GLP2 receptor agonists and methods of use

Abstract

The present invention provides peptide conjugates comprising peptides that modulate the GLP-2 receptor. The peptide conjugates can be used to treat conditions that respond to modulation of the GLP-2 receptor. The present invention also provides stapled GLP-2 peptide conjugates.

Description

[0001] Cross-references

[0002] This application claims the benefit of U.S. Provisional Application Serial No. 62 / 943,667, filed December 4, 2019, and U.S. Provisional Application Serial No. 62 / 994,791, filed March 25, 2020; which are incorporated herein by reference in their entireties. Background Art

[0003] The development of therapeutic agents is often hampered by short half-lives. The biological half-life of a drug is the time it takes for the drug to lose half of its pharmacological, physiological, or radiological activity. As a result, patients often take higher doses of therapeutic agents more frequently, which can lead to reduced compliance, higher costs, and increased risk of side effects. Therefore, there is a need to develop therapeutic agents with extended half-lives. Summary of the Invention

[0004] Disclosed herein is a peptide conjugate comprising:

[0005] a) peptides that modulate the GLP-2 receptor; and

[0006] b) A staple linked to the peptide at the first and second amino acids.

[0007] In some embodiments, the stapled compound is a compound of formula (I):

[0008]

[0009] in:

[0010] A is optionally substituted alkylene, optionally substituted arylene, optionally substituted heteroarylene, optionally substituted -NR 3 -alkylene-NR 3 -or-N-;

[0011] X 1 and X 2 are independently a bond, -C(=O)-, -alkylene-C(=O)-, -C(=O)-alkylene, or -alkylene-C(=O)NR 3 -, -alkylene-C(=O)NR 3 -alkylene-;

[0012] where X 1 is linked to the first amino acid of the peptide, and X 2 linked to a second amino acid of the peptide;

[0013] R is hydrogen or -(L) s -Y;

[0014] Each L is independently -(CR1 R 2 ) v -, -alkylene-O-, -O-alkylene-, -C(=O)-alkylene-, -alkylene-C(=O)-, -NR 3 -alkylene-, -alkylene-NR 3 -, -S-alkylene-, -alkylene-S-, -S(=O)-alkylene-, -alkylene-S(=O)-, -S(=O)2-alkylene, -alkylene-S(=O)2-, -C(=O)-, -C(=O)NR 3 -、-NR 3 C(=O)-、-NR 3 C(=O)NR 3 -、-NR 3 C(=O)NR 3 -alkylene-, -NR 3 C(=O)-alkylene-NR 3 -, -alkylene-C(=O)NR 3 -, -C(=O)NR 3 -alkylene-, -alkylene-NR 3 C(=O)- or -NR 3 C(=O)-alkylene-;

[0015] v is 2-20;

[0016] R 1 or R 2 are independently hydrogen, halogen, -CN, -OR a 、-SR a 、-S(=O)R b 、-NO2、-NR c R d 、-S(=O)2R d 、-NR a S(=O)2R d 、-S(=O)2NR c R d 、-C(=O)R b 、-OC(=O)R b 、-CO2R a 、-OCO2R a 、-C(=O)NR c R d 、-OC(=O)NR c R d 、-NR a C(=O)NR c R d 、-NR a C(=O)Rb 、-NR a C(=O)OR a , C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C3-C8 cycloalkyl, C2-C8 heterocycloalkyl, aryl or heteroaryl; wherein alkyl, alkenyl, alkynyl and heteroalkyl are optionally substituted by halogen, -OR a or -NR c R d One, two or three of; and cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally substituted by halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR a 、-NR c R d One, two or three substitutions;

[0017] or R 1 and R 2 Together they form a C1-C6 cycloalkyl group or a C1-C6 heterocycloalkyl group;

[0018] Each R 3 are independently hydrogen, -S(=O)R b 、-S(=O)2R a 、-S(=O)2NR c R d 、-C(=O)R b 、-CO2R a 、-C(=O)NR c R d , C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C3-C8 cycloalkyl, C2-C8 heterocycloalkyl, aryl or heteroaryl; wherein alkyl, alkenyl, alkynyl and heteroalkyl are optionally substituted by halogen, -OR a or -NR c R d One, two or three of; and cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally substituted by halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR a or -NR c R d One, two or three substitutions;

[0019] Y is hydrogen, C1-C6 alkyl, -CO2H, -CO2(C1-C6 alkyl), -CO2NH2, -CO2N(alkyl)2, or -CO2NH(alkyl); and

[0020] s is 0-20;

[0021] R ais hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C3-C8 cycloalkyl, C2-C8 heterocycloalkyl, aryl or heteroaryl; wherein alkyl, alkenyl, alkynyl and heteroalkyl are optionally substituted with one, two or three of halogen, -OH, -OMe or -NH2; and cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally substituted with one, two or three of halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OH, -OMe or -NH2;

[0022] R b is C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C3-C8 cycloalkyl, C2-C8 heterocycloalkyl, aryl or heteroaryl; wherein alkyl, alkenyl, alkynyl and heteroalkyl are optionally substituted with one, two or three of halogen, -OH, -OMe or -NH2; and cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally substituted with one, two or three of halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OH, -OMe or -NH2;

[0023] R c and R d each independently represents hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C3-C8 cycloalkyl, C2-C8 heterocycloalkyl, aryl or heteroaryl; wherein alkyl, alkenyl, alkynyl and heteroalkyl are optionally substituted with one, two or three of halogen, -OH, -OMe or -NH2; and cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally substituted with one, two or three of halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OH, -OMe or -NH2;

[0024] or R c and R d Together with the nitrogen atom to which they are attached, they form a heterocycloalkyl or heteroaryl group; wherein the heterocycloalkyl and heteroaryl groups are optionally substituted by one, two or three of halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OH, -OMe or -NH2.

[0025] Also disclosed herein are pharmaceutical compositions comprising the peptide conjugates described herein and a pharmaceutically acceptable excipient.

[0026] Also disclosed herein are methods for treating a disease or condition in a subject in need thereof, comprising administering to the subject a composition comprising a therapeutically effective amount of a peptide conjugate described herein.

[0027] Also described herein are staples of the formula:

[0028]

[0029] in:

[0030] A is optionally substituted alkylene, optionally substituted arylene, optionally substituted heteroarylene, optionally substituted -NR 3 -alkylene-NR 3 -or-N-;

[0031] X 1 and X 2 are independently a bond, -C(=O)-, -alkylene-C(=O)-, -C(=O)-alkylene, -alkylene-C(=O)NR 3 -or-Alkylene-C(=O)NR 3 -alkylene-;

[0032] Y 1 and Y 2 are independently halogen, -COOH, or -S- or -CONH-, independently of a sulfhydryl-containing amino acid, wherein -NH- is part of an amine-containing amino acid in a peptide that modulates the GLP-2 receptor;

[0033] R is hydrogen or -(L) s -Y;

[0034] Each L is independently -(CR 1 R 2 ) v -, -alkylene-O-, -O-alkylene-, -C(=O)-alkylene-, -alkylene-C(=O)-, -NR 3 -alkylene-, -alkylene-NR 3 -, -S-alkylene-, -alkylene-S-, -S(=O)-alkylene-, -alkylene-S(=O)-, -S(=O)2-alkylene, -alkylene-S(=O)2-, -C(=O)-, -C(=O)NR 3 -、-NR 3 C(=O)-、-NR 3 C(=O)NR 3 -、-NR 3 C(=O)NR 3 -alkylene-, -NR 3 C(=O)-alkylene-NR 3 -, -alkylene-C(=O)NR 3 -, -C(=O)NR 3 -alkylene-, -alkylene-NR 3 C(=O)- or -NR 3 C(=O)-alkylene-;

[0035] v is 2-20;

[0036] R 1 or R 2 are independently hydrogen, halogen, -CN, -OR a 、-SR a 、-S(=O)R b 、-NO2、-NR c R d 、-S(=O)2R d 、-NR a S(=O)2R d 、-S(=O)2NR c R d 、-C(=O)R b 、-OC(=O)R b 、-CO2R a 、-OCO2R a 、-C(=O)NR c R d 、-OC(=O)NR c R d 、-NR a C(=O)NR c R d 、-NR a C(=O)R b 、-NR a C(=O)OR a , C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C3-C8 cycloalkyl, C2-C8 heterocycloalkyl, aryl or heteroaryl; wherein alkyl, alkenyl, alkynyl and heteroalkyl are optionally substituted by halogen, -OR a or -NR c R d One, two or three of; and cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally substituted by halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR a 、-NR c R d One, two or three substitutions;

[0037] or R 1 and R 2 Together they form a C1-C6 cycloalkyl group or a C1-C6 heterocycloalkyl group;

[0038] Each R 3 are independently hydrogen, -S(=O)R b 、-S(=O)2R a 、-S(=O)2NR c R d 、-C(=O)Rb 、-CO2R a 、-C(=O)NR c R d , C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C3-C8 cycloalkyl, C2-C8 heterocycloalkyl, aryl or heteroaryl; wherein alkyl, alkenyl, alkynyl and heteroalkyl are optionally substituted by halogen, -OR a or -NR c R d One, two or three of; and cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally substituted by halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR a or -NR c R d One, two or three substitutions;

[0039] Y is hydrogen, C1-C6 alkyl, -CO2H, -CO2(C1-C6 alkyl), -CO2NH2, -CO2N(alkyl)2, or -CO2NH(alkyl); and

[0040] s is 0-20;

[0041] R a is hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C3-C8 cycloalkyl, C2-C8 heterocycloalkyl, aryl or heteroaryl; wherein alkyl, alkenyl, alkynyl and heteroalkyl are optionally substituted with one, two or three of halogen, -OH, -OMe or -NH2; and cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally substituted with one, two or three of halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OH, -OMe or -NH2;

[0042] R b is C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C3-C8 cycloalkyl, C2-C8 heterocycloalkyl, aryl or heteroaryl; wherein alkyl, alkenyl, alkynyl and heteroalkyl are optionally substituted with one, two or three of halogen, -OH, -OMe or -NH2; and cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally substituted with one, two or three of halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OH, -OMe or -NH2;

[0043] R c and R deach independently represents hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C3-C8 cycloalkyl, C2-C8 heterocycloalkyl, aryl or heteroaryl; wherein alkyl, alkenyl, alkynyl and heteroalkyl are optionally substituted with one, two or three of halogen, -OH, -OMe or -NH2; and cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally substituted with one, two or three of halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OH, -OMe or -NH2;

[0044] or R c and R d Together with the nitrogen atom to which they are attached, they form a heterocycloalkyl or heteroaryl group; wherein the heterocycloalkyl and heteroaryl groups are optionally substituted by one, two or three of halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OH, -OMe or -NH2.

[0045] In some embodiments, A is optionally substituted alkylene. In some embodiments, A is -(CH2) t -, wherein t is 1-12. In some embodiments, A is an optionally substituted arylene. In some embodiments, A is -NR 3 -alkylene-NR 3 -. In some embodiments, A is -N-.

[0046] In some embodiments, X 1 and X 2 In some embodiments, X 1 and X 2 In some embodiments, X 1 and X 2 is -CH2-C(=O)-. In some embodiments, X 1 and X 2 are independently -alkylene-C(=O)NR 3 -. In some embodiments, X 1 and X 2 are independently -CH2-C(=O)NR 3 -. In some embodiments, X 1 and X 2 are independently -alkylene-C(=O)NR 3 -alkylene-. In some embodiments, X 1 and X 2 are independently -CH2-C(=O)NR 3 -CH2CH2-.

[0047] In some embodiments, >AR has the following structure:

[0048]

[0049] wherein r1 and r2 are each independently 0-4.

[0050] In some embodiments, >AR has the following structure:

[0051]

[0052] In some embodiments, >AR has the following structure:

[0053]

[0054] Where p1 is 1-5.

[0055] In some embodiments, >AR has the following structure:

[0056]

[0057] In some embodiments, >AR has the following structure:

[0058]

[0059] In some embodiments, s is 1-15. In some embodiments, s is 1-10. In some embodiments, s is 5-15. In some embodiments, s is 5-10.

[0060] In some embodiments, Y is hydrogen or -CO2H.

[0061] In some embodiments, each L is independently -(CR 1 R 2 ) v -, -alkylene-O-, -C(=O)-, -C(=O)NR 3 -、-NR 3 C(=O)-, -alkylene-C(=O)NR 3 -or-alkylene-NR 3 C(=O)-; and v is 2-20.

[0062] In some embodiments, Y 1 and Y 2 In some embodiments, Y 1 and Y 2 In some embodiments, Y 1 and Y 2It is the -S- of two sulfhydryl-containing amino acids in a peptide that regulates the GLP-2 receptor.

[0063] In some embodiments, Y 1 and Y 2 It is the -S- of two sulfhydryl-containing amino acids in the peptide.

[0064] In some embodiments, Y 1 and Y 2 is -CONH-, wherein -NH- is part of two amine-containing amino acids in a peptide that modulates the GLP-2 receptor.

[0065] In some embodiments, Y 1 and Y 2 is -CONH-, wherein -NH- is part of two amine-containing amino acids separated by 7 amino acids in a peptide that modulates the GLP-2 receptor. BRIEF DESCRIPTION OF THE DRAWINGS

[0066] Figure 1A Concentration-response curves of teduglutide and long-acting GLP2R agonists at the human GLP2R are described without serum addition.

[0067] Figure 1B Concentration-response curves of teduglutide and long-acting GLP2R agonists spiked with serum at the human GLP2R are described.

[0068] Figure 2 Concentration-response curves of teduglutide, a long-acting GLP2R agonist, and apraglutide at the mouse GLP2R are described.

[0069] Figure 3 Concentration-response curves of teduglutide and long-acting GLP2R agonists at the GLP2R in cynomolgus monkeys are described.

[0070] Figure 4A Concentration-response curves of teduglutide and long-acting GLP2R agonists at GLP1R are described.

[0071] Figure 4B Concentration-response curves for teduglutide and long-acting GLP2R agonists on GCGR are described.

[0072] Figure 4C Concentration-response curves of teduglutide and long-acting GLP2R agonists on GIPR are described.

[0073] Figure 5AThe thermal stability of GLP2-2G-10Nle-1K-Ex4-K5 (GLP2-K5) and GLP2-2G-1-EX4-L5A (GLP2-L5A) at 4°C over 4 days is described.

[0074] Figure 5B The thermal stability of GLP2-2G-10Nle-1K-Ex4-K5 (GLP2-K5) and GLP2-2G-1-EX4-L5A (GLP2-L5A) at 25°C over 4 days is described.

[0075] Figure 5C The thermal stability of GLP2-2G-10Nle-1K-Ex4-K5 (GLP2-K5) and GLP2-2G-1-EX4-L5A (GLP2-L5A) at 37°C over 4 days is described.

[0076] Figure 5D The thermal stability of GLP2-2G-10Nle-1K-Ex4-K5 (GLP2-K5) and GLP2-2G-1-EX4-L5A (GLP2-L5A) at 70°C over 4 days is described.

[0077] Figure 6A The stability of GLP2-2G-10Nle-1K-Ex4-K5 (GLP2-K5) and GLP2-2G-1-EX4-L5A (GLP2-L5A) at pH 3.3 and 4°C over 4 days is described.

[0078] Figure 6B The stability of GLP2-2G-10Nle-1K-Ex4-K5 (GLP2-K5) and GLP2-2G-1-EX4-L5A (GLP2-L5A) at pH 3.3 and room temperature over 4 days is described.

[0079] Figure 6C The stability of GLP2-2G-10Nle-1K-Ex4-K5 (GLP2-K5) and GLP2-2G-1-EX4-L5A (GLP2-L5A) at pH 37.5 and 4°C over 4 days is described.

[0080] Figure 6D The stability of GLP2-2G-10Nle-1K-Ex4-K5 (GLP2-K5) and GLP2-2G-1-EX4-L5A (GLP2-L5A) at pH 37.5 and room temperature over 4 days is described.

[0081] Figure 6EThe stability of GLP2-2G-10Nle-1K-Ex4-K5 (GLP2-K5) and GLP2-2G-1-EX4-L5A (GLP2-L5A) at pH 8.9 and 4°C over 4 days is described.

[0082] Figure 6F The stability of GLP2-2G-10Nle-1K-Ex4-K5 (GLP2-K5) and GLP2-2G-1-EX4-L5A (GLP2-L5A) at pH 8.9 and room temperature over 4 days is described.

[0083] Figure 7A The hepatic stability of the long-acting GLP2-2G-1-EX4-L5A over 120 minutes was described.

[0084] Figure 7B The hepatic stability of the long-acting GLP2-2G-10Nle-1-EX4-L5A over 120 minutes was described.

[0085] Figure 7C The hepatic stability of the long-acting GLP2-2G-10Nle-1K-EX4-K5 over 120 minutes was described.

[0086] Figure 8 Depicted are mean plasma concentrations of GLP2-2G-1-EX4-L5A in mice over 96 hours.

[0087] Figure 9 Depicted are mean plasma concentrations of GLP2-2G-1-EX4-L5A in cynomolgus monkeys over 504 hours.

[0088] Figure 10 Depicted are mean plasma concentrations of GLP2-2G-10Nle-1-EX4-L5A in mice over 96 hours.

[0089] Figure 11 Depicted are mean plasma concentrations of GLP2-2G-10Nle-1-EX4-L5A in cynomolgus monkeys over 504 hours.

[0090] Figure 12 Depicted are mean plasma concentrations of GLP2-2G-10Nle-1K-EX4-K5 in mice over 96 hours.

[0091] Figure 13 Depicted are mean plasma concentrations of GLP2-2G-10Nle-1K-EX4-K5 in cynomolgus monkeys over 504 hours.

[0092] Figure 14ADepicted are the normalized lengths of the small intestine in wild-type mice that received no treatment, GLP2-2G-1-L5A (GLP2-2G-1-EX4-L5A), and GLP2-2G-5-L5A (GLP2-2G-10Nle-1-EX4-L5A).

[0093] Figure 14B Depicted are the normalized lengths of the small intestine in wild-type mice that received no treatment, GLP2-2G-1-L5A (GLP2-2G-1-EX4-L5A), and GLP2-2G-5-L5A (GLP2-2G-10Nle-1-EX4-L5A).

[0094] Figure 14C Depicted are body weights over 11 days in wild-type mice that received no treatment, received GLP2-2G-1-L5A (GLP2-2G-1-EX4), and received GLP2-2G-5-L5A (GLP2-2G-10Nle-1-EX4).

[0095] Figure 15A Depicted are the small intestine lengths of wild-type mice treated with GLP2-2G-10Nle-1K-EX4-K5 and untreated wild-type mice.

[0096] Figure 15B Depicted are small intestinal weights of wild-type mice treated with GLP2-2G-10Nle-1K-EX4-K5 and untreated wild-type mice.

[0097] Figure 15C Depicted are colon lengths of wild-type mice treated with GLP2-2G-10Nle-1K-EX4-K5 and untreated wild-type mice.

[0098] Figure 15D Depicted are colon weights of wild-type mice treated with GLP2-2G-10Nle-1K-EX4-K5 and untreated wild-type mice.

[0099] Figure 16A Depicted are body weights over 12 days in untreated, GLP2-2G-1-L5A (GLP2-2G-1-EX4-L5), and cyclosporin A-treated mice with induced acute colitis.

[0100] Figure 16B Depicted are normalized colon weights of untreated, GLP2-2G-1-L5A (GLP2-2G-1-EX4-L5), and cyclosporin A-treated mice with induced acute colitis.

[0101] Figure 16C Depicted are normalized small intestinal weights of untreated, GLP2-2G-1-L5A (GLP2-2G-1-EX4-L5), and cyclosporin A-treated mice with induced acute colitis.

[0102] Figure 16D Depicted are crypt depths in the colon of untreated and GLP2-2G-1-L5A (GLP2-2G-1-EX4-L5)-treated wild-type mice and mice with induced acute colitis.

[0103] Figure 16E Jejunal villus length of wild-type mice and mice with induced acute colitis, both untreated and treated with GLP2-2G-1-L5A (GLP2-2G-1-EX4-L5), is depicted.

[0104] Figure 17A Depicted are body weights over 10 days in untreated, GLP2-2G-10Nle-1K-EX4-K5, teduglutide, and cyclosporin A-treated mice with induced acute colitis.

[0105] Figure 17B Depicted are colon lengths of untreated, GLP2-2G-10Nle-1K-EX4-K5, teduglutide, and cyclosporin A-treated mice with induced acute colitis.

[0106] Figure 17C Depicted are the small intestine lengths of untreated, GLP2-2G-10Nle-1K-EX4-K5, teduglutide, and cyclosporin A-treated mice with induced acute colitis.

[0107] Figure 17D Depicted are small intestinal weights of untreated, GLP2-2G-10Nle-1K-EX4-K5, teduglutide, and cyclosporin A-treated mice with induced acute colitis.

[0108] Figure 17E Jejunal villus height of untreated, GLP2-2G-10Nle-1K-EX4-K5, teduglutide, and cyclosporin A-treated mice with induced acute colitis is depicted.

[0109] Figure 17F Depicted are the proliferation indices in the jejunum of untreated, GLP2-2G-10Nle-1K-EX4-K5, teduglutide, and cyclosporin A-treated mice with induced acute colitis.

[0110] Figure 17GThe pharmacokinetics of GLP2-2G-10Nle-1K-EX4-K5 and teduglutide in mice are described.

[0111] Figure 18A Depicted are the changes in percent body weight over 8 days in untreated, GLP2-2G-10Nle-L5A- and cyclosporin A-treated mice with induced acute colitis.

[0112] Figure 18B Depicted are the changes in percent body weight over 8 days in untreated, GLP2-2G-1-EX4-L5A-treated, and cyclosporin A-treated mice with induced acute colitis.

[0113] Figure 18C Depicted are the changes in percent body weight over 8 days in untreated, GLP2-2G-10Nle-1K-EX4-K5, and cyclosporin A-treated mice with induced acute colitis.

[0114] Figure 18D Depicted are the colon lengths of untreated mice, mice treated with different long-acting GLP2R agonists, and mice with induced acute colitis.

[0115] Figure 18E Depicted are colon weights of untreated mice, mice treated with a long-acting GLP2R agonist, and mice with induced acute colitis.

[0116] Figure 18F Depicted are the small intestine lengths of untreated mice and mice with induced acute colitis treated with different long-acting GLP2R agonists, cyclosporin A.

[0117] Figure 18G Depicted are small intestinal weights of untreated mice, mice treated with different long-acting GLP2R agonists, and mice with induced acute colitis.

[0118] Figure 18H Gallbladder enlargement in untreated mice and mice treated with different long-acting GLP2R agonists, cyclosporin A, with induced acute colitis is described.

[0119] Figure 18I The amount of occult blood in feces of untreated mice and mice treated with different long-acting GLP2R agonists, cyclosporin A, with induced acute colitis is described.

[0120] Figure 18J The pharmacokinetics of a long-acting GLP2R agonist at a dose of 0.03 mg / kg are described.

[0121] Figure 18K The pharmacokinetics of a long-acting GLP2R agonist at a dose of 0.1 mg / kg are described.

[0122] Figure 18L The pharmacokinetics of two doses of GLP2-2G-10Nle-L5A in a mouse model of acute colitis were described.

[0123] Figure 18M The pharmacokinetics of two doses of GLP2-2G-1-EX4-L5A in a mouse model of acute colitis were described.

[0124] Figure 18N The pharmacokinetics of two doses of GLP2-2G-10Nle-1K-EX4-K5 in a mouse model of acute colitis were described.

[0125] Figure 19A Depicted are the absolute changes in body weight in untreated, GLP2-2G-1-EX4-L5A-treated, cyclosporine-treated, and teduglutide-treated mice with induced chronic colitis.

[0126] Figure 19B Depicted are colon lengths of untreated, GLP2-2G-1-EX4-L5A-, cyclosporine-, and teduglutide-treated mice with induced chronic colitis.

[0127] Figure 19C Depicted are colon weights of untreated, GLP2-2G-1-EX4-L5A, cyclosporine, and teduglutide-treated mice with induced chronic colitis.

[0128] Figure 19D Depicted are small intestinal weights of untreated, GLP2-2G-1-EX4-L5A, cyclosporine, and teduglutide-treated mice with induced chronic colitis.

[0129] Figure 20A Depicted are colon lengths of untreated, GLP2-2G-10Nle-1-EX4-L5A-, cyclosporine-, and teduglutide-treated mice with induced chronic colitis.

[0130] Figure 20B Depicted are colon weights of untreated, GLP2-2G-10Nle-1-EX4-L5A, cyclosporine, and teduglutide-treated mice with induced chronic colitis.

[0131] Figure 20CDepicted are the small intestine lengths of untreated, GLP2-2G-10Nle-1-EX4-L5A, cyclosporine, and teduglutide-treated mice with induced chronic colitis.

[0132] Figure 21A ALT serum levels are described in untreated and GLP2-2G-5-EX4-L5A-treated choline-deficient mice, as well as mice fed a normal diet.

[0133] Figure 21B Described are AST serum levels in untreated and GLP2-2G-5-EX4-L5A-treated choline-deficient mice, as well as mice fed a normal diet.

[0134] Figure 21C Fibrosis scores are described for choline-deficient mice that were untreated and treated with GLP2-2G-5-EX4-L5A, as well as mice fed a normal diet.

[0135] Figure 21D Steatosis is described in choline-deficient mice that were untreated and treated with GLP2-2G-5-EX4-L5A, as well as mice fed a normal diet.

[0136] Figure 21E Lobular inflammation is described in choline-deficient mice that were untreated and treated with GLP2-2G-5-EX4-L5A, as well as mice fed a normal diet.

[0137] Figure 22A Depicted are body weights of male mice weaned on a regular diet that received no treatment, teduglutide, or GLP2-2G-10Nle-1K-EX4-K5.

[0138] Figure 22B Depicted are body weights of female mice weaned on a regular diet that received no treatment, teduglutide, or GLP2-2G-10Nle-1K-EX4-K5.

[0139] Figure 22C Depicted are body weights of male mice weaned on a deficient diet that received no treatment, teduglutide, or GLP2-2G-10Nle-1K-EX4-K5.

[0140] Figure 22D Depicted are body weights of untreated, teduglutide-treated, or GLP2-2G-10Nle-1K-EX4-K5-treated female mice weaned on a deficient diet.

[0141] Figure 22E Depicted are normalized small intestinal weights of male mice weaned on a regular diet that received no treatment, teduglutide, or GLP2-2G-10Nle-1K-EX4-K5.

[0142] Figure 22F Depicted are normalized small intestinal weights of female mice weaned on a regular diet that received no treatment, teduglutide, or GLP2-2G-10Nle-1K-EX4-K5. DETAILED DESCRIPTION

[0143] Glucagon-like peptide 2 (GLP-2) is a hormone secreted from intestinal endocrine cells. GLP-2 stimulates intestinal growth, increases nutrient absorption and blood flow, reduces intestinal permeability and motility, and reduces epithelial cell apoptosis and inflammation. Due to the enterotrophic effects of GLP-2, GLP-2 and related analogs can be used to treat gastrointestinal (GI) disorders. In humans, the short plasma half-life of native GLP-2 requires higher doses and frequent injections or infusions to achieve clinical efficacy, which can negatively impact patient compliance. Several approaches have been used to extend the half-life of GLP-2, including pegylation and fusion to polypeptides to increase molecular weight and hydrodynamic radius and reduce clearance through renal filtration. However, the resulting analogs have reduced in vitro potency, and therefore require higher doses to be effective in vivo.

[0144] Peptide conjugates

[0145] In one aspect, disclosed herein are peptide conjugates comprising peptides that modulate the GLP-2 receptor. In exemplary embodiments, the peptide that modulates the GLP-2 receptor comprises two amino acids linked by a stapler. Non-limiting examples of amino acids for conjugation include cysteine, homocysteine, 2-amino-5-mercaptopentanoic acid, 2-amino-6-mercaptohexanoic acid, lysine, ornithine, diaminobutyric acid, diaminopropionic acid, homolysine, other sulfhydryl-containing amino acids, or other amine-containing amino acids. For peptides that modulate the GLP-2 receptor comprising two amino acids linked by a stapler, the two amino acids are separated by about or at least about 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, or more amino acids. For example, the first amino acid is at position i, and the second amino acid is at position i+7, i+11, i+13, i+15, or i+16. For example, the first amino acid is at position i in the peptide, and the second amino acid is at position i+n in the peptide, where n is 4-16. For example, the first amino acid is at position i in the peptide, and the second amino acid is at position i + 7 in the peptide. For example, the first amino acid is at position i in the peptide, and the second amino acid is at position i + 11 in the peptide. For example, the first amino acid is at position i in the peptide, and the second amino acid is at position i + 15 in the peptide. For example, the first amino acid is at position i in the peptide, and the second amino acid is at position i + 16 in the peptide.

[0146] Peptides that modulate the GLP-2 receptor

[0147] In one aspect, provided herein are peptide conjugates comprising a peptide that modulates the GLP-2 receptor. In some embodiments, the peptide that modulates the GLP-2 receptor is a GLP-2 receptor agonist.

[0148] The binding affinity of the peptide conjugates described herein may be within about 5% of the binding affinity of an unmodified form of a GLP-2 peptide (e.g., an unconjugated GLP-2 peptide). The binding affinity of the peptide conjugates described herein may be within about 10% of the binding affinity of an unmodified form of a GLP-2 peptide. The binding affinity of the peptide conjugates described herein may be within about 15% of the binding affinity of an unmodified form of a GLP-2 peptide. The binding affinity of the peptide conjugates described herein may be within about 20% of the binding affinity of an unmodified form of a GLP-2 peptide.

[0149] A peptide that modulates the GLP-2 receptor may comprise at least a portion of a wild-type GLP-2 peptide and may comprise one or more amino acid mutations. The one or more amino acid mutations may comprise deletions, substitutions, additions, or a combination thereof. The one or more amino acid mutations may comprise additions of one or more amino acid residues to a wild-type GLP-2 peptide. The one or more amino acid mutations may comprise deletions of one or more amino acid residues of a wild-type GLP-2 peptide. The one or more amino acid mutations may comprise substitutions of one or more amino acid residues of a wild-type GLP-2 peptide. The one or more amino acid mutations may comprise substitutions of one or more amino acid residues of a wild-type GLP-2 peptide with one or more cysteine, lysine, or other sulfhydryl or amine-containing residues. The one or more amino acid mutations may comprise substitutions of one or more amino acid residues of a wild-type GLP-2 peptide with one or more D-amino acid residues. The one or more amino acid residues of a wild-type GLP-2 peptide may comprise one or more alanine, methionine, arginine, serine, threonine, and tyrosine.

[0150] Peptides that modulate the GLP-2 receptor can be modified, for example, by acetylation, phosphorylation, and methylation. Peptide modifications can include chemical modifications. Peptide modifications can occur at the N-terminus of the peptide. Peptide modifications can include acetylation of the amino group at the N-terminus of the peptide. Alternatively or additionally, peptide modifications can occur at the C-terminus of the peptide. Peptide modifications can occur at one or more internal amino acids of the peptide. Peptide modifications can include replacement of the carboxyl group at the C-terminus of the peptide. Peptide modifications can include modification of the carboxyl group at the C-terminus of the peptide. The carboxyl group at the C-terminus of the peptide can be modified to produce an amide group. The carboxyl group at the C-terminus of the peptide can be modified to produce an amine group.

[0151] Non-limiting examples of peptides that modulate the GLP-2 receptor are shown in Table 1.

[0152] In some embodiments, the peptide that modulates the GLP-2 receptor comprises the amino acid sequence of any one of SEQ ID NOs: 1-40. In some cases, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to any one of SEQ ID NOs: 1-40. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence that has up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to any one of SEQ ID NOs: 1-40.

[0153] In some embodiments, the peptide that modulates the GLP-2 receptor comprises the amino acid sequence of any one of SEQ ID NOs: 1-9. In some cases, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to any one of SEQ ID NOs: 1-9. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence that has up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to any one of SEQ ID NOs: 1-9.

[0154] In some embodiments, the GLP-2 receptor modulating peptide comprises the amino acid sequence of SEQ ID NO: 1. In some cases, the GLP-2 receptor modulating peptide comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 1. In some embodiments, the GLP-2 receptor modulating peptide comprises an amino acid sequence that has up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 1.

[0155] In some embodiments, the GLP-2 receptor modulating peptide comprises the amino acid sequence of SEQ ID NO: 2. In some cases, the GLP-2 receptor modulating peptide comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 2. In some embodiments, the GLP-2 receptor modulating peptide comprises an amino acid sequence that has up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 2.

[0156] In some embodiments, the GLP-2 receptor modulating peptide comprises the amino acid sequence of SEQ ID NO: 3. In some cases, the GLP-2 receptor modulating peptide comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 3. In some embodiments, the GLP-2 receptor modulating peptide comprises an amino acid sequence that has up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 3.

[0157] In some embodiments, the GLP-2 receptor modulating peptide comprises the amino acid sequence of SEQ ID NO: 4. In some cases, the GLP-2 receptor modulating peptide comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 4. In some embodiments, the GLP-2 receptor modulating peptide comprises an amino acid sequence that has up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 4.

[0158] In some embodiments, the GLP-2 receptor modulating peptide comprises the amino acid sequence of SEQ ID NO: 5. In some cases, the GLP-2 receptor modulating peptide comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 5. In some embodiments, the GLP-2 receptor modulating peptide comprises an amino acid sequence that has up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 5.

[0159] In some embodiments, the GLP-2 receptor modulating peptide comprises the amino acid sequence of SEQ ID NO: 6. In some cases, the GLP-2 receptor modulating peptide comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 6. In some embodiments, the GLP-2 receptor modulating peptide comprises an amino acid sequence that has up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 6.

[0160] In some embodiments, the GLP-2 receptor modulating peptide comprises the amino acid sequence of SEQ ID NO: 7. In some cases, the GLP-2 receptor modulating peptide comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 7. In some embodiments, the GLP-2 receptor modulating peptide comprises an amino acid sequence that has up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 7.

[0161] In some embodiments, the GLP-2 receptor modulating peptide comprises the amino acid sequence of SEQ ID NO: 8. In some cases, the GLP-2 receptor modulating peptide comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 8. In some embodiments, the GLP-2 receptor modulating peptide comprises an amino acid sequence that has up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 8.

[0162] In some embodiments, the GLP-2 receptor modulating peptide comprises the amino acid sequence of SEQ ID NO: 9. In some cases, the GLP-2 receptor modulating peptide comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 9. In some embodiments, the GLP-2 receptor modulating peptide comprises an amino acid sequence that has up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 9.

[0163] In some embodiments, the peptide that modulates the GLP-2 receptor comprises the amino acid sequence of any one of SEQ ID NOs: 10-20. In some cases, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to any one of SEQ ID NOs: 10-20. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence that has up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to any one of SEQ ID NOs: 10-20.

[0164] In some embodiments, the GLP-2 receptor modulating peptide comprises the amino acid sequence of SEQ ID NO: 10. In some cases, the GLP-2 receptor modulating peptide comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 10. In some embodiments, the GLP-2 receptor modulating peptide comprises an amino acid sequence that has up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 10.

[0165] In some embodiments, the GLP-2 receptor modulating peptide comprises the amino acid sequence of SEQ ID NO: 11. In some cases, the GLP-2 receptor modulating peptide comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 11. In some embodiments, the GLP-2 receptor modulating peptide comprises an amino acid sequence that has up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 11.

[0166] In some embodiments, the GLP-2 receptor modulating peptide comprises the amino acid sequence of SEQ ID NO: 12. In some cases, the GLP-2 receptor modulating peptide comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 12. In some embodiments, the GLP-2 receptor modulating peptide comprises an amino acid sequence that has up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 12.

[0167] In some embodiments, the GLP-2 receptor modulating peptide comprises the amino acid sequence of SEQ ID NO: 13. In some cases, the GLP-2 receptor modulating peptide comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 13. In some embodiments, the GLP-2 receptor modulating peptide comprises an amino acid sequence that has up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 13.

[0168] In some embodiments, the GLP-2 receptor modulating peptide comprises the amino acid sequence of SEQ ID NO: 14. In some cases, the GLP-2 receptor modulating peptide comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 14. In some embodiments, the GLP-2 receptor modulating peptide comprises an amino acid sequence that has up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 14.

[0169] In some embodiments, the GLP-2 receptor modulating peptide comprises the amino acid sequence of SEQ ID NO: 15. In some cases, the GLP-2 receptor modulating peptide comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 15. In some embodiments, the GLP-2 receptor modulating peptide comprises an amino acid sequence that has up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 15.

[0170] In some embodiments, the GLP-2 receptor modulating peptide comprises the amino acid sequence of SEQ ID NO: 16. In some cases, the GLP-2 receptor modulating peptide comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 16. In some embodiments, the GLP-2 receptor modulating peptide comprises an amino acid sequence that has up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 16.

[0171] In some embodiments, the GLP-2 receptor modulating peptide comprises the amino acid sequence of SEQ ID NO: 17. In some cases, the GLP-2 receptor modulating peptide comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 17. In some embodiments, the GLP-2 receptor modulating peptide comprises an amino acid sequence that has up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 17.

[0172] In some embodiments, the GLP-2 receptor modulating peptide comprises the amino acid sequence of SEQ ID NO: 18. In some cases, the GLP-2 receptor modulating peptide comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 18. In some embodiments, the GLP-2 receptor modulating peptide comprises an amino acid sequence that has up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 18.

[0173] In some embodiments, the GLP-2 receptor modulating peptide comprises the amino acid sequence of SEQ ID NO: 19. In some cases, the GLP-2 receptor modulating peptide comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 19. In some embodiments, the GLP-2 receptor modulating peptide comprises an amino acid sequence that has up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 19.

[0174] In some embodiments, the GLP-2 receptor modulating peptide comprises the amino acid sequence of SEQ ID NO: 20. In some cases, the GLP-2 receptor modulating peptide comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 20. In some embodiments, the GLP-2 receptor modulating peptide comprises an amino acid sequence that has up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 20.

[0175] In some embodiments, the peptide that modulates the GLP-2 receptor comprises the amino acid sequence of any one of SEQ ID NOs: 21-29. In some cases, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to any one of SEQ ID NOs: 21-29. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence that has up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to any one of SEQ ID NOs: 21-29.

[0176] In some embodiments, the GLP-2 receptor modulating peptide comprises the amino acid sequence of SEQ ID NO: 21. In some cases, the GLP-2 receptor modulating peptide comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 21. In some embodiments, the GLP-2 receptor modulating peptide comprises an amino acid sequence that has up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 21.

[0177] In some embodiments, the GLP-2 receptor modulating peptide comprises the amino acid sequence of SEQ ID NO: 2. In some cases, the GLP-2 receptor modulating peptide comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 22. In some embodiments, the GLP-2 receptor modulating peptide comprises an amino acid sequence that has up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 22.

[0178] In some embodiments, the GLP-2 receptor modulating peptide comprises the amino acid sequence of SEQ ID NO: 23. In some cases, the GLP-2 receptor modulating peptide comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 23. In some embodiments, the GLP-2 receptor modulating peptide comprises an amino acid sequence that has up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 23.

[0179] In some embodiments, the GLP-2 receptor modulating peptide comprises the amino acid sequence of SEQ ID NO: 24. In some cases, the GLP-2 receptor modulating peptide comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 24. In some embodiments, the GLP-2 receptor modulating peptide comprises an amino acid sequence that has up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 24.

[0180] In some embodiments, the GLP-2 receptor modulating peptide comprises the amino acid sequence of SEQ ID NO: 25. In some cases, the GLP-2 receptor modulating peptide comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 25. In some embodiments, the GLP-2 receptor modulating peptide comprises an amino acid sequence that has up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 25.

[0181] In some embodiments, the GLP-2 receptor modulating peptide comprises the amino acid sequence of SEQ ID NO: 26. In some cases, the GLP-2 receptor modulating peptide comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 26. In some embodiments, the GLP-2 receptor modulating peptide comprises an amino acid sequence that has up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 26.

[0182] In some embodiments, the GLP-2 receptor modulating peptide comprises the amino acid sequence of SEQ ID NO: 27. In some cases, the GLP-2 receptor modulating peptide comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 27. In some embodiments, the GLP-2 receptor modulating peptide comprises an amino acid sequence that has up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 27.

[0183] In some embodiments, the GLP-2 receptor modulating peptide comprises the amino acid sequence of SEQ ID NO: 28. In some cases, the GLP-2 receptor modulating peptide comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 28. In some embodiments, the GLP-2 receptor modulating peptide comprises an amino acid sequence that has up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 28.

[0184] In some embodiments, the GLP-2 receptor modulating peptide comprises the amino acid sequence of SEQ ID NO: 29. In some cases, the GLP-2 receptor modulating peptide comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 29. In some embodiments, the GLP-2 receptor modulating peptide comprises an amino acid sequence that has up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 29.

[0185] In some embodiments, the peptide that modulates the GLP-2 receptor comprises the amino acid sequence of any one of SEQ ID NOs: 30-40. In some cases, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to any one of SEQ ID NOs: 30-40. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence that has up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to any one of SEQ ID NOs: 30-40.

[0186] In some embodiments, the GLP-2 receptor modulating peptide comprises the amino acid sequence of SEQ ID NO: 30. In some cases, the GLP-2 receptor modulating peptide comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 30. In some embodiments, the GLP-2 receptor modulating peptide comprises an amino acid sequence that has up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 30.

[0187] In some embodiments, the GLP-2 receptor modulating peptide comprises the amino acid sequence of SEQ ID NO: 31. In some cases, the GLP-2 receptor modulating peptide comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 31. In some embodiments, the GLP-2 receptor modulating peptide comprises an amino acid sequence that has up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 31.

[0188] In some embodiments, the GLP-2 receptor modulating peptide comprises the amino acid sequence of SEQ ID NO: 32. In some cases, the GLP-2 receptor modulating peptide comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 32. In some embodiments, the GLP-2 receptor modulating peptide comprises an amino acid sequence that has up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 32.

[0189] In some embodiments, the GLP-2 receptor modulating peptide comprises the amino acid sequence of SEQ ID NO: 33. In some cases, the GLP-2 receptor modulating peptide comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 33. In some embodiments, the GLP-2 receptor modulating peptide comprises an amino acid sequence that has up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 33.

[0190] In some embodiments, the GLP-2 receptor modulating peptide comprises the amino acid sequence of SEQ ID NO: 34. In some cases, the GLP-2 receptor modulating peptide comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 34. In some embodiments, the GLP-2 receptor modulating peptide comprises an amino acid sequence that has up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 34.

[0191] In some embodiments, the GLP-2 receptor modulating peptide comprises the amino acid sequence of SEQ ID NO: 35. In some cases, the GLP-2 receptor modulating peptide comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 35. In some embodiments, the GLP-2 receptor modulating peptide comprises an amino acid sequence that has up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 35.

[0192] In some embodiments, the GLP-2 receptor modulating peptide comprises the amino acid sequence of SEQ ID NO: 36. In some cases, the GLP-2 receptor modulating peptide comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 36. In some embodiments, the GLP-2 receptor modulating peptide comprises an amino acid sequence that has up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 36.

[0193] In some embodiments, the GLP-2 receptor modulating peptide comprises the amino acid sequence of SEQ ID NO: 37. In some cases, the GLP-2 receptor modulating peptide comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 37. In some embodiments, the GLP-2 receptor modulating peptide comprises an amino acid sequence that has up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 37.

[0194] In some embodiments, the GLP-2 receptor modulating peptide comprises the amino acid sequence of SEQ ID NO: 38. In some cases, the GLP-2 receptor modulating peptide comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 38. In some embodiments, the GLP-2 receptor modulating peptide comprises an amino acid sequence that has up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 38.

[0195] In some embodiments, the GLP-2 receptor modulating peptide comprises the amino acid sequence of SEQ ID NO: 39. In some cases, the GLP-2 receptor modulating peptide comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 39. In some embodiments, the GLP-2 receptor modulating peptide comprises an amino acid sequence that has up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 39.

[0196] In some embodiments, the GLP-2 receptor modulating peptide comprises the amino acid sequence of SEQ ID NO: 40. In some cases, the GLP-2 receptor modulating peptide comprises an amino acid sequence that is at least about 80%, about 81%, about 82%, about 83%, about 84%, about 85%, about 86%, about 87%, about 88%, about 89%, 90%, about 91%, about 92%, about 93%, about 94%, about 95%, about 96%, about 97%, about 98%, or about 99% identical to SEQ ID NO: 40. In some embodiments, the GLP-2 receptor modulating peptide comprises an amino acid sequence that has up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 40.

[0197] Table 1: SEQ ID Table

[0198]

[0199]

[0200]

[0201]

[0202]

[0203]

[0204] stapled body

[0205] Disclosed herein are peptide conjugates comprising staplers.

[0206] In some embodiments, the stapled complex linked to the peptide is a compound of Formula (I):

[0207]

[0208] in:

[0209] A is optionally substituted alkylene, optionally substituted arylene, optionally substituted heteroarylene, optionally substituted -NR 3 -alkylene-NR 3 -or-N-;

[0210] X 1 and X 2 are independently a bond, -C(=O)-, -alkylene-C(=O)-, -C(=O)-alkylene-, -alkylene-C(=O)NR 3 -, -alkylene-NR 3 C(=O)-, -C(=O)NR 3 -alkylene-, -NR 3 C(=O)-alkylene-, -alkylene-C(=O)NR 3 -alkylene- or -alkylene-NR 3 C(=O)-alkylene-;

[0211] where X 1 is linked to the first amino acid of the peptide, and X 2 linked to a second amino acid of the peptide;

[0212] R is hydrogen or -X 3 -(L) s -Y;

[0213] X 3 is a bond, -C(=O)-, -alkylene-C(=O)-, -C(=O)-alkylene, -alkylene-C(=O)NR 3 -or-Alkylene-C(=O)NR 3 -alkylene-;

[0214] Each L is independently -(CR 1 R 2 ) v -, -alkylene-O-, -O-alkylene-, -C(=O)-alkylene-, -alkylene-C(=O)-, -NR 3 -alkylene-, -alkylene-NR 3 -, -S-alkylene-, -alkylene-S-, -S(=O)-alkylene-, -alkylene-S(=O)-, -S(=O)2-alkylene, -alkylene-S(=O)2-, -C(=O)-, -C(=O)NR 3 -、-NR 3 C(=O)-、-NR 3 C(=O)NR 3 -、-NR 3 C(=O)NR3 -alkylene-, -NR 3 C(=O)-alkylene-NR 3 -, -alkylene-C(=O)NR 3 -, -C(=O)NR 3 -alkylene-, -alkylene-NR 3 C(=O)- or -NR 3 C(=O)-alkylene-;

[0215] v is 2-20;

[0216] R 1 or R 2 are independently hydrogen, halogen, -CN, -OR a 、-SR a 、-S(=O)R b 、-NO2、-NR c R d 、-S(=O)2R d 、-NR a S(=O)2R d 、-S(=O)2NR c R d 、-C(=O)R b 、-OC(=O)R b 、-CO2R a 、-OCO2R a 、-C(=O)NR c R d 、-OC(=O)NR c R d 、-NR a C(=O)NR c R d 、-NR a C(=O)R b 、-NR a C(=O)OR a , C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C3-C8 cycloalkyl, C2-C8 heterocycloalkyl, aryl or heteroaryl; wherein alkyl, alkenyl, alkynyl and heteroalkyl are optionally substituted by halogen, -OR a or -NR c R d One, two or three of; and cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally substituted by halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR a 、-NR c R d One, two or three substitutions;

[0217] or R 1 and R 2 Together they form a C1-C6 cycloalkyl group or a C1-C6 heterocycloalkyl group;

[0218] Each R 3 are independently hydrogen, -S(=O)R b 、-S(=O)2R a 、-S(=O)2NR c R d 、-C(=O)R b 、-CO2R a 、-C(=O)NR c R d , C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C3-C8 cycloalkyl, C2-C8 heterocycloalkyl, aryl or heteroaryl; wherein alkyl, alkenyl, alkynyl and heteroalkyl are optionally substituted by halogen, -OR a or -NR c R d One, two or three of; and cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally substituted by halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR a or -NR c R d One, two or three substitutions;

[0219] Y is hydrogen, C1-C6 alkyl, -CO2H, -CO2(C1-C6 alkyl), -CO2NH2, -CO2N(alkyl)2, or -CO2NH(alkyl); and

[0220] s is 0-20;

[0221] R a is hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C3-C8 cycloalkyl, C2-C8 heterocycloalkyl, aryl or heteroaryl; wherein alkyl, alkenyl, alkynyl and heteroalkyl are optionally substituted with one, two or three of halogen, -OH, -OMe or -NH2; and cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally substituted with one, two or three of halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OH, -OMe or -NH2;

[0222] R bis C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C3-C8 cycloalkyl, C2-C8 heterocycloalkyl, aryl or heteroaryl; wherein alkyl, alkenyl, alkynyl and heteroalkyl are optionally substituted with one, two or three of halogen, -OH, -OMe or -NH2; and cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally substituted with one, two or three of halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OH, -OMe or -NH2;

[0223] R c and R d each independently represents hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C3-C8 cycloalkyl, C2-C8 heterocycloalkyl, aryl or heteroaryl; wherein alkyl, alkenyl, alkynyl and heteroalkyl are optionally substituted with one, two or three of halogen, -OH, -OMe or -NH2; and cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally substituted with one, two or three of halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OH, -OMe or -NH2;

[0224] or R c and R d Together with the nitrogen atom to which they are attached, they form a heterocycloalkyl or heteroaryl group; wherein the heterocycloalkyl and heteroaryl groups are optionally substituted by one, two or three of halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OH, -OMe or -NH2.

[0225] In some embodiments, the stapled complex linked to the peptide is a compound of Formula (I):

[0226]

[0227] in:

[0228] A is optionally substituted alkylene, optionally substituted arylene, optionally substituted heteroarylene, optionally substituted -NR 3 -alkylene-NR 3 -or-N-;

[0229] X 1 and X 2 are independently a bond, -C(=O)-, -alkylene-C(=O)-, -C(=O)-alkylene, -alkylene-C(=O)NR 3 -or-Alkylene-C(=O)NR 3 -alkylene-;

[0230] where X 1 is linked to the first amino acid of the peptide, and X 2linked to a second amino acid of the peptide;

[0231] R is hydrogen or -X 3 -(L) s -Y;

[0232] X 3 is a bond, -C(=O)-, -alkylene-C(=O)-, -C(=O)-alkylene, -alkylene-C(=O)NR 3 -or-Alkylene-C(=O)NR 3 -alkylene-;

[0233] Each L is independently -(CR 1 R 2 ) v -, -alkylene-O-, -O-alkylene-, -C(=O)-alkylene-, -alkylene-C(=O)-, -NR 3 -alkylene-, -alkylene-NR 3 -, -S-alkylene-, -alkylene-S-, -S(=O)-alkylene-, -alkylene-S(=O)-, -S(=O)2-alkylene, -alkylene-S(=O)2-, -C(=O)-, -C(=O)NR 3 -、-NR 3 C(=O)-、-NR 3 C(=O)NR 3 -、-NR 3 C(=O)NR 3 -alkylene-, -NR 3 C(=O)-alkylene-NR 3 -, -alkylene-C(=O)NR 3 -, -C(=O)NR 3 -alkylene-, -alkylene-NR 3 C(=O)- or -NR 3 C(=O)-alkylene-;

[0234] v is 2-20;

[0235] R 1 or R 2 are independently hydrogen, halogen, -CN, -OR a 、-SR a 、-S(=O)R b 、-NO2、-NR c R d 、-S(=O)2R d 、-NR a S(=O)2R d 、-S(=O)2NR c Rd 、-C(=O)R b 、-OC(=O)R b 、-CO2R a 、-OCO2R a 、-C(=O)NR c R d 、-OC(=O)NR c R d 、-NR a C(=O)NR c R d 、-NR a C(=O)R b 、-NR a C(=O)OR a , C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C3-C8 cycloalkyl, C2-C8 heterocycloalkyl, aryl or heteroaryl; wherein alkyl, alkenyl, alkynyl and heteroalkyl are optionally substituted by halogen, -OR a or -NR c R d One, two or three of; and cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally substituted by halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OR a 、-NR c R d One, two or three substitutions;

[0236] or R 1 and R 2 Together they form a C1-C6 cycloalkyl group or a C1-C6 heterocycloalkyl group;

[0237] Each R 3 are independently hydrogen, -S(=O)R b 、-S(=O)2R a 、-S(=O)2NR c R d 、-C(=O)R b 、-CO2R a 、-C(=O)NR c R d , C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C3-C8 cycloalkyl, C2-C8 heterocycloalkyl, aryl or heteroaryl; wherein alkyl, alkenyl, alkynyl and heteroalkyl are optionally substituted by halogen, -OR a or -NR c R d One, two or three of; and cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally substituted by halogen, C1-C6 alkyl, C1-C6 haloalkyl, -ORa or -NR c R d One, two or three substitutions;

[0238] Y is hydrogen, C1-C6 alkyl, -CO2H, -CO2(C1-C6 alkyl), -CO2NH2, -CO2N(alkyl)2, or -CO2NH(alkyl); and

[0239] s is 0-20;

[0240] R a is hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C3-C8 cycloalkyl, C2-C8 heterocycloalkyl, aryl or heteroaryl; wherein alkyl, alkenyl, alkynyl and heteroalkyl are optionally substituted with one, two or three of halogen, -OH, -OMe or -NH2; and cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally substituted with one, two or three of halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OH, -OMe or -NH2;

[0241] R b is C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C3-C8 cycloalkyl, C2-C8 heterocycloalkyl, aryl or heteroaryl; wherein alkyl, alkenyl, alkynyl and heteroalkyl are optionally substituted with one, two or three of halogen, -OH, -OMe or -NH2; and cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally substituted with one, two or three of halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OH, -OMe or -NH2;

[0242] R c and R d each independently represents hydrogen, C1-C6 alkyl, C2-C6 alkenyl, C2-C6 alkynyl, C1-C6 heteroalkyl, C3-C8 cycloalkyl, C2-C8 heterocycloalkyl, aryl or heteroaryl; wherein alkyl, alkenyl, alkynyl and heteroalkyl are optionally substituted with one, two or three of halogen, -OH, -OMe or -NH2; and cycloalkyl, heterocycloalkyl, aryl and heteroaryl are optionally substituted with one, two or three of halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OH, -OMe or -NH2;

[0243] or R c and R d Together with the nitrogen atom to which they are attached, they form a heterocycloalkyl or heteroaryl group; wherein the heterocycloalkyl and heteroaryl groups are optionally substituted by one, two or three of halogen, C1-C6 alkyl, C1-C6 haloalkyl, -OH, -OMe or -NH2.

[0244] In some embodiments, A is optionally substituted alkylene. In some embodiments, A is -(CH2) t -, wherein t is 1-12. In some embodiments, A is -(CH2) t -, wherein t is 1-10. In some embodiments, A is -(CH2) t -, wherein t is 1-8. In some embodiments, A is -(CH2) t -, wherein t is 1-6. In some embodiments, A is -(CH2) t -, where t is 1-4.

[0245] In some embodiments, A is optionally substituted arylene. In some embodiments, A is arylene optionally substituted with halogen, alkyl, or haloalkyl. In some embodiments, A is arylene.

[0246] In some embodiments, A is -NR 3 -alkylene-NR 3 -.

[0247] In some embodiments, A is -N-.

[0248] In some embodiments, X 1 and X 2 In some embodiments, X 1 and X 2 different.

[0249] In some embodiments, X 1 and X 2 In some embodiments, X 1 and X 2 is independently -alkylene-C(=O)- or -C(=O)alkylene-. In some embodiments, X 1 and X 2 is independently -CH2-C(=O)- or -C(=O)-CH2-. In some embodiments, X 1 and X 2 are independently -alkylene-C(=O)NR 3 or -C(=O)NR 3 -alkylene-. In some embodiments, X 1 and X 2 are independently -CH2-C(=O)NR 3 -or-C(=O)NR 3 -CH2-. In some embodiments, X 1 and X 2 are independently -alkylene-C(=O)NR3 -alkylene- or -alkylene-NR 3 C(=O)-alkylene-. In some embodiments, X 1 and X 2 are independently CH2-C(=O)NR 3 -CH2CH2- or -CH2-NR 3 C(=O)-CH2CH2-. In some embodiments, X 1 and X 2 are independently -CH2-C(=O)NH-CH2CH2- or -CH2-NHC(=O)-CH2CH2-.

[0250] In some embodiments, each R 3 is independently hydrogen or C1-C6 alkyl. 3 For hydrogen.

[0251] In some embodiments, >AR has the following structure:

[0252] wherein r1 and r2 are each independently 0-4.

[0253] In some embodiments, R1 and R2 are each independently 0 to 2. In some embodiments, R1 and R2 are each 0. In some embodiments, R1 and R2 are each 1. In some embodiments, R1 and R2 are each 3.

[0254] In some embodiments, >AR has the following structure:

[0255]

[0256] In some embodiments, >AR has the following structure:

[0257] Where p1 is 1-5.

[0258] In some embodiments, p1 is 1 to 3. In some embodiments, p1 is 1 to 2. In some embodiments, p1 is 1. In some embodiments, p1 is 2. In some embodiments, p1 is 3. In some embodiments, p1 is 4. In some embodiments, p1 is 5.

[0259] In some embodiments, >AR has the following structure:

[0260]

[0261] In some embodiments, >AR has the following structure:

[0262]

[0263] In some embodiments, s is 1-15. In some embodiments, s is 1-10. In some embodiments, s is 5-15. In some embodiments, s is 5-10. In some embodiments, s is 5-20.

[0264] In some embodiments, Y is hydrogen or -CO2H. In some embodiments, Y is hydrogen. In some embodiments, Y is -CO2H.

[0265] In some embodiments, each L is independently -(CR 1 R 2 ) v -, -alkylene-O-, -C(=O)-, -C(=O)NR 3 -、-NR 3 C(=O)-, -alkylene-C(=O)NR 3 -or-alkylene-NR 3 C(=O)-; and v is 2-20.

[0266] In some embodiments, each L is independently -(CR 1 R 2 ) v -, -alkylene-O-, -C(=O)-, -C(=O)NR 3 -、-NR 3 C(=O)-, -alkylene-C(=O)NR 3 -or-alkylene-NR 3 C(=O)-; and v is 2-16.

[0267] In some embodiments, v is 2 to 16. In some embodiments, v is 2 to 5. In some embodiments, v is 5 to 16. In some embodiments, v is 5 or 16. In some embodiments, v is 2 or 16.

[0268] In some embodiments, R 1 or R 2 are independently hydrogen, halogen, -CN, -OR a 、-NR c R d 、-C(=O)R b 、-CO2R a 、-C(=O)NR c R d or C1-C6 alkyl.

[0269] In some embodiments, R 1 or R2 Each independently is hydrogen, halogen, -CO2R a 、-C(=O)NR c R d or C1-C6 alkyl. In some embodiments, R 1 or R 2 Each independently is hydrogen, -CO2R a or -C(=O)NR c R d In some embodiments, R 1 or R 2 Each independently is hydrogen or -CO2R a .

[0270] In some embodiments, the stapler is of the formula

[0271] In some embodiments, the stapler attached to the peptide is Each L 1 Independently -(CR 1 R 2 ) v -, -alkylene-O-, -O-alkylene-, -C(=O)NR 3 -、-NR 3 C(=O)-, -alkylene-C(=O)NR 3 -or-alkylene-NR 3 C(═O)—; v is 2-20; and s1 is 1-15.

[0272] In some embodiments, the stapler attached to the peptide is Each L 2 Independently -(CR 1 R 2 ) v -, -alkylene-O-, -O-alkylene-, -C(=O)NR 3 -、-NR 3 C(=O)-, -alkylene-C(=O)NR 3 -or-alkylene-NR 3 C(=O)-; v is 2-20; and s2 is 1-15.

[0273] In some embodiments, the stapler attached to the peptide is Each L 3 Independently -(CR 1 R 2 ) v -, -alkylene-O-, -O-alkylene-, -C(=O)NR 3 -、-NR 3C(=O)-, -alkylene-C(=O)NR 3 -or-alkylene-NR 3 C(=O)-; v is 2-20; and s3 is 1-15.

[0274] In some embodiments, the stapler attached to the peptide is Each L 4 Independently -(CR 1 R 2 ) v -, -alkylene-O-, -O-alkylene-, -C(=O)NR 3 -、-NR 3 C(=O)-, -alkylene-C(=O)NR 3 -or-alkylene-NR 3 C(=O)-; v is 2-20; and s4 is 1-15.

[0275] In some embodiments, the stapler attached to the peptide is Each L 5 Independently -(CR 1 R 2 ) v -, -C(=O)NR 3 -、-NR 3 C(=O)-, -alkylene-C(=O)NR 3 -or-alkylene-NR 3 C(=O)-; v is 2-20; and s5 is 1-10.

[0276] In some embodiments, the stapler attached to the peptide is Each L 6 Independently -(CR 1 R 2 ) v -, -C(=O)NR 3 -、-NR 3 C(=O)-, -alkylene-C(=O)NR 3 -or-alkylene-NR 3 C(=O)-; v is 2-20; and s6 is 1-5.

[0277] In some embodiments, the stapler attached to the peptide is Each L 7 Independently -(CR 1 R 2 ) v -, -C(=O)NR 3 -or-NR 3C(=O)-; v is 2-20; and s7 is 1-5.

[0278] In some embodiments, the stapler attached to the peptide is Among them L 8 For-(CR 1 R 2 ) v - and v is 10-20.

[0279] In some embodiments, the stapler attached to the peptide is Each L 9 Independently -(CR 1 R 2 ) v -, -C(=O)NR 3 -、-NR 3 C(=O)-, -alkylene-C(=O)NR 3 -or-alkylene-NR 3 C(=O)-; v is 2-20; and s9 is 1-5.

[0280] In some embodiments, the stapler attached to the peptide is Among them L 10 For-(CR 1 R 2 ) v - and v is 10-20.

[0281] In some embodiments, the stapler attached to the peptide is

[0282] In some embodiments, the stapler attached to the peptide is Each L 11 Independently -(CR 1 R 2 ) v -, -alkylene-O-, -O-alkylene-, -C(=O)NR 3 -、-NR 3 C(=O)-, -alkylene-C(=O)NR 3 -or-alkylene-NR 3 C(═O)—; v is 2-20; and s11 is 1-15.

[0283] In some embodiments, the stapler attached to the peptide is Each L 12 Independently -(CR 1 R 2 ) v -, -alkylene-O-, -O-alkylene-, -C(=O)NR 3-、-NR 3 C(=O)-, -alkylene-C(=O)NR 3 -or-alkylene-NR 3 C(=O)-; v is 2-20; and s12 is 1-15.

[0284] In some embodiments, the stapler attached to the peptide is Each L 13 Independently -(CR 1 R 2 ) v -, -alkylene-O-, -O-alkylene-, -C(=O)NR 3 -、-NR 3 C(=O)-, -alkylene-C(=O)NR 3 -or-alkylene-NR 3 C(=O)-; v is 2-20; and s13 is 1-15.

[0285] In some embodiments, the stapler attached to the peptide is Each L 14 Independently -(CR 1 R 2 ) v -, -alkylene-O-, -O-alkylene-, -C(=O)NR 3 -、-NR 3 C(=O)-, -alkylene-C(=O)NR 3 -or-alkylene-NR 3 C(=O)-; v is 2-20; and s14 is 1-15.

[0286] In some embodiments, the stapler attached to the peptide is Each L 15 Independently -(CR 1 R 2 ) v -, -C(=O)NR 3 -、-NR 3 C(=O)-, -alkylene-C(=O)NR 3 -or-alkylene-NR 3 C(=O)-; v is 2-20; and s15 is 1-10.

[0287] In some embodiments, the stapler attached to the peptide is Each L 16 Independently -(CR 1 R 2 ) v -, -C(=O)NR3 -or-NR 3 C(=O)-; v is 2-20; and s16 is 1-5.

[0288] In some embodiments, the stapler attached to the peptide is Each L 17 Independently -(CR 1 R 2 ) v -, -C(=O)NR 3 -or-NR 3 C(=O)-; v is 2-20; and s17 is 1-5.

[0289] In some embodiments, the stapler attached to the peptide is Among them L 18 For-(CR 1 R 2 ) v - and v is 10-20.

[0290] In some embodiments, the stapler attached to the peptide is Each L1 9 Independently -(CR 1 R 2 ) v -, -C(=O)NR 3 -、-NR 3 C(=O)-, -alkylene-C(=O)NR 3 -or-alkylene-NR 3 C(=O)-; v is 2-20; and s19 is 1-5.

[0291] In some embodiments, the stapler attached to the peptide is Among them L 20 For-(CR 1 R 2 ) v - and v is 10-20.

[0292] In some embodiments, the stapler attached to the peptide is

[0293]

[0294]

[0295] is part of a cysteine, homocysteine, 2-amino-5-mercaptopentanoic acid or 2-amino-6-mercaptohexanoic acid residue, and It is part of a lysine, ornithine, diaminobutyric acid, diaminopropionic acid or homolysine residue.

[0296] In some embodiments, the stapler attached to the peptide is

[0297] Where n is 1-4 and m is 6-20; is part of a cysteine, homocysteine, 2-amino-5-mercaptopentanoic acid or 2-amino-6-mercaptohexanoic acid residue, and It is part of a lysine, ornithine, diaminobutyric acid, diaminopropionic acid or homolysine residue.

[0298] In some embodiments, the stapler attached to the peptide is

[0299] is part of a cysteine, homocysteine, 2-amino-5-mercaptopentanoic acid or 2-amino-6-mercaptohexanoic acid residue, and It is part of a lysine, ornithine, diaminobutyric acid, diaminopropionic acid or homolysine residue.

[0300] In some embodiments, the stapler attached to the peptide is

[0301] It is part of a cysteine, homocysteine, 2-amino-5-mercaptopentanoic acid or 2-amino-6-mercaptohexanoic acid residue.

[0302] In some embodiments, the stapler attached to the peptide is

[0303] It is part of a lysine, ornithine, diaminobutyric acid, diaminopropionic acid or homolysine residue.

[0304] In some embodiments, the stapler attached to the peptide is

[0305] It is part of a lysine, ornithine, diaminobutyric acid, diaminopropionic acid or homolysine residue.

[0306] In some embodiments, the stapler attached to the peptide is:

[0307]

[0308] It is part of a cysteine, homocysteine, 2-amino-5-mercaptopentanoic acid or 2-amino-6-mercaptohexanoic acid residue.

[0309] In some embodiments, the stapler attached to the peptide is:

[0310] It is part of a cysteine, homocysteine, 2-amino-5-mercaptopentanoic acid or 2-amino-6-mercaptohexanoic acid residue.

[0311] In some embodiments, the stapler attached to the peptide is:

[0312] It is part of a lysine, ornithine, diaminobutyric acid, diaminopropionic acid or homolysine residue.

[0313] In some embodiments, the stapler attached to the peptide is:

[0314] It is part of a lysine, ornithine, diaminobutyric acid, diaminopropionic acid or homolysine residue.

[0315] In some embodiments, the peptide conjugate comprises:

[0316] a) a peptide that modulates the GLP-2 receptor, comprising the sequence of any one of SEQ ID NOs: 1-9; and

[0317] b) A stapler having the following structure ("S" is part of a cysteine ​​residue) attached to the peptide at the first and second cysteine ​​residues:

[0318]

[0319] In some embodiments, the peptide conjugate comprises:

[0320] a) a peptide that modulates the GLP-2 receptor, comprising the sequence of SEQ ID NO: 1; and

[0321] b) A stapler having the following structure ("S" is part of a cysteine ​​residue) attached to the peptide at the first and second cysteine ​​residues:

[0322]

[0323] In some embodiments, the peptide conjugate comprises:

[0324] a) a peptide that modulates the GLP-2 receptor, comprising the sequence of SEQ ID NO: 2; and

[0325] b) A stapler having the following structure ("S" is part of a cysteine ​​residue) attached to the peptide at the first and second cysteine ​​residues:

[0326]

[0327] In some embodiments, the peptide conjugate comprises:

[0328] a) a peptide that modulates the GLP-2 receptor, comprising the sequence of any one of SEQ ID NOs: 10-20; and

[0329] b) a stapler having the following structure ("NH" is part of a lysine residue) attached to the peptide at the first and second lysine residues:

[0330]

[0331] In some embodiments, the peptide conjugate comprises:

[0332] a) a peptide that modulates the GLP-2 receptor, comprising the sequence of SEQ ID NO: 10; and

[0333] b) a stapler having the following structure ("NH" is part of a lysine residue) attached to the peptide at the first and second lysine residues:

[0334]

[0335] In some embodiments, the peptide conjugate comprises:

[0336] a) a peptide that modulates the GLP-2 receptor, comprising the sequence of any one of SEQ ID NOs: 21-29; and

[0337] b) A stapler having the following structure ("S" is part of a cysteine ​​residue) attached to the peptide at the first and second cysteine ​​residues:

[0338]

[0339]

[0340] In some embodiments, the peptide conjugate comprises:

[0341] a) a peptide that modulates the GLP-2 receptor, comprising the sequence of SEQ ID NO: 21; and

[0342] b) A stapler having the following structure ("S" is part of a cysteine ​​residue) attached to the peptide at the first and second cysteine ​​residues:

[0343]

[0344] In some embodiments, the peptide conjugate comprises:

[0345] a) a GLP-2 receptor modulating peptide comprising the sequence of SEQ ID NO: 22; and

[0346] b) A stapler having the following structure ("S" is part of a cysteine ​​residue) attached to the peptide at the first and second cysteine ​​residues:

[0347]

[0348] In some embodiments, the peptide conjugate comprises:

[0349] a) a peptide that modulates the GLP-2 receptor, comprising the sequence of any one of SEQ ID NOs: 30-40; and

[0350] b) a stapler having the following structure ("NH" is part of a lysine residue) attached to the peptide at the first and second lysine residues:

[0351]

[0352]

[0353] In some embodiments, the peptide conjugate comprises:

[0354] a) a GLP-2 receptor modulating peptide comprising the sequence of SEQ ID NO: 30; and

[0355] b) a stapler having the following structure ("NH" is part of a lysine residue) attached to the peptide at the first and second lysine residues:

[0356]

[0357] Pharmacokinetics

[0358] Mechanisms by which the peptide conjugates positively affect pharmacokinetic or pharmacodynamic behavior include, but are not limited to, (i) prevention or reduction of in vivo proteolytic degradation or other activity-reducing chemical modifications of the peptide that modulates the GLP-2 receptor; (ii) improvement of half-life or other pharmacokinetic properties by reducing renal filtration, reducing receptor-mediated clearance, or increasing bioavailability; (iii) reduction of toxicity; (iv) increase in solubility; and / or (v) increase in biological activity and / or target selectivity of the peptide or unmodified peptide.

[0359] The peptide conjugates can enhance one or more pharmacokinetic properties of a peptide that modulates the GLP-2 receptor when linked to the peptide. The peptide conjugates disclosed herein can enhance one or more pharmacokinetic properties of a peptide that modulates the GLP-2 receptor by at least about 200% when compared to the peptide alone or an unmodified peptide, as measured by pharmacodynamics. The peptide conjugates disclosed herein can enhance one or more pharmacokinetic properties of a therapeutic agent by at least about 300%, 400%, 500%, 600%, 700%, 800%, 900%, or 1000% when compared to the peptide alone or an unmodified peptide, as measured by pharmacodynamics.

[0360] Pharmacokinetic properties may include half-life. The half-life of the peptide conjugate may be at least about 2 times the half-life of the unmodified peptide alone. The half-life of the peptide conjugates disclosed herein may be at least about 3, 4, 5, or 10 times the half-life of the therapeutic agent or unmodified therapeutic peptide alone. The half-life of the peptide conjugates disclosed herein may be at least about 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, or 50 times the half-life of the unmodified peptide alone.

[0361] In some embodiments, the half-life of the peptide conjugate is at least about 2 times the half-life of the unmodified form of the peptide. In some embodiments, the half-life of the peptide conjugate is at least about 5 times the half-life of the unmodified form of the peptide. In some embodiments, the half-life of the peptide conjugate is at least about 10 times the half-life of the unmodified form of the peptide.

[0362] Furthermore, the peptide conjugates described herein may have a positive impact on increasing the manufacturability of the peptide and / or reducing the immunogenicity of the peptide compared to the unconjugated, unmodified therapeutic peptide.

[0363] Therapeutic uses

[0364] In one aspect, the peptide conjugates disclosed herein can be used to treat, alleviate, inhibit, and / or prevent one or more diseases and / or conditions. The disease and / or condition can be a chronic disease or condition. Alternatively, the disease and / or condition is an acute disease or condition. The disease or condition can be recurrent, refractory, accelerated, or alleviated. The disease or condition can affect one or more cell types. The one or more diseases and / or conditions can be autoimmune diseases, inflammatory diseases, or metabolic diseases.

[0365] Disclosed herein is a method for treating a disease or condition in a subject in need thereof, the method comprising administering a peptide conjugate as described herein to the subject. The above-mentioned disease or condition may be diabetes or obesity, or a medical condition associated with diabetes or obesity. The above-mentioned disease or condition may be non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), or cardiovascular disease. The above-mentioned disease or condition may be an autoimmune condition. The above-mentioned disease or condition may be Crohn's disease or ulcerative colitis. The above-mentioned disease or condition may be short bowel syndrome (SBS). The above-mentioned disease or condition may be inflammatory bowel disease (IBD), inflammatory bowel syndrome (IBS), or psoriasis. The above-mentioned disease or condition may be Alzheimer's disease, Parkinson's disease, or Huntington's disease. PLC may be administered together with one or more additional therapeutic agents. Disclosed herein is a method for treating a disease or condition in a subject in need thereof, the method comprising administering a composition comprising one or more peptide conjugates disclosed herein to the subject.

[0366] Provided herein are methods for preventing or treating a metabolic disease or condition in a subject in need thereof, the methods comprising administering to the subject a peptide conjugate as described herein. The metabolic disease or condition may be diabetes. The metabolic disease or condition may be obesity. The metabolic disease or disorder can be glycogen storage disease, phenylketonuria, maple syrup urine disease, glutaric acidemia type 1, carbamoyl phosphate synthetase I deficiency, alcaptonuria, medium-chain acyl-coenzyme A dehydrogenase deficiency (MCADD), acute intermittent porphyria, Lesch-Nyhan syndrome, lipoid congenital adrenal hyperplasia, congenital adrenal hyperplasia, POMPC deficiency, LEPR deficiency, Bardet-Biedl syndrome, syndrome, Alstrome syndrome, Prader-Willi Syndrome, Kearns-Sayre syndrome, Zellweger syndrome, Gaucher's disease, or Niemann-Pick disease.

[0367] Provided herein are methods of preventing or treating NAFLD, NASH, or cardiovascular disease in a subject in need thereof, the methods comprising administering to the subject a peptide conjugate described herein.

[0368] Provided herein are methods of preventing or treating short bowel syndrome (SBS) in a subject in need thereof, the methods comprising administering to the subject a peptide conjugate described herein.

[0369] Provided herein are methods of preventing or treating inflammatory bowel disease (IBD), inflammatory bowel syndrome (IBS), or psoriasis in a subject in need thereof, the methods comprising administering to the subject a peptide conjugate described herein.

[0370] Provided herein are methods of preventing or treating Crohn's disease or ulcerative colitis in a subject in need thereof, the methods comprising administering to the subject a peptide conjugate described herein.

[0371] This article provides a method for preventing or treating sleep disorders.

[0372] Provided herein is a method for preventing or treating absence seizures. Provided herein is a method for preventing or treating chronic kidney disease (e.g., a complication of diabetes). Provided herein is a method for preventing or treating diabetic heart disease. Provided herein is a method for preventing or treating cardiovascular events.

[0373] Provided herein is a method of preventing or treating Alzheimer's disease, Parkinson's disease, or Huntington's disease in a subject in need thereof, the method comprising administering to the subject a peptide conjugate described herein.

[0374] Provided herein is a method for preventing or treating gastric and intestinal related disorders, such as treating newborns with impaired intestinal function, osteoporosis, and DPP-IV (Dipeptidyl Peptidase-IV) mediated conditions. By way of example, stomach and intestinal related disorders include ulcers, gastritis, digestion disorders, malabsorption syndrome, short-gut syndrome, cul-de-sac syndrome, inflammatory bowel disease, celiac sprue (e.g., caused by gluten-induced enteropathy or celiac disease), tropical sprue, hypogammaglobulinemia sprue, enteritis, regional enteritis (Crohn's disease), ulcerative colitis, irritable bowel syndrome associated with diarrhea, small intestinal damage, and short bowel syndrome.

[0375] Provided herein is a method for preventing or treating radiation enteritis, infectious or post-infectious enteritis, and small intestinal damage caused by toxic or other chemotherapeutic agents. This may require administering a peptide conjugate before, during, or after a course of chemotherapy or radiotherapy to reduce chemotherapy side effects such as diarrhea, abdominal cramps, and vomiting, and to reduce structural and functional damage to the intestinal epithelium caused by chemotherapy or radiotherapy.

[0376] Provided herein is a method of preventing or treating malnutrition, for example, conditions such as wasting syndrome cachexia and anorexia.

[0377] Provided herein is a method of preventing or treating a disease or condition that would benefit from a GLP-2 receptor modulator in a subject in need thereof, comprising administering to the subject a peptide conjugate described herein.

[0378] combination

[0379] Disclosed herein is a pharmaceutical composition comprising a peptide conjugate described herein and one or more additional therapeutic agents.

[0380] Additional therapeutic agents can include one or more other diabetes drugs, DPP4 inhibitors, SGLT2 inhibitors, hypoglycemic drugs and biguanidine drugs, insulin secretagogues and sulfonylurea drugs, TZD drugs, insulin and insulin analogs, FGF21 and analogs, leptin and leptin analogs, amylin and amylin analogs, anti-inflammatory drugs, cyclosporine A or FK506, 5-ASA or statins, or any combination thereof. Additional therapeutic agents can be aspirin.

[0381] The additional therapeutic agent may comprise a therapeutic incretin or a derivative thereof. Non-limiting examples of incretins or derivatives thereof include GLP-1, glucagon, oxyntomodulin, exendin-4, GLP-2, GIP, and combinations thereof.

[0382] Composition

[0383] Disclosed herein are pharmaceutical compositions comprising a peptide conjugate as described herein and a pharmaceutically acceptable excipient or vehicle. Pharmaceutically acceptable excipients or vehicles can include carriers, excipients, diluents, antioxidants, preservatives, colorants, flavorings, and diluting agents, emulsifying agents, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, tonicity agents, cosolvents, wetting agents, complexing agents, buffers, antimicrobials, and surfactants.

[0384] Neutral buffered saline or saline mixed with serum albumin is an exemplary suitable carrier. The pharmaceutical composition may include antioxidants such as ascorbic acid; low molecular weight polypeptides; proteins such as serum albumin, gelatin or immunoglobulins; hydrophilic polymers such as polyvinyl pyrrolidone; amino acids such as glycine, glutamine, asparagine, arginine or lysine; monosaccharides, disaccharides and other carbohydrates, including glucose, mannose or dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol or sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as Tween, pluronics or polyethylene glycol (PEG). For example, suitable tonicity enhancers include alkali metal halides (preferably sodium chloride or potassium chloride), mannitol, sorbitol, etc. Suitable preservatives include benzalkonium chloride, thimerosal, phenylethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, etc. Hydrogen peroxide can also be used as a preservative. Suitable cosolvents include glycerol, propylene glycol, and PEG. Suitable complexing agents include caffeine, polyvinyl pyrrolidone, β-cyclodextrin, or hydroxypropyl-β-cyclodextrin. Suitable surfactants or wetting agents include sorbitan esters, polysorbates such as polysorbate 80, tromethamine, lecithin, cholesterol, tyloxapal, and the like. The buffer can be a conventional buffer, such as acetate, borate, citrate, phosphate, bicarbonate, or Tris-HCl. Acetate buffer can be about pH 4-5.5, and Tris buffer can be about pH 7-8.5. Other agents are described in Remington's Pharmaceutical Sciences, 18th Edition, edited by A.R. Gennaro, ed., published by Mack Publishing Company, 1990.

[0385] The composition can be in liquid form or freeze-dried or lyophilized form, and can include one or more lyoprotectants, excipients, surfactants, high molecular weight structural additives and / or swelling agents. In one embodiment, a lyoprotectant is included, which is a non-reducing sugar such as sucrose, lactose or trehalose. The amount of the lyoprotectant typically included is such that when reconstituted, the resulting preparation will be isotonic, although hypertonic or slightly hypotonic preparations may also be suitable. In addition, the amount of the lyoprotectant should be enough to prevent degradation and / or aggregation of the unacceptable amount of protein during lyophilization. The exemplary lyoprotectant concentration of sugar (e.g., sucrose, lactose, trehalose) in the pre-lyophilized formulation is from about 10mM to about 400mM. In another embodiment, surfactants are included, for example, nonionic surfactants and ionic surfactants such as polysorbate (e.g., polysorbate 20, polysorbate 80); poloxamer (e.g., poloxamer 188); poly(ethylene glycol) phenyl ether (e.g., Triton); sodium dodecyl sulfate (SDS); sodium laurel sulfate; sodium octyl glucoside (e.g., PEG-60); sodium octyl succinate ... glycoside; lauryl-sulfobetaine, myristyl-sulfobetaine, linoleyl-sulfobetaine, or stearyl-sulfobetaine; lauryl-sarcosine, myristyl-sarcosine, linoleyl-sarcosine, or stearyl-sarcosine; linoleyl-betaine, myristyl-betaine, or cetyl-betaine;lauroamidopropyl-betaine, cocamidopropyl-betaine, linoleamidopropyl-betaine, myristamidopropyl-betaine, palmitopropyl-betaine, or isostearamidopropyl-betaine (e.g., lauroamidopropyl); myristamidopropyl-dimethylamine, palmitopropyl-dimethylamine, or isostearamidopropyl-dimethylamine; sodium methyl cocoyl-taurate or disodium methyl cocoyl taurate. cocoyl-taurate); and MONAQUAT; TMseries (Mona Industries, Inc., Paterson, NJ), polyethylene glycol, polypropylene glycol, and copolymers of ethylene and propylene glycol (e.g., Pluronics, PF68, etc.). An exemplary amount of surfactant that may be present in the pre-lyophilized formulation is about 0.001-0.5%.High molecular weight structural additives (e.g., fillers, binders) can include, for example, acacia, albumin, alginic acid, calcium phosphate (dibasic), cellulose, carboxymethylcellulose, carboxymethylcellulose sodium, hydroxyethylcellulose, hydroxypropylcellulose, hydroxypropylmethylcellulose, microcrystalline cellulose, dextran, dextrin, dextrate, sucrose, tylose, pregelatinized starch, calcium sulfate, amylose, glycine, bentonite, maltose, sorbitol, ethylcellulose, disodium hydrogen phosphate, disodium phosphate,

[0014] Examples of the high molecular weight structural additives include cellulose, maltodextrin, disodium pyrosulfite, polyvinyl alcohol, gelatin, glucose, guar gum, liquid glucose, compressible sugar, magnesium aluminum silicate, maltodextrin, polyethylene oxide, polymethacrylate, povidone, sodium alginate, tragacanth, microcrystalline cellulose, starch, and zein. Exemplary concentrations of the high molecular weight structural additives are 0.1-10% by weight.In other embodiments, bulking agents (eg, mannitol, glycine) may be included.

[0386] The composition may be suitable for parenteral administration. Exemplary compositions are suitable for injection or infusion into animals by any route available to the skilled person, such as intra-articular, subcutaneous, intravenous, intramuscular, intraperitoneal, intracerebral (intracerebral parenchyma), intracerebroventricular, intramuscular, intraocular, intraarterial or intralesional routes. Parenteral formulations may typically be sterile, pyrogen-free, isotonic aqueous solutions, optionally containing a pharmaceutically acceptable preservative.

[0387] Examples of non-aqueous solvents are propylene glycol, polyethylene glycol, vegetable oils such as olive oil, and injectable organic esters such as ethyl oleate. Aqueous carriers include water, alcohol / water solutions, emulsions or suspensions, including saline and buffered media. Parenteral carriers include sodium chloride solution, Ringer's dextrose, glucose and sodium chloride, lactated Ringer's solution, or fixed oils. Intravenous carriers include fluid and nutrient supplements, electrolyte supplements (e.g., those based on Ringer's dextrose), etc. Preservatives and other additives, such as antimicrobials, antioxidants, chelating agents, and inert gases, etc., may also be present. Generally, see Remington's Pharmaceutical Science, 16th Ed., published by Mack Eds., 1980.

[0388] The pharmaceutical compositions described herein can be formulated for controlled or sustained delivery in a manner that provides a local concentration (e.g., bolus, depot effect) and / or increased stability or half-life of the product in a specific local environment. The compositions may include formulations of peptide conjugates disclosed herein having granular formulations of polymeric compounds such as polylactic acid, polyglycolic acid, and agents such as biodegradable matrices, injectable microspheres, microcapsule particles, microcapsules, bioerodible granule beads, liposomes, and implantable delivery devices providing controlled or sustained release of active agents, which can then be delivered as depot injections. The technology for preparing such sustained or controlled delivery methods is known, and a variety of polymers have been developed and used for controlled release and delivery of drugs. Such polymers are typically biodegradable and biocompatible. Polymer hydrogels, including those formed by complexation of enantiomeric polymers or polypeptide fragments, and hydrogels with temperature or pH sensitivity, may be desirable for providing a drug depot effect due to the mild and aqueous conditions involved in capturing bioactive protein reagents (e.g., antibodies comprising ultralong CDR3s).

[0389] According to the expected route of administration, delivery form and required dosage, suitable and / or preferred pharmaceutical formulations can be determined in view of the general knowledge of the present disclosure and formulation technology. Regardless of the mode of administration, the effective dose can be calculated based on the patient's weight, body surface area or organ size. Further refinement of the calculation for determining the appropriate dose for the treatment of each preparation described herein is routinely performed in the art and is within the scope of the tasks routinely performed in the art. Appropriate dosage can be determined by using appropriate dose-response data.

[0390] definition

[0391] As used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "an agent" includes a plurality of such agents, reference to "the cell" includes reference to one or more cells (or reference to a plurality of cells) and equivalents thereof known to those skilled in the art, and so forth. When ranges are used herein for physical properties (e.g., molecular weight) or chemical properties (e.g., chemical formulae), all combinations and subcombinations of ranges and specific embodiments thereof are intended to be included. When referring to a number or numerical range, the term "about" means that the referenced number or numerical range is an approximation within the experimental variability (or within the statistical experimental error), and thus, in some cases, the number or numerical range will vary between 1% and 15% of the number or numerical range. The term "comprising" (and related terms such as "comprise," "comprises," or "having," or "including") is not intended to exclude certain other embodiments, e.g., embodiments of any composition of matter, composition, method, process, etc. described herein "consisting of" or "consisting essentially of" the recited features.

[0392] As used in the specification and appended claims, the following terms have the following indicated meanings unless indicated to the contrary.

[0393] "Alkyl" refers to a straight or branched chain hydrocarbon monovalent group, which may be fully saturated or unsaturated, having from 1 to about 10 carbon atoms or from 1 to 6 carbon atoms, in which the sp3 hybridized carbon of the alkyl residue is attached to the rest of the molecule by a single bond. Examples of saturated hydrocarbon monovalent groups include, but are not limited to, methyl, ethyl, n-propyl, isopropyl, 2-methyl-1-propyl, 2-methyl-2-propyl, 2-methyl-1-butyl, 3-methyl-1-butyl, 2-methyl-3-butyl, 2,2-dimethyl-1-propyl, 2-methyl-1-pentyl, 3-methyl-1-pentyl, 4-methyl-1-pentyl, 2-methyl-2-pentyl, 3-methyl-2-pentyl, 4-methyl-2-pentyl, 2,2-dimethyl-1-butyl, 3,3-dimethyl-1-butyl, 2-ethyl-1-butyl, n-butyl, isobutyl, sec-butyl, tert-butyl, n-pentyl, isopentyl, neopentyl, tert-pentyl, and hexyl, as well as longer alkyl groups such as heptyl, octyl, and the like. Whenever it appears herein, a numerical range such as "C1-C6 alkyl" means that the alkyl group consists of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms, although this definition also encompasses occurrences of the term "alkyl" where no numerical range is specified. In some embodiments, the alkyl group is C1-C6. 10 Alkyl, C1-C9 alkyl, C1-C8 alkyl, C1-C7 alkyl, C1-C6 alkyl, C1-C5 alkyl, C1-C4 alkyl, C1-C3 alkyl, C1-C2 alkyl or C1 alkyl. When alkyl refers to an unsaturated straight or branched hydrocarbon monovalent radical, it is referred to as an "alkenyl" or "alkynyl". Alkenyl can be in cis or trans conformation about the double bond and should be understood to include both isomers. Examples of alkenyl include, but are not limited to, vinyl (-CH=CH2), 1-propenyl (-CH2CH=CH2), isopropenyl [-C(CH3)=CH2], butenyl, 1,3-butadienyl, etc. Whenever it appears herein, a numerical range such as "C2-C6 alkenyl" means that the alkenyl group can be composed of 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms or 6 carbon atoms, although this definition also covers the occurrence of the term "alkenyl" in which no numerical range is specified. In some embodiments, alkenyl is C2-C 10 In some embodiments, the term "alkynyl" refers to a C2-C9 alkenyl, a C2-C8 alkenyl, a C2-C7 alkenyl, a C2-C6 alkenyl, a C2-C5 alkenyl, a C2-C4 alkenyl, a C2-C3 alkenyl or a C2 alkenyl. Examples of alkenyl groups include, but are not limited to, ethynyl, 2-propynyl, 2-, etc. Whenever it appears herein, a numerical range such as "C2-C6 alkynyl" means that the alkynyl group can be composed of 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms or 6 carbon atoms, although this definition also covers the occurrence of the term "alkynyl" where no numerical range is specified. In some embodiments, the alkenyl group is C2-C 10Alkyl, C2-C9 alkynyl, C2-C8 alkynyl, C2-C7 alkynyl, C2-C6 alkynyl, C2-C5 alkynyl, C2-C4 alkynyl, C2-C3 alkynyl or C2 alkynyl.Unless specifically stated otherwise in the specification sheets, alkyl is optionally replaced by, for example, oxo, halogen, amino, nitrile, nitro, hydroxyl, haloalkyl, alkoxy, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, as described below.In some embodiments, alkyl is optionally replaced by oxo, halogen ,-CN ,-CF ,-OH ,-OMe ,-NH or-NO .In some embodiments, alkyl is optionally replaced by oxo, halogen ,-CN ,-CF ,-OH or-OMe.In some embodiments, alkyl is optionally replaced by halogen.

[0394] "Alkylene" refers to a straight or branched divalent hydrocarbon chain. Whenever it appears herein, a numerical range such as "C1-C6 alkylene" means that the alkylene consists of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms, although this definition also encompasses occurrences of the term "alkylene" where no numerical range is specified. In some embodiments, the alkylene is C1-C6. 10 Alkylene, C1-C9 alkylene, C1-C8 alkylene, C1-C7 alkylene, C1-C6 alkylene, C1-C5 alkylene, C1-C4 alkylene, C1-C3 alkylene, C1-C2 alkylene or C1 alkylene.Unless otherwise specifically stated in the specification, alkylene can be optionally replaced by, for example, oxo, halogen, amino, nitrile, nitro, hydroxyl, haloalkyl, alkoxy, aryl, cycloalkyl, heterocycloalkyl, heteroaryl etc. In some embodiments, alkylene is optionally replaced by oxo, halogen ,-CN ,-CF ,-OH ,-OMe ,-NH or-NO .In some embodiments, alkylene is optionally replaced by oxo, halogen ,-CN ,-CF ,-OH or-OMe.In some embodiments, alkylene is optionally replaced by halogen.

[0395] "Alkoxy" refers to a group of the formula -OR a A group in which R a For alkyl as defined. Unless specifically stated otherwise in the specification, alkoxy can be optionally substituted by, for example, oxo, halogen, amino, nitrile, nitro, hydroxyl, haloalkyl, alkoxy, aryl, cycloalkyl, heterocycloalkyl, heteroaryl etc. In some embodiments, alkoxy is optionally substituted by oxo, halogen, -CN, -CF , -OH, -OMe, -NH or -NO . In some embodiments, alkoxy is optionally substituted by oxo, halogen, -CN, -CF , -OH or -OMe. In some embodiments, alkoxy is optionally substituted by halogen.

[0396] "Aryl" refers to a group derived from a hydrocarbon ring system comprising hydrogen, 6 to 30 carbon atoms and at least one aromatic ring. Aryl can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which can include a fused ring (when fused to a cycloalkyl or heterocycloalkyl ring, the aryl is bonded by aromatic ring atoms) or a bridged ring system. In some embodiments, aryl is a 6-10 yuan aryl. In some embodiments, aryl is a 6 yuan aryl. Aryl includes but is not limited to an aryl derived from anthracene, naphthylene, phenanthrenyl, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, asymmetric indacene (as-indacene), symmetric indacene (s-indacene), indane, indene, naphthalene, phenanthren, phenanthren, pleiadene, pyrene and triphenylene hydrocarbon ring system. In some embodiments, aryl is phenyl. Unless otherwise specifically stated in the specification, aryl can be optionally substituted by, for example, halogen, amino, nitrile, nitro, hydroxyl, alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, etc. In some embodiments, aryl is optionally substituted by halogen, methyl, ethyl, -CN, -CF , -OH, -OMe, -NH or -NO . In some embodiments, aryl is optionally substituted by halogen, methyl, ethyl, -CN, -CF , -OH or -OMe. In some embodiments, aryl is optionally substituted by halogen.

[0397] "Cycloalkyl" refers to a stable, partially or fully saturated monocyclic or polycyclic carbocyclic ring which may include a fused ring (when fused to an aryl or heteroaryl ring, the cycloalkyl is bonded through a non-aromatic ring atom) or a bridged ring system. Representative cycloalkyl groups include, but are not limited to, groups having 3-15 carbon atoms (C3-C 15 Cycloalkyl), 3-10 carbon atoms (C3-C 10In some embodiments, the cycloalkyl group is a 3-6 membered cycloalkyl group. In some embodiments, the cycloalkyl group is a 5-6 membered cycloalkyl group. Monocyclic cycloalkyl groups include, for example, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, and cyclooctyl. Polycyclic cycloalkyl or carbocycle include, for example, adamantyl, norbornyl, decahydronaphthalene, bicyclo [3.3.0] octane, bicyclo [4.3.0] nonane, cis-decalin, trans-decalin, bicyclo [2.1.1] hexane, bicyclo [2.2.1] heptane, bicyclo [2.2.2] octane, bicyclo [3.2.2] nonane and bicyclo [3.3.2] decane and 7,7-dimethyl-bicyclo [2.2.1] heptane. Partially saturated cycloalkyl includes, for example, cyclopentenyl, cyclohexenyl, cycloheptenyl and cyclooctenyl. Unless otherwise specified in the specification, cycloalkyl is optionally substituted by, for example, oxo, halogen, amino, nitrile, nitro, hydroxyl, alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, etc. In some embodiments, cycloalkyl is optionally substituted with oxo, halogen, methyl, ethyl, -CN, -CF , -OH, -OMe, -NH or -NO . In some embodiments, cycloalkyl is optionally substituted with oxo, halogen, methyl, ethyl, -CN, -CF , -OH or -OMe. In some embodiments, cycloalkyl is optionally substituted with halogen.

[0398] "Halo" or "halogen" refers to bromo, chloro, fluoro, or iodo. In some embodiments, halogen is fluoro or chloro. In some embodiments, halogen is fluoro.

[0399] "Haloalkyl" refers to an alkyl group as defined above substituted with one or more halo groups as defined above, for example, trifluoromethyl, difluoromethyl, fluoromethyl, trichloromethyl, 2,2,2-trifluoroethyl, 1,2-difluoroethyl, 3-bromo-2-fluoropropyl, 1,2-dibromoethyl, and the like.

[0400] "Heterocycloalkyl" refers to a stable 3-24 membered partially or fully saturated ring group containing 2-23 carbon atoms and 1-8 heteroatoms selected from nitrogen, oxygen, phosphorus and sulfur. Representative heterocycloalkyls include, but are not limited to, heterocycloalkyls (C2-C 15 Heterocycloalkyl), heterocycloalkyl with 2 to 10 carbon atoms (C2-C 10In some embodiments, the heterocycloalkyl group is a 3-6 membered heterocycloalkyl group. In some embodiments, the heterocycloalkyl group is a 5-6 membered heterocycloalkyl group. Unless otherwise specifically stated in the specification, the heterocycloalkyl group may be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which may include a fused ring (when fused to an aryl or heteroaryl ring, the heterocycloalkyl group is bonded through a non-aromatic ring atom) or a bridged ring system; and the nitrogen, carbon or sulfur atoms in the heterocycloalkyl group may be optionally oxidized; the nitrogen atom may be optionally quaternized. Examples of such heterocycloalkyl groups include, but are not limited to, aziridinyl, azetidinyl, dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolinyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidon ... ), pyrrolidinyl, pyrazolidinyl, quinuclidine, thiazolidinyl, tetrahydrofuranyl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiomorpholinyl, 1-oxo-thiomorpholinyl, 1,1-dioxo-thiomorpholinyl, 1,3-dihydroisobenzofuran-1-yl, 3-oxo-1,3-dihydroisobenzofuran-1-yl, methyl-2-oxo-1,3-dioxol-4-yl and 2-oxo-1,3-dioxol-4-yl. The term heterocycloalkyl also includes all ring forms of carbohydrates, including but not limited to monosaccharides, disaccharides and oligosaccharides. Unless otherwise specified, heterocycloalkyl has 2-10 carbons in the ring. It should be understood that when referring to the number of carbon atoms in heterocycloalkyl, the number of carbon atoms in heterocycloalkyl is different from the total number of atoms (including heteroatoms) constituting heterocycloalkyl (i.e., the skeleton atoms of heterocycloalkyl ring). Partially saturated heterocycloalkyl includes, for example, dihydropyrrolyl or tetrahydropyridine. Unless otherwise specifically stated in the specification, heterocycloalkyl is optionally substituted by, for example, oxo, halogen, amino, nitrile, nitro, hydroxyl, alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, etc. In some embodiments, heterocycloalkyl is optionally substituted by oxo, halogen, methyl, ethyl, -CN, -CF , -OH, -OMe, -NH or -NO . In some embodiments, heterocycloalkyl is optionally substituted by oxo, halogen, methyl, ethyl, -CN, -CF , -OH or -OMe. In some embodiments, heterocycloalkyl is optionally substituted by halogen.

[0401] " assorted alkyl " refers to an alkyl group, wherein the one or more skeleton atoms of the alkyl are selected from the atom other than carbon, such as oxygen, nitrogen (such as-NH-,-N (alkyl)-), sulphur or its combination. Assorted alkyl is connected to the rest of the molecule at the carbon atom of assorted alkyl. In one aspect, assorted alkyl is C1-C6 assorted alkyl, wherein assorted alkyl comprises 1-6 carbon atoms and one or more atoms other than carbon, such as oxygen, nitrogen (such as-NH-,-N (alkyl)-), sulphur or its combination, wherein assorted alkyl is connected to the rest of the molecule at the carbon atom of assorted alkyl. Unless otherwise specifically stated in the specification, assorted alkyl is optionally substituted by such as oxo, halogen, amino, nitrile, nitro, hydroxyl, alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, aryl, cycloalkyl, heterocycloalkyl, heteroaryl etc. In some embodiments, assorted alkyl is optionally substituted by oxo, halogen, methyl, ethyl ,-CN ,-CF3,-OH,-OMe,-NH2 or-NO2. In some embodiments, heteroalkyl is optionally substituted with oxo, halogen, methyl, ethyl, -CN, -CF 3 , -OH or -OMe. In some embodiments, heteroalkyl is optionally substituted with halogen.

[0402] "Heteroaryl" refers to a 5-14 membered ring system radical comprising hydrogen atoms, 1-13 carbon atoms, 1-6 heteroatoms selected from nitrogen, oxygen, phosphorus and sulfur, and at least one aromatic ring. The heteroaryl group can be a monocyclic, bicyclic, tricyclic or tetracyclic ring system, which can include a fused ring (when fused to a cycloalkyl or heterocycloalkyl ring, the heteroaryl group is bonded through an aromatic ring atom) or a bridged ring system; and the nitrogen, carbon or sulfur atoms in the heteroaryl group can be optionally oxidized; the nitrogen atom can be optionally quaternized. In some embodiments, the heteroaryl group is a 5-10 membered heteroaryl group. In some embodiments, the heteroaryl group is a 5-6 membered heteroaryl group. In some embodiments, the heteroaryl group is a 5 membered heteroaryl group. In some embodiments, the heteroaryl group is a 6 membered heteroaryl group. Examples include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepanyl, 1,4-benzodioxanyl, benzonaphthofuranyl, yl), benzoxazolyl, benzodioxazolyl, benzodioxinyl, benzopyranyl, benzopyrone, benzofuranyl, benzofuranone, benzothiophenyl (benzophenylthio), benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzophenylthio, furanyl, furanone, isothiazolyl, imidazolyl, indazolyl, indole 1-H-pyrrolyl, 1-phenyl-1 ... The heteroaryl group may be substituted with halogen, amino, nitrile, nitro, hydroxy, alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, etc.In some embodiments, heteroaryl is optionally substituted by halogen, methyl, ethyl, -CN, -CF , -OH, -OMe, -NH , or -NO . In some embodiments, heteroaryl is optionally substituted by halogen, methyl, ethyl, -CN, -CF , -OH, or -OMe. In some embodiments, heteroaryl is optionally substituted by halogen.

[0403] The term "percent identity" refers to the comparison between two nucleic acid or amino acid sequences. This comparison is measured using any number of alignment methods known in the art, including but not limited to global (e.g., Needleman-Wunsch algorithm) or local alignment (e.g., Smith-Waterman, Sellers or other algorithms). Percent identity generally refers to the percentage of matching positions of two sequences for a continuous portion of a position, wherein the two sequences are arranged in a manner that maximizes the matching position and minimizes the gap (gap) of the non-matching position. In some cases, an alignment is performed in which there is no gap between the two sequences. In some cases, the alignment results in a gap that is less than 5%, less than 3%, or less than 1%. Other methods of sequence comparison or alignment are also consistent with the present disclosure.

[0404] As used herein, the term "homology" can be used to calculate the "homology" or "homology percentage" between two or more amino acid sequences, which can be determined by aligning the sequences for optimal comparison purposes (e.g., gaps can be introduced into the sequence of the first sequence). The amino acids at the corresponding positions can then be compared, and the percentage identity between the two sequences can be a function of the number of identical positions shared by the sequences (i.e., % homology = number of identical positions # / total number of positions # × 100). For example, a position in the first sequence can be occupied by the same amino acid as the corresponding position in the second sequence, and the molecules are identical at that position. The percentage homology between the two sequences can be a function of the number of identical positions shared by the sequences, taking into account the number of gaps and the length of each gap, which need to be introduced for optimal alignment of the two sequences. In some embodiments, the length of sequences aligned for comparison purposes can be at least about 30%, 40%, 50%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 95% of the length of the reference sequence. The search can determine the homology between two sequences. The homology can be between the full length of the two sequences or between portions of the full length of the two sequences. The two sequences can be peptide sequences, amino acid sequences, or fragments thereof. The actual comparison of the two sequences can be accomplished by known methods, such as using a mathematical algorithm. Non-limiting examples of such mathematical algorithms can be described in Karlin, S. and Altschul, S., Proc. Natl. Acad. Sci. USA, 90-5873-5877 (1993). Such algorithms can be incorporated into the NBLAST and XBLAST programs (version 2.0), described in Altschul, S. et al., Nucleic Acids Res., 25:3389-3402 (1997). When using BLAST and Gapped BLAST programs, any relevant parameters of the corresponding program (e.g., NBLAST) can be used. For example, the parameters for sequence comparison can be set to score = 100, word length = 12, or can be varied (e.g., W = 5 or W = 20). Other examples include Myers and Miller, CABIOS (1989), ADVANCE, ADAM, BLAT and FASTA algorithms. In another embodiment, the percent identity between two amino acid sequences can be determined using, for example, the GAP program in the GCG software package (Accelrys, Cambridge, UK).

[0405] "Pharmaceutically acceptable" means approved or approved by a regulatory agency of the Federal or a state government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeia for use in animals, including humans.

[0406] "Pharmaceutically acceptable salt" refers to a salt of a compound that is pharmaceutically acceptable and that possesses the desired pharmacological activity of the parent compound.

[0407] A "pharmaceutically acceptable excipient, carrier, or adjuvant" refers to an excipient, carrier, or adjuvant that can be administered to a subject with at least one antibody of the present disclosure and that does not destroy its pharmacological activity and is non-toxic when administered in a dose sufficient to deliver a therapeutic amount of the compound.

[0408] "Pharmaceutically acceptable vehicle" refers to a diluent, adjuvant, excipient, or carrier with which at least one antibody of the present disclosure is administered.

[0409] Terms such as "treating / treatment / to treat" or "alleviating / to alleviate" and the like may refer to: 1) therapeutic measures that cure, slow down, alleviate the symptoms of, and / or block the progression of, a diagnosed pathological condition or disorder; and / or 2) prophylactic or preventative measures that prevent and / or slow the development of a target pathological condition or disorder. "Treatment" refers to clinical intervention that attempts to alter the natural course of the individual or cell being treated, and may be performed for prevention or during the course of clinical pathology. Desirable effects of treatment include preventing the occurrence or recurrence of the disease, alleviating symptoms and reducing any direct or indirect pathological consequences of the disease, preventing metastasis, reducing the rate of disease progression, ameliorating or alleviating the disease state, and alleviating or improving prognosis. Thus, persons in need of treatment may include persons already suffering from such a disorder; persons susceptible to such a disorder; and persons for whom such a disorder is to be prevented.

[0410] "Amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function similarly to naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, such as hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acid analogs refer to compounds that have the same basic chemical structure as naturally occurring amino acids, such as an alpha carbon bound to hydrogen, a carboxyl group, an amino group, and an R group, such as homoserine, norleucine, methionine sulfoxide, and methionine methylsulfonium. Such analogs may have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but function similarly to a naturally occurring amino acid.

[0411] "Disorder" or "disease" refers to a condition that would benefit from treatment with a substance / molecule (e.g., a peptide conjugate disclosed herein) or method disclosed herein. This includes chronic and acute disorders or diseases, including those pathological conditions that predispose a mammal to the disorder.

[0412] "Mammal" for therapeutic purposes refers to any animal classified as a mammal, including humans, rodents (e.g., mice and rats), and monkeys; livestock and farm animals; and zoo, sport, laboratory, or pet animals, such as dogs, cats, cows, horses, sheep, pigs, goats, rabbits, etc. In some embodiments, the mammal is selected from humans, rodents, or monkeys.

[0413] "Unmodified peptide" refers to the unmodified sequence (wild-type peptide) or the modified sequence without a stapler.

[0414] Example

[0415] Peptides were synthesized by standard solid phase peptide synthesis (SPPS) techniques and purified by HPLC (as described).

[0416] Unless otherwise stated, all reagents were purchased from commercial suppliers (Sigma Aldrich, Fisher, Oakwood) and used without further purification. Peptides were purchased from Cellmano Biotech Limited (Hefei), InnoPep (San Diego), Shanghai Apeptide Co. (Shanghai), or Shanghai Dechi Biosciences Co. (Shanghai). All reactions involving air- or moisture-sensitive reagents or intermediates were carried out under an inert atmosphere of nitrogen or argon. All solvents used were HPLC grade. Reactions were monitored by LC-MS or by thin layer chromatography (TLC) on Merck 50×100 mm silica gel 60 aluminum sheets stained with an aqueous solution of KMnO4.

[0417] Flash chromatography purification was performed on a pre-packed silica gel column (40 μm, from Teledyne Isco Rf) The purified final compound eluted as a single and symmetrical peak (thus confirming a purity of > 95%).

[0418] The HPLC was carried out on a Shimadzu HPLC system using a Phenomenex Luna column (C18, pore size Particle size 10 μm, 250 × 10.0 mm, flow rate: 4 mL / min) or on an Agilent 1200 HPLC using a Phenomenex Luna column (C18, pore size Semi-preparative chromatography was performed using a 5 μm particle size column (150 × 21.2 mm, flow rate: 20 mL / min).

[0419] Recorded on a Bruker 400 system in d6-DMSO, CDCl3 or CD3OD 1 H and 13C NMR spectra. Chemical shifts are given in parts per million (ppm) with tetramethylsilane as the internal standard. Abbreviations used are as follows: s = singlet, d = doublet, t = triplet, q = quartet, p = quintet, m = multiplet, dd = doublet of doublets, br = broad. Coupling constants (J values) are given in Hertz (Hz). Analyses were performed on a Phemomenex Luna Omega C18 column (C18, pore size Low-resolution mass spectra were recorded on a Waters Acquity UPLC (particle size 1.6 μm, 50×2.1 mm, flow rate: 0.4 mL / min). Solvent: A—H₂O + 0.1% formic acid, B—MeCN + 0.1% formic acid, gradient: 0-1 min 10-90% B, 1-1.6 min 90% B, 1.6-1.7 min 90-10% B, 1.7-2 min 10% B.

[0420] High-resolution mass spectra (HRMS) were recorded on an Agilent 1200 Series precision time-of-flight mass spectrometer (TOF) equipped with an Aeris Widepore column (XB-C8, particle size 3.6 μm, 150×2.1 mm, flow rate: 0.5 mL / min). Solvent: A—H 2 O + 0.1% formic acid, B—MeCN + 0.1% formic acid, gradient: 0-2 min 5% B, 2-12 min 5-60% B, 12-13 min 60-80% B, 13-14 min 80-20% B, 14-15 min 20-80% B, 15-16 min 80-20% B, 16-17 min 20-95% B, 17-20 min 95% B, 20-21 min 95-5% B.

[0421] General Protocol A for loading chlorotrityl chloride resin

[0422] Fmoc-Lys(ivDde)-OH (60 mg, 100 μmol) was coupled to 2-chlorotrityl chloride resin (Novabiochem) (100 mg, 80 μmol) by mixing the amino acid, resin, and DIEA (70 μL, 400 μmol) in 5 mL DMF and stirring for 30 min. The resin was then washed with DMF (3×), DCM (3×), and treated with CH OH / DCM / DIEA (8:1:1) for 10 min to block unreacted trityl chloride sites, dried under vacuum, and stored in a desiccator.

[0423] General Scheme B for Deprotection of Fmoc Protecting Groups

[0424] A 20% solution of piperidine in DMF was added to the resin. The mixture was shaken for 5 minutes and drained. Fresh 20% piperidine was added and the mixture was shaken for 15 minutes. A positive ninhydrin and / or TNBS test was observed. The resin was then washed with DMF (3x) and DCM (3x).

[0425] General Scheme C for Deprotection of ivDde Protecting Group

[0426] After washing with DMF and DCM, the resin was treated with 2% hydrazine in DMF (5 mL, 2 x 15 min). A positive ninhydrin and / or TNBS test was observed. The resin was then washed with DMF (3 x), DCM (3 x).

[0427] General Scheme D for Peptide Coupling

[0428] The resin was treated with the indicated carboxylic acid derivative (3 eq) using a solution of coupling agent HATU (3.3 eq) and DIEA (3.3 eq) in DMF (5 mL) for 2 h or repeatedly until a negative ninhydrin and / or TNBS test was observed. The resin was then washed with DMF (3×), DCM (3×).

[0429] General Protocol E for On-Resin Bromoacetylation

[0430] The resin was then treated with a solution of bromoacetic anhydride (2.4 eq.) and DIEA (2.6 eq.) in 200 mL of DCM for 30 min.

[0431] General Protocol F for Cleavage of Peptides from Chlorotrityl Chloride Resin

[0432] The resin was washed with DCM (3x) and the product was cleaved from the resin using 5 mL of 10% TFA in DCM containing 10% H2O and 10% triisopropylsilane for 1 h.

[0433] Example 1: Synthesis of L1

[0434]

[0435] To a solution of 1,4-diaminobutane (80 μL, 0.795 mmol, 1 eq) in DCM (10 mL) was added DIEA (276 μL, 1.59 mmol, 2 eq) at 0 ° C., followed by the addition of bromoacetic anhydride (413 g, 1.59 mmol, 2 eq) dissolved in 1 mL of DCM. The reaction mixture was then stirred at 0 ° C. for 30 minutes, at room temperature for 1.5 h, and the solvent was removed. Purification by silica gel flash column chromatography gave L1 (162 mg, 0.49 mmol, 61%) as a white solid. MS (ES) + )m / z 331.0([M+H]+ ). 1 H NMR (400 MHz, methanol-d4) δ 3.94 (s, 4H), 3.40-3.30 (m, 4H), 1.68 (p, J = 3.5 Hz, 4H).

[0436] Example 2: Synthesis of L1B

[0437]

[0438] To a solution of 1,2-ethylenediamine (30 μL, 0.448 mmol, 1 eq) in DCM (5 mL) was added DIEA (172 μL, 0.985 mmol, 2.2 eq) at 0 ° C., followed by bromoacetic anhydride (233 mg, 0.897 mmol, 2 eq) dissolved in 1 mL of DCM. The reaction mixture was then stirred at 0 ° C. for 30 minutes, at room temperature for 1.5 h, and the solvent was removed. Purification by silica gel flash column chromatography gave L1B (43.9 mg, 0.145 mmol, 32%) as a white solid. MS (ES) + )m / z 302.55([M+H] + ),304.54([M+H] + ). 1 H NMR (400 MHz, methanol-d4) δ 2.49 (s, 4H), 2.06 (s, 4H).

[0439] Example 3: Synthesis of L1C

[0440]

[0441] To a solution of 1,3-diaminopropane (30 μL, 0.359 mmol, 1 eq) in DCM (5 mL) was added DIEA (138 μL, 0.789 mmol, 2.2 eq) at 0 ° C., followed by the addition of bromoacetic anhydride (186 mg, 0.718 mmol, 2 eq) dissolved in 1 mL of DCM. The reaction mixture was then stirred at 0 ° C. for 30 minutes, at room temperature for 1.5 h, and the solvent was removed. Purification by silica gel flash column chromatography gave L1C (60.8 mg, 0.19 mmol, 53%) as a white solid. MS (ES) + )m / z316.32([M+H] + ),318.6([M+H] + ). 1 H NMR (400MHz, methanol-d4) δ3.86 (s, 4H), 3.27 (t, J = 6.8 Hz, 4H), 1.74 (p, J = 6.8 Hz, 2H).

[0442] Example 4: Synthesis of L1D

[0443]

[0444] To a solution of 1,7-diaminohexane (65 mg, 0.499 mmol, 1 eq) in DCM (15 mL) was added DIEA (208 μL, 1.197 mmol, 2.4 eq) at 0 ° C., followed by bromoacetic anhydride (259 mg, 0.998 mmol, 2 eq) dissolved in 1 mL of DCM. The reaction mixture was then stirred at 0 ° C. for 30 minutes, at room temperature for 1.5 h, and the solvent was removed. Purification by silica gel flash column chromatography gave L1D (120 mg, 0.322 mmol, 64%) as a white solid. MS (ES) + )m / z372.71([M+H] + ),374.70([M+3H] + ). 1 H NMR (400 MHz, CHLOROFORM-d) δ 6.55 (s, 2H), 3.91 (s, 4H), 3.30 (q, J = 7.1 Hz, 4H), 1.56 (p, J = 7.1 Hz, 4H), 1.45-1.29 (m, 6H).

[0445] Example 5: Synthesis of L1E

[0446]

[0447] To a solution of 1,11-diaminoundecane (48 mg, 0.257 mmol, 1 eq) in DCM (10 mL) was added DIEA (108 μL, 0.616 mmol, 2.4 eq) at 0 ° C., followed by the addition of bromoacetic anhydride (134 mg, 0.515 mmol, 2 eq) dissolved in 1 mL of DCM. The reaction mixture was then stirred at 0 ° C. for 30 minutes, at room temperature for 1.5 h, and the solvent was removed. Purification by silica gel flash column chromatography gave L1E (62.3 mg, 0.145 mmol, 56%) as a white solid. MS (ES) + )m / z428.33([M+H] + ). 1 H NMR (400MHz, chloroform-d) δ6.53 (s, 2H), 3.91 (s, 4H), 3.30 (q, J = 6.8Hz, 4H), 1.57 (q, J = 7.2Hz, 4H), 1.42-1.20 (m, 14H).

[0448] Example 6: Synthesis of L1F

[0449]

[0450] To a solution of cadaverine (48 mg, 0.257 mmol, 1 eq) in DCM (20 mL) was added DIEA (284 μL, 1.63 mmol, 2.4 eq) at 0 ° C., followed by bromoacetic anhydride (353 mg, 1.36 mmol, 2 eq) dissolved in 1 mL of DCM. The reaction mixture was then stirred at 0 ° C. for 30 minutes, at room temperature for 1.5 h, and the solvent was removed. Purification by silica gel flash column chromatography gave L1F (156 mg, 0.453 mmol, 66%) as a white solid. MS (ES) + )m / z 344.65([M+H] + ),346.64([M+H] + ). 1 H NMR (400 MHz, methanol-d4) δ 3.83 (s, 4H), 3.23 (q, J = 6.8 Hz, 4H), 1.57 (p, J = 7.2 Hz, 4H), 1.44-1.33 (m, 2H).

[0451] Example 7: Synthesis of L1G

[0452]

[0453] Intermediate L1Ga

[0454] To a DCM (20 mL) solution of tert-butyl bis (2-aminoethyl) carbamate (167 mg, 0.82 mmol, 1 equivalent) was added DIEA (342 μL, 11.96 mmol, 2.4 equivalents) at 0 ° C., followed by the addition of bromoacetic anhydride (426 mg, 1.64 mmol, 2 equivalents) dissolved in 1 mL of DCM. The reaction mixture was then stirred at 0 ° C. for 30 minutes, stirred at room temperature for 1.5 h, and the solvent was removed. Purification by silica gel flash column chromatography gave L1Ga (289 mg, 0.65 mmol, 79%) as a white solid. MS (ES) + )m / z 445.71([M+H] + ),447.7([M+H] + ). 1 H NMR (400 MHz, methanol-d4) δ 3.85 (s, 4H), 3.39 (s, 9H), 1.50 (s, 10H).

[0455] L1G

[0456] Compound L1Ga (20 mg) was dissolved in TFA / DCM (1:1, v / v, 2 mL), stirred at room temperature for 30 minutes and evaporated (co-evaporated with hexane) to give compound L1G as an oil. The product was used directly in the other steps. MS (ES + )m / z 345.2([M+H] + ).

[0457] Example 8: Synthesis of L3

[0458]

[0459] Intermediate L3a

[0460] Myristic acid (184 mg, 0.805 mmol, 1 equivalent) was dissolved in 4 mL of DMF. HATU (321 mg, 0.845 mmol, 1.1 equivalents) and DIEA (154 μL, 0.885 mmol, 1.1 equivalents) were added, followed by the addition of Boc-NH-PEG2-COOH (200 mg, 0.805 mmol, 1 equivalent). The reaction mixture was then stirred for 1.5 h and the solvent removed. The product was dissolved in EtOAc. The organic layer was washed with 1 M HCl, saturated NaHCO3 and brine, dried over Na2SO4, filtered, and concentrated. Purification by flash column chromatography on silica gel afforded the desired compound L3a (254 mg, 0.55 mmol, 69%) as a white solid. 1 H NMR (400 MHz, chloroform-d) δ 3.66-3.54 (m, 8H), 3.49 (q, J = 5.2 Hz, 2H), 3.35 (d, J = 6.1 Hz, 2H), 2.20 (t, J = 7.7 Hz, 2H), 1.63-1.58 (m, 2H), 1.47 (s, 8H), 1.33-1.24 (m, 21H), 0.90 (t, J = 6.9 Hz, 3H). R =2.21min (Agilent). MS (ES + )m / z 459.6([M+H] + )

[0461] Intermediate L3b

[0462] A solution of compound L3a (242 mg, 0.527 mmol, 1 equivalent) in DCM (2 mL) was treated with TFA (2 mL) for 30 minutes. The mixture was concentrated and co-evaporated with hexane. HATU (224 mg, 0.59 mmol, 1.1 equivalents) was added to a solution of BocNH-PEG2-CO2H (146 mg, 0.527 mol, 1 equivalent) dissolved in DMF (5 mL). A DMF solution of deprotected compound L3a and DIEA (183 μL, 1.05 mmol, 2 equivalents) was added to the reaction mixture. The reaction mixture was stirred at room temperature for 2 h. The product was diluted with EtOAc. The organic layer was washed with 1M HCl, saturated NaHCO3 and brine in sequence, dried over Na2SO4, filtered, and concentrated. Purification by flash column chromatography on silica gel gave the desired oily compound L3b (129 mg, 0.209 mmol, 40%). 1 H NMR (400MHz, chloroform-d) δ6.76(s,1H),6.19(s,1H),5.29(s,1H),3.76(t,J=5. 8Hz, 2H), 3.69-3.62 (m, 8H), 3.57 (dt, J = 12.3, 5.0Hz, 6H), 3.48 (dt, J = 10. 4,5.5Hz,4H),3.33(s,2H),2.51(t,J=5.8Hz,2H),2.20(t,J=7.0Hz,2H),1 .90-1.75(m,4H),1.64(p,J=7.3Hz,2H),1.46(s,9H),1.33-1.22(m,17H).

[0463] Intermediate L3c

[0464] A solution of compound L3b (129 mg, 0.209 mmol, 1 equivalent) in DCM (2 mL) was treated with TFA (2 mL) for 30 minutes. The mixture was concentrated and co-evaporated with hexane. HATU (88 mg, 0.23 mmol, 1.1 equivalents) was added to a solution of Boc-Orn(Boc)-OH (69 mg, 0.209 mmol, 1 equivalent) dissolved in DMF (5 mL). A DMF solution of deprotected compound L3b and DIEA (73 μL, 0.419 mmol, 2 equivalents) was added to the reaction mixture. The reaction mixture was stirred at room temperature for 2 h. The product was diluted with EtOAc. The organic layer was washed with 1M HCl, saturated NaHCO3 and brine in sequence, dried over Na2SO4, filtered, and concentrated. Purification by silica gel flash column chromatography gave the desired oily compound L3c (137 mg, 0.164 mmol, 78%). R =4.07 min (Agilent). MS (ES+ )m / z832.9([M+H] + ). 1 H NMR (400MHz, chloroform-d) δ7.12(s,1H),6.80(s,1H),6.30(s,1H),4.87(s,1H), 3.85-3.73(m,2H),3.68-3.61(m,7H),3.58(p,J=6.1,5.5Hz,7H),3.53-3. 36(m,6H),3.29-3.00(m,2H),2.51(t,J=5.8Hz,2H),2.20(t,J=7.7Hz,2H) ,2.00-1.74(m,6H),1.71-1.51(m,5H),1.45(s,18H),1.35-1.22(m,21H).

[0465] L3

[0466] A solution of compound L3c (137 mg, 0.165 mmol, 1 equivalent) in DCM (2 mL) was treated with TFA (2 mL) for 30 minutes. The mixture was concentrated, co-evaporated with hexane and dissolved in 10 mL of DCM and cooled at 0 ° C. DIEA (115 μL, 0.66 mmol, 4 equivalents) was added, followed by bromoacetic anhydride (85.8 g, 0.33 mmol, 2 equivalents) dissolved in 1 mL of DCM. The reaction mixture was then stirred at 0 ° C for 30 minutes, stirred at room temperature for 1.5 h, and the solvent was removed. Purification by flash column chromatography on silica gel gave L3 (56 mg, 0.064 mmol, 39%) as a white solid. R =3.4 min (Agilent). MS (ES + )m / z 872.4([M+H] + ),874.3([M+H] + ).

[0467] Example 9: Synthesis of L4

[0468]

[0469] Intermediate L4a

[0470] To a solution of Boc-Orn(Boc)-OH (595 mg, 1.79 mmol, 1 eq) dissolved in DMF (5 mL) was added HATU (750 mg, 1.79 mmol, 1.1 eq), DIEA (343 μL, 1.97 mmol, 1.1 eq) and amine-PEG3-N3 (391 mg, 1.79 mmol, 1 eq) dissolved in 1 mL DMF. The reaction mixture was stirred at room temperature for 16 h. The product was diluted with EtOAc. The organic layer was washed with 1 M HCl, saturated NaHCO3 and brine in sequence, dried over Na2SO4, filtered, and concentrated. Purification by silica gel flash column chromatography gave the desired oily compound L4a (558 mg, 1.05 mmol, 58%). MS (ES) + )m / z 533.13([M+H] + ). 1 H NMR (400 MHz, CHLOROFORM-d) δ 6.82 (s, 1H), 5.25 (d, J = 8.3 Hz, 1H), 4.75 (s, 1H), 4.19 (s, 1H), 3.76-3.60 (m, 10H), 3.57 (t, J = 5.1 Hz, 2H), 3.43 (t, J = 4.6 Hz, 2H), 3.30-3.19 (m, 1H), 3.18-3.03 (m, 1H), 1.85 (s, 4H), 1.68-1.49 (m, 2H), 1.45 (s, 18H).

[0471] Intermediate L4b

[0472] To a solution of compound L4a (548 mg, 1.02 mmol, 1 eq) in anhydrous MeOH (10 mL) was added Pd / C (10.9 mg, 0.102 mmol, 0.1 eq) under argon and the argon was replaced with H . The reaction mixture was stirred at room temperature for 6 h, filtered on celite and evaporated to afford compound L4b (516 mg, 1.02 mmol, quantitative) as an oil. The product was used without further purification.

[0473] Intermediate L4c

[0474] To a solution of tert-butyl octadecanoate (370 mg, 1.02 mmol, 1 equivalent) dissolved in DMF (5 mL) was added HATU (387 mg, 1.02 mmol, 1.1 equivalents), DIEA (186 μL, 1.07 mmol, 2 equivalents) and compound L4b (516 mg, 1.02 mmol, 1 equivalent) dissolved in 1 mL DMF. The reaction mixture was stirred at room temperature for 3 h. The product was diluted with EtOAc. The organic layer was washed with 1 M HCl, saturated NaHCO 3 and brine in sequence, dried over Na 2 SO 4 , filtered, and concentrated. Purification by flash column chromatography on silica gel gave the desired oily compound L4c (697 mg, 0.81 mmol, 79%). 1 H NMR (400MHz, chloroform-d) δ6.94(s,1H),6.42(s,1H),4.81(s,1H),4.20(s,1H),3.65(d,J=6.7Hz,8H),3.59(dt,J=9.7,5.1Hz,4H),3.51-3. 35(m,4H),3.31-3.18(m,1H),3.17-3.06(m,1H),2.20(q,J=8.0Hz,4H),1.87(s,4H),1.71-1.53(m,6H),1.45(s,26H),1.26(s,24H).

[0475] L4

[0476] L4c (422mg, 0.49mmol, 1 equivalent) in DCM (2mL) solution was treated with TFA (2mL) for 30 minutes. The mixture was concentrated, co-evaporated with hexane and dissolved in 20mL DCM and cooled at 0°C. DIEA (327 μL, 1.96mmol, 4 equivalents) was added, followed by bromoacetic anhydride (254mg, 0.98mmol, 2 equivalents) dissolved in 1mL DCM. The reaction mixture was then stirred at 0°C for 30 minutes, stirred at room temperature for 1.5h, and the solvent was removed. Purified by flash column chromatography on silica gel to obtain L4 (53mg, 0.063mmol, 12%) as a white solid. MS (ES) + )m / z 845.08([M+H] + ),847.07([M+H] + ) 1H NMR (400 MHz, methanol-d4) δ 3.68-3.60 (m, 8H), 3.54 (td, J = 5.4, 3.4 Hz, 4H), 3.43-3.35 (m, 4H), 3.30-3.16 (m, 2H), 2.27 (t, J = 7.5 Hz, 2H), 2.17 (t, J = 7.6 Hz, 2H), 1.86-1.73 (m, 1H), 1.72-1.45 (m, 8H), 1.37-1.19 (m, 28H).

[0477] Example 10: Synthesis of L4A

[0478]

[0479] Intermediate L4Aa

[0480] At 0 DEG C, bromoacetic acid anhydride (1.31g, 5.04mmol, 2.05 equivalents, 1mL DCM solution) was added dropwise to tert-butyl bis(2-aminoethyl)carbamate (500mg, 2.45mmol, 1 equivalent) and DCM (20mL) solution of DIEA (1.02mL, 5.88mmol, 2 equivalents). The reaction mixture was stirred at 0 DEG C for 30 minutes, stirred for 2h and evaporated in vacuo at RT. Oily product (883mg, 81%) was obtained by purification by flash chromatography. 1 H NMR (400 MHz, methanol-d4) δ 1.50 (s, 9H), 3.39 (s, 8H), 3.85 (s, 4H). t R =1.04min.MS(ES + )m / z 445.71 / 447.70([M+H] + ).

[0481] Intermediate L4Ab

[0482] A solution of compound L4Aa (1 equivalent) in DCM / TFA (1:1, v / v) was stirred at room temperature for 30 minutes and concentrated in vacuo (co-evaporated with heptane). Compound L4Ab was used directly in the other steps without purification. R =0.58min.MS(ES + )m / z345.65 / 347.67([M+H] + ).

[0483] Intermediate L4Ac

[0484] To a solution of mono-tert-butyl succinate (1.05 equivalents) in DMF was added HATU (1.05 equivalents). The reaction mixture was stirred at room temperature for 5 minutes. Compound L4Ab and DIEA (4 equivalents) were dissolved in DMF (1 mL) and added to the reaction mixture. The reactants were stirred at room temperature overnight and diluted with AcOEt. The organic phase was washed with HCl 1N, saturated NaHCO3 solution, dried over MgSO4 and evaporated. The product was purified by flash chromatography to give an oily product. R =1.07min.MS(ES + )m / z501.52 / 503.80([M+H] + ).

[0485] Intermediate L4Ad

[0486] A solution of compound L4Ac (1 equivalent) in DCM / TFA (1:1, v / v) was stirred at room temperature for 30 minutes and concentrated in vacuo (co-evaporated with heptane). Compound L4Ad was used directly in the other steps without purification. R =0.57min.MS(ES + )m / z445.71 / 447.73([M+H] + ).

[0487] Intermediate L4Ae

[0488] Octadecanedioic acid mono-tert-butyl ester (200 mg, 0.54 mmol, 1 equivalent) was dissolved in 5 mL of DMF. HATU (225 mg, 0.59 mmol, 1.1 equivalents) and DIEA (103 μL, 0.59 mmol, 1.1 equivalents) were added, followed by the addition of Boc-NH-PEG3-NH2 (157.8 g, 0.54 mmol, 1 equivalent). The reaction mixture was then stirred for 3 h and the solvent removed. The product was dissolved in EtOAc. The organic layer was washed with saturated NaHCO3, 1 M HCl, and brine, dried over Na2SO4, filtered, and concentrated. Purification by flash column chromatography on silica gel gave the desired product, L4Ae (281 mg, 0.43 mmol, 81%) as a white solid. MS (ES) + )m / z645.5([M+H] + ). 1H NMR (400MHz, chloroform-d) δ3.76-3.61(m,8H),3.63-3.54(m,4H),3.48(q,J=5.1Hz,2H),3.34(s,2H),2.2 0(dt,J=9.8,7.6Hz,4H),1.67-1.55(m,4H),1.49-1.44(m,17H),1.30(s,6H),1.30-1.24(m,19H).

[0489] L4A

[0490] A solution of compound L4Ae in DCM was treated with TFA for 30 minutes. The mixture was concentrated, co-evaporated with heptane, dissolved in DMF and added to a solution of compound L4Ad, HATU and DIEA in DMF. The reaction mixture was stirred for 3 hours and purified by semi-preparative HPLC to give the desired product L4A.

[0491] Example 11: Synthesis of L5

[0492]

[0493] General scheme A, B, D (octadecane dioic acid mono-tert-butyl ester); C, D (Fmoc-PEG2-propionic acid); B, D (Fmoc-PEG2-propionic acid); B, D (Fmoc-Orn(Fmoc)-OH); B, E, F.

[0494] The crude product was purified by semi-preparative HPLC with mass detection to give the product L5 (73 mg, 0.065 mmol, 11%) as a white solid. 1 H NMR (400MHz, methanol-d4) δ4.36 (td, J=8.9, 5.1Hz, 2H), 3.89 (q, J=11.4Hz, 2H), 3.82 (s,2H),3.74(t,J=6.2Hz,2H),3.60(s,4H),3.54(t,J=5.5Hz,2H),3.37(q,J=5. 2Hz,2H),3.29-3.11(m,5H),2.44(t,J=6.2Hz,2H),2.26(dt,J=12.3,7.5Hz,4H) ,1.89-1.77(m,2H),1.76-1.49(m,10H),1.48-1.38(m,2H),1.37-1.25(m,25H).

[0495] Example 12: Synthesis of L5A

[0496]

[0497] Intermediate L5Aa

[0498] A solution of Fmoc-OSu (131 g, 388 mmol) in DCM (200 mL) was added dropwise to a solution of diethylenetriamine (20 g, 194 mmol) in DCM (200 mL) at -40 °C under N2 and stirred for 2 h. LCMS showed the reaction was complete. The crude product in the solution was used directly in the next step without purification. 1 H NMR(400MHz,DMSO-d6)δ7.88(d,J=7.6Hz,4H),7.68(d,J=7.6Hz,4H),7.43-7.24(m,10H), 4.30(d,J=6.4Hz,4H), 4.21(d,J=6.4Hz,2H), 3.06(d,J=5.6Hz,4H), 2.57(d,J=7.6Hz,4H). MS(ES + )m / z 548.2([M+H] + ).

[0499] Intermediate L5Ab

[0500] To a solution of compound L5Aa (106 g, 194 mmol) in DCM (400 mL) were added DMAP (4.74 g, 38.8 mmol) and tetrahydrofuran-2,5-dione (67.9 g, 678 mmol) and stirred at 25 ° C for 14 h. LCMS showed that the reaction was complete. 1N HCl was added to the reaction mixture until pH = 5-6, stirred for 15 minutes, the organic phase was separated, and then the organic phase was washed with water and saturated NaCl (500 mL), and the aqueous phase was extracted twice with DCM (500 mL). The combined DCM was dried over anhydrous Na2SO4 and concentrated in vacuo. The crude product was purified by silica gel column chromatography using DCM / MeOH (80:0-5:1) as eluent to give compound L5Ab (57.6 g, 45% yield) as a white solid powder. 1 H NMR (400MHz, DMSO-d6) δ12.09(s,1H),7.87(d,J=7.5Hz,4H),7.66(d,J=7.0Hz,4H),7.23-7.48(m ,10H),4.24-4.33(m,4H),4.14-4.22(m,2H),3.27(s,4H),2.95-3.19(m,4H),2.37-2.44(m,4H). MS(ES + )m / z 648.2([M+H] + ).

[0501] L5A

[0502] General scheme A, B, D (octadecane dioic acid mono-tert-butyl ester); C, D (Fmoc-PEG2-propionic acid); B, D (Fmoc-PEG2-propionic acid); B, D (compound L5Ab); B, E, F.

[0503] The crude product was purified by HPLC to give the white solid product L5A (5.2 g, 11% yield). MS (ES + )m / z1188.5([M+H] + ).

[0504] Example 13: Synthesis of L6

[0505]

[0506] Intermediate L6a

[0507] Palmitic acid (235 mg, 0.919 mmol, 1.05 equivalents) was dissolved in 4 mL of DMF. HATU (349 mg, 0.919 mmol, 1.1 equivalents) and DIEA (167 μL, 0.963 mmol, 1.1 equivalents) were added, followed by the addition of Boc-NH-PEG2-NH2 (200 mg, 0.875 mmol, 1 equivalent). The reaction mixture was then stirred for 2 h and the solvent removed. The product was dissolved in EtOAc. The organic layer was washed sequentially with 1 M HCl, saturated NaHCO3, HCl, and brine, dried over Na2SO4, filtered, and concentrated to give the desired compound L6a (412 mg, 0.84 mmol, 97%) as a white solid. 1 H NMR (400 MHz, CHLOROFORM-d) δ 6.17 (s, 1H), 5.07 (s, 1H), 3.58 (s, 4H), 3.53 (t, J = 5.0 Hz, 3H), 3.43 (q, J = 5.3 Hz, 2H), 3.36-3.21 (m, 2H), 2.15 (t, J = 7.5 Hz, 2H), 1.66-1.54 (m, 2H), 1.32-1.15 (m, 26H), 0.84 (t, J = 6.6 Hz, 3H).

[0508] Intermediate L6b

[0509] A solution of compound L6a (412 mg, 0.84 mmol, 1 equivalent) in DCM (2 mL) was treated with TFA (2 mL) for 30 minutes. The mixture was concentrated and co-evaporated with hexane. HATU (353 mg, 0.931 mmol, 1.1 equivalents) was added to a solution of BocNH-PEG2-CO2H (258 mg, 0.931 mmol, 1.1 equivalents) dissolved in DMF (5 mL). A DMF solution of deprotected compound L6a and DIEA (294 μL, 1.69 mmol, 2 equivalents) was added to the reaction mixture. The reaction mixture was stirred at room temperature for 2 h. The product was diluted with EtOAc. The organic layer was washed with 1 M HCl, saturated NaHCO3, HCl and brine, dried over Na2SO4, filtered, and concentrated. Purification by flash column chromatography on silica gel gave the desired oily compound L6b (329 mg, 0.51 mmol, 60%). 1 H NMR (400MHz, chloroform-d) δ6.79(s,1H),6.28(s,1H),5.28(s,1H),3.68(t,J=5.8Hz,2H),3.61-3.44(m,14H),3.38(p,J=5.6Hz,4H),3.24(q,J= 5.5Hz,2H),2.42(t,J=5.8Hz,2H),2.11(t,J=7.9Hz,2H),1.55(p,J=7.2Hz,2H),1.38(s,9H),1.32-1.10(m,24H),0.81(t,J=6.7Hz,3H).

[0510] Intermediate L6c

[0511] A solution of compound L6b (329 mg, 0.51 mmol, 1 equivalent) in DCM (2 mL) was treated with TFA (2 mL) for 30 minutes. The mixture was concentrated and co-evaporated with hexane. HATU (213 mg, 0.56 mmol, 1.1 equivalents) was added to a solution of Boc-Orn(Boc)-OH (186 mg, 0.56 mmol, 1.1 equivalents) dissolved in DMF (5 mL). A DMF solution of deprotected compound L6b and DIEA (177 μL, 1.02 mmol, 2 equivalents) was added to the reaction mixture. The reaction mixture was stirred at room temperature for 2 h. The product was diluted with EtOAc. The organic layer was washed with saturated NaHCO 3 , 1M HCl and brine in sequence, dried over Na 2 SO 4 , filtered, and concentrated. Purification by flash column chromatography on silica gel gave the desired oily compound L6c (326 mg, 0.37 mmol, 94%). 1HNMR (400 MHz, chloroform-d) δ7.18 (s, 1H), 6.92 (s, 1H), 6.48 (s, 1H), 5.61 (d, J = 8.4 Hz, 1H), 5.08 (t, J = 5.9 Hz, 1H), 4.13 (s, 1H), 3.73-3.65 (m, 2H), 3.59-3.44 (m, 14H), 3.42-3.2 9(m,8H),3.19-2.86(m,2H),2.42(t,J=5.9Hz,2H),2.10(d,J=7.3Hz,2H),1.78-1.63 (m,1H),1.60-1.40(m,5H),1.35(s,18H),1.26-1.09(m,22H),0.80(t,J=6.7Hz,3H).

[0512] L6

[0513] Compound L6c (100mg, 0.116mmol, 1 equivalent) DCM (2mL) solution TFA (2mL) is processed 30 minutes.Mixture is concentrated, co-evaporated with hexane and dissolved in 10mL DCM and cooled at 0 ℃.DIEA (80.8 μ L, 0.46mmol, 4 equivalents) is added, followed by the addition of bromoacetic anhydride (61.9mg, 0.238mmol, 2.05 equivalents) dissolved in 1mL DCM.Then the reaction mixture was stirred 30 minutes at 0 ℃, stirred at room temperature for 1.5h, and desolvated. By flash column chromatography on silica gel, white solid L6 (50.1mg, 0.055mmol, 40%) is obtained. 1 H NMR (400 MHz, methanol-d4) δ 4.39 (dd, J = 8.4, 5.5 Hz, 1H), 3.91 (q, J = 11.4 Hz, 2H), 3.84 (s, 2H), 3.76 (t, J = 6.2 Hz, 2H), 3.63 (d, J = 7.1 Hz, 8H), 3.57 (q, J = 5.5 Hz, 6H), 3.43-3.3 6(m,6H),3.25(t,J=13.9,6.8Hz,2H),2.49(t,J=6.2Hz,2H),2.21(t,J=7.5Hz,2H) ,1.91-1.79(m,1H),1.75-1.53(m,5H),1.42-1.25(m,24H),0.92(t,J=6.7Hz,3H).

[0514] Example 14: Synthesis of L7

[0515]

[0516] Intermediate L7a

[0517] Stearic acid (261 mg, 0.919 mmol, 1.05 equiv) was dissolved in 4 mL of DMF. HATU (349 mg, 0.919 mmol, 1.1 equiv) and DIEA (167 μL, 0.963 mmol, 1.1 equiv) were added, followed by Boc-NH-PEG2-NH2 (200 mg, 0.875 mmol, 1 equiv). The reaction mixture was then stirred for 2 h and the solvent removed. The product was dissolved in EtOAc. The organic layer was washed sequentially with 1 M HCl, saturated NaHCO3, and brine, dried over Na2SO4, filtered, and concentrated to give the desired compound L7a (430 mg, 0.83 mmol, 95%) as a white solid. 1 H NMR (400MHz, chloroform-d) δ3.69-3.59(m,4H),3.56(t,J=5.1Hz,4H),3.46(q,J=5.2Hz,2H),3.40-3.23(m,2H) ,2.18(t,J=7.6Hz,2H),1.62(t,J=7.3Hz,2H),1.45(s,9H),1.35-1.19(m,30H),0.88(t,J=6.7Hz,4H).

[0518] Intermediate L7b

[0519] A solution of compound L7a (426 mg, 0.87 mmol, 1 equivalent) in DCM (2 mL) was treated with TFA (2 mL) for 30 minutes. The mixture was concentrated and co-evaporated with hexane. HATU (366 mg, 0.96 mmol, 1.1 equivalents) was added to a solution of BocNH-PEG2-CO2H (266 mg, 0.96 mmol, 1.1 equivalents) dissolved in DMF (5 mL). A DMF solution of deprotected compound L7a and DIEA (304 μL, 1.75 mmol, 2 equivalents) was added to the reaction mixture. The reaction mixture was stirred at room temperature for 2 h. The product was diluted with EtOAc. The organic layer was washed with 1 M HCl, saturated NaHCO3 and brine, dried over Na2SO4, filtered, and concentrated. Purified by flash column chromatography on silica gel, the desired oily compound L7b (360 mg, 0.53 mmol, 61%) was obtained. 1HNMR (400MHz, chloroform-d) δ6.75(s,1H),6.18(s,1H),5.26(s,1H),3.75(t,J=5.8Hz,2H),3.69-3.52(m,14H),3.47(p,J=5.4Hz,4H),3.33(q,J=5.5Hz ,2H),2.50(t,J=5.8Hz,2H),2.19(t,J=7.5Hz,2H),2.07(s,1H),1.63(p ,J=7.3Hz,2H),1.46(s,9H),1.37-1.19(m,29H),0.89(t,J=6.7Hz,3H).

[0520] Intermediate L7c

[0521] A solution of compound L7b (360 mg, 0.53 mmol, 1 equivalent) in DCM (2 mL) was treated with TFA (2 mL) for 30 minutes. The mixture was concentrated and co-evaporated with hexane. HATU (223 mg, 0.58 mmol, 1.1 equivalents) was added to a solution of Boc-Orn(Boc)-OH (195 mg, 0.58 mmol, 1.1 equivalents) dissolved in DMF (5 mL). A DMF solution of deprotected compound L7b and DIEA (186 μL, 1.07 mmol, 2 equivalents) was added to the reaction mixture. The reaction mixture was stirred at room temperature for 2 h. The product was diluted with EtOAc. The organic layer was washed with 1 M HCl, saturated NaHCO 3 and brine in sequence, dried over Na 2 SO 4 , filtered, and concentrated. Purified by flash column chromatography on silica gel, the desired oily compound L7c (373 mg, 0.42 mmol, 78%) was obtained. 1 HNMR (400MHz, chloroform-d) δ7.14 (s, 1H), 6.84 (s, 1H), 6.35 (s, 1H), 5.53 (d, J = 8.2Hz, 1H),5.05-4.88(m,1H),4.20(s,1H),3.82-3.69(m,2H),3.65-3.31(m,22H),3. 23-3.00(m,2H),2.48(t,J=5.8Hz,2H),2.17(t,J=7.8Hz,2H),1.87-1.72(m,1H ),1.67-1.48(m,5H),1.42(s,18H),1.34-1.14(m,29H),0.87(t,J=6.9Hz,3H).

[0522] L7

[0523] A solution of compound L7c (100 mg, 0.112 mmol, 1 equivalent) in DCM (2 mL) was treated with TFA (2 mL) for 30 minutes. The mixture was concentrated, co-evaporated with hexane and dissolved in 10 mL DCM and cooled at 0 ° C. DIEA (78 μ L, 0.44 mmol, 4 equivalents) was added, followed by bromoacetic anhydride (62 mg, 0.24 mmol, 2.05 equivalents) dissolved in 1 mL DCM. The reaction mixture was then stirred at 0 ° C for 30 minutes, stirred at room temperature for 1.5 h, and the solvent was removed. The product was dissolved in EtOAc. The organic layer was washed with 1 M HCl, saturated NaHCO 3 and brine in sequence, dried over Na 2 SO 4 , filtered, and concentrated. Purified by flash column chromatography on silica gel to give L7 (95 mg, 0.10 mmol, 91%) as a white solid. MS (ES) + )m / z 931.31([M+H] + ),933.25([M+H] + ). 1 H NMR (400MHz, methanol-d4) δ4.39 (dd, J=8.5, 5.4Hz, 1H), 3.91 (q, J=11.3Hz, 2H), 3.84 ( s,2H),3.76(t,J=6.2Hz,2H),3.63(d,J=7.0Hz,8H),3.57(t,J=5.5Hz,6H),3.42 -3.35(m,6H),3.31-3.13(m,4H),2.49(t,J=6.2Hz,2H),2.20(t,J=7.4Hz,2H),1 .91-1.79(m,1H),1.75-1.56(m,6H),1.39-1.26(m,26H),0.92(t,J=6.3Hz,3H).

[0524] Example 15: Synthesis of L8

[0525]

[0526] General scheme A, B, D (mono-tert-butyl hexadecanedioate); C, D (Fmoc-PEG2-propionic acid); B, D (Fmoc-PEG2-propionic acid); B, D (Fmoc-Orn(Fmoc)-OH); B, E, F.

[0527] The crude product was purified by semi-preparative HPLC with mass detection to give the product L8 (42.6 mg, 0.038 mmol, 22%) as a white solid. 1H NMR (400 MHz, methanol-d4) δ 4.38 (td, J = 8.6, 5.1 Hz, 2H), 3.91 (q, J = 11.3 Hz, 2H), 3.84 (s, 2H), 3.76 (q, J = 6.1 Hz, 4H), 3.65-3.59 (m, 8H), 3.56 (td, J = 5.5, 1.7 Hz, 4H), 3.43-3.3 7(m,4H),3.31-3.16(m,4H),2.48(dt,J=15.7,6.2Hz,4H),2.28(dt,J=12.6,7.5Hz, 4H),1.95-1.79(m,1H),1.77-1.51(m,10H),1.49-1.41(m,2H),1.40-1.26(m,31H).

[0528] Example 16: Synthesis of L9

[0529]

[0530] General scheme A, B, D (mono-tert-butyl heptadecanedioate); C, D (Fmoc-PEG2-propionic acid); B, D (Fmoc-PEG2-propionic acid); B, D (Fmoc-Orn(Fmoc)-OH); B, E, F.

[0531] The crude product was purified by semi-preparative HPLC with mass detection to give the product L9 (49 mg, 0.089 mmol, 9%) as a white solid. 1 H NMR (400MHz, methanol-d4) δ4.45-4.33(m,2H),3.92(t,J=10.9Hz,2H),3.85(d,J=1.1Hz,2 H),3.77(q,J=6.0Hz,4H),3.63(s,8H),3.57(t,J=5.6Hz,4H),3.40(t,J=5.5Hz,4H), 3.25(dq,J=22.7,6.7Hz,4H),2.48(dt,J=15.6,6.2Hz,4H),2.29(dt,J=13.2,7.4Hz ,4H),1.95-1.79(m,2H),1.80-1.50(m,10H),1.51-1.41(m,2H),1.40-1.27(m,20H).

[0532] Example 17: Synthesis of L12

[0533]

[0534] General scheme A, B, D (octadecane dioic acid); C, D (Fmoc-PEG2-propionic acid); B, D (Fmoc-Orn(Fmoc)-OH); B, E, F.

[0535] The crude product was purified by semi-preparative HPLC with mass detection to give the product L12 (51.7 mg, 0.054 mmol, 3%) as a white solid. 1 H NMR (400 MHz, methanol-d4) δ 4.39 (td, J = 9.2, 5.1 Hz, 2H), 3.92 (qd, J = 11.4, 1.2 Hz, 2H), 3.85 (s, 2H), 3.76 (t, J = 6.2 Hz, 2H), 3.63 (s, 4H), 3.57 (t, J = 5.5 Hz, 2H), 3.40 (q, J = 5.1Hz,2H),3.30-3.12(m,6H),2.47(t,J=6.1Hz,2H),2.29(dt,J=12.1,7.4Hz,4H ),1.95-1.77(m,2H),1.78-1.50(m,10H),1.48-1.40(m,2H),1.39-1.26(m,22H).

[0536] Example 18: Synthesis of L14

[0537]

[0538] Intermediate L14a

[0539] To a solution of hexadecanedioic acid mono-tert-butyl ester (102 mg, 0.30 mmol, 1 equivalent) dissolved in DMF (5 mL) was added HATU (125 mg, 0.33 mmol, 1.1 equivalents), DIEA (51 μL, 0.33 mmol, 1.1 equivalents) and compound L4b (151.9 mg, 0.3 mmol, 1 equivalent) dissolved in 1 mL DMF. The reaction mixture was stirred at room temperature for 3 h. The product was diluted with EtOAc. The organic layer was washed with 1 M HCl, saturated NaHCO 3 and brine in sequence, dried over Na 2 SO 4 , filtered, and concentrated. Purification by flash column chromatography on silica gel gave the desired oily compound L14a (147 mg, 0.176 mmol, 59%). 1H NMR (400MHz, chloroform-d) δ6.87(s,1H),6.40(s,1H),5.32(s,2H),4.79(s,1H),4. 20(s,1H),3.66(d,J=7.0Hz,8H),3.60(dt,J=10.0,5.1Hz,4H),3.49-3.45(m ,3H),3.31-3.18(m,1H),3.13-3.06(m,1H),2.21(td,J=7.8,6.0Hz,4H),1. 88-1.78(m,1H),1.66-1.53(m,7H),1.51-1.42(m,27H),1.36-1.19(m,20H).

[0540] L14

[0541] A solution of compound L14a (40 mg, 0.048 mmol, 1 equivalent) in DCM (2 mL) was treated with TFA (2 mL) for 30 minutes. The mixture was concentrated, co-evaporated with hexane and dissolved in 20 mL DCM and cooled at 0 ° C. DIEA (34 μ L, 0.1924 mmol, 4 equivalents) was added, followed by the addition of bromoacetic anhydride (23.63 mg, 0.098 mmol, 2.05 equivalents) dissolved in 1 mL DCM. The reaction mixture was then stirred at 0 ° C for 30 minutes, stirred at room temperature for 1.5 h, and the solvent was removed. Purified by flash column chromatography on silica gel to give L14 (18.3 mg, 0.022 mmol, 46%) as a white solid. MS (ES) + )m / z 817.1([M+H] + ),819.09([M+H] + ). 1 H NMR (400MHz, methanol-d4) δ4.38 (dd, J=8.4, 5.5Hz, 1H), 3.92 (q, J=11.2, 10.6Hz, 2H),3.84(s,2H),3.69-3.61(m,8H),3.56(td,J=5.5,2.6Hz,4H),3.44-3.3 6(m,4H),3.30-3.14(m,2H),2.29(t,J=7.4Hz,2H),2.21(t,J=7.5Hz,2H),1 .91-1.78(m,1H),1.76-1.67(m,1H),1.67-1.54(m,6H),1.40-1.29(m,20H).

[0542] Example 19: Synthesis of L15

[0543]

[0544] Intermediate L15a

[0545] To a solution of 20-(tert-butoxy)-20-oxoeicosanoic acid (360 mg, 0.90 mmol, 1.05 equivalents) dissolved in DMF (5 mL) was added HATU (343 mg, 0.90 mmol, 1.05 equivalents), DIEA (300 μL, 1.71 mmol, 2 equivalents) and compound L4b (435 mg, 0.858 mmol, 1 equivalent) dissolved in 1 mL of DMF. The reaction mixture was stirred at room temperature for 3 h. The product was diluted with EtOAc. The organic layer was washed with 1 M HCl, saturated NaHCO 3 and brine in sequence, dried over Na 2 SO 4 , filtered, and concentrated. Purification by flash column chromatography on silica gel gave the desired oily compound L15a (555 mg, 0.625 mmol, 72%). 1 HNMR (400 MHz, chloroform-d) δ 6.87 (s, 1H), 6.40 (s, 1H), 4.79 (s, 1H), 4.21 (s, 1H), 3.76-3.53 (m, 15H), 3.47 (s, 5H), 3.32-3.05 (m, 3H), 2.29-2.17 (m, 4H), 1.90-1.76 (m, 4H), 1.69-1.53 ​​(m, 2H), 1.52-1.41 (m, 33H), 1.36-1.20 (m, 29H).

[0546] L15

[0547] A solution of compound L15a (100 mg, 0.112 mmol, 1 equivalent) in DCM (2 mL) was treated with TFA (2 mL) for 30 minutes. The mixture was concentrated, co-evaporated with hexane and dissolved in 20 mL of DCM and cooled at 0 ° C. DIEA (79 μ L, 0.45 mmol, 4 equivalents) was added, followed by bromoacetic anhydride (60 mg, 0.231 mmol, 2.05 equivalents) dissolved in 1 mL of DCM. The reaction mixture was then stirred at 0 ° C for 30 minutes, stirred at room temperature for 1.5 h, and the solvent was removed. Purified by flash column chromatography on silica gel to give L15 (17.5 mg, 0.02 mmol, 18%) as a white solid. MS (ES) + )m / z 873.21([M+H] + ),875.20([M+H] + ) 1H NMR (400MHz, methanol-d4) δ4.38 (dd, J=8.4, 5.5Hz, 1H), 3.91 (q, J=11.4Hz, 2H), 3.84 (s, 2H), 3.72-3.61 (m, 8H), 3.56 (td, J=5.5, 2.7Hz, 4H), 3.4 4-3.35(m,5H),3.30-3.17(m,2H),2.29(t,J=7.4Hz,2H),2.21(t,J=7.5Hz,2H),1.92-1.77(m,1H),1.75-1.53(m,7H),1.40-1.27(m,27H).

[0548] Example 20: Synthesis of L16

[0549]

[0550] Intermediate L16a

[0551] To a solution of Boc-Orn(Boc)-OH (400 mg, 1.2 mmol, 1 equivalent) dissolved in DMF (10 mL) was added HATU (504 mg, 1.32 mmol, 1.1 equivalents), DIEA (230 μL, 1.32 mmol, 1.1 equivalents) and amine-PEG2-N3 (210 mg, 1.20 mmol, 1 equivalent) dissolved in 1 mL DMF. The reaction mixture was stirred at room temperature for 4 h. The product was diluted with EtOAc. The organic layer was washed with 1 M HCl, saturated NaHCO3 and brine, dried over Na2SO4, filtered, and concentrated. Purification by silica gel flash column chromatography gave the desired oily compound L16a (471 mg, 0.96 mmol, 80%). 1 H NMR (400 MHz, methanol-d4) δ 4.01 (t, J = 6.6 Hz, 1H), 3.71-3.60 (m, 6H), 3.55 (t, J = 5.5 Hz, 2H), 3.41-3.37 (m, 3H), 3.04 (t, J = 6.2 Hz, 2H), 1.78-1.66 (m, 1H), 1.62-1.48 (m, 3H), 1.48-1.39 (m, 18H).

[0552] Intermediate L16b

[0553] To a solution of compound L16a (471 mg, 0.9 mmol, 1 eq) in anhydrous MeOH (10 mL) under argon was added Pd / C (10.2 mg, 0.09 mmol, 0.1 eq) and the argon was replaced with H 2 . The reaction mixture was stirred at room temperature for 6 h, filtered on celite and evaporated to give compound L16b (295.5 mg, 0.64 mmol, 71%) as an oil. The product was used without further purification. MS (ES) + )m / z 462.51([M+H] + ).

[0554] Intermediate L16c

[0555] To a solution of tert-butyl octadecanoate (281 mg, 0.76 mmol, 1 equivalent) dissolved in DMF (5 mL), HATU (288 mg, 0.76 mmol, 1 equivalent), DIEA (132 μ L, 0.76 mmol, 1 equivalent) and compound L16b (351 mg, 0.76 mmol, 1 equivalent) dissolved in 1 mL DMF were added. The reaction mixture was stirred at room temperature for 3 h. The product was diluted with EtOAc. The organic layer was washed with 1 M HCl, saturated NaHCO 3 and salt water in sequence, dried over Na 2 SO 4 , filtered, and concentrated. Purification by flash column chromatography on silica gel gave the desired oily compound L16c (351 mg, 0.43 mmol, 57%). 1 H NMR (400MHz, methanol-d4) δ3.61(s,4H),3.54(td,J=5.6,2.3Hz,4H),3.40-3.34(m,4H),3.04(t,J=6.6Hz,2H),2 .20(td,J=7.6,5.9Hz,4H),1.77-1.68(m,2H),1.64-1.48(m,2H),1.48-1.42(m,28H),1.35-1.26(m,26H).

[0556] L16

[0557] A solution of compound L16c (31 mg, 0.038 mmol, 1 equivalent) in DCM (2 mL) was treated with TFA (2 mL) for 30 minutes. The mixture was concentrated, co-evaporated with hexane and dissolved in 20 mL DCM and cooled at 0 ° C. DIEA (27 μ L, 0.152 mmol, 4 equivalents) was added, followed by bromoacetic anhydride (21 mg, 0.078 mmol, 2.05 equivalents) dissolved in 1 mL DCM. The reaction mixture was then stirred at 0 ° C for 30 minutes, stirred at room temperature for 1.5 h, and the solvent was removed. Purified by flash column chromatography on silica gel to give L16 (12.6 mg, 0.015 mmol, 41%) as a white solid. MS (ES) + )m / z 801.13([M+H] + ),803.12([M+H] + ). 1 H NMR (400MHz, methanol-d4) δ4.37(dd,J=8.5,5.4Hz,1H),3.91(q,J=11.3Hz,2H),3.84(s,2H),3.63(s,4H),3.57(td,J=5.6,2.6Hz,4H),3.43- 3.36(m,4H),3.31-3.17(m,1H),2.29(t,J=7.4Hz,2H),2.21(t,J=7.5Hz,2H),1.90-1.79(m,1H),1.76-1.54(m,7H),1.41-1.30(m,26H).

[0558] Example 21: Synthesis of L17

[0559]

[0560] Intermediate L17a

[0561] To a solution of Boc-Orn(Boc)-OH (400 mg, 1.2 mmol, 1 equivalent) dissolved in DMF (10 mL) was added HATU (504 mg, 1.32 mmol, 1.1 equivalents), DIEA (230 μL, 1.32 mmol, 1.1 equivalents) and amine-PEG2-N3 (316 mg, 1.20 mmol, 1 equivalent) dissolved in 1 mL DMF. The reaction mixture was stirred at room temperature for 4 h. The product was diluted with EtOAc. The organic layer was washed with 1 M HCl, saturated NaHCO3 and brine, dried over Na2SO4, filtered, and concentrated. Purification by flash column chromatography on silica gel gave the desired oily compound L17a (454 mg, 0.78 mmol, 66%). 1H NMR (400 MHz, methanol-d4) δ 4.04-3.97 (m, 1H), 3.71-3.58 (m, 14H), 3.54 (t, J = 5.4 Hz, 2H), 3.37 (t, J = 5.0 Hz, 4H), 3.04 (t, J = 6.6 Hz, 2H), 1.75-1.67 (m, 1H), 1.62-1.48 (m, 3H), 1.48-1.41 (m, 18H).

[0562] Intermediate L17b

[0563] To a solution of compound L17a (454 mg, 0.9 mmol, 1 eq) in anhydrous MeOH (10 mL) under argon was added Pd / C (8.3 mg, 0.078 mmol, 0.1 eq) and the argon atmosphere was replaced with H . The reaction mixture was stirred at room temperature for 6 h, filtered through celite and evaporated to afford compound L17b (192 mg, 0.35 mmol, 45%) as an oil. The product was used without further purification.

[0564] Intermediate L17c

[0565] To a solution of tert-butyl octadecanoate (225 mg, 0.61 mmol, 1 equivalent) dissolved in DMF (5 mL) was added HATU (231 mg, 0.61 mmol, 1 equivalent), DIEA (106 μL, 0.61 mmol, 1 equivalent) and compound L17b (335 mg, 0.61 mmol, 1 equivalent) dissolved in 1 mL DMF. The reaction mixture was stirred at room temperature for 2 h. The product was diluted with EtOAc. The organic layer was washed with 1 M HCl, saturated NaHCO 3 and brine in sequence, dried over Na 2 SO 4 , filtered, and concentrated. Purification by flash column chromatography on silica gel gave the desired oily compound L17c (178 mg, 0.20 mmol, 32%). 1 H NMR (400MHz, chloroform-d) δ5.32 (s, 2H), 3.74-3.63 (m, 11H), 3.59 (dt, J = 10.9, 5.0Hz, 4H), 3.52-3.43 (m, 4 H), 3.27-3.08 (m, 2H), 2.22 (d, J = 7.6Hz, 4H), 1.69-1.52 (m, 6H), 1.51-1.42 (m, 27H), 1.27 (s, 26H).

[0566] L17

[0567] A solution of compound L17c (45.6 mg, 0.05 mmol, 1 equivalent) in DCM (2 mL) was treated with TFA (2 mL) for 30 minutes. The mixture was concentrated, co-evaporated with hexane and dissolved in 20 mL DCM and cooled at 0 ° C. DIEA (36 μ L, 0.202 mmol, 4 equivalents) was added, followed by bromoacetic anhydride (27 mg, 0.103 mmol, 2.05 equivalents) dissolved in 1 mL DCM. The reaction mixture was then stirred at 0 ° C for 30 minutes, stirred at room temperature for 1.5 h, and the solvent was removed. Purified by flash column chromatography on silica gel to give L17 (14.9 mg, 0.017 mmol, 33%) as a white solid. MS (ES) + )m / z 889.18([M+H] + ),891.17([M+H] + ) 1 H NMR (400MHz, methanol-d4) δ4.38 (dd, J=8.3, 5.5Hz, 1H), 3.92 (q, J=11.3Hz, 2H), 3.84 (s, 2H), 3.67-3.60 (m, 7H), 3.56 (td, J=5.5, 3.5Hz, 4H), 3.4 5-3.35(m,5H),3.32-3.15(m,3H),2.29(t,J=7.4Hz,2H),2.21(t,J=7.5Hz,2H),1.90-1.76(m,1H),1.74-1.57(m,7H),1.41-1.26(m,25H).

[0568] Example 22: Synthesis of L18

[0569]

[0570] General scheme A, B, D (octadecane dioic acid); C, D (Fmoc-PEG2-propionic acid); B (Fmoc-PEG2-propionic acid), B (Fmoc-PEG2-propionic acid); B, D (Fmoc-Orn(Fmoc)-OH); B, E, F.

[0571] The crude product was purified by semi-preparative HPLC with mass detection to give the product L18 as a white solid (47 mg, 0.036 mmol, 10%). MS (ES + )m / z 1276.39([M+H] + ),1278.37([M+H] + ).

[0572] General protocol for bromoacetyl peptide stapling / conjugation

[0573] The peptide was dissolved in 1:3 (v / v) MeCN / 30mM NH4HCO3 buffer (pH 8.5) at a concentration of 2mM using 1.5 equivalents of bromoacetyl stapler. The pH of the reaction mixture was readjusted with ammonium hydroxide to correct for the pH drop caused by the peptide TFA counterion. For particularly insoluble peptides, more MeCN was added. The reaction was stirred at room temperature for 2-4h and then acidified to pH 5 by the dropwise addition of acetic acid. The resulting solution was lyophilized and purified by reverse phase HPLC.

[0574] General solid-phase protocol for lactam stapling

[0575] The peptide-resin, bearing orthogonal protection of amine side chains (Dde / Mmt) at each stapled position, was swollen in DMF for 1 h. The Dde protecting group was removed from the first side chain by treatment with 2% hydrazine in DMF (2 x 15 min). A positive TNBS test was observed. The linker building blocks specified below were coupled as described below and a negative TNBS test was observed. The solvent was exchanged to DCM and the Mmt group was removed from the second side chain by treatment with 1% TFA in DCM containing 5% TIPS for 5 x 2 min. The resin was washed with DCM, 10% DIEA in DMF, and DMF and a positive TNBS test was observed. The linker was cyclized and the PEG-fatty acid portion of the stapled body (if applicable) was extended as described below. The intact stapled peptide was cleaved from the resin using 95% TFA, 2.5% TIPS, 2.5% H2O for 3 h. The peptide cleavage mixture was evaporated to an oil, triturated, washed with ether, and purified by reverse phase HPLC. A Dde / Alloc protection scheme can also be used in this method, which requires the addition of allyl alcohol as a scavenger to the Dde deprotection mixture to prevent the simultaneous reduction of the Alloc allylic moiety.

[0576] Synthesis of K(Fmoc) linker

[0577]

[0578] Intermediate Ka

[0579] Fmoc-β-Ala-OH (1.00 g, 3.21 mmol) and di-tert-butyl iminodiacetate (0.461 g, 2.68 mmol) were suspended in 100 mL of DCM. HATU (1.02 g, 2.68 mmol) and DIEA (3.32 mL, 12.8 mmol) were added and the reaction was stirred at room temperature for 3.5 h. The solvent was evaporated and the residue was dissolved in methanol and purified by flash silica gel column chromatography (hexane / EtOAc) to give the product as a white solid (0.802 g, 56%). 1H NMR (400MHz, chloroform-d) δ7.78(d,J=7.4Hz,2H),7.62(d,J=7.4Hz,2H),7.42(t,J=7.4Hz,2H),7.33(t,J=7.4Hz,2H),5.66(t,J=5.7Hz,1H) ,4.35(d,J=7.3Hz,2H),4.23(t,J=7.3Hz,1H),4.10(s,2H),4.02(s,2H),3.56(q,J=5.7Hz,2H),2.55(t,J=5.7Hz,2H),1.49(s,18H).

[0580] K(Fmoc) connector

[0581] Compound Ka was treated with 20 mL 1:1 TFA / DCM for 2 h. The solvent was evaporated, the residue was triturated, and washed with ether to give K(Fmoc) linker as a white solid (0.371 g, 58%). MS (ES) + )m / z 427.15([M+H] + ).

[0582] Synthesis of A(Fmoc) linker

[0583]

[0584] A solution of 5-aminoisophthalic acid (1.00 g, 5.5 mmol) in 10 mL of dioxane was added to a degassed solution of Na2CO3 (1.46 g, 5.5 mmol) in 15 mL of water. The solution was cooled on ice, and then a solution of Fmoc chloride (1.42 g, 5.5 mmol) in 10 mL of dioxane was added dropwise over 15 minutes with stirring. The reaction was then stirred for 1 hour and then at room temperature for 24 hours. The dioxane was removed in vacuo, and the remaining aqueous solution was acidified with 1 M HCl. The resulting solid precipitate was then washed with ether (4 x 10 mL), redissolved in EtOAc, filtered, washed with brine, dried over Na2SO4, filtered, and concentrated to afford the A(Fmoc) linker (119 mg, 5%) as a white solid. 1 H NMR (500MHz, DMSO-d6) δ13.24(s,2H),10.12(s,1H),8.33(d,J=1.5Hz,2H),8.12(t,J=1.5Hz,1H),7.91(d,J=7.6Hz,2H),7. 76 (dd, J = 7.6, 1.2 Hz, 2H), 7.43 (t, J = 7.6 Hz, 2H), 7.36 (td, J = 7.6, 1.2 Hz, 2H), 4.50 (d, J = 6.8 Hz, 2H), 4.33 (t, J = 6.8 Hz, 1H).

[0585] General Scheme G for the “A1” and “K1” Series of Simple Lactam Staplers

[0586] For linker coupling, the appropriate diacid building block (2 eq) was attached using HATU (4 eq) and DIEA (4 eq) in DMF for 1 x 2 h. The cyclization step was achieved using HATU (1 eq) and DIEA (2 eq) in DMF for 1 x 2 h.

[0587] General Protocol H for 'K'PEG-Fatty Acid Trifunctional Lactam Stapled Conjugates

[0588] For the joint coupling, DIC (2 equivalents) and catalytic DMAP were used to preform the intramolecular symmetrical anhydride of the building block K (Fmoc) joint (2 equivalents) in dry DCM at room temperature for 10 minutes. The peptide-resin solvent was replaced with DCM, and then the anhydride was added and stirred overnight. The resin was drained and washed with DCM and DMF. The joint was cyclized overnight by treatment with a DMF solution of DIC (1 equivalent) and HOBt or HOAt (1 equivalent), and a TNBS negative result was observed. The remaining uncyclized joint was capped by treatment with a 10% acetic anhydride solution in DMF (30 min). The joint Fmoc group was deprotected by treatment with a 20% piperidine solution in DMF (2×10 min). A TNBS positive result was observed. Subsequent stapler PEG and fatty acid building blocks were sequentially attached to the linker free amine via standard coupling chemistry: building block (3 eq), HATU (3 eq) and DIEA (6 eq) in DMF at room temperature for 1 h, followed by a deprotection cycle using 20% ​​piperidine in DMF (5 + 10 min, RT).

[0589] General Protocol I for 'A' PEG-Fatty Acid Trifunctional Lactam Stapled Conjugates

[0590] For the linker coupling, the building block A (Fmoc) linker (2 equiv) was ligated using HATU (4 equiv) and DIEA (4 equiv) in DMF for 1 x 2 h. The cyclization step was achieved using HATU (1 equiv) and DIEA (2 equiv) in DMF for 1 x 2 h. The remaining uncyclized linker was capped by treatment with 10% acetic anhydride in DMF (30 min). The linker Fmoc group was deprotected by treatment with 20% piperidine in DMF (2 x 10 min). A TNBS positive reaction was not observed for the aniline nitrogen. Fmoc-β-Ala-OH (3 equiv) was coupled using HATU (3 equiv) and DIEA (6 equiv) in DMF for 4 x 1 h at room temperature or as a symmetrical anhydride using DIC / DMAP in DCM (2 h, RT). Subsequent stapler PEG and fatty acid building blocks were sequentially attached to the linker free amine via standard coupling chemistry: building block (3 eq), HATU (3 eq) and DIEA (6 eq) in DMF at room temperature for 1 h, followed by a deprotection cycle using 20% ​​piperidine in DMF (5 + 10 min, RT).

[0591] In some embodiments, a peptide conjugate described herein comprises a stapler of Table 2.

[0592] Table 2

[0593]

[0594]

[0595]

[0596]

[0597] is part of a cysteine, homocysteine, 2-amino-5-mercaptopentanoic acid or 2-amino-6-mercaptohexanoic acid residue, and It is part of a lysine, ornithine, diaminobutyric acid, diaminopropionic acid or homolysine residue.

[0598] In some embodiments, the peptide conjugates described herein are as shown in Table 3.

[0599] Table 3: Peptide conjugates

[0600]

[0601]

[0602]

[0603]

[0604]

[0605]

[0606]

[0607] Example A: In vitro GLP-2 receptor activation reporter gene assay (receptor-mediated cAMP synthesis)

[0608] The activity and potency of peptides against GLP-2R activation were determined using a stable HEK293 cell line overexpressing a cAMP response element (CRE)-driven luciferase reporter gene and human GLP-2R in the presence of 10% FBS. GLP2-2G (teduglutide) was used as a positive control.

[0609] HEK293-GLP-2R-CRE cells were seeded in 384-well plates at a density of 5000 cells per well and cultured for 18 h in DMEM with 10% FBS at 37 ° C and 5% CO2. Cells were treated with peptides for 16 h in a dose-dependent manner, and receptor activation was reported by luminescence intensity using One-Glo (Promega, WI) luciferase reagent according to the manufacturer's instructions. The EC50 of each peptide was determined using GraphPad Prism 6 software (GraphPad, San Diego, CA). The assay was performed in triplicate and the results were obtained in three independent experiments. The results are shown in Table 4.

[0610] Table 4

[0611]

[0612] Example B: Pharmacokinetics (half-life) of peptides in mice

[0613] To determine the in vivo half-life of the GLP-2 agonist, pharmacokinetic (PK) studies were performed by intravenously or subcutaneously injecting the peptide at 10 nmol / kg in CD1 female mice (n=4 per group). In vitro GLP-2R-mediated cell-based reporter gene assays were used to determine plasma levels of the peptide at different time points (5 min, 30 min, 1 h, 3 h, 7 h, and 24 h). The estimated terminal half-life after intravenous or subcutaneous administration is shown in Table 5 below.

[0614] Briefly, female CD-1 mice (n=4 per group) from Charles River Laboratories were fasted overnight and administered 100 μL of each peptide in phosphate-buffered saline via intravenous (iv) or subcutaneous (sc) routes. Mice were immediately fed after 30 minutes of bleeding. Blood was extracted into heparin tubes and centrifuged at 3000 g for 15 minutes. The resulting supernatant plasma was then stored at -80°C for peptide concentration determination. The concentration of the peptide in plasma at each time point was determined by an in vitro cell-based activity assay. HEK293-GLP-2R-CRE cells were treated with plasma samples at different time points (a five-point dose response, starting with a 1:10 to 1:100 dilution of each plasma sample) and incubated in DMEM containing 10% FBS at 37°C and 5% CO₂ for 16 hours before measuring firefly luciferase activity. Simultaneously, standard curves and parameters for bottom (input data), top (output data), EC50, and Hill slope were generated using the same peptides. The peptide concentration in plasma (nmol / L) was calculated using the relative light units (RLU) of each plasma sample using the parameters obtained from the standard curve (RLU = Bottom + (Top-Bottom) / (1 + 10 ((logEC50-concentration) Hill slope)). The peptide concentration in plasma was obtained and plotted against time points using WinNonLin Phoenix software (Pharsight Corp, St. Louis, MO) to obtain the in vivo half-life of each peptide.

[0615] Table 5

[0616]

[0617]

[0618] Example C: In vitro efficacy of long-acting GLP2R agonists at human GLP2R

[0619] This example evaluates the efficacy of long-acting GLP2 at GLP2R. A decrease in the 665 / 615 ratio indicates an increase in free cAMP due to increased GLP2R activity.

[0620] GLP2-2G (teduglutide) was used as a positive control. Figure 1A As shown, as the concentration of teduglutide increased, the 665 / 615 ratio decreased, indicating increased GLP2R activity. Varying the concentrations of GLP2-2G-10Nle-1K-EX4-K5, GLP2-2G-1-EX4-L5A, and GLP2-2G-10Nle-1-EX4-L5A produced similar activity levels compared to teduglutide. Based on this data, the IC 50 The values ​​are shown in Table 6. Figure 1BAs shown, when 10% fetal bovine serum was added to the assay, the teduglutide curve became steeper than the curves of the long-acting GP2R agonists. As listed in Table 6, the resulting IC values ​​for all three long-acting GLP2R agonists were 50 The values ​​are all higher.

[0621] Table 6: IC of long-acting GLP2R agonists 50 value

[0622]

[0623] Example D: In vitro efficacy of long-acting GLP2R agonists on mouse GLP2R

[0624] Determine the potency of long-acting GLP2R agonists at mouse GLP2R. A decrease in the 665 / 615 ratio indicates an increase in free cAMP due to increased GLP2R activity.

[0625] like Figure 2 As shown, the ratio of 665 fluorescence to 615 fluorescence was plotted against the molecular concentration, and this data was used to calculate the IC 50 The values ​​are listed in Table 7. Teduglutide (GLP2-2G) and apurglutide (a synthetic GLP-2 analogue) were used as positive controls. The IC values ​​of long-acting GLP2R agonists were found to be 50 The values ​​were in a similar range to those of teduglutide and apurglutide, indicating that this long-acting GLP2R agonist is a relatively potent agonist of the mouse GLP2R.

[0626] Table 7: IC values ​​of long-acting GLP2R agonists for mouse GLP2R 50 value

[0627]

[0628] Example E: In vitro efficacy of long-acting GLP2R agonists on cynomolgus monkey GLP2R

[0629] To determine the efficacy of long-acting GLP2R agonists at the GLP2R in cynomolgus monkeys. Figure 3 As shown, the ratio of 665 to 615 was plotted against concentration (nM) to determine the potency of long-acting GLP2R agonists at the cynomolgus monkey GLP2R. Figure 3 Calculate EC from data in 50 , and the values ​​are listed in Table 8. The EC50 values ​​of the long-acting GLP2R agonists ranged from 0.119 to 0.156 nM, indicating that the long-acting GLP2R agonists were relatively potent agonists of cynomolgus monkey GLP2R.

[0630] Table 8: EC in cynomolgus monkeys 50 value

[0631]

[0632] Example F: Long-acting GLP2R agonists are selective for GLP2R over other G protein-coupled receptors

[0633] This example evaluates the effects of stable GLP2R agonists on other G protein-coupled receptors (GPCRs).

[0634] A decrease in the 665 / 615 ratio indicates an increase in free cAMP due to increased GLP2R activity.

[0635] like Figure 4A As shown, neither GLP2 nor the tested stabilized molecules (GLP2-2G-10Nle-1K-EX4-K5 or GLP2-2G-10Nle-1-L5A) produced any significant changes in GLP-1R activity levels when compared to the changes produced by different concentrations of semaglutide, a GLP-1R agonist. 50 When the values ​​were obtained, as listed in Table 9, these values ​​were very high compared to semaglutide (positive control). This suggests that very high concentrations of GLP2 and long-acting GLP2R agonists are required before GLP-1R is activated.

[0636] Table 9: IC values ​​of stable GLP2R agonists relative to other GPCRs 50

[0637]

[0638] like Figure 4B As shown, long-acting GLP2R agonists -2 and 10 2 nm concentration range did not result in changes in GCGR activity levels. However, increasing glucagon concentrations affected GCGR activity levels. As shown in Table 9, although the IC 50 was 0.04, indicating that it affects the activity level of GCGR at a lower concentration, but the IC 50 The value is greater than 500.

[0639] like Figure 4C As shown, long-acting GLP2R agonists -2 and 10 2 The concentration range of 100 nm did not result in changes in GIPR activity levels. However, increasing the concentration of GIP affected the activity levels of GIPR. IC 50 The values ​​are listed in Table 9. GIP IC 50was 0.04, indicating that it was relatively effective in affecting the activity level of GIPR; however, the IC 50 The value is greater than 500.

[0640] In addition, GLP2-2G-1-EX4-L5A and GLP2-2G-10Nle-1K-EX4-K5 were profiled by DiscoverRx relative to the gpcrMAX Panel. 168 GPCR targets were tested using agonist and antagonist primary screens. Assays were performed using PathHunter β-arrestin fragment complementation (EFC) technology. In agonist mode, no targets other than GLP2 were identified with activity >30%. In antagonist mode: no targets were identified with inhibitory activity >35%. Example G: Stability of long-acting GLP2R agonists at different temperatures

[0641] This example evaluates the stability of stabilized GLP2R agonists at various temperatures over extended periods of time.

[0642] like Figure 5A As shown, GLP2-2G-1-EX4-L5A (GLP2-L5A) and GLP2-2G-10Nle-1K-EX4-L5A (GLP2-K5) were stable at 4°C for 4 days. Figure 5B As shown, at 25°C, 3% oxidation of GLP2-2G-1-EX4-L5A was observed, while GLP2-2G-10Nle-1K-EX4-K5 remained quite intact for 4 days. At 37°C, 11% oxidation of GLP2-2G-1-EX4-L5A was observed, and the +12Da impurity increased by 4% on day 2. Figure 5C As shown, GLP2-2G-10Nle-1K-EX4-K5 had a higher percentage of intact peptide than GLP2-2G-1-EX4-L5A at day 4. At 70°C (forced degradation), many racemic products of both peptides were present. Figure 5D As shown, after 4 days at 70°C, the percentage of intact peptides of GLP2-2G-1-EX4-L5A and GLP2-2G-10Nle-1K-EX4-K5 were both less than 50%.

[0643] Example H: Stability of long-acting GLP2R agonists in different solutions

[0644] As listed in Table 10, the stability of the compounds in various solutions was measured at 0 h. For GLP2-2G-1-EX4-L5A and GLP2-2G-10Nle-1-EX4-L5A, no target compound was detected at 24 h. For GLP2-2G-10Nle-1K-EX4-K5, the glutathione group showed good protection at 24 h. Overall, this indicates that the most stable peptide is GLP2-2G-10Nle-1-EX4-L5A, while the least stable peptide is GLP2-2G-1-EX4-L5A.

[0645] Table 10: Stability of compounds in different solutions at 0 h

[0646]

[0647]

[0648] Example E: Long-term stability of thioether peptides in liquid and solid form

[0649] In this example the long term stability of the thioether peptides was tested.

[0650] The stability of the thioether peptides to wet air oxidation was determined. Met oxidation of GLP2-2G-1-EX4-L5A was observed after 10 days, with 16% degradation observed. GLP2-2G-10Nle-1-EX4-L5A was more stable to wet air oxidation. This indicates that the thioether bridge is stable to oxidation for at least 10 days.

[0651] The powder was stored as the HCl salt at 4° C. After 4 months, GLP2-2G-1-EX4-L5A (GLP2-L5A) showed no signs of Met oxidation. Similarly, after 7 months, GLP2-2G-1-EX4-L5A (GLP2-L5A) showed no signs of Met oxidation.

[0652] Example F: Stability of long-acting GLP2R agonists at different pH values

[0653] The stability of the peptide was determined over a range of pH and temperature.

[0654] At pH 3.3 and room temperature, GLP2-2G-1-EX4-L5A (GLP2-L5A) was 100% stable over 4 days. Figure 6A As shown, GLP2-2G-10Nle-1K-EX4-K5 was also stable, with 95% of the peptide remaining intact over 5 days. Figure 6BAs shown, GLP2-2G-10Nle-1K-EX4-K5 and GLP2-2G-1-EX4-L5A (GLP2-L5A) were less stable at pH 3.3 and 37°C than at room temperature. At 37°C, GLP2-2G-10Nle-1K-EX4-K5 and GLP2-2G-1-EX4-L5A (GLP2-L5A) were primarily hydrolyzed at pH 3.4 (-18 Da and -775 Da). In addition, GLP2-2G-1-EX4-L5A (GLP2-L5A) was insoluble at pH 4.6.

[0655] like Figure 6C As shown, GLP2-2G-10Nle-1K-EX4-K5 and GLP2-2G-1-EX4-L5A (GLP2-L5A) were 100% stable at pH 7.5 and room temperature for 4 days. Figure 6D As shown in , at 37°C, GLP2-2G-10Nle-1K-EX4-K5 was degraded by 1% and L5A was degraded by 1%. Figure 6E As shown, GLP2-2G-10Nle-1K-EX4-K5 and GLP2-2G-1-EX4-L5A (GLP2-L5A) were 100% stable at pH 8.9 and room temperature for 4 days. Figure 6F As shown in , at 37° C. and pH 8.9, GLP2-2G-10Nle-1K-EX4-K5 was degraded by 1% and GLP2-2G-1-EX4-L5A (GLP2-L5A) was degraded by 1%.

[0656] Example G: Stability of long-acting GLP2R agonists in hepatocytes

[0657] Measuring the hepatocyte stability of long-acting GLP2R agonists over time. Figure 7A As shown, for GLP2-2G-1-EX4-L5A, the mouse and MC values ​​were slightly above 100% after 120 min. Figures 7B-7C As shown, for both GLP2-2G-10Nle-1-EX4-L5A and GLP2-2G-10Nle-1K-EX4-K5, although the mouse values ​​increased to slightly above 100%, the MC values ​​decreased to approximately 60% after 120 minutes.

[0658] As listed in Table 11, the biological half-life (T 1 / 2) and intrinsic clearance (CLint) values. In hepatocytes and liver, GLP2-2G-1-EX4-L5A had the highest half-life and the lowest CLint. In hepatocytes and liver, GLP2-2G-10Nle-1-EX4-L5A had the lowest half-life, while GLP2-2G-10Nle-1-EX4-L5A had the highest CLint value.

[0659] Table 11: Half-life and CLint values ​​in hepatocytes

[0660]

[0661] Example H: GLP2-2G-1-EX4-L5A exhibits prolonged in vivo half-life in mice

[0662] Male C57BL / 6 mice were dosed with 1.5 mg / kg of GLP2-2G-1-EX4-L5A in PBS (pH 7.5, clear solution), and the plasma concentration of the agonist was followed for 96 h. Figure 8 Plasma concentrations were analyzed using an LC-MS assay with a limit of quantification of 20 ng / mL. These values ​​were also used to calculate other pharmacokinetic properties of the compound in mice administered by intravenous and subcutaneous injection, as shown in Table 12. A long in vivo half-life of approximately 8.4 hours was observed, similar to the 8-hour half-life of semaglutide in rodents.

[0663] Table 12: Pharmacokinetics of GLP2-Met-L5A in mice

[0664]

[0665] Example 1: GLP2-2G-1-EX4-L5A exhibits prolonged in vivo half-life in cynomolgus monkeys

[0666] Male cynomolgus monkeys were administered 1.0 mg / kg of GLP2-2G-1-EX4-L5A in PBS (pH 7.5, clear solution) and the plasma concentration of the agonist was followed for 504 h. Figure 9 The pharmacokinetics of GLP2-2G-1-EX4-L5A were analyzed for intravenous and subcutaneous drug delivery, as listed in Table 13. Plasma concentrations were analyzed using an LC-MS assay with a limit of quantification of 10 ng / mL. A long in vivo half-life of approximately 70 hours was observed, which is longer than the approximately 50-hour half-life of semaglutide in rodents. This long in vivo half-life suggests potential translation to once-weekly dosing in humans.

[0667] Table 13: Pharmacokinetic properties of GLP2-2G-1-EX4-L5A in cynomolgus monkeys

[0668]

[0669] Example J: GLP2-2G-10Nle-1-EX4-L5A exhibits a long in vivo half-life in mice

[0670] Male C57BL / 6 mice were administered 1.5 mg / kg of GLP2-2G-10Nle-1-EX4-L5A in PBS (pH 7.5) subcutaneously (SC) or intravenously (IV). Figure 10 As shown in Table 14, the plasma concentration of the agonist was tracked 96 hours after administration. The plasma concentration was analyzed using an LC-MS assay with a lower limit of quantification of 5 ng / mL. The pharmacokinetic properties of the drug in mice, including half-life, were calculated from the data and are presented in Table 14. An in vivo half-life of approximately 8 hours was observed for the drug in mice.

[0671] Table 14: Pharmacokinetic properties of GLP2-2G-10Nle-1-EX4-L5A in mice

[0672]

[0673] Example K: GLP2-2G-10Nle-1-EX4-L5A shows prolonged half-life in cynomolgus monkeys

[0674] Male cynomolgus monkeys were administered 1.0 mg / kg of GLP2-2G-10Nle-1-EX4-L5A in PBS (pH 7.5) subcutaneously (SC) or intravenously (IV). Figure 11 As shown in Table 15, the plasma concentration of the agonist was tracked for 504 h after administration. Plasma concentrations were analyzed using an LC-MS assay with a limit of quantification of 5 ng / mL. The pharmacokinetic properties of the drug in cynomolgus monkeys, including half-life, were calculated from this data and the values ​​are listed in Table 15. An in vivo half-life of approximately 57 h was observed for the drug in cynomolgus monkeys.

[0675] Table 15: Pharmacokinetic properties of GLP2-2G-10Nle-1-EX4-L5A in cynomolgus monkeys

[0676]

[0677] Example L: GLP2-2G-10Nle-1K-EX4-K5 exhibits a long in vivo half-life in mice

[0678] Male C57BL / 6 mice were administered 1.5 mg / kg of GLP2-2G-10Nle-1K-EX4-K5 in PBS (pH 7.5) subcutaneously (SC) or intravenously (IV). Figure 12 As shown in Table 16, the plasma concentration of the agonist was tracked 72 hours after administration. The plasma concentration was analyzed using an LC-MS assay with a lower limit of quantification of 5 ng / mL. The pharmacokinetic properties of the drug in mice, including half-life, were calculated from the data and are presented in Table 16. An in vivo half-life of approximately 7 hours was observed for the drug in mice.

[0679] Table 16: Pharmacokinetic properties of GLP2-2G-10Nle-1K-EX4-K5 in mice

[0680]

[0681] Example M: ​​GLP2-2G-10Nle-1K-EX4-K5 shows prolonged half-life in cynomolgus monkeys

[0682] Male cynomolgus monkeys were administered 1.0 mg / kg of GLP2-2G-10Nle-1K-EX4-K5 in PBS (pH 7.5) subcutaneously (SC) or intravenously (IV). Figure 13 As shown in Table 17, plasma concentrations of the agonist were tracked for 504 h after administration. Plasma concentrations were analyzed using an LC-MS assay with a limit of quantification of 5 ng / mL. The pharmacokinetic properties of the drug in cynomolgus monkeys, including half-life, were calculated from these data and are listed in Table 17. An in vivo half-life of approximately 36 h was observed for the drug in cynomolgus monkeys.

[0683] Table 17: Pharmacokinetic properties of GLP2-2G-10Nle-1K-EX4-K5 in cynomolgus monkeys

[0684]

[0685] Example N: GLP2-L5A produces enterotrophic effects in mice

[0686] 13-week-old female CD1 mice were divided into 5 treatment groups as follows: A (vehicle PBS, SC, QD); B (GLP-C14, 0.05 mg / kg, BID); C (GLP2-2G-1-EX4-L5A, 0.1 mg / kg, QD); D (GLP2-2G-1-EX4-L5A, 1 mg / kg, QD); E (GLP2-2G-10Nle-1-EX4-L5A, 0.1 mg / kg, QD) and F (GLP2-2G-10Nle-1-EX4-L5A, 1 mg / kg, QD). Each group consisted of 5 mice. The relevant doses were administered subcutaneously to the mice daily (QD) or twice daily (BID) using DPBS as a vehicle, and body weight was monitored daily.

[0687] Gastrointestinal (GI) measurements were collected 10 days after dosing. These measurements included collecting peripheral blood, dissecting the small intestine, and measuring the length and weight of the small intestine; and recording the length and weight of the empty large intestine.

[0688] like Figure 14A As shown, the small intestine weight of mice treated with at least 0.1 mg / kg of either long-acting GLP2R agonist (CF group) was significantly increased compared to untreated mice (A group). Figure 14B As shown, the small intestine length of all mice treated with long-acting GLP2R agonists increased compared to untreated mice (Group A), and the small intestine length of mice treated with 0.1 mg / kg of GLP2-2G-5-L5A (Group E) was significantly increased. Figure 14C As shown, none of the treatment groups produced significant changes in body weight over the 10 days.

[0689] Example O: GLP2-2G-10Nle-1-EX4-L5A effectively treats a mouse model of acute colitis

[0690] This example evaluates the enterotrophic effects of GLP2-2G-10Nle-1-Ex4-L5A in mice.

[0691] Male C57B6 mice aged 7-8 weeks were divided into 6 experimental groups: A (vehicle PBS, QD); B (teduglutide, 0.5 mg / kg, BID); C (GLP2-2G-10Nle-1K-EX4-K5, 0.03 mg / kg, QD); D (GLP2-2G-10Nle-1K-EX4-K5, 0.1 mg / kg, QD); E (GLP2-2G-10Nle-1K-EX4-K5, 0.3 mg / kg, QD); and F (GLP2-2G-10Nle-1K-EX4-K5, 1 mg / kg, QD). Except for group A, which contained 4 mice, each treatment group contained 6 mice. Using FPBS as a vehicle, the mice were subcutaneously administered with a dosing volume of 5 mL / kg, once a day (QD) or twice a day (BID). Body weight was monitored daily, and gastrointestinal (GI) measurements were collected after 10 days of dosing. These measurements included collecting peripheral blood, dissecting the small intestine, measuring the length and weight of the small intestine, and recording the length and weight of the empty large intestine.

[0692] like Figures 15C-15DAs shown, all treatment groups (BF) resulted in significant increases in small intestine length and weight compared to the control group. Even the lowest dose (0.03 mg / kg) of GLP2-2G-10Nle-1K-EX4-K5 (Group C) produced significant effects on both measures of small intestine strength. Furthermore, a 0.1 mg / kg dose of GLP2-2G-10Nle-1-EX4-K5 (Group D) showed comparable effects to treatment with 0.5 mg / kg of teduglutide (Group B).

[0693] Mice that received a high dose of GLP2-2G-10Nle-1-EX4-K5 (Groups EF) showed a significant increase in colon length compared to the untreated control group (Group A). ​​All treated groups showed a significant increase in colon weight compared to the untreated control group. However, mice in Groups E and F, treated with the highest dose of GLP2-2G-10Nle-1-EX4-K5, showed the greatest increase.

[0694] Example P: GLP2-2G-1-EX4-L5A effectively treats a mouse model of acute colitis

[0695] Acute colitis was induced in mice by a single 5-day course of treatment with 3% dextran sodium sulfate (DSS). Mice were divided into the following 4 treatment groups: A (control mice that did not receive DSS); B (mice that received DSS and subcutaneous injection of PBS); C (mice that received DSS and 1 mg / kg GLP2-2G-1-EX4-L5 subcutaneously); and D (mice that received DSS and 20 mg.kg cyclosporine intraperitoneally).

[0696] like Figure 16A As shown, body weight was measured over 12 days. Compared with untreated mice with induced colitis (group B), mice treated with acute colitis (groups C and D) did not lose much weight. Figures 16B-16C As shown, mice in group C treated with GLP2-2G-1-EX4-L5A showed a significant increase in colon and small intestine weight compared to untreated mice with induced acute colitis (group B). Mice treated with GLP2-2G-1-EX4-L5A also showed a significant increase in colon and small intestine length when compared to untreated controls with induced acute colitis (figure not shown). GLP2-2G-1-EX4-L5A treatment also improved colon and small intestine histopathology in the DSS-induced colitis model. Figure 16D As shown, histopathology of mice that received both DSS and L5A showed that the depth of crypts in the colon was similar to that of mice that did not receive DSS treatment, whereas the length of crypts in mice that received only DSS was significantly reduced. Figure 16EAs shown, in the jejunum, the length of the jejunal villi in Group C mice was greater than that in Groups A or B. When comparing mice treated with GLP2-2G-1-EX4-L5A with untreated mice, the length of the jejunal villi was significantly increased. Furthermore, Group C mice did not exhibit the villus distortion and abscesses seen in Group B mice.

[0697] Example Q: GLP2-2G-10Nle-1K-EX4-K5 effectively treats a mouse model of acute colitis

[0698] Eight-week-old male C57BL / 6 mice were divided into seven treatment groups as follows: A (control mice that did not receive DSS); B (mice that received DSS and subcutaneous injection of PBS); C (mice that received DSS and 0.1 mg / kg GLP2-2G-10Nle-1K-EX4-K5 subcutaneously); D (mice that received DSS and 0.3 mg / kg GLP2-2G-10Nle-1K-EX4-K5 subcutaneously); E (mice that received DSS and 1 mg / kg GLP2-2G-10Nle-1K-EX4-K5 subcutaneously); F (mice that received DSS and 0.5 mg / kg teduglutide subcutaneously) and G (mice that received DSS and 20 mg.kg cyclosporine intraperitoneally). Each group contained six mice, except for group A, which contained four mice.

[0699] like Figure 17A As shown, acute colitis was induced in mice by a single 5-day course of treatment with 3% dextran sodium sulfate (DSS). Animals were treated daily with the appropriate dose for each treatment group for 11 days. Body weight was monitored daily. If the animal lost more than 20% body weight, it was euthanized. On days 10-11, samples were collected at 0, 1, 3, 7, and 24 h after administration for pharmacokinetic analysis. On day 11, the animals were euthanized and autopsied. Terminal blood was collected into heparinized collection tubes and processed into plasma. The small intestine and colon were collected to measure weight and length. Gastrointestinal tissue was collected for histological examination.

[0700] When weight is checked over time, e.g. Figure 17A As shown, mice in group G that received cyclosporine showed a greater percentage of body weight loss during DSS treatment than mice in any other treatment group, but gained weight after the end of DSS treatment. Mice that received DSS without treatment (group B) showed the greatest percentage of body weight loss after the end of DSS treatment compared to all other treatment groups. Mice that received GLP2-2G-10Nle-1K-EX4-K5 (groups CE) or teduglutide (group F) did not show significant changes in body weight during this time compared to mice that did not receive DSS (group A).

[0701] GLP2-2G-10Nle-1K-EX4-K5 treatment protected mice from weight loss and restored colon shortening. Figure 17B As shown in Figure 3, the colon length of mice receiving high-dose GLP2-2G-10Nle-1K-EX4-K5 or teduglutide (DF group) was significantly increased compared with the untreated group (B group). There was no change in colon weight. Figures 17C-17D As shown, in the small intestine, the length and weight of the groups receiving teduglutide or GLP2-2G-10Nle-1K-EX4-K5 (CF group) were significantly increased compared to untreated mice. The lowest dose (0.1 mg / ml) of GLP2-2G-10Nle-1K-EX4-K5 (C group) showed an effect comparable to teduglutide at 0.5 mg / kg BID.

[0702] Treatment with GLP2-2G-10Nle-1K-EX4-K5 also affected the histological characteristics of the intestine. Figure 17E As shown, the teduglutide and GLP2-2G-10Nle-1K-EX4-K5 treated group (CF group) showed a significant increase in villus height compared to untreated mice (AB group). The lowest dose (0.1 mg / kg) of GLP2-2G-10Nle-1K-EX4-K5 showed an enterotrophic effect comparable to 0.5 mg / kg of teduglutide administered twice daily.

[0703] In addition, if Figure 17F As shown, Ki67 staining showed no increased proliferation in any of the treatment groups, indicating that there was no evidence of abnormal proliferation associated with GLP2-2G-10Nle-1K-EX4-K5 treatment.

[0704] The pharmacokinetic properties of the treated group CF are plotted on Figure 17G Teduglutide was undetectable 3 hours after treatment. However, all doses of GLP2-2G-10Nle-1K-EX4-K5 were detectable up to 24 hours after treatment.

[0705] Example R: Long-acting GLP2R agonists are effective in treating acute colitis

[0706] Mice were divided into 9 treatment groups as follows: A (no DSS: vehicle), B (DSS: vehicle (PBS)), C (DSS: GLP2-2G-1-EX4-L5A, 0.03 mg / kg), D (DSS: GLP2-2G-1-EX4-L5A, 0.1 mg / kg), E (DSS: GLP2-2G-10Nle-1-EX4-L5A, 0.03 mg / kg), F (DSS: GLP2-2G-10Nle-1-EX4-L5A, 0.1 mg / kg), G (DSS: GLP2-2G-10Nle-1K-EX4-K5, 0.03 mg / kg), H (DSS: GLP2-2G-10Nle-1K-EX4-K5, 0.1 mg / kg), and I (DSS: cyclosporine A, 20 mg / kg, IP). Each group consisted of six 8-week-old C57BL / 6 male mice. Mice were administered 3% DSS for 7 days and the appropriate treatment for 8 days to induce acute colitis. All animals except Group I were administered 5 ml / kg subcutaneously with a vehicle of DPBS, where the vehicle was olive oil. Pharmacokinetic samples were collected from the CH group on days 6-7 at 0, 1, 3, 7, and 24 hours after dosing. Measurements were performed on day 9. These measurements included collecting peripheral blood; dissecting the small intestine; measuring the length and weight of the small intestine; and recording the length and weight of the empty large intestine.

[0707] All long-acting GLP-2 agonists showed dose-related protection against weight loss compared to untreated animals, and this protection was significant at a dose of 0.1 mg / kg. Figure 18A As shown, animals treated with either dose of GLP2-2G-10Nle-1-Ex4-L5A (Groups C and D) had higher total body weights than untreated animals (Group B). Figure 18B As shown, animals treated with either dose of GLP2-2G-1-EX4-L5A (Groups E, F) had higher total body weights than untreated animals (Group B). Figure 18C As shown, animals treated with either dose of GLP2-2G-10Nle-1K-Ex4-K5A (Groups G and H) had higher total body weights than untreated animals (Group B). In addition, at a dose of 0.03 mg / kg, GLP2-2G-10Nle-1-Ex4-L5A and GLP2-2G-10Nle-1K-Ex4-K5 were more effective than GLP2-2G-1-EX4-L5A in protecting against weight loss.

[0708] like Figure 18DAs shown in Figure 2, all three long-acting GLP-2 agonists significantly increased colon length at 0.1 mg / kg in the acute DSS-induced colitis model. Figure 18E As shown in Figure 2, there was a trend toward non-significant increases in colon weight when animals treated with a long-acting GLP2 agonist were compared to untreated DSS animals. Figures 18F-18G As shown, all three long-acting GLP2 agonists showed dose-related trophic effects on small intestine weight and length.

[0709] like Figure 18H As shown in , treatment with GLP2R agonists also increases gallbladder size. Figure 18I As shown, fecal occult blood was measured using the Hemoccult II test. Several treatment parameters (e.g., two doses of 1L5A) reduced occult blood levels compared to untreated DSS model mice.

[0710] Measuring levels of long-acting GLP2 agonists over time. Figure 18J As shown in Figure 2, at a dose of 0.03 mg / kg, the concentration increased until 7 h after administration, but was still detectable 24 h after administration. Figure 18K As shown in the figure, at a dose of 0.1 mg / kg, the concentration increased until 7 h after administration and was still detectable 24 h after administration. For both doses, GLP2-2G-1-EX4-L5A had the highest content, followed by GLP2-2G-10Nle-1-Ex4-L5A, and then GLP2-2G-10Nle-1K-Ex4-K5. Figures 18L-18N As shown, for all three drugs tested, higher doses resulted in higher drug concentrations at all time points tested.

[0711] A significant decrease in the mRNA levels of inflammatory cytokines was observed in colon tissue.

[0712] Example S: GLP2-2G-1-EX4-L5A effectively treats chronic colitis

[0713] Chronic DSS-induced colitis was induced in C57BL / 6 mice (male, 10-12 weeks old) by administering 2.5% DSS in drinking water for 5 consecutive days for 3 cycles, followed by 7 days of recovery. During the final DSS-induced cycle, animals were treated daily for 7 days. Treatment was administered subcutaneously (S) or intraperitoneally (IP) once daily (QD) or twice daily (BID). Mice were treated according to the treatment groups listed as follows: A (no DSS: vehicle, SC, QD (n=6)), B (DSS: vehicle (PBS), SC, QD (n=8)), C (DSS: GLP2-2G-1-EX4-L5A, 0.1 mg / kg, SC, QD (n=8)), D (DSS: GLP2-2G-1-EX4-L5A, 0.3 mg / kg, SC, QD (n=8)), E (DSS: cyclosporine, 20 mg / kg, IP (n=6)), and F (DSS: teduglutide, 0.3 mg / kg, SC, QD (n=6)).

[0714] Body weight was monitored three times weekly. Pharmacokinetic data were collected three and four days before necropsy. On day 33, a necropsy was performed, and measurements were taken. These measurements included collecting peripheral blood, dissecting the small intestine, measuring the length and weight of the small intestine, and recording the length and weight of the empty large intestine.

[0715] GLP2-2G-1-EX4-L5A effectively treats weight loss in a mouse model of chronic colitis. Figure 19A As shown, mice treated with GLP2-2G-1-EX4-L5A or teduglutide (Groups C, D, and F) did not lose the same percentage or total body weight as untreated mice (Group B). The protective effect against weight loss was dose-dependent, with higher doses resulting in enhanced protection. Furthermore, these effects were equivalent to treatment with 0.3 mg / kg teduglutide once daily (QD).

[0716] like Figure 19B As shown, GLP2-2G-1-EX4-L5A showed dose-related restoration of colon length when compared to untreated mice. Furthermore, when 0.3 mg / kg GLP2-2G-1-EX4-L5A treatment was compared to an equivalent dose of teduglutide, the effects of GLP2-2G-1-EX4-L5A were less variable than those of teduglutide treatment. Figure 19C As shown, GLP2-2G-1-EX4-L5A and teduglutide treatment also affected colon weight. Mice treated with a low dose of GLP2-2G-1-EX4-L5A had colon weights similar to those of mice treated with teduglutide.

[0717] like Figure 19DAs shown, higher doses of GLP2-2G-1-EX4-L5A showed a significant increase in small intestine weight compared to untreated mice, an effect equivalent to that produced by teduglutide.

[0718] Example T: GLP2-2G-10Nle-1-Ex4-L5A effectively treats a mouse model of chronic colitis

[0719] Chronic DSS-induced colitis was induced in C57BL / 6 mice (male, 10-12 weeks old) by administering 2.5% DSS in drinking water for 5 consecutive days for 3 cycles, followed by 7 days of recovery. During the final DSS-induced cycle, animals were treated daily for 7 days. Treatment was administered subcutaneously (S) or intraperitoneally (IP) once daily (QD) or twice daily (BID). Mice were treated according to the treatment groups listed below: A (no DSS: vehicle, SC, QD (n=4)), B (DSS: vehicle (PBS), SC, QD (n=6)), C (DSS: GLP2-2G-10Nle-1-L5A, 0.03 mg / kg, SC, QD (n=6)), D (DSS: GLP2-2G-10Nle-1-L5A, 0.1 mg / kg, SC, QD (n=6)), E (DSS: GLP2-2G-10Nle-1-L5A, 0.3 mg / kg, SC, QD (n=6)), F (DSS: GLP2-2G-10Nle-1-L5A, 1 mg / kg, SC, QD (n=6)), and G (DSS: teduglutide, 0.5 mg / kg, SC, BID (n=6)).

[0720] Body weight was monitored three times per week. Pharmacokinetic data were collected three and four days before necropsy. On day 33, an necropsy was performed and measurements were taken. These measurements included collecting peripheral blood, dissecting the small intestine, measuring the length and weight of the small intestine, and recording the length and weight of the empty large intestine.

[0721] Low doses of GLP2-2G-10Nle-1-Ex4-L5A showed a modest effect on weight loss. Treatment with doses of 0.03 mg / kg and 0.3 mg / kg was protective against weight loss when compared to untreated mice (not shown).

[0722] like Figures 20A-20BAs shown, GLP2-2G-10Nle-1-Ex4-L5A can increase colon length and weight in a chronic DSS-induced colitis model. When compared with untreated animals (Group B), the colon length of animals treated with 0.1 mg / kg and higher doses of GLP2-2G-10Nle-1-Ex4-L5A and animals treated with teduglutide (Group DG) was significantly increased. The colon weight of animals treated with 0.3 mg / kg or higher doses of GLP2-2G-10Nle-1-Ex4-L5A and animals treated with teduglutide was increased compared with untreated animals.

[0723] GLP2-2G-10Nle-1-Ex4-L5A also significantly affected the weight and length of the small intestine. Figure 20C As shown, treatment with 0.1 mg / kg and higher doses of GLP2-2G-10Nle-1-Ex4-L5A and treatment with teduglutide resulted in a significant increase in small intestine length compared to untreated mouse models. Treatment with 0.3 mg / kg or higher doses of GLP2-2G-10Nle-1-Ex4-L5A resulted in a significant increase in small intestine weight compared to untreated mice (not shown).

[0724] Example U: GLP-2-2G-5-L5A Treatment in a NASH Model

[0725] Five-week-old C57BL / 6 mice were placed on a choline-deficient diet (CDAA, Dyets#518753) or an AA-supplemented control diet (CSAA, Dyets#518754) for 19 weeks. The mice were divided into three treatment groups of 8 mice each as follows: CSAA control diet, treated with vehicle only; CDAA diet treated with vehicle (MCT, PO; saline, SC); and CDAA diet treated subcutaneously with 1 mg / kg GLP2-2G-5-EX4-L5A. After 15 weeks, mice were treated with vehicle or compound for 4 weeks. Body weight was monitored weekly during the diet induction phase and 3 times a week during the treatment phase. After 19 weeks, the animals were euthanized and peripheral blood and liver samples were collected for serum analysis, histology, and gene expression.

[0726] Chronic treatment with GLP2-2G-5-EX4-L5A improves markers of liver function. Figures 21A-21B As shown, in mice fed a choline-deficient diet, serum ALT and serum AST were significantly reduced compared to untreated mice on the same diet. Total serum bilirubin was also reduced in mice treated with GLP2-2G-5-EX4-L5A compared to untreated mice on the same diet. Gallbladder enlargement was observed in 7 out of 8 GLP-2-treated mice.

[0727] like Figure 21C As shown, treatment with GLP2-2G-5-EX4-L5A resulted in a 20% reduction in liver fibrosis scores. Collagen deposition / fibrosis was observed using picrosirius red, and severity was graded using the following scale: 0 = absent; 1 = minimal; 2 = mild; 3 = moderate; 4 = marked; 5 = severe. The treatment had no significant effect on body weight, indicating that the treatment was tolerable.

[0728] The effects of this treatment on hepatic steatosis and inflammation were also analyzed. Steatosis was analyzed by the percentage of hepatocyte vacuolization, determined by crisp, round, unstained lipid vacuoles, and was assigned a grade based on the following scale: 0 for less than 5%; 1 for 5-33%; 2 for 33-66%; and 3 for greater than 66%. Figure 21D As shown, treatment with GLP2-2G-5-EX4-L5A did not significantly affect the grade of steatosis in the liver. Lobular inflammation was analyzed by assessing the infiltration of neutrophils, lymphocytes, and macrophages in the inflammatory lesions. Lobular inflammation was scored using the following scale: 0 for no lesions; 1 for 2 lesions / 200x field; 2 for 2-4 lesions / 200x field; and 3 for more than 4 lesions / 200x field. Figure 21E As shown, treatment with GLP-2-2G-5-L5A reduced the level of lobular inflammation compared to animals on an untreated CDAA diet.

[0729] This example shows that treatment with GLP2-2G-5-EX4-L5A improves markers of liver injury and prevents progression of liver fibrosis in the CDAA-NASH model.

[0730] Example V: Long-acting GLP2 agonist treatment of a mouse model of environmental enteric dysfunction (EED)

[0731] The ability of GLP2-2G-10Nle-1K-EX4-K5 to treat environmental enteric dysfunction (EED) was evaluated using a weaning malnutrition model. All dams were placed on an isocaloric Northeastern Brazilian diet (Regional Basic Diet - RBD) and their pups were moderately deficient in protein, fat, and minerals when they were 10 days old. At weaning (3 weeks of age), the pups were placed on a standard control diet (CD) or continued on RBD. At 4 weeks of age, weaned pups were given drug or placebo (0.1 mg / kg, formulated in PBS (vehicle)) subcutaneously once a day for 2-3 weeks. Body weight and food consumption were measured twice a week. Feces were collected for calorimetry and microbiome analysis at weaning, 6 weeks of age, and 8 weeks of age. Oral FITC-dextran was used as a measure of barrier function. At 6 weeks of age, mice were euthanized and jejunal tissue was collected for morphology, immunohistochemistry, and compartmental analysis of transmucosal resistance and permeability.

[0732] like Figures 22A-22B As shown in Figure 2, both male and female RBD mice weaned as CD and treated with teduglutide or GLP2-2G-10Nle-1K-EX4-K5 showed a trend toward increased body weight. Figures 22C-22D As shown, male and female mice weaned with RBD showed a trend toward worsening body weight when administered teduglutide or a long-acting GLP2 agonist.

[0733] Treatment with teduglutide and a long-acting GLP2 agonist had a profound effect on intestinal wet weight and length. Figure 22E As shown, CD males treated with teduglutide or GLP2-2G-10Nle-1K-EX4-K5 showed a significant increase in small intestine wet weight / body weight. Figure 22F As shown, CD females treated with teduglutide or GLP2-2G-10Nle-1K-EX4-K5 showed a significant increase in small intestinal wet weight / body weight compared to untreated females. In addition, treatment with GLP2-2G-10Nle-1K-EX4-K5 also resulted in a significant increase when compared to treatment with teduglutide. RBD males treated with teduglutide or GLP2-2G-10Nle-1K-EX4-K5 showed a significant increase in small intestinal wet weight / body weight compared to untreated males. In RBD females, only animals treated with GLP2-2G-10Nle-1K-EX4-K5 showed a significant increase in small intestinal wet weight / body weight compared to untreated animals.

[0734] For animals weaned on the CD diet and animals weaned on the RBD diet, treatment with GLP2 or teduglutide resulted in a significant increase in small intestine length when compared to untreated animals.

[0735] Treatment with GLP2-2G-10Nle-1K-EX4-K5 also had an enterotrophic effect on the animals. CD males treated with teduglutide or GLP2-2G-10Nle-1K-EX4-K5 had significantly longer villus height than untreated males. CD females treated with GLP2-2G-10Nle-1K-EX4-K5 also had significantly longer villus length than untreated females. Crypt depth of treated and untreated CD animals. Males treated with teduglutide or GLP2-2G-10Nle-1K-EX4-K5 had longer crypt depth than untreated males.

[0736] Intestinal permeability was also measured in these mice, where greater FITC-dextran relative fluorescence indicates greater intestinal permeability. Compared to untreated mice, CD males treated with teduglutide or GLP2-2G-10Nle-1K-EX4-K5 showed a trend toward decreased permeability in treated mice. When compared to untreated CD female mice, CD female mice treated with teduglutide or GLP2-2G-10Nle-1K-EX4-K5 showed a significant decrease in permeability. Treatment with teduglutide or GLP2-2G-10Nle-1K-EX4-K5 did not significantly affect permeability in RBD females or RBD males when compared to untreated mice.

[0737] Untreated CD mice showed higher intestinal permeability levels than untreated RBD mice. When treated with teduglutide, CD and RBD mice had similar overall permeability levels. Female mice treated with GLP2-2G-10Nle-1K-EX4-K5 may have slightly higher permeability levels when compared to male mice fed the same diet.

Claims

1. A peptide conjugate comprising: a) a peptide that modulates the GLP-2 receptor, said peptide being the sequence: HGDGSFSDEMNTILDNCAARDFICWLIQTKITDPSSGAPPPS (SEQ ID NO: 1); and b) a stapler linked to the peptide at the first cysteine ​​and the second cysteine, wherein the stapler has the structure: wherein each "S" is a sulfur atom of the first cysteine ​​or the second cysteine.

2. A peptide conjugate comprising: a) a peptide that modulates the GLP-2 receptor, the peptide being the sequence: HGDGSFSDE(Nle)NTILDNCAARDFICWLIQTKITDPSSGAPPPS (SEQ ID NO: 2); and b) a stapler linked to the peptide at the first cysteine ​​and the second cysteine, wherein the stapler has the structure: wherein each "S" is a sulfur atom of the first cysteine ​​or the second cysteine.

3. A peptide conjugate comprising: a) a peptide that modulates the GLP-2 receptor, the peptide being the sequence: HGDGSFSDE(Nle)NTILDNKAARDFIKWLIQTKITDPSSGAPPPS (SEQ ID NO: 10), wherein the sequence is L-norleucine at position 10; and b) a stapler having the following structure attached to the peptide at the first lysine and the second lysine: wherein each "NH" is the amine of the first lysine or the second lysine. A pharmaceutical composition comprising the peptide conjugate according to claim 1 and a pharmaceutically acceptable excipient.

5. Use of the peptide conjugate according to claim 1 in the preparation of a medicament for treating acute colitis or chronic colitis.

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