is -C(O)-CH2-O-(CH2)2-O-(CH2)2-NH-.

[0055] In some embodiments, X21 is Lys, and the side chain amino(ε-amino) group of Lys The following part is acylated: [ka] In the formula, W is -C(O)-C(CH3)2-NH-.

[0056] In some embodiments, X21 is lipid-modified Lys, and the side chain amino(ε) of Lys The mino group is, [ka] The portion represented by the portion provided in Table 1 is acylated. [Table 1]

[0057] In another embodiment, the present disclosure is a prior embodiment selected from the peptides provided in Table 2. The polypeptide described in any one of the application forms is provided. [Table 2] TIFF2026075626000033.tif224164TIFF2026075626000034.tif208163

[0058] Unless otherwise specified, this disclosure covers both the L and D isomers of amino acids in the sequence. It is intended to do so.

[0059] As described herein, Ser(OMe) is a hydroxyl group with methyl It is a serine amino acid that has been molten and has the following structure. [ka]

[0060] The polypeptide sequences referred to in this disclosure are single amino acid sequences approved by IUPAC. It is represented by a character code.

[0061] When used herein to define the acylated portion of an embodiment of the present disclosure, Q, T U, W, Y, and Z are single amino acids used to indicate polypeptide sequences. It is different from character encoding.

[0062] The polypeptides of this disclosure, surprisingly, showed results in oral glucose loading tests in SD rats. When subjected to an oral glucose tolerance test (OGTT), it showed a significant decrease in blood glucose. If an additional Leu or Ile is used at the X33 position, the rate of blood glucose reduction in SD rats is... It was significantly lower than the lacking corresponding polypeptide.

[0063] The present invention will be described in further detail with reference to the following embodiments. The embodiments are described in all respects. It is preferable to consider these as illustrative examples and to limit the scope of the claimed invention. That was not the intention. [Examples]

[0064] General preparation method: Polypeptide compounds of this disclosure are prepared by the methods described below herein. This can be done. The process involves preparing the parent linear peptide and the parent peptide of the fatty acid chain thereafter. Two steps are involved, including adhesion to the material.

[0065] The peptides described herein are produced using solid-phase techniques, e.g., G. Barany and RBM. errifield's "The Peptide:Analysis,Synthesis" s, Biology, Volume 2, "Special Methods in Peptide "Synthesis, Part A," pp. 3-284, E. Gross and J. Me Edited by Ienhofer, Academic Press, New York, 1980. Rabini JMStewart and JDYoung's "Solid-Phase "Peptide Synthesis," 2nd edition, Pierce Chemical Co. Chemical compounds using those described in Rockford, Ill., 1984, etc. It can be prepared by [method]. The preferred strategy is tert for the transient protection of amino acid side chains. -Butyl (-tBu), tert-butyloxycarbonyl (-Boc), trityl (- Fmoc( for temporary protection of α-amino groups in combination with protecting groups such as Trt) groups Based on the 9-fluorenylmethyl-oxycarbonyl group (for example, E. Atherto n and RCSheppard's "The Fluorenylmethoxyca rbonyl Amino Protecting Group”, “The Pept ides: Analysis, Synthesis, Biology, Volume 9 "Spec ial Methods in Peptide Synthesis,Part C” Pages 1-38, edited by S. Undenfriend and J. Meienhofer, Aca (See Democracy Press, San Diego, 1987).

[0066] Peptides are mediated stepwise in an insoluble polymer support (also called "resin"), The synthesis can be started from the C-terminus of the plutide. The synthesis involves amide or ester bonds. This process begins by attaching the C-terminal amino acid of the peptide to the resin through its formation. This allows for the final release of the peptides obtained as C-terminal amides or carboxylic acids, respectively. It becomes possible to exit.

[0067] The C-terminal amino acids and all other amino acids used in the synthesis are α-amino acids. To allow protective groups to be selectively removed during synthesis, their α-amino groups and side-chain functional groups ( It is necessary to differentially protect the amino acid bond, active ester (if present). Activation of its carboxyl group, and non-blocking of the N-terminal amino acid attached to the resin. This is carried out by a reaction with an α-amino group. Deprotection and coupling of the α-amino group Repeat the column until the entire peptide sequence is assembled. Then, the peptide is processed normally as follows: In the presence of a scavenger, the side chain functional groups are released from the resin using simultaneous deprotection, and side reactions occur. The process is restricted. The resulting peptide is finally purified by reverse-phase HPLC.

[0068] Next, by binding the activated fatty acid chain to the parent peptide, the parent peptide becomes the fatty acid chain It can be bonded to. Fatty acid chains can be prepared by well-known organic chemical methods. For example, fatty acid chains are prepared using solid-phase synthesis methods that enable the preparation of straight fatty acid chains. It is possible.

[0069] The synthesized linear peptide was prepared by a preparative HPLC procedure, as outlined below. Ta. Preparative HPLC: WATERS 2555 Quaternary Gradient M Odule (Maximum total flow rate: 300 mL / min, Maximum pressure: 3000 psi) Shimadzu LC-8A (maximum total flow rate: 150mL, maximum pressure: 20Mpa) Column: C18, 10μ Flow rate: 75mL / min Mobile phase: In the case of the first purification Mobile phase A: Phosphate buffer at pH 7.5 Mobile phase B: Acetonitrile Gradient: Mobile phase B with a gradient of 10-40% over 300 minutes Second purification case Mobile phase A: 1% acetic acid in water Mobile phase B: 1% acetic acid:n-propanol in acetonitrile (50:50) Gradient: Mobile phase B with a gradient of 15-45% over 300 minutes

[0070] The final compound of this disclosure was purified by a preparative HPLC procedure, as outlined below. Preparative HPLC: WATERS 2555 Quaternary Gradient M Odule (Maximum total flow rate: 300 mL / min, Maximum pressure: 3000 psi) Shimadzu LC-8A (maximum total flow rate: 150mL, maximum pressure: 20Mpa) Column: C18, 10μ Flow rate: 75mL / min [Table 3]

[0071] The purity of the compounds disclosed herein was analyzed by RP-HPLC, as outlined below. . HPLC method B1: Column: YMC Pack-Ph (4.6mm x 150mm, 3μ) Eluent: Mobile phase A: 0.1% trifluoroacetic acid in water Mobile phase B: 0.1% trifluoroacetic acid in acetonitrile Flow rate: 1.5 mL / min Detection: UV detection at 210 nm Column temperature: 50 °C Run time: 50 minutes

Table 4

Table 5

Table 6

[0072] The compounds of the present disclosure were analyzed by LCMS as outlined below.​​

[0073] The mass spectrum is from Waters Acquity® QDa®. Waters Micromass Quattro Micro API, or Th Using ermo Scientific LCQ Fleet®, on LCMS This was recorded. The test solution contained a suitable amount of analyte, at a concentration of 1 μg / ml depending on the ionization of the analyte. The test solution was prepared by dissolving it in a diluent at a final concentration of ~50 μg / ml. The sample was injected into the LCMS at a rate of approximately 10 μl to 50 μl per minute for 1 minute, and the mass spectrum was obtained. Recorded in positive or negative trospray ionization (ESI) mode within an appropriate mass range. Ta.

[0074] Example 1: Preparation of activated fatty acid side chains: 1.18-[[(1S)-1-carboxy-4-[2-[2-[2-[2-[2-[2- (2,5-Dioxopyrrolidine-1-yl)oxy-2-oxo-ethoxy]ethoxy] Ethylamino]-2-oxo-ethoxy]ethoxy]ethylamino]-4-oxo-buty Preparation of [L]amino]-18-oxo-octadecanoic acid (partially A-OSu, intermediate 3) The activated fatty acid side chain, part A-OSu, is schematically represented in Figure 1A as 2-k It was prepared by solid-phase synthesis using lorotrityl chloride resin. 2-[2-(2-Fm oc-aminoethoxy)ethoxy]acetic acid (intermediate 1) is converted to N,N'-di-isopropyl acetate It is attached to a 2-chlorotrityl chloride resin in the presence of chloroamine (DIPEA), -[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid-2-Cl-Trt-resin Obtained. Intermediate 1 is Fmoc N of 2-[2-(2-aminoethoxy)-ethoxy]acetic acid. -It can be prepared by bonding with hydroxysuccinimide ester. Intermediate 1 is commercially available and can be procured in that manner. The Fmoc protecting group is pi 2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid-2- The amino groups of the Cl-Trt- resin are removed by selective deblockage, and then the free amino groups are removed. This is done using 1-hydroxybenztriazole (HOBt) and N,N'-diisopropyl Using carbodiimide (DIPC), 2-[2-(2-Fmoc-aminoethoxy) It combines with toxyacetic acid to form 2-[2-[2-[[2-[2-(2-Fmoc-aminoethyl [xy)ethoxy]acetyl]amino]ethoxy]ethoxy]ethoxy]acetic acid-2-Cl- Trt-resin was obtained. Next, the Fmoc group was treated with piperidine using 2-[2-[2-[ [2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetyl]amino]ethoxy The amino groups of ethoxyacetic acid-2-Cl-Trt resin are removed by selective deblockage. Next, the free amino groups are removed using HOBt and DIPC with Fmoc-Glu-Ot Binding to Bu, 2-[2-[2-[[2-[2-[2-[[(4S)-4-Fmoc- Amino-5-tert-butoxy-5-oxo-pentanoyl]amino]ethoxy]eth A resin was obtained from which xy[acetyl]amino[ethoxy]ethoxy]acetic acid-2-Cl-Trt-resin was obtained. The obtained 2-[2-[2-[[2-[2-[2-[[(4S)-4-Fmoc-amino- 5-tert-butoxy-5-oxopentanoyl]amino]ethoxy]ethoxy]a Cetyl]amino]ethoxy]ethoxy]acetic acid-2-Cl-Trt-resin, piperidine Used to selectively deblock and then combine with octadecanedioic acid monoter-butyl ester to obtain Intermediate 2, namely [2-[2-[2-[[2-[2-[2-[[(4S)-5 -tert-butoxy-4-[(18-tert-butoxy-18-oxo-octadeca no-yl)amino]-5-oxo-pentanoyl]amino]ethoxy]ethoxy]acetyl amino]ethoxy]ethoxy]acetic acid]-2-Cl-Trt resin. Then, Intermediate 2 was cleaved from the 2-Cl-Trt resin using trifluoroethanol:DCM (1:1). Subsequently, the resulting compound was reacted with N-hydroxysuccinimide (HOSu in the presence of isobutyl chloroformate (IBCF) and N-methylmorpholine (NMM), followed by deprotection with trifluoroacetic acid to obtain the title compound (Part A-OS u, Intermediate 3). The entire process can also be shown as schematically represented in Figure 1B.

[0075] 2. Preparation of N-palmitoyl-L-γ-glutamyl succinimide ester (Part B-OSu)

Chemical Structure

[0076] ​​​3.18-[[(1S)-1-carboxy-4-[4-[2-[2-[2-(2,5-di Oxopyrrolidine-1-yl)oxy-2-oxo-ethoxy]ethoxy]ethylcarb [moylamino]butylamino]-4-oxo-butyl]amino]-18-oxo-octa Preparation of Decanoic Acid (Partially C-OSu) The activated fatty acid side chain, the C-OSu portion, is converted to 2-clo, as schematically shown in Figure 2. It was prepared using solid-phase synthesis with rotrityl chloride resin. 2-[2-(2-Fm oc-aminoethoxy)ethoxyacetic acid is converted to 2-chlorotritil acetate in the presence of DIPEA. Adhering to a lorido resin, 2-[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid A -2-Cl-Trt- resin was obtained. The Fmoc protecting group was modified using piperidine. The free amino groups are removed by selective deblockage, and then the free amino groups are removed from THF and DIPEA. Activation using p-nitrophenyl chloroformate, followed by THF:DMAc and Reacting with Fmoc-aminobutylamine hydrochloride in DIPEA, 2-[2-[2-( 4-Fmoc-aminobutylcarbamoylamino)ethoxy]ethoxy]acetic acid-2-Cl -Trt- resin was obtained. The Fmoc group was removed by selective deblockage using piperidine. Then, the free amino groups are removed using HOBt and DIPC, and Fmoc-Glu- Binds to OtBu, 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 obtained. [4-[[(4S)-4-Fmoc-amino-5-tert-butoxy-5-oxo-pe [Hantanoyl]amino]-butylcarbamoylamino]ethoxy]ethoxy]acetate-2-C l-Trt-resin is selectively deblocked using piperidine, followed by octadecanediol. It combines with monotert-butyl ester to form the intermediate 2-[2-[2-[4-[[(4S) -5-tert-butoxy-4-[(18-tert-butoxy-18-oxo-octa Decanoyl)amino]-5-oxo-pentanoyl]amino]butylcarbamoylamino [Ethoxy]ethoxy]acetic acid]-2-Cl-Trt-resin was obtained. Next, the intermediate was t 2-Cl-Trt resin was cleaved using difluoroethanol:DCM (1:1). te, 2-[2-[2-[4-[[(4S)-5-tert-butoxy-4-[(18-t ert-butoxy-18-oxo-octadecanoyl)amino]-5-oxo-pentano Il-amino-butylcarbamoylamino-ethoxy-ethoxy-acetic acid (LCMS=m / z:814.56(M+H + Next, the obtained compound was divided into dicyclohexylka. The succinimide protected intermediate is obtained by reacting it with HOSu in the presence of rubodiimide (DCC). This is then deprotected with trifluoroacetic acid to obtain the title compound (partially C-OSu). Ta.

[0077] 4.18-[[(1S)-1-carboxy-4-[[2-[2-[2-(2,5-dioxy Sopyrolidine-1-yl)oxy-2-oxo-ethoxy]ethoxy]ethylamino]- 1,1-dimethyl-2-oxoethyl]amino]-4-oxobutyl]amino]-1 Preparation of 8-oxo-octadecanoic acid (partially D-OSu) As schematically shown in Figure 3, the fatty acid side chains are made using 2-chlorotrityl chloride resin. It was prepared using solid-phase synthesis. 2-[2-(2-Fmoc-aminoethoxy)eth [Xy]acetic acid is attached to a 2-chlorotrityl chloride resin in the presence of DIPEA, and 2 -[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid-2-Cl-Trt-resin The Fmoc protecting group was removed by selective deblockage of the amino group using piperidine. Then, using DIPC and HOBt, Fmoc-Aib- in THF:DMAc It combines with OH to form 2-[2-[2-[(2-Fmoc-amino-2-methyl-propanoid A resin was obtained using the Fmoc group. The free amino group is removed by selective deblockage using piperidine, and HOBt and By using DIPC, it binds with Fmoc-Glu-OtBu, and 2-[2-[2--[[ 2-[[(4S)-4-Fmoc-amino-5-tert-butoxy-5-oxo-pene Tanoyl]amino]-2-methyl-propanoyl]amino]ethoxy]ethoxy]acetic acid- A 2-Cl-Trt resin was obtained. The Fmoc group of the obtained compound was modified using piperidine. Selectively deblocking, and then the free amino group, octadecane dioxide monotert-butyl It combines with tert to form 2-[2-[2-[[2-[[(4S)-5-tert-butoxy-4 -[(18-tert-butoxy-18-oxo-octadecanoyl)amino]-5-o Xo-pentanoyl]amino]-2-methyl-propanoyl]-amino]ethoxy]eth A resin was obtained from xyacetic acid-2-Cl-Trt. Next, the intermediate was trifluoroethanol Cut from 2-Cl-Trt-resin using DCM(1:1), 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 (LCMS=m / z:7 86.39(M+H + Next, the obtained compound was prepared in the presence of DCC using HOSu This is reacted with to obtain a succinimide protected intermediate, which is then deprotected using trifluoroacetic acid. By following the procedure, the title compound (partially D-OSu) was obtained.

[0078] 5.18-[[(1S)-1-carboxy-4-[3-[2-[2-[2-(2,5-di Oxopyrrolidine-1-yl)oxy-2-oxo-ethoxy]ethoxy]ethylcarb [moylaminopropylamino]-4-oxo-butyl]amino]-18-oxo-oxy Preparation of tadecanoic acid (partially E-OSu) As schematically shown in Figure 4, the fatty acid side chain is made using 2-chlorotrityl chloride resin. It was prepared using solid-phase synthesis. 2-[2-(2-Fmoc-aminoethoxy)eth [Xy]acetic acid is attached to a 2-chlorotrityl chloride resin in the presence of DIPEA, and 2 -[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid-2-Cl-Trt-resin The Fmoc protecting group was removed by selective deblockage of the amino group using piperidine. Next, the free amino group is converted to p-nitrophenylchlorophosphate in THF and DIPEA. Activate using Lumate, then use HOBt in the presence of DIPEA to activate THF:D It reacts with 1,3-diaminopropane in MAc, and NH2-(CH2)3-NH-C( O)-{(2-(2-amino-ethoxy)-ethoxy}-acetic acid-2-Cl-Trt-resin A was formed. Then, the free amino group was removed using HOBt and DIPC to form Fmoc-G lu-OtBu combines with this, resulting in 2-[2-[2-[3-[[(4S)-4-Fmo c-amino-5-tert-butoxy-5-oxo-pentanoyl]amino]propyl A rubamoylamino]ethoxy]ethoxy]acetic acid-2-Cl-Trt resin was obtained. ta2-[2-[2-[3-[[(4S)-4-Fmoc-amino-5-tert-butoki C-5-oxo-pentanoyl]amino]propylcarbamoylamino]ethoxy]-e Toxy]acetic acid-2-Cl-Trt resin is selectively deblocked using piperidine, and then Then it binds with octadecane diacitate monotertbutyl ester, and 2-[2-[2-[3- [[(4S)-5-tert-butoxy-4-[(18-tert-butoxy-18-o Xo-octadecanoyl)amino]-5-oxo-pentanoyl]amino]propylcal Bamoylamino]ethoxy]ethoxy]acetic acid]-2-Cl-Trt-resin was obtained. The intermediate is prepared using trifluoroethanol:DCM (1:1) and 2-Cl-Trt- Cut from the resin, 2-[2-[2-[[3-[[(4S)-5-tert-butoxy- 4-[(18-tert-butoxy-18-oxo-octadecanoyl)amino]-5- Oxo-pentanoyl amino propylcarbamoyl amino ethoxy ethoxy Acetic acid (LCMS=m / z:801.41(M+H + )) was obtained. Next, the obtained compound It is reacted with HOSu in the presence of dicyclohexylcarbodiimide (DCC), succin An imide-protected intermediate is obtained, which is then deprotected with trifluoroacetic acid to obtain the title compound ( Partial E-OSu was obtained.

[0079] 6.18-[[(1S)-1-carboxy-4-[4-[2-[2-[2-(2,5-di Oxopyrrolidine-1-yl)oxy-2-oxo-ethoxy]ethoxy]ethylcarb [moylamino]-1-piperidyl]-4-oxo-butyl]amino]-18-oxo-o Preparation of kutadecanoic acid (partially F-OSu) As schematically shown in Figure 5, the fatty acid side chain is made using 2-chlorotrityl chloride resin. It was prepared using solid-phase synthesis. 2-[2-(2-Fmoc-aminoethoxy)eth [Xy]acetic acid is attached to a 2-chlorotrityl chloride resin in the presence of DIPEA, and 2 -[2-(2-Fmoc-aminoethoxy)ethoxy]acetic acid-2-Cl-Trt-resin The Fmoc protecting group was removed by selective deblockage of the amino group using piperidine. Next, the free amino group is converted to p-nitrophenylchlorophosphate in THF and DIPEA. Activate using Lumate, then use HOBt in the presence of DIPEA to activate THF:D It reacts with 4-amino-Boc-piperidine in MAc, (2-[2-[2-(4-B oc-piperidylcarbamoylamino)ethoxy]ethoxy]acetate-2-Cl-Trt- A resin was obtained. The compound obtained by cleavage using trifluoroacetic acid was 2-[2-[ This yields 2-(4-piperidylcarbamoylamino)ethoxy]ethoxy]acetic acid, and this This is further reacted with Fmoc-OSu in the presence of triethylamine (TEA). Therefore, 2-[2-[2-(4-Fmoc-piperidylcarbamoylamino)ethoxy] Ethoxyacetic acid was obtained. The obtained compound was then 2-chlorotized in the presence of DIPEA. Further adhering to the lithylchloride resin, 2-[2-[2-(4-Fmoc-piperidyl A carbamoylamino)ethoxy]ethoxy]acetic acid-2-Cl-Trt resin was obtained. Fm The oc group is removed by selective deprotection using piperidine, and then the free amino group is removed. This is combined with Fmoc-Glu-OtBu using HOBt and DIPC, and 2-[ 2-[2-[[1-[(4S)-4-amino-5-tert-butoxy-5-oxo-pe [Hentanoyl]-4-piperidyl]carbamoylamino]ethoxy]ethoxy]acetic acid-2- Cl-Trt-resin was obtained. The obtained compound was selectively deblocked using piperidine. Next, it combines with octadecane dioxide monotert-butyl ester, and 2-[2-[2 -[[1-[(4S)-5-tert-butoxy-4-[(18-tert-butoxy- 18-Oxo-octadecanoyl)amino]-5-Oxo-pentanoyl]-4-piperine A resin was obtained from [zyl]carbamoylamino]ethoxy]ethoxy]acetic acid-2-Cl-Trt-resin. Next, the intermediate is converted to 2-Cl- using trifluoroethanol:DCM (1:1). Cut from Trt resin, 2-[2-[2-[[1-[(4S)-5-tert-but Xy-4-[(18-tert-butoxy-18-oxo-octadecanoyl)amino] -5-oxo-pentanoyl]-4-piperidyl]carbamoylamino]ethoxy]eth xy]acetic acid (LCMS=m / z:827.40(M+H + )) was obtained. Next, obtained The compound was reacted with HOSu in the presence of DCC to obtain a succinimide protected intermediate, and this The compound was deprotected using trifluoroacetic acid to obtain the title compound, partial F-OSu.

[0080] Example 2: Compound 1: N-ε 26-[2-(2-[2-(2-[2-(2-[4-(17-Carboxyheptade Canoylamino)-4(S)carboxybutyrylamino]ethoxy)ethoxy]acetyl [Amino)ethoxy]Ethoxy)acetyl][(Aib 8 Arg 34 , Leu 38 GLP -1(7-38) Peptide Synthesis Part A. Parent linear peptide Aib 8 Arg 34 , Leu 38 GLP-1(7-38) synthesis The parent peptide was synthesized by a solid-phase method. The starting resin used in the synthesis was Wang resin. Fmoc-protected leucine was used for bonding with Wang resin. The bond was formed by 4-dimethylamine. In the presence of minopyridine (DMAP), diisopropylcarbodiimide, as a binding agent This is performed using N-hydroxybenzotriazole (DIC-HOBt). Fmoc-Leu-Wang resin was obtained using piperidine. Following the selective deblockage of amino groups in the ang resin, HOBt and DIPC are used to perform F By bonding with moc-Gly-OH, Fmoc-Gly-Leu-Wang resin This yielded [result]. This completes one cycle. Acetate anhydride and diisopropylethyl [result] Min / pyridine was used to terminate the unbound amino groups in each amino acid bond.

[0081] The two steps described above, namely, selective de-barriering of Fmoc protection of amino acids attached to the resin. Breakdown, and the bond with the Fmoc-protected amino group of the adjacent amino acid residue in the sequence, the remaining 30 The process was repeated for each amino acid residue. Selective deblockage, i.e., deprotection of the Fmoc group, This is done using piperidine, and the binding to the adjacent Fmoc-protecting amino acid is performed using HOBt / DIPC. The procedure was carried out using the following: The side chains of Fmoc-protected amino acids are protected orthogonally, for example, serine, cyanoacrylate. The hydroxyl group of rosine or threonine is protected with a tert-butyl (-tBu) group. The amino and guanide groups of lysine and arginine are, respectively, tert-butyl. Oxycarbonyl (-Boc) and 2,2,4,6,7-pentamethyldihydrobenzo Protected by a furan-5-sulfonyl (-Pbf) group, the histidine imidazole is tri Protected with til (-Trt), the carboxylic acid group of aspartic acid or glutamic acid is t Protected with Bu group. The two steps described above, namely selective deblocking, and then adjacent The Fmoc-His(Trt)-Aib-G was formed by binding with an Fmoc-protecting amino acid. lu(OtBu)-Gly-Thr(tBu)-Phe-Thr(tBu)-Ser(t Bu)-Asp(OtBu)-Val-Ser(tBu)-Ser(tBu)-Tyr( tBu)-Leu-Glu(OtBu)-Gly-Gln-Ala-Ala-Lys(B oc)-Glu(OtBu)-Phe-Ile-Ala-Trp-Leu-Val-Ar g(Pbf)-Gly-Arg(Pbf)-Gly-Leu-resin was obtained.

[0082] Fmoc-His(Trt)-Aib-Glu(OtBu)-G using piperidine ly-Thr(tBu)-Phe-Thr(tBu)-Ser(tBu)-Asp(Ot Bu)-Val-Ser(tBu)-Ser(tBu)-Tyr(tBu)-Leu-G lu(OtBu)-Gly-Gln-Ala-Ala-Lys(Boc)-Glu(Ot Bu)-Phe-Ile-Ala-Trp-Leu-Val-Arg(Pbf)-Gly De-blocking of the -Arg(Pbf)-Gly-Leu- resin, followed by trifluoroacetic acid and ethane. Crude H-His-Aib was obtained by cleavage and deprotection using -1,2-dithiols. -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 38 GLP-1(7-38) peptide was obtained, This was purified by HPLC.

[0083] Part B: The activated fatty acid chain, part A-OSu, is purified (linear peptide) H obtained in part A. -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 is transplanted in acetonitrile with a pH of approximately 10. The title peptide was obtained and purified by preparative HPLC. The compound's characteristics are shown in Table 3. To provide.

[0084] Example 3: Preparation of compounds 2, 3, 5, 9, 10, and 12 Linear peptides of compounds 2, 3, 5, 9, 10, and 12 are described in Part A of Example 1. It was prepared by a solid-phase method using a similar process as described above. Following the process of Part B of Example 1 This involves transplanting the A-OSu portion, which is an activated fatty acid chain, onto each linear peptide. Compounds 2, 3, 5, 9, 10, and 12 were obtained.

[0085] Example 4: Preparation of compounds 4 and 11: The linear peptides of compounds 4 and 11 were obtained by a similar process described in Part A of Example 2. It was prepared by a solid-phase method, however, here, Fmoc-protected D-leucine was initially used. The ang resin was bonded, and then other amino acids were bonded sequentially. The process of part B of Example 2 According to the procedure, the activated fatty acid chain portion A-OSu is transplanted onto each linear peptide. By doing so, compounds 4 and 11 were obtained.

[0086] Example 5: Preparation of Compound 8 Linear peptides were prepared by a solid-phase method using a similar process described in Part A of Example 2. However, in this case, Fmoc-protected isoleucine is first bonded to the Wang resin. Next, other amino acids were sequentially attached. Following the process of Part B of Example 2, the activated lipid Compound 8 was obtained by transplanting the A-OSu portion, which is a malformed acid chain, onto a linear peptide.

[0087] Example 6: Preparation of Compound 6 Linear peptides were prepared by a solid-phase method using a similar process described in Part A of Example 2. It was prepared by following a similar process to Part B of Example 2, which involves the activated fatty acid chain, part BO Compound 6 was obtained by transferring Su onto a linear peptide.

[0088] Example 7: Preparation of Compound 7 Following a similar process to Part B of Example 2, the activated fatty acid chain, part B-OSu, is obtained. Compound 7 was obtained by transferring it onto the linear peptide of Part A of Example 2.

[0089] Example 8: Preparation of Compound 13 Following a process similar to Part B of Example 1, the activated fatty acid chain, part C-OSu, is obtained. Compound 13 was obtained by transferring it onto the linear peptide of Part A of Example 1.

[0090] Example 9: Preparation of Compound 14 Linear peptides were prepared by a solid-phase method using a similar process described in Part A of Example 2. It was prepared by following a similar process to Part B of Example 2, which involves the activation of a partial CO2 fatty acid chain. Compound 14 was obtained by transferring Su onto a linear peptide.

[0091] Example 10: Preparation of Compound 15 Linear peptides were prepared by a solid-phase method using a similar process described in Part A of Example 2. The process involves first binding Fmoc-protected isoleucine to the Wang resin, and then sequentially bonding it to other amino acids. The process was initiated by binding an acid. Following a similar process to that in Part B of Example 2, the activation By transplanting the fatty acid chain portion C-OSu onto a linear peptide, compound 15 is obtained. I got it.

[0092] Example 11: Preparation of Compound 16 Following a similar process to Part B of Example 2, the activated fatty acid chain, partial D-OSu, is obtained. Compound 16 was obtained by transferring it onto the linear peptide of Part A of Example 2.

[0093] Example 12: Preparation of Compound 17 Following a similar process to Part B of Example 2, the activated fatty acid chain, part E-OSu, is obtained. Compound 17 was obtained by transferring it onto the linear peptide of Part A of Example 2.

[0094] Example 13: Preparation of Compound 18 Following a similar process to Part B of Example 2, the activated fatty acid chain, partial F-OSu, is obtained. Compound 18 was obtained by transferring it onto the linear peptide of Part A of Example 2.

[0095] Characterization data for the synthetic compounds disclosed herein are provided in Table 3 below. [Table 7]

[0096] Example 14: Oral glucose tolerance test (OGTT) in rats, single injection, 1 mg / kg The animals were divided into three groups (normal control group, test group, and third semaglutide group), and 4 were added to each group. The animals were fasted for 12 hours before the start of the OGTT. Compound 1 was administered to the test group animals. The semaglutide group was administered a dose of 1 mg / kg by subcutaneous injection. The results were obtained 22 hours, 166 hours, and 334 hours after subcutaneous injection of the test drug or semaglutide. After a certain time, blood glucose levels were measured using a blood glucose meter (measurement at time 0). Then, all Animals were orally administered a 2 g / kg glucose solution. Blood glucose levels were measured using a glucose test at 20°C. Measurements were taken at 40, 60, 90, and 120 minutes. Body weight and food intake were recorded. Blood The glucose data was analyzed using two-way analysis of variance (ANOVA), and then... Using PRISM (Graph Pad version 5.03) with Bonferron Post-test correction was performed for i. Blood glucose AUC. 0~120分 The data was analyzed using a t-test. did.

[0097] The polypeptides disclosed herein were studied in oral glucose tolerance tests (OGTT) in rats. In this case, a significant glucose-lowering effect was observed compared to the control group. For example, Figure 6 shows, In the test group administered compound 1 and the semaglutide treatment group, at 22 hours and 46 hours Provides changes in blood glucose levels 0-120 minutes after administration. 22 hours after a single dose, normal control. For this, an ANOVA with p<0.001 was performed, followed by Bonferroni's post-test correction. The experiment was conducted, and compound 1 showed a statistically significant decrease in blood glucose levels. The effect was superior to that observed with semaglutide in terms of glucose reduction (see Figure 6A). Even 46 hours after subcutaneous administration, the superiority of compound 1's glucose-lowering effect was observed. (Figure 6B). Furthermore, both compound 1 and semaglutide were observed on day 2 and day 4. Compared to the control group at the time of detection, there was a statistically significant decrease in food intake (Table 4 and (See 5). The reduction in food intake shown by compound 1 on day 4 was greater than that of semaglutide. The effect was significant (see Table 5). Regarding weight loss, only the test compound showed a significant decrease in body weight on day 4. It showed a decrease. [Table 8] [Table 9]

[0098] Surprisingly, compounds having X33 as Leu and Ile were found in the given study. While it showed a significant decrease in blood glucose, compounds containing amino acids other than Leu and Ile... It was found that it has a significantly smaller effect in lowering blood glucose. The polypeptide of this disclosure The group showed a significant decrease in blood glucose compared to the control group. Also, at position X33, Le Compounds containing amino acids other than u and Ile were also tested. For example, compound 1 (SEQ ID NO: The Leu at the 32nd position of 05) is replaced with Lys and Ser, respectively, and the compound labels are formed. Pre-1 and Standard 2 were obtained. Standard 1 and Standard 2 were defined as blood glucose AUC. 0~120分 Approximately 3 The decrease was only 5% and 15% (Table 6). [Table 10]

[0099] Similarly, compound 6 differs from liraglutide in that it has an additional Leu at the 32nd position. , and the second amino acid Ala is replaced with Aib, and Le is added as the additional 32nd amino acid. Compound 7, which differs from liraglutide in that it contains u, shows a blood glucose-lowering effect in 24 hours. This was significantly higher than that of liraglutide (Table 6).

[0100] Compound 1 was significantly better in terms of glucose reduction, food intake, and weight loss. Once it was determined that the compound of the present invention was effective, experiments were conducted to determine the duration of action of the compound. Representative compounds of the present invention (Compound 1) after 66 hours (7 days) and 334 hours (14 days). The effects of compounds 13 and 16) were studied and compared with the effects of semaglutide. The compounds are described below. The method provided was used for testing.

[0101] The animals were divided into three groups (normal control group, test group, and third semaglutide group), and 4 were added to each group. The animals were fasted for 12 hours before the start of the OGTT. Compound 1 was administered to the test group animals. Compounds 13 and 16 were administered by subcutaneous injection at a dose of 1 mg / kg. In the semaglutide group... A dose of 1 mg / kg was administered by subcutaneous injection. 22 doses of the test compound or semaglutide were administered by subcutaneous injection. Blood glucose levels were measured using a blood glucose meter at 1 hour, 166 hours, and 334 hours after the initial time. (Time 0 measurement). Subsequently, all animals were orally administered a 2 g / kg glucose solution. Blood glucose levels were measured at 20, 40, 60, 90, and 120 minutes after the glucose test. Weight and food intake were recorded. Blood glucose data was analyzed using two-way ANOVA. The analysis was performed using ANOVA, followed by PRISM (GraphPad version 5). Bonferroni's post-test correction was performed using 03). Blood glucose AUC 0~120 分 The data was analyzed using a t-test.

[0102] Table 7 shows the reduction in blood glucose AUC for representative compounds of the present invention (compounds 1, 13, and 16). The results are provided in comparison to a control group 1 day, 7 days, and 14 days after administration. [Table 11]

[0103] Compounds 1 and 13 were studied and compared with semaglutide in an experiment (Experiment 1). Compound 16 was studied and compared with semaglutide in a separate experiment (Experiment 2). Injection of compound 16 After 8 hours, compounds 1, 13, and 16 of the present invention, compared to zero-time blood glucose levels, showed a blood glucose level of 8 hours later. It showed a decrease of approximately 60% in glucose AUC. On the other hand, semaglutide showed a decrease in blood glucose relative to zero-time glucose levels. It showed a decrease of approximately 25% in sugar levels.

[0104] Similar observations were made regarding food consumption and weight changes. As can be seen in Table 8 below. Animals administered representative compounds (compounds 1, 13, and 16) were given semaglutide. Compared to the animals given food, they consumed significantly less food. Compound 16 increased the amount of body weight. It showed a significant decrease, demonstrating its potential usefulness in treating obesity. [Table 12] TIFF2026075626000047.tif44146

[0105] Example 15: Reduction of HbA1c in db / db2 diabetic mice after chronic treatment This study was conducted in a diabetic mouse model. The animals were divided into a diabetic control group, a test group, and... The patients were divided into three treatment groups: a semaglutide treatment group and a semaglutide treatment group. Compound 1 of this disclosure was administered at 0.3 mg / k The treatment involves taking a g dose once daily for 3 days (qd x 3), followed by a 0.1 mg / kg dose every other day for 7 doses. (q2d*7), followed by 0.1 mg / kg once every 4 days for 2 dose cycles (q4d ×2) Subcutaneous injection was administered. The same administration regimen was given to the semaglutide treatment group. Blood glucose levels and Weight was measured daily. %HbA1c was measured by column chromatography on day 0. Measurements were taken on days 7, 14, and 27. Cumulative food intake was calculated on day 27. The %HbA1C data was analyzed using two-way ANOVA. Analyze the data, and then use PRISM (Graph Pad version 5.03) to perform the Bonf analysis. Erroni's post-test correction was performed.

[0106] Animals in the test group administered compound 1 showed statistically significant blood glucose levels compared to the diabetic control group. It showed a significant decrease (see Figure 7), and the effect was superior to that of the semaglutide treatment group in the later stages of the study. The animals in the test group administered compound 1 showed the following results, as can be seen in the results provided in Figure 8. A significant decrease in food intake was observed. Figure 8 shows the db / d ratios treated with the control and the test compound. This report provides cumulative food intake data from day 0 to day 27 for B mice. It also includes the test compound and semaglu. Both groups showed a statistically significant decrease in food intake compared to the diabetic control group. Furthermore, the test compound showed significantly lower food intake compared to semaglutide. Compound 1 also showed a significant reduction in body weight compared to the diabetic control group. (Figure 9) This provides the percentage change in body weight for the control and test groups from day 0 to day 27. Item 1 showed a significant reduction of -16% compared to -8% observed in the semaglutide treatment group. The result was small (see Figure 9).

[0107] In diabetes, higher HbA1c levels indicate poorer control of blood glucose levels. It is associated with cardiovascular disease, nephropathy, neuropathy, and retinopathy. In a 27-day study... Compound 1 controls HbA1c levels in db / db2 diabetic mice after chronic treatment. A statistically significant decrease was observed. Table 9 and Figure 10 below show the results for the diabetes control group and compound 1. The HbA1c levels from 0 to 27 days in the group that received chronic treatment are provided. The effect is This was statistically significant even when compared to semaglutide. [Table 13]

[0108] In separate studies, test compounds 1, 13, and 16 were studied, and HbA1c and insulin Phosphorus levels, as well as their effects on cumulative food consumption, weight change, and blood glucose AUC. The results were compared with semaglutide. This study was applied to a diabetic mouse model as described above. The study was conducted in the following format. Animals were divided into three groups: a diabetes control group, an experimental group, and a semaglutide treatment group. The treatment groups were divided into the following: Compound 1, Compound 13, and Compound 13, which are representative compounds of this disclosure. 16 was administered by subcutaneous injection at a dose of 3.04 or 6.078 nM (every other day for up to 28 days). (q2d x 15). The same administration regimen was administered to the semaglutide treatment group. Blood glucose levels and body weight were measured. Measurements were taken daily: %HbA1c and insulin, on day 0, day 14, and day 29. Measurements were taken. Cumulative food intake and weight change were calculated on days 14 and 29. % HbA1c and insulin data are used in a two-way ANOVA. Therefore, we performed the analysis, and then used PRISM (Graph Pad version 5.03) Bonferroni's post-test correction was performed. Meanwhile, blood glucose AUC, weight change, and cumulative Total food intake data was analyzed using one-way analysis of variance (ANOVA). Next, using PRISM (Graph Pad version 5.03), Bonfe Post-test correction of rroni was performed. Animals were kept in the recovery period from day 29 to day 45. No drug treatment was administered during this period. Blood glucose levels and weight were measured during this time. Day 45 In addition, weight change, %HbA1c, and insulin levels were measured.

[0109] The results are provided in Tables 10, 11, and 12 below. [Table 14] [Table 15] [Table 16]

[0110] Representative compounds of this disclosure at doses of approximately 3 nM and 6 nM (compounds 1, 13, and 16) Compared to the control group, the group showed significant improvements in HbA1c, blood glucose levels, food consumption, and body weight. A significant decrease was observed (Table 12). The decrease was demonstrated by semaglutide at a dose of approximately 12 nM. It was equivalent to that of [another product]. Furthermore, the effect was still visible even after 29 days (Tables 13 and 14), and frequently This project aims to develop long-acting drugs that do not require administration and, consequently, increase patient adherence to medication. To demonstrate the potential of the compound in the invention. [Table 17] [Table 18]

[0111] These results indicate that the compounds of the present invention have potential use for the treatment of diabetes and obesity. To demonstrate that it can be found.

Claims

1. A polypeptide comprising the following amino acid sequence, H-X2-X3-X4-G-TFT-S-DV-S-SY-L-X16-G- Q-A-A-X21-E-F-X24-A-W-L-V-R-G-R-G-X33-X3 ４ In the formula, X2 is Ser, Ser(OMe), D-Ser, D-Ser(OMe), Ala , or Aib, X3 does not exist, or it is Gln. X4 is Glu, X16 is Glu, X24 is Ile, X33 is Leu, -D-Leu, D-Ile, or Ile, X34 does not exist. X21 is Lys, and the amino (ε-amino) group in the side chain of Lys is acylated at the following point. It is 【Chemistry 1】 In the formula, Q and T are absent. U is either absent or -C(O)-CH ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ } is -NH-, where} is the attachment point with the base W, W is absent or -C(O)-CH ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -NHH-]、-C(O)-NHH-(CH ２ ) ３－４ -NHH-]、-C(O)-C(CH ３ ) ２ -NH-], and 【Chemistry 2】 A selection is made from the group consisting of, where in the formula, ] is the attachment point with base Y, Y is -C(O)-(CH ２ ) ２ -CH(COOH)NH--, where -- is attached to the base Z. The destination, Z is -C(O)-(CH ２ ) ｎ -COOH or -C(O)-(CH ２ ) ｎ -CH ３ in Yes, a polypeptide in which n is an integer between 14 and 20.

2. X2 is Aib, X3 does not exist, X33 is Leu, U is -C(O)-CH ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -NH-} W is -C(O)-CH ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -NH-] Z is -C(O)-(CH ２ ) ｎ -COOH, where n is an integer 16, claim The polypeptide described in 1.

3. X2 is Aib, X3 does not exist, X33 is Leu, U is -C(O)-CH ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -NH-} W is -C(O)-C(CH ３ ) ２ -NH-] Z is -C(O)-(CH ２ ) ｎ -COOH, where n is an integer 16, claim The polypeptide described in 1.

4. X2 is Aib, X3 does not exist, X33 is Leu, U is -C(O)-CH ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -NH-} W is -C(O)-NH-(CH ２ ) ４ -NH-] Z is -C(O)-(CH ２ ) ｎ -COOH, where n is an integer 16, claim The polypeptide described in 1.

5. X2 is Aib, X3 does not exist, X33 is Leu, U is -C(O)-CH ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -NH-} W 【Transformation 3】 And, Z is -C(O)-(CH ２ ) ｎ -COOH, where n is an integer 16, claim The polypeptide described in 1.

6. X2 is Aib, X3 does not exist, X33 is Leu, U is -C(O)-CH ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -NH-} W is -C(O)-NH-(CH ２ ) ３ -NH-] Z is -C(O)-(CH ２ ) ｎ -COOH, where n is an integer 16, claim The polypeptide described in 1.

7. X2 is Ser, Ser(OMe), D-Ser, D-Ser(OMe), X3 does not exist, X33 is Leu, U is -C(O)-CH ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -NH-} Wが-C(O)-CH ２ -O-(CH ２ ) ２ -O-(CH ２ ) ２ -NH-]、-C(O )-NH-(CH ２ ) ３－４ -NH-], -C(O)-C(CH ３ ) ２ -NH-] 、 Z is -C(O)-(CH ２ ) ｎ -COOH or -C(O)-(CH ２ ) ｎ -CH ３ in The polypeptide according to claim 1, wherein n is an integer from 14 to 20 in the formula.

8. Wが、-C(O)-NH-(CH ２ ) ３－４ -NH-]、-C(O)-C(CH ３ ) ２ A polypeptide according to claim 1, selected from the group consisting of -NH- and the following formulas. 。 【Chemistry 4】

9. X2 is Ala, X3 does not exist, X33 is Leu, W does not exist, Z is -C(O)-(CH ２ ) ｎ -CH ３ Claim 1, wherein n is an integer 14. Polypeptides as described.

10. X2 is Aib, X3 does not exist, X33 is Leu, W does not exist, Z is -C(O)-(CH ２ ) ｎ -CH ３ Claim 1, wherein n is an integer 14. Polypeptides as described.

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