GLP2 Receptor Agonist and Method for Using the Same

Abstract

Peptide conjugates are provided that include peptides that modulate the GLP-2 receptor. The peptide conjugates can be used to treat diseases that depend on modulation of the GLP-2 receptor. Stapled GLP-2 peptide conjugates are also provided.

Description

Technical Field

[0001] Cross-reference This application claims the benefit of U.S. Provisional Patent Application No. 62 / 943,667, filed on December 4, 2019, and U.S. Provisional Patent Application No. 62 / 994,791, filed on March 25, 2020, which are hereby incorporated by reference in their entirety.

Background Art

[0002] The development of therapeutic agents is often hampered by their 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 are often administered higher doses of the therapeutic agent more frequently, which can lead to decreased compliance, increased costs, and an increased risk of side effects. Therefore, there is a need to produce therapeutic agents with extended half-lives.

Summary of the Invention

[0003] Disclosed herein is a) a peptide that modulates the GLP-2 receptor, and b) a staple that binds to the peptide at a first amino acid and a second amino acid comprising a peptide conjugate.

[0004] In some embodiments, the staple has the formula (I):

[0005]

Chemical formula

[0006] Also disclosed herein is a pharmaceutical composition comprising the peptide conjugate described herein and a pharmaceutically acceptable excipient.

[0007] Also disclosed herein is a method of treating a disease or disorder in a subject, the method comprising administering to the subject a composition comprising a therapeutically effective amount of the peptide conjugate described herein.

[0008] Also disclosed herein is the following formula:

[0009]

Chemical formula

[0010]

Chemical formula

[0011] In some embodiments, A is optionally substituted alkylene. In some embodiments, A is -(CH 2 ) t -, where t is from 1 to 12. In some embodiments, A is optionally substituted arylene. In some embodiments, A is -NR 3 -alkylene-NR 3 -. In some embodiments, A is -N-.

[0012] In some embodiments, X 1 and X2 is -C(=O)-. In some embodiments, X 1 and X 2 is -alkylene-C(=O)-. In some embodiments, X 1 and X 2 is -CH 2 -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 -CH 2 -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 -CH 2 -C(=O)NR 3 -CH 2 CH 2 -.

[0013] In some embodiments, >A-R has the following structure:

[0014]

Chemical formula

[0015] In some embodiments, >A-R has the following structure:

[0016]

Chemical formula

[0017] In some embodiments, >A-R has the following structure:

[0018] [Chemical formula] having, wherein p1 is from 1 to 5.

[0019] In some embodiments, >A-R has the following structure:

[0020] [Chemical formula] having.

[0021] In some embodiments, >A-R has the following structure:

[0022] [Chemical formula] having.

[0023] In some embodiments, s is from 1 to 15. In some embodiments, s is from 1 to 10. In some embodiments, s is from 5 to 15. In some embodiments, s is from 5 to 10.

[0024] In some embodiments, Y is hydrogen or -CO 2 H.

[0025] 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 from 2 to 20.

[0026] In some embodiments, Y 1 and Y 2 are halogen. In some embodiments, Y 1 and Y 2is -COOH. In some embodiments, Y 1 and Y 2 are -S- of two sulfhydryl-containing amino acids in a peptide that modulates the GLP-2 receptor.

[0027] In some embodiments, Y 1 and Y 2 are -S- of two sulfhydryl-containing amino acids in a peptide.

[0028] In some embodiments, Y 1 and Y 2 is -CONH-, and -NH- is part of two amine-containing amino acids in a peptide that modulates the GLP-2 receptor.

[0029] In some embodiments, Y 1 and Y 2 is -CONH-, and -NH- is part of two amine-containing amino acids in a peptide that modulates the GLP-2 receptor, and the two amine-containing amino acids are separated by seven amino acids.

Brief Description of the Drawings

[0030]

Figure 1

Figure 2

Figure 3

Figure 4A

Figure 4B

Figure 4C

Figure 5A

Figure 5B

Figure 5C

Figure 5D

Figure 6A

Figure 6B

Figure 6C

Figure 6D

Figure 6E

Figure 6F

Figure 7A

Figure 7B

Figure 7C

Figure 8

Figure 9

Figure 10

Figure 11

Figure 12

Figure 13

Figure 14A

Figure 14B

Figure 14C

Figure 15A

Figure 15B

Figure 15C

Figure 15D

Figure 16A

Figure 16B

Figure 16C

Figure 16D

Figure 16E

Figure 17A

Figure 17B

Figure 17C

Figure 17D

Figure 17E

Figure 17F

Figure 17G

Figure 18A

Figure 18B

Figure 18C

Figure 18D

Figure 18E

Figure 18F

Figure 18G

Figure 18H

Figure 18I

Figure 18J

Figure 18K

Figure 18L

Figure 18M

Figure 18N

Figure 19A

Figure 19B

Figure 19C

Figure 19D

Figure 20A

Figure 20B

Figure 20C

Figure 21A

Figure 21B

Figure 21C

Figure 21D

Figure 21E

Figure 22A

Figure 22B

Figure 22C

Figure 22D

Figure 22E

Figure 22F

Mode for Carrying Out the Invention

[0031] Glucagon-like peptide 2 (GLP-2) is a hormone secreted from enteroendocrine cells. GLP-2 stimulates intestinal growth, increases nutrient absorption and blood flow, decreases intestinal permeability and motility, and reduces epithelial cell apoptosis and inflammation. Due to the intestinotrophic effects of GLP-2, GLP-2 and related analogs may be useful in the treatment of GI disorders. In humans, the short plasma half-life of native GLP-2 requires more frequent injections or infusions at higher doses to achieve clinical efficacy, which may negatively impact patient compliance. Methods for extending the half-life of GLP-2, including PEGylation and fusion to polypeptides, are utilized to increase the molecular weight and hydrodynamic radius and decrease the clearance rate via renal filtration. However, the resulting analogs are plagued by a decrease in potency in vitro, and as a result, higher doses are required to be effective in vivo.

[0032] Peptide conjugate In one aspect, the present specification discloses a peptide conjugate comprising a peptide that modulates the GLP-2 receptor. In an exemplary example, the peptide that modulates the GLP-2 receptor comprises two amino acids connected by a staple. Non-limiting examples of amino acids for use in 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. In a peptide that modulates the GLP-2 receptor and comprises two amino acids connected by a staple, 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 has the i position, and the second amino acid has the i + 7 position, i + 11 position, i + 13 position, i + 15 position, or i + 16 position. For example, the first amino acid has the i position in the peptide, and the second amino acid has the i + n position in the peptide, where n is from 4 to 16. For example, the first amino acid has the i position in the peptide, and the second amino acid has the i + 7 position in the peptide. For example, the first amino acid has the i position in the peptide, and the second amino acid has the i + 11 position in the peptide. For example, the first amino acid has the i position in the peptide, and the second amino acid has the i + 15 position in the peptide. For example, the first amino acid has the i position in the peptide, and the second amino acid has the i + 16 position in the peptide.

[0033] Peptide that modulates the GLP-2 receptor In one aspect, the present specification provides a peptide conjugate 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.

[0034] The binding affinity of the peptide conjugates described herein may be within about 5% of the binding affinity of the unmodified form of the GLP-2 peptide (e.g., the unconjugated GLP-2 peptide). The binding affinity of the peptide conjugates described herein may be within about 10% of the binding affinity of the unmodified form of the GLP-2 peptide. The binding affinity of the peptide conjugates described herein may be within about 15% of the binding affinity of the unmodified form of the GLP-2 peptide. The binding affinity of the peptide conjugates described herein may be within about 20% of the binding affinity of the unmodified form of the GLP-2 peptide.

[0035] Peptides that modulate the GLP-2 receptor include at least a portion of the wild-type GLP-2 peptide and may include one or more amino acid mutations. The one or more amino acid mutations may include deletions, substitutions, additions, or combinations thereof. The one or more amino acid mutations may include the addition of one or more amino acid residues to the wild-type GLP-2 peptide. The one or more amino acid mutations may include the deletion of one or more amino acid residues of the wild-type GLP-2 peptide. The one or more amino acid mutations may include the substitution of one or more amino acid residues of the wild-type GLP-2 peptide. The one or more amino acid mutations may include replacing one or more amino acid residues of the 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 include replacing one or more amino acid residues of the wild-type GLP-2 peptide with one or more D-amino acid residues. One or more amino acid residues of the GLP-2 wild-type peptide may include one or more alanine, methionine, arginine, serine, threonine, and tyrosine.

[0036] Peptides that regulate the GLP-2 receptor may be modified, for example, by acetylation, phosphorylation, or methylation. Peptide modification may include chemical modification. Peptide modification may occur at the N-terminus of the peptide. Peptide modification may include acetylating the amino group at the N-terminus of the peptide. Alternatively or additionally, peptide modification may occur at the C-terminus of the peptide. Peptide modification may occur at one or more internal amino acids of the peptide. Peptide modification may include replacing the carboxyl group at the C-terminus of the peptide. Peptide modification may include modifying the carboxyl group at the C-terminus of the peptide. The carboxyl group at the C-terminus of the peptide may be modified to produce an amide group. The carboxyl group at the C-terminus of the peptide may be modified to produce an amine group.

[0037] Non-limiting examples of peptides that regulate the GLP-2 receptor are shown in Table 1.

[0038] In some embodiments, a peptide that regulates the GLP-2 receptor comprises an amino acid sequence of any one of SEQ ID NOs: 1-40. Optionally, the peptide that regulates 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, a peptide that regulates the GLP-2 receptor comprises an amino acid sequence having 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.

[0039] In some embodiments, the peptide that modulates the GLP-2 receptor comprises any one of the amino acid sequences of SEQ ID NOs: 1-9. Optionally, 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 having 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.

[0040] In some embodiments, the peptide that modulates the GLP-2 receptor comprises the amino acid sequence of SEQ ID NO: 1. Optionally, 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 SEQ ID NO: 1. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 1.

[0041] In some embodiments, the peptide that modulates the GLP-2 receptor comprises the amino acid sequence of SEQ ID NO: 2. Optionally, 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 SEQ ID NO: 2. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 2.

[0042] In some embodiments, the peptide that modulates the GLP-2 receptor comprises the amino acid sequence of SEQ ID NO: 3. Optionally, 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 SEQ ID NO: 3. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 3.

[0043] In some embodiments, the peptide that modulates the GLP-2 receptor comprises the amino acid sequence of SEQ ID NO: 4. Optionally, 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 SEQ ID NO: 4. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 4.

[0044] In some embodiments, the peptide that modulates the GLP-2 receptor comprises the amino acid sequence of SEQ ID NO: 5. Optionally, 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 SEQ ID NO: 5. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 5.

[0045] In some embodiments, the peptide that modulates the GLP-2 receptor comprises the amino acid sequence of SEQ ID NO: 6. Optionally, 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 SEQ ID NO: 6. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 6.

[0046] In some embodiments, the peptide that modulates the GLP-2 receptor comprises the amino acid sequence of SEQ ID NO: 7. Optionally, 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 SEQ ID NO: 7. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 7.

[0047] In some embodiments, the peptide that modulates the GLP-2 receptor comprises the amino acid sequence of SEQ ID NO: 8. Optionally, 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 SEQ ID NO: 8. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 8.

[0048] In some embodiments, the peptide that modulates the GLP-2 receptor comprises the amino acid sequence of SEQ ID NO: 9. Optionally, 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 SEQ ID NO: 9. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 9.

[0049] In some embodiments, the peptide that modulates the GLP-2 receptor comprises any one of the amino acid sequences of SEQ ID NOs: 10-20. Optionally, 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 having 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.

[0050] In some embodiments, the peptide that modulates the GLP-2 receptor comprises the amino acid sequence of SEQ ID NO: 10. Optionally, 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 SEQ ID NO: 10. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 10.

[0051] In some embodiments, the peptide that modulates the GLP-2 receptor comprises the amino acid sequence of SEQ ID NO: 11. Optionally, 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 SEQ ID NO: 11. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 11.

[0052] In some embodiments, the peptide that modulates the GLP-2 receptor comprises the amino acid sequence of SEQ ID NO: 12. Optionally, 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 SEQ ID NO: 12. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 12.

[0053] In some embodiments, the peptide that modulates the GLP-2 receptor comprises the amino acid sequence of SEQ ID NO: 13. Optionally, 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 SEQ ID NO: 13. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 13.

[0054] In some embodiments, the peptide that modulates the GLP-2 receptor comprises the amino acid sequence of SEQ ID NO: 14. Optionally, 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 SEQ ID NO: 14. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 14.

[0055] In some embodiments, the peptide that modulates the GLP-2 receptor comprises the amino acid sequence of SEQ ID NO: 15. Optionally, 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 SEQ ID NO: 15. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 15.

[0056] In some embodiments, the peptide that modulates the GLP-2 receptor comprises the amino acid sequence of SEQ ID NO: 16. Optionally, 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 SEQ ID NO: 16. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 16.

[0057] In some embodiments, the peptide that modulates the GLP-2 receptor comprises the amino acid sequence of SEQ ID NO: 17. Optionally, 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 SEQ ID NO: 17. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 17.

[0058] In some embodiments, the peptide that modulates the GLP-2 receptor comprises the amino acid sequence of SEQ ID NO: 18. Optionally, 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 SEQ ID NO: 18. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 18.

[0059] In some embodiments, the peptide that modulates the GLP-2 receptor comprises the amino acid sequence of SEQ ID NO: 19. Optionally, 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 SEQ ID NO: 19. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 19.

[0060] In some embodiments, the peptide that modulates the GLP-2 receptor comprises the amino acid sequence of SEQ ID NO: 20. Optionally, 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 SEQ ID NO: 20. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 20.

[0061] In some embodiments, the peptide that modulates the GLP-2 receptor comprises any one of the amino acid sequences of SEQ ID NOs: 21 to 29. Optionally, 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 to 29. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having at most about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to any one of SEQ ID NOs: 21 to 29.

[0062] In some embodiments, the peptide that modulates the GLP-2 receptor comprises the amino acid sequence of SEQ ID NO: 21. Optionally, 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 SEQ ID NO: 21. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having at most about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 21.

[0063] In some embodiments, the peptide that modulates the GLP-2 receptor comprises the amino acid sequence of SEQ ID NO: 22. Optionally, 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 SEQ ID NO: 22. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having at most about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 22.

[0064] In some embodiments, the peptide that modulates the GLP-2 receptor comprises the amino acid sequence of SEQ ID NO: 23. Optionally, 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 SEQ ID NO: 23. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 23.

[0065] In some embodiments, the peptide that modulates the GLP-2 receptor comprises the amino acid sequence of SEQ ID NO: 24. Optionally, 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 SEQ ID NO: 24. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 24.

[0066] In some embodiments, the peptide that modulates the GLP-2 receptor comprises the amino acid sequence of SEQ ID NO: 25. Optionally, 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 SEQ ID NO: 25. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 25.

[0067] In some embodiments, the peptide that modulates the GLP-2 receptor comprises the amino acid sequence of SEQ ID NO: 26. Optionally, 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 SEQ ID NO: 26. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 26.

[0068] In some embodiments, the peptide that modulates the GLP-2 receptor comprises the amino acid sequence of SEQ ID NO: 27. Optionally, 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 SEQ ID NO: 27. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 27.

[0069] In some embodiments, the peptide that modulates the GLP-2 receptor comprises the amino acid sequence of SEQ ID NO: 28. Optionally, 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 SEQ ID NO: 28. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 28.

[0070] In some embodiments, the peptide that modulates the GLP-2 receptor comprises the amino acid sequence of SEQ ID NO: 29. Optionally, 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 SEQ ID NO: 29. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 29.

[0071] In some embodiments, the peptide that modulates the GLP-2 receptor comprises any one of the amino acid sequences of SEQ ID NOs: 30 to 40. Optionally, 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 to 40. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having a maximum of about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to any one of SEQ ID NOs: 30 to 40.

[0072] In some embodiments, the peptide that modulates the GLP-2 receptor comprises the amino acid sequence of SEQ ID NO: 30. Optionally, 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 SEQ ID NO: 30. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having a maximum of about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 30.

[0073] In some embodiments, the peptide that modulates the GLP-2 receptor comprises the amino acid sequence of SEQ ID NO: 31. Optionally, 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 SEQ ID NO: 31. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having a maximum of about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 31.

[0074] In some embodiments, the peptide that modulates the GLP-2 receptor comprises the amino acid sequence of SEQ ID NO: 32. Optionally, 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 SEQ ID NO: 32. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 32.

[0075] In some embodiments, the peptide that modulates the GLP-2 receptor comprises the amino acid sequence of SEQ ID NO: 33. Optionally, 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 SEQ ID NO: 33. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions as compared to SEQ ID NO: 33.

[0076] In some embodiments, the peptide that modulates the GLP-2 receptor comprises the amino acid sequence of SEQ ID NO: 34. Optionally, 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 SEQ ID NO: 34. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 34.

[0077] In some embodiments, the peptide that modulates the GLP-2 receptor comprises the amino acid sequence of SEQ ID NO: 35. Optionally, 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 SEQ ID NO: 35. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 35.

[0078] In some embodiments, the peptide that modulates the GLP-2 receptor comprises the amino acid sequence of SEQ ID NO: 36. Optionally, 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 SEQ ID NO: 36. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 36.

[0079] In some embodiments, the peptide that modulates the GLP-2 receptor comprises the amino acid sequence of SEQ ID NO: 37. Optionally, 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 SEQ ID NO: 37. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 37.

[0080] In some embodiments, the peptide that modulates the GLP-2 receptor comprises the amino acid sequence of SEQ ID NO: 38. Optionally, 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 SEQ ID NO: 38. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 38.

[0081] In some embodiments, the peptide that modulates the GLP-2 receptor comprises the amino acid sequence of SEQ ID NO: 39. Optionally, 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 SEQ ID NO: 39. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 39.

[0082] In some embodiments, the peptide that modulates the GLP-2 receptor comprises the amino acid sequence of SEQ ID NO: 40. Optionally, 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 SEQ ID NO: 40. In some embodiments, the peptide that modulates the GLP-2 receptor comprises an amino acid sequence having up to about 1, 2, 3, 4, or 5 amino acid insertions, deletions, modifications, or substitutions compared to SEQ ID NO: 40.

[0083]

Table 1-1

[0084]

Table 1-2

[0085]

Table 1-3

[0086] Staple Peptide conjugates comprising staples are disclosed herein.

[0087] In some embodiments, the staple attached to the peptide is of formula (I):

[0088]

Chemical formula

[0089] In some embodiments, the staple attached to the peptide has the formula (I):

[0090]

Chemical formula

[0091] In some embodiments, A is optionally substituted alkylene. In some embodiments, A is -(CH 2 ) t -, where t is from 1 to 12. In some embodiments, A is -(CH 2 )t - and here t is from 1 to 10. In some embodiments, A is -(CH 2 ) t - and here t is from 1 to 8. In some embodiments, A is -(CH 2 ) t - and here t is from 1 to 6. In some embodiments, A is -(CH 2 ) t - and here t is from 1 to 4.

[0092] In some embodiments, A is an optionally substituted arylene. In some embodiments, A is an arylene optionally substituted by halogen, alkyl, or haloalkyl. In some embodiments, A is arylene.

[0093] In some embodiments, A is -NR 3 -alkylene-NR 3 -.

[0094] In some embodiments, A is -N-.

[0095] In some embodiments, X 1 and X 2 are the same. In some embodiments, X 1 and X 2 are different.

[0096] In some embodiments, X 1 and X 2 are -C(=O)-. In some embodiments, X 1 and X 2 are independently -alkylene-C(=O)- or -C(=O)alkylene-. In some embodiments, X 1 and X 2 are independently -CH 2 -C(=O)- or -C(=O)-CH 2 -. In some embodiments, X 1 and X 2 are independently -alkylene-C(=O)NR3 -or -C(=O)NR 3 -alkylene-. In some embodiments, X 1 and X 2 are independently -CH 2 -C(=O)NR 3 -or -C(=O)NR 3 -CH 2 -. In some embodiments, X 1 and X 2 are independently -alkylene-C(=O)NR 3 -alkylene- or -alkylene-NR 3 C(=O)-alkylene-. In some embodiments, X 1 and X 2 are independently -CH 2 -C(=O)NR 3 -CH 2 CH 2 -or -CH 2 -NR 3 C(=O)-CH 2 CH 2 -. In some embodiments, X 1 and X 2 are independently -CH 2 -C(=O)NH-CH 2 CH 2 -or -CH 2 -NHC(=O)-CH 2 CH 2 -.

[0097] In some embodiments, each R 3 is independently hydrogen or C 1 -C 6 alkyl. In some embodiments, each R 3 is hydrogen.

[0098] In some embodiments, >A-R has the following structure:

[0099]

Chemical formula

[0100] In some embodiments, R 1 and R 2 are each independently from 0 to 2. In some embodiments, R 1 and R 2 are each 0. In some embodiments, R 1 and R 2 are each 1. In some embodiments, R 1 and R 2 are each 3.

[0101] In some embodiments, >A-R has the following structure:

[0102]

Chemical formula

[0103] In some embodiments, >A-R has the following structure:

[0104]

Chemical formula

[0105] In some embodiments, p1 is from 1 to 3. In some embodiments, p1 is from 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.

[0106] In some embodiments, >A-R has the following structure:

[0107]

Chemical formula

[0108] In some embodiments, >A-R has the following structure:

[0109]

Chemical formula

[0110] In some embodiments, s is from 1 to 15. In some embodiments, s is from 1 to 10. In some embodiments, s is from 5 to 15. In some embodiments, s is from 5 to 10. In some embodiments, s is from 5 to 20.

[0111] In some embodiments, Y is hydrogen or -CO 2 H. In some embodiments, Y is hydrogen. In some embodiments, Y is -CO 2 H.

[0112] 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 from 2 to 20.

[0113] 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 from 2 to 16.

[0114] In some embodiments, v is from 2 to 16. In some embodiments, v is from 2 to 5. In some embodiments, v is from 5 to 16. In some embodiments, v is 5 or 16. In some embodiments, v is 2 or 16.

[0115] In some embodiments, R 1 or R 2 is independently hydrogen, halogen, -CN, -OR a , -NR c R d , -C(=O)R b , -CO 2 R a , -C(=O)NR c R d , or C 1 -C 6 alkyl.

[0116] In some embodiments, R 1 or R 2 is independently hydrogen, halogen, -CO 2 R a , -C(=O)NR c R d , or C 1 -C 6 alkyl. In some embodiments, R 1 or R 2 is independently hydrogen, -CO 2 R a , or -C(=O)NR c R d . In some embodiments, R 1 or R 2 is independently hydrogen or -CO 2 R a .

[0117] In some embodiments, the staple is

[0118]

Chemical formula

[0119] In some embodiments, the staple attached to the peptide is

[0120]

Chemical formula

[0121] In some embodiments, the staple attached to the peptide is

[0122]

Chemical formula

[0123] In some embodiments, the staple attached to the peptide is

[0124]

Chemical formula

[0125] In some embodiments, the staple attached to the peptide is

[0126]

Chemical formula

[0127] In some embodiments, the staple attached to the peptide is

[0128]

Chemical formula

[0129] In some embodiments, the staple attached to the peptide is

[0130] [Chemical formula] and each L 6 is independently -(CR 1 R 2 ) v -, -C(=)NR 3 -, -NR 3 C(=O)-, -alkylene-C(=O)NR 3 -, or -alkylene-NR 3 C(=O)-, v is from 2 to 20, and s6 is from 1 to 5.

[0131] In some embodiments, the staple attached to the peptide is

[0132] [Chemical formula] and each L 7 is independently -(CR 1 R 2 ) v -, -C(=)NR 3 (-), or -NR 3 C(=O)-, v is from 2 to 20, and s7 is from 1 to 5.

[0133] In some embodiments, the staple attached to the peptide is

[0134] [Chemical formula] and each L 8 is -(CR 1 R 2 ) v -, and v is from 10 to 20.

[0135] In some embodiments, the staple attached to the peptide is

[0136] [Chemical formula] and each L 9 is independently -(CR 1 R 2 ) v -, -C(=)NR 3 -, -NR 3 C(=O)-, -alkylene-C(=O)NR 3 -, or -alkylene-NR 3 C(=O)-, where v is from 2 to 20, and s9 is from 1 to 5.

[0137] In some embodiments, the staple attached to the peptide is

[0138]

Chemical formula

[0139] In some embodiments, the staple attached to the peptide is

[0140]

Chemical formula

[0141] In some embodiments, the staple attached to the peptide is

[0142]

Chemical formula

[0143] In some embodiments, the staple attached to the peptide is

[0144]

Chemical Structure

[0145] In some embodiments, the staple attached to the peptide is

[0146]

Chemical Structure

[0147] In some embodiments, the staple attached to the peptide is

[0148]

Chemical Structure

[0149] In some embodiments, the staple attached to the peptide is

[0150]

Chemical formula

[0151] In some embodiments, the staple attached to the peptide is

[0152]

Chemical formula

[0153] In some embodiments, the staple attached to the peptide is

[0154] [Chemical formula] and each L 17 is independently -(CR 1 R 2 ) v -, -C(=O)NR 3 -, or -NR 3 C(=O)-, where v is from 2 to 20, and s17 is from 1 to 5.

[0155] In some embodiments, the staple attached to the peptide is

[0156] [Chemical formula] and each L 18 is -(CR 1 R 2 ) v -, where v is from 10 to 20.

[0157] In some embodiments, the staple attached to the peptide is

[0158] [Chemical formula] and each L 19 is 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)-, where v is from 2 to 20, and s19 is from 1 to 5.

[0159] In some embodiments, the staple attached to the peptide is

[0160] [Chemical formula] and L 20 is -(CR1 R 2 ) v - wherein v is from 10 to 20.

[0161] In some embodiments, the staple attached to the peptide is

[0162]

Chemical formula

[0163]

Chemical formula

[0164]

Chemical formula

[0165]

Chemical formula

[0166]

Chemical formula

[0167] In some embodiments, the staple attached to the peptide is

[0168]

Chemical formula

[0169]

Chemical formula

[0170] [Chemical formula] is a moiety of a lysine, ornithine, diaminobutyric acid, diaminopropionic acid, or homolysine residue.

[0171] In some embodiments, the staple attached to the peptide is,

[0172] [Chemical formula] and,

[0173] [Chemical formula] is a moiety of a cysteine, homocysteine, 2-amino-5-mercaptopentanoic acid, or 2-amino-6-mercaptohexanoic acid residue,

[0174] [Chemical formula] is a moiety of a lysine, ornithine, diaminobutyric acid, diaminopropionic acid, or homolysine residue.

[0175] In some embodiments, the staple attached to the peptide is,

[0176] [Chemical formula] and,

[0177] [Chemical formula] is a moiety of a cysteine, homocysteine, 2-amino-5-mercaptopentanoic acid, or 2-amino-6-mercaptohexanoic acid residue.

[0178] In some embodiments, the staple attached to the peptide is

[0179] [Chemical formula] and is

[0180] [Chemical formula] a moiety of a lysine, ornithine, diaminobutyric acid, diaminopropionic acid, or homolysine residue.

[0181] In some embodiments, the staple attached to the peptide is

[0182] [Chemical formula] and is

[0183] [Chemical formula] a moiety of a lysine, ornithine, diaminobutyric acid, diaminopropionic acid, or homolysine residue.

[0184] In some embodiments, the staple attached to the peptide is

[0185] [Chemical formula] and is

[0186] [Chemical formula] a moiety of a cysteine, homocysteine, 2-amino-5-mercaptopentanoic acid, or 2-amino-6-mercaptohexanoic acid residue.

[0187] In some embodiments, the staple attached to the peptide is

[0188]

Chemical formula

[0189]

Chemical formula

[0190] In some embodiments, the staple attached to the peptide is

[0191]

Chemical formula

[0192]

Chemical formula

[0193] In some embodiments, the staple attached to the peptide is

[0194]

Chemical formula

[0195]

Chemical formula

[0196] In some embodiments, the peptide conjugate is a) A peptide that modulates the GLP-2 receptor and contains an array selected from any one of SEQ ID NOs: 1-9, 21-29, b) A staple that binds to the peptide at a first cysteine and a second cysteine and has the following structure (where "S" is part of a cysteine residue):

[0197]

Chemical formula

[0198] In some embodiments, the peptide conjugate is a) A peptide that modulates the GLP-2 receptor and contains the array of SEQ ID NO: 1, b) A staple that binds to the peptide at a first cysteine and a second cysteine and has the following structure (where "S" is part of a cysteine residue):

[0199]

Chemical formula

[0200] In some embodiments, the peptide conjugate is a) A peptide that modulates the GLP-2 receptor and contains the array of SEQ ID NO: 2, b) A staple that binds to the peptide at a first cysteine and a second cysteine and has the following structure (where "S" is part of a cysteine residue):

[0201]

Chemical formula

[0202] In some embodiments, the peptide conjugate is a) A peptide that modulates the GLP-2 receptor and contains an array selected from any one of SEQ ID NOs: 10 to 20, b) A staple that binds to the peptide at a first lysine and a second lysine and has the following structure (where "NH" is part of a lysine residue):

[0203]

Chemical formula

[0204] In some embodiments, the peptide conjugate is a) A peptide that modulates the GLP-2 receptor and contains the array of SEQ ID NO: 10, b) A staple that binds to the peptide at a first lysine and a second lysine and has the following structure (where "NH" is part of a lysine residue):

[0205]

Chemical formula

[0206] In some embodiments, the peptide conjugate is a) A peptide that modulates the GLP-2 receptor and contains an array selected from any one of SEQ ID NOs: 21 to 29, b) A staple that binds to the peptide at a first cysteine and a second cysteine and has the following structure (where "S" is part of a cysteine residue):

[0207]

Chemical formula

[0208] In some embodiments, the peptide conjugate is a) A peptide that modulates the GLP-2 receptor and contains the array of SEQ ID NO: 21, b) A staple that binds to the peptide at the first cysteine and the second cysteine and has the following structure (where "S" is part of a cysteine residue):

[0209]

Chemical formula

[0210] In some embodiments, the peptide conjugate is a) A peptide that modulates the GLP-2 receptor and includes the sequence of SEQ ID NO: 22, and b) A staple that binds to the peptide at the first cysteine and the second cysteine and has the following structure (where "S" is part of a cysteine residue):

[0211]

Chemical formula

[0212] In some embodiments, the peptide conjugate is a) A peptide that modulates the GLP-2 receptor and includes any one of the sequences of SEQ ID NOs: 30 to 40, and b) A staple that binds to the peptide at the first lysine and the second lysine and has the following structure (where "NH" is part of a lysine residue):

[0213]

Chemical formula

[0214] In some embodiments, the peptide conjugate is a) A peptide that modulates the GLP-2 receptor and includes the sequence of SEQ ID NO: 30, and b) A staple that binds to the peptide with a first lysine and a second lysine, having the following structure (where "NH" is part of a lysine residue):

[0215]

Chemical formula

[0216] Pharmacokinetics As mechanisms by which the peptide conjugate positively affects pharmacokinetic or pharmacodynamic behavior, (i) preventing or reducing proteolysis in vivo or other activity-diminishing chemical modifications in peptides that regulate the GLP-2 receptor, (ii) improving the half-life or pharmacokinetic properties by reducing renal filtration, reducing receptor-mediated clearance, or increasing bioavailability, (iii) reducing toxicity, (iv) improving solubility, and / or (v) increasing the biological activity and / or target selectivity of the peptide or unmodified peptide, but not limited thereto.

[0217] The peptide conjugate can enhance one or more pharmacokinetic properties of a peptide that regulates the GLP-2 receptor when bound to the peptide. The peptide conjugates disclosed herein can enhance one or more pharmacokinetic properties of a peptide that regulates the GLP-2 receptor by at least about 200% as measured by pharmacodynamic methods compared to the peptide or unmodified peptide alone. 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%, 1000% as measured by pharmacodynamic methods compared to the peptide or unmodified peptide alone.

[0218] Pharmacokinetic properties may include a half-life. The half-life of a peptide conjugate may be at least about 2-fold compared to the half-life of the unmodified peptide alone. The half-life of the peptide conjugates disclosed herein may be at least about 3-fold, 4-fold, 5-fold, or 10-fold compared to the half-life of the therapeutic agent or the unmodified therapeutic agent alone. The half-life of the peptide conjugates disclosed herein may be at least about 6-fold, 7-fold, 8-fold, 9-fold, 10-fold, 15-fold, 20-fold, 25-fold, 30-fold, 35-fold, 40-fold, 45-fold, or 50-fold compared to the half-life of the unmodified peptide alone.

[0219] In some embodiments, the half-life of the peptide conjugate is at least about 2-fold 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-fold 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-fold the half-life of the unmodified form of the peptide.

[0220] In addition, the peptide conjugates described herein may have a positive effect in increasing the manufacturability of the peptide and / or reducing the immunogenicity of the peptide compared to the unconjugated form of the unmodified therapeutic peptide.

[0221] Therapeutic Use In one aspect, the peptide conjugates disclosed herein are useful for treating, alleviating, inhibiting, and / or preventing one or more diseases and / or disorders. The disease and / or disorder can be a chronic disease or disorder. Alternatively, the disease and / or disorder is an acute disease or disorder. The disease or disorder can be recurrent, refractory, progressive, or in remission. The disease or disorder can affect one or more cell types. The one or more diseases and / or disorders can be an autoimmune disease, an inflammatory disease, or a metabolic disease.

[0222] Disclosed herein are methods of treating a desired target disease or disorder, the methods comprising administering to a subject a peptide conjugate described herein. The disease or disorder can be diabetes or obesity, or a medical condition associated with diabetes or obesity. The disease or disorder can be non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), or a cardiovascular disease. The disease or disorder can be an autoimmune disorder. The disease or disorder can be Crohn's disease or ulcerative colitis. The disease or disorder can be short bowel syndrome (SBS). The disease or disorder can be inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), or psoriasis. The disease or disorder can be Alzheimer's disease, Parkinson's disease, or Huntington's disease. The PLC can be administered with one or more additional therapeutic agents. Disclosed herein are methods of treating a desired target disease or disorder, the methods comprising administering to a subject a composition disclosed herein comprising one or more peptide conjugates.

[0223] Disclosed herein are methods of preventing or treating a metabolic disease or disorder in a subject in need thereof, the methods comprising administering to the subject a peptide conjugate described herein. The metabolic disease or disorder can be diabetes. The metabolic disease or disorder can be obesity. The metabolic disease or disorder can be glycogen storage disease, phenylketonuria, maple syrup urine disease, glutaric acidemia type I, carbamoyl phosphate synthetase I deficiency, alkaptonuria, medium-chain acyl-CoA dehydrogenase deficiency (MCADD), acute intermittent porphyria, Lesch-Nyhan syndrome, lipoid congenital adrenal hyperplasia, congenital adrenal hyperplasia, POMPC deficiency, LEPR deficiency, Bardet-Biedl syndrome, Alström syndrome, Prader-Willi syndrome, Kearns-Sayre syndrome, Zellweger syndrome, Gaucher's disease, or Niemann-Pick disease.

[0224] Provided herein is a method for preventing or treating NAFLD, NASH, or cardiovascular disease in a subject in need thereof, the method comprising administering to the subject a peptide conjugate described herein.

[0225] Provided herein is a method for preventing or treating short bowel syndrome (SBS) in a subject in need thereof, the method comprising administering to the subject a peptide conjugate described herein.

[0226] Provided herein is a method for preventing or treating inflammatory bowel disease (IBD), irritable bowel syndrome (IBS), or psoriasis in a subject in need thereof, the method comprising administering to the subject a peptide conjugate described herein.

[0227] Provided herein is a method for preventing or treating Crohn's disease or ulcerative colitis in a subject in need thereof, the method comprising administering to the subject a peptide conjugate described herein.

[0228] Provided herein is a method for preventing or treating sleep disorders.

[0229] 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.

[0230] Provided herein is a method for 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.

[0231] This specification provides methods for preventing or treating gastrointestinal-related disorders, such as the treatment of newborns with impaired intestinal function, suffering from osteoporosis, and suffering from DPP-IV (dipeptidyl peptidase-IV)-mediated diseases. By way of example, gastrointestinal-related disorders include ulcers, gastritis, digestive disorders, malabsorption syndrome, short bowel syndrome, blind loop syndrome, inflammatory bowel disease, celiac disease (e.g., gluten-induced enteropathy or resulting from celiac disease), tropical sprue, hypogammaglobulinemic sprue, enteritis, regional enteritis (Crohn's disease), ulcerative colitis, irritable bowel syndrome with diarrhea, small intestine injury, short bowel syndrome.

[0232] This specification provides methods for preventing or treating radiation enteritis, infectious or post-infectious enteritis, and small intestine injury caused by toxic or other chemotherapeutic agents. This method may require the administration of a peptide conjugate before, simultaneously with, or after chemotherapy or radiation therapy in order to reduce the side effects of chemotherapy such as diarrhea, abdominal cramps, vomiting, etc., and to reduce the structural and functional damage of the intestinal epithelium resulting from chemotherapy or radiation therapy.

[0233] This specification provides methods for preventing or treating nutritional disorders, such as diseases such as cachexia syndrome, cachexia, and anorexia nervosa.

[0234] This specification provides a method for preventing or treating a disease or disorder that benefits from a modulator of the GLP-2 receptor in a subject in need thereof, the method comprising the step of administering the peptide conjugate described herein to the subject.

[0235] Combination This specification provides a pharmaceutical composition comprising the peptide conjugate described herein and one or more additional therapeutic agents.

[0236] The additional therapeutic agent may include one or more other anti-diabetic drugs, DPP4 inhibitors, SGLT2 inhibitors, hypoglycemic agents, insulin secretagogues, TZD drugs, insulin and insulin analogs, FGF21 and its analogs, leptin or leptin analogs, amylin and amylin analogs, anti-inflammatory drugs, cyclosporin A or FK506, 5-ASA, or statins, or any combination thereof. The additional therapeutic agent may be aspirin.

[0237] The additional therapeutic agent may include 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.

[0238] Composition Disclosed herein are pharmaceutical compositions comprising the peptide conjugates described herein and a pharmaceutically acceptable excipient or vehicle. Pharmaceutically acceptable excipients or vehicles include carriers, excipients, diluents, antioxidants, preservatives, coloring agents, flavoring agents, diluents, emulsifiers, suspending agents, solvents, fillers, bulking agents, buffers, delivery vehicles, isotonic agents, co-solvents, wetting agents, complexing agents, buffering agents, antibacterial agents, and surfactants.

[0239] Neutral buffered saline or saline mixed with serum albumin is an exemplary and suitable carrier. As pharmaceutical compositions, antioxidants such as ascorbic acid; low molecular weight polypeptides; proteins such as serum albumin, gelatin, immunoglobulins; hydrophilic polymers such as polyvinylpyrrolidone, amino acids such as glycine, glutamine, asparagine, arginine, lysine; monosaccharides and disaccharides, other carbohydrates including glucose, mannose, dextrin; chelating agents such as EDTA; sugar alcohols such as mannitol and sorbitol; salt-forming counterions such as sodium; and / or nonionic surfactants such as Tween, Pluronic® or polyethylene glycol (PEG) can be mentioned. As further examples, suitable isotonicity promoters include alkali metal halides (preferably sodium chloride or potassium chloride), mannitol, sorbitol, etc. Suitable preservatives include benzalkonium chloride, thimerosal, phenethyl alcohol, methylparaben, propylparaben, chlorhexidine, sorbic acid, etc. Hydrogen peroxide can also be used as a preservative. Suitable co-solvents include glycerin, propylene glycol, PEG. Suitable complexing agents include caffeine, polyvinylpyrrolidone, beta-cyclodextrin, hydroxy-propyl-beta-cyclodextrin. Suitable surfactants or wetting agents include sorbitan esters, polysorbates such as polysorbate 80, tromethamine, lecithin, cholesterol, tyloxapol, etc. The buffer may be acetate, borate, citrate, phosphate, bicarbonate, or a conventional buffer such as Tris-HCl. The pH of the acetate buffer may be about 4 to 5.5, and the pH of the Tris buffer may be about 7 to 8.5. Further pharmaceuticals are specified in Remington’s Pharmaceutical Sciences, 18th Edition, A.R. Gennaro, ed., Mack Publishing Company, 1990.

[0240] The composition may be in liquid form or in lyophilized or freeze-dried form, and may contain one or more lyoprotectants, excipients, surfactants, high molecular weight structural additives, and / or fillers. In one embodiment, lyoprotectants that are non-reducing sugars such as sucrose, lactose, trehalose are included. Generally, the amount of lyoprotectant included is an amount such that the formulation obtained upon reconstitution is isotonic, although hypertonic or slightly hypotonic formulations are also suitable. In addition, the amount of lyoprotectant must be sufficient to prevent unacceptable amounts of protein degradation and / or aggregation during lyophilization. Exemplary lyoprotectant concentrations of sugars (such as sucrose, lactose, trehalose) in a pre-lyophilized formulation are from about 10 mM to about 400 mM. In another embodiment, surfactants include, for example, nonionic surfactants, ionic surfactants such as polysorbates (such as polysorbate 20, polysorbate 80); poloxamers (such as poloxamer 188); poly(ethylene glycol) phenyl ether (such as Triton); sodium dodecyl sulfate (SDS); sodium lauryl sulfate; sodium octyl glucoside; lauryl-, myristyl-, linoleyl-, or stearyl-sulfobetaine; lauryl-, myristyl-, linoleyl-, or stearyl-sarcosine; linoleyl-, myristyl-, or cetyl-betaine; lauramidopropyl-, cocamidopropyl-, linoleamidopropyl-, myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-betaine (such as lauramidopropyl); myristamidopropyl-, palmidopropyl-, or isostearamidopropyl-dimethylamine; sodium cocoyl methyl taurate- or disodium methyl oleyl-taurate; MONAQUAT™ series (Mona Industries, Inc., Paterson, N.J.), polyethylene glycol, polypropylene glycol, and copolymers of ethylene and propylene glycol (such as Pluronic®, PF68, etc.).Exemplary amounts of surfactants that may be present in a lyophilized formulation may be present in an amount of about 0.001 to 0.5%. As high molecular weight structural additives (e.g., fillers, binders), for example, acacia, albumin, alginic acid, calcium phosphate (dibasic), cellulose, carboxymethyl cellulose, sodium carboxymethyl cellulose, hydroxyethyl cellulose, hydroxypropyl cellulose, hydroxypropyl methyl cellulose, microcrystalline cellulose, dextran, dextrin, dextrose, sucrose, tyrothricin, pregelatinized starch, calcium sulfate, amylose, glycine, bentonite, maltose, sorbitol, ethyl cellulose, disodium hydrogen phosphate, disodium phosphate, sodium 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, zein can be mentioned. Exemplary concentrations of high molecular weight structural additives are from 0.1% to 10% by weight. In other embodiments, bulking agents (e.g., mannitol, glycine) may be included.

[0241] The composition may be suitable for parenteral administration. Exemplary compositions are suitable for injection or infusion into an animal by any route available to those skilled in the art, for example, intra-articular, subcutaneous, intravenous, intramuscular, intraperitoneal, intracerebral (parenchymal), intraventricular, intramuscular, intraocular, intra-arterial, intra-lesional routes. Parenteral formulations may generally be sterile, pyrogen-free isotonic aqueous solutions and may optionally contain pharmaceutically acceptable preservatives.

[0242] Examples of non-aqueous solvents are vegetable oils such as propylene glycol, polyethylene glycol, olive oil, and injectable organic esters such as ethyl oleate. Examples of aqueous carriers include water, alcohol / aqueous solutions, emulsions or suspensions containing saline or buffered media. Examples of parenteral vehicles include sodium chloride solution, Ringer's dextrose, dextrose and sodium chloride, lactated Ringer's, and fixed oils. Examples of vehicles for intravenous administration include fluid and nutrient replenishers, electrolyte replenishers such as Ringer's dextrose-based ones. Preservatives and other additives, such as antibacterial agents, antioxidants, chelating agents, inert gases, etc. may also be present. Generally, see Remington’s Pharmaceutical Science, 16th Ed., Mack Eds., 1980.

[0243] The pharmaceutical compositions described herein may be formulated for controlled or sustained delivery in such a way as to provide local concentrations of the product (e.g., high-dose, depot effects), and / or increased stability or half-life in a particular local environment. The compositions may include formulations of the peptides disclosed herein, in addition to particulate preparations of polymer compounds such as polylactic acid and polyglycolic acid, biodegradable matrices, injectable microspheres, microcapsule particles, microcapsules, bioerodible particle beads, liposomes, and implantable delivery devices that provide controlled or sustained release of the active agent that can be delivered as a depot injection later. Techniques for formulating such sustained or controlled delivery means are known, and various polymers have been developed and used for the controlled release and delivery of drugs. Such polymers are generally biodegradable and biocompatible. Polymer hydrogels, including those formed by complexation of enantiomeric polymers or polypeptide segments, and hydrogels having temperature- or pH-sensitive properties, may be desirable for providing a drug depot effect due to the mild aqueous conditions involved in the capture of bioactive protein drugs (e.g., antibodies containing ultra-long CDR3).

[0244] Appropriate and / or preferred pharmaceutical formulations can be determined by taking into account the present disclosure and general knowledge of formulation techniques, depending on the intended route of administration, delivery format, and desired dosage. Regardless of the method of administration, an effective amount can be calculated according to the patient's body weight, body surface area, or organ size. Further improvements to the calculations for determining the appropriate dosage for each of the formulations described herein for the treatments that require them are routinely made in the art and are within the scope of work routinely done in the art. The appropriate dosage can be confirmed using appropriate dose-response data.

[0245] Definitions As used herein and in the appended claims, the singular forms "a", "an", and "the" mean, unless the context clearly dictates otherwise, including the plural. Thus, for example, references to "agent" include references to multiple such agents, references to "cell" include references to one or more cells (or multiple cells), and equivalents thereof known to those skilled in the art, etc. When ranges regarding physical properties such as molecular weight or chemical properties such as chemical formula are used herein, combinations of the range and specific embodiments therein, and sub-combinations, are all intended to be included. The term "about", when referring to a number or a range thereof, means that the number or range thereof being referred to is an approximation within the range of experimental variation (or within statistical experimental error), and thus in some examples, it means varying by 1% to 15% of the number or range described. The term "comprising" (and related terms such as "comprise", "comprises", "having", or "including") is intended not to exclude, in other specific embodiments, for example, embodiments of any substance composition, composition, method, or process described herein from "consisting of" or "consisting essentially of" the recited features.

[0246] As used in this specification and the appended claims, unless specified to the contrary, the following terms have the meanings set forth below.

[0247] "Alkyl" represents a straight-chain or branched hydrocarbon monoradical having 1 to 10 carbon atoms, or 1 to 6 carbon atoms, which may be fully saturated or unsaturated, and the sp 3 -hybridized carbon of the alkyl residue is bonded to the remainder of the molecule by a single bond. Examples of saturated hydrocarbon monoradicals include 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, t-butyl, n-pentyl, isopentyl, neopentyl, tert-amyl, hexyl, heptyl, octyl, and other long alkyl groups, but are not limited thereto. Whenever it appears in this specification, the numerical range such as "C 1 -C 6 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. However, when no numerical range is indicated, this definition also includes the occurrence of the term "alkyl". In some embodiments, alkyl is C 1 -C 10 alkyl, C 1 -C 9 alkyl, C 1 -C 8 alkyl, C 1 -C 7 alkyl, C 1 -C 6 alkyl, C 1 -C 5 alkyl, C 1 -C 4 alkyl, C1 -C 3 alkyl, C 1 -C 2 alkyl, or C 1 is alkyl. When alkyl refers to an unsaturated straight-chain or branched hydrocarbon monoradical, it is known as "alkenyl" or "alkynyl". Alkenyl is understood to be either in the cis configuration or the trans configuration with respect to the double bond and to include both isomers. Examples of alkenyl include ethenyl (-CH=CH 2 ), 1-propenyl (-CH 2 CH=CH 2 ), isopropenyl [-C(CH 3 )=CH 2 , butenyl, 1,3-butadienyl, etc., but are not limited thereto. Whenever it appears in this specification, the numerical range such as "C 2 -C 6 alkenyl" means that the alkenyl group consists of 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms. When no numerical range is indicated, this definition also includes the occurrence of the term "alkenyl". In some embodiments, alkenyl is C 2 -C 10 alkenyl, C 2 -C 9 alkenyl, C 2 -C 8 alkenyl, C 2 -C 7 alkenyl, C 2 -C 6 alkenyl, C 2 -C 5 alkenyl, C 2 -C 4 alkenyl, C 2 -C 3 alkenyl, or C 2 is alkenyl. Examples of alkynyl include ethynyl, 2-propynyl, 2-, etc., but are not limited thereto. Whenever it appears in this specification, "C 2 -C 6The numerical range such as "alkynyl" means that the alkynyl group consists of 2 carbon atoms, 3 carbon atoms, 4 carbon atoms, 5 carbon atoms, or 6 carbon atoms. However, when no numerical range is indicated, this definition also encompasses the occurrence of the term "alkynyl". In some embodiments, alkynyl is C 2 -C 10 alkynyl, C 2 -C 9 alkynyl, C 2 -C 8 alkynyl, C 2 -C 7 alkynyl, C 2 -C 6 alkynyl, C 2 -C 5 alkynyl, C 2 -C 4 alkynyl, C 2 -C 3 alkynyl, or C 2 is alkynyl. Unless otherwise indicated herein, the alkyl group is optionally substituted, for example, by oxo, halogen, amino, nitrile, nitro, hydroxyl, haloalkyl, alkoxy, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, etc., as described hereinafter. In some embodiments, alkyl is optionally substituted by oxo, halogen, -CN, -CF 3 , -OH, -OMe, -NH 2 , or -NO 2 . In some embodiments, alkyl is optionally substituted by oxo, halogen, -CN, -CF 3 , -OH, or -OMe. In some embodiments, alkyl is optionally substituted by halogen.

[0248] "Alkylene" refers to a straight-chain or branched-chain divalent hydrocarbon chain. Whenever it appears in this specification, "C 1 -C 6The numerical range such as "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. However, when no numerical range is indicated, this definition also includes the occurrence of the term "alkylene". In some embodiments, the alkylene is C 1 -C 10 alkylene, C 1 -C 9 alkylene, C 1 -C 8 alkylene, C 1 -C 7 alkylene, C 1 -C 6 alkylene, C 1 -C 5 alkylene, C 1 -C 4 alkylene, C 1 -C 3 alkylene, C 1 -C 2 alkylene, or C 1 alkylene. Unless otherwise indicated herein, the alkylene group is optionally substituted, for example, by oxo, halogen, amino, nitrile, nitro, hydroxyl, haloalkyl, alkoxy, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, etc., as described below. In some embodiments, the alkylene is optionally substituted by oxo, halogen, -CN, -CF 3 , -OH, -OMe, -NH 2 , or -NO 2 . In some embodiments, the alkylene is optionally substituted by oxo, halogen, -CN, -CF 3 , -OH, or -OMe. In some embodiments, the alkylene is optionally substituted by halogen.

[0249] "Alkoxy" represents a radical of the formula -OR a , where R ais an alkyl radical as defined. Unless otherwise indicated herein, an alkoxy group is optionally substituted, as described hereinafter, by, for example, oxo, halogen, amino, nitrile, nitro, hydroxyl, haloalkyl, alkoxy, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, and the like. In some embodiments, the alkoxy is optionally substituted by oxo, halogen, -CN, -CF 3 , -OH, -OMe, -NH 2 , or -NO 2 . In some embodiments, the alkoxy is optionally substituted by oxo, halogen, -CN, -CF 3 , -OH, or -OMe. In some embodiments, the alkoxy is optionally substituted by halogen.

[0250] "Aryl" represents a radical derived from a hydrocarbon ring system containing hydrogen, 6 to 30 carbon atoms, and at least one aromatic ring. The aryl radical may be of a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may include a fused ring system (when fused with a cycloalkyl or heterocycloalkyl ring, the aryl is bonded through an aromatic ring atom) or a bridged ring system. In some embodiments, the aryl is a 6- to 10-membered aryl. In some embodiments, the aryl is a 6-membered aryl. Examples of aryl radicals include, but are not limited to, aryl radicals derived from hydrocarbon ring systems such as anthrylene, naphthylene, phenanthrylene, anthracene, azulene, benzene, chrysene, fluoranthene, fluorene, as-indacene, s-indacene, indane, indene, naphthalene, phenalene, phenanthrene, preiadene, pyrene, and triphenylene. In some embodiments, the aryl is phenyl. Unless otherwise indicated herein, the aryl is optionally substituted, for example, by halogen, amino, nitrile, nitro, hydroxyl, alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, etc. In some embodiments, the aryl is optionally substituted by halogen, methyl, ethyl, -CN, -CF 3 , -OH, -OMe, -NH 2 , or -NO 2 . In some embodiments, the aryl is optionally substituted by halogen, methyl, ethyl, -CN, -CF 3 , -OH, or -OMe. In some embodiments, the aryl is optionally substituted by halogen.

[0251] "Cycloalkyl" refers to a stable, partially or fully saturated, monocyclic or polycyclic cyclic carbon, which may include a fused ring system (when fused with an aryl or heteroaryl ring, the cycloalkyl is bonded through a non-aromatic ring atom) or a bridged ring system. Representative cycloalkyls include those having 3 to 15 carbon atoms (C 3 -C15 cycloalkyl), 3 to 10 carbon atoms (C 3 -C 10 cycloalkyl), 3 to 8 carbon atoms (C 3 -C 8 cycloalkyl), 3 to 6 carbon atoms (C 3 -C 6 cycloalkyl), 3 to 5 carbon atoms (C 3 -C 5 cycloalkyl), or 3 to 4 carbon atoms (C 3 -C 4 Examples of the cycloalkyl having cycloalkyl) include, but are not limited to, those having cycloalkyl). In some embodiments, the cycloalkyl is a 3- to 6-membered cycloalkyl. In some embodiments, the cycloalkyl is a 5- to 6-membered cycloalkyl. Examples of monocyclic cycloalkyl include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, cyclooctyl. Examples of polycyclic cycloalkyl or carbocyclic compounds include, but are not limited to, adamantyl, norbornyl, decalinyl, 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, bicyclo[3.3.2]decane, 7,7-dimethyl-bicyclo[2.2.1]heptanyl. Examples of partially saturated cycloalkyl include, but are not limited to, cyclopentenyl, cyclohexenyl, cycloheptenyl, cyclooctenyl. Unless otherwise indicated herein, the cycloalkyl is optionally substituted, for example, by oxo, halogen, amino, nitrile, nitro, hydroxyl, alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, etc. In some embodiments, the cycloalkyl is oxo, halogen, methyl, ethyl, -CN, -CF 3 ,, -OH, -OMe, -NH 2 , or -NO 2is optionally substituted. In some embodiments, the cycloalkyl is optionally substituted with oxo, halogen, methyl, ethyl, -CN, -CF 3 , -OH, -OMe, -OH, or -OMe. In some embodiments, the cycloalkyl is optionally substituted with halogen.

[0252] "Halo" or "halogen" refers to bromo, chloro, fluoro, or iodo. In some embodiments, the halogen is fluoro or chloro. In some embodiments, the halogen is fluoro.

[0253] "Haloalkyl" represents an alkyl radical as defined above, which is substituted with one or more halo radicals 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.

[0254] "Heterocycloalkyl" represents a stable 3- to 24-membered partially or fully saturated ring radical containing 2 to 23 carbon atoms and 1 to 8 heteroatoms selected from nitrogen, oxygen, phosphorus, and sulfur. Representative heterocycloalkyls include those having 2 to 15 carbon atoms (C 2 -C 15 heterocycloalkyl), 2 to 10 carbon atoms (C 2 -C 10 heterocycloalkyl), 2 to 8 carbon atoms (C 2 -C 8 heterocycloalkyl), 2 to 6 carbon atoms (C 2 -C 6 heterocycloalkyl), 2 to 5 carbon atoms (C 2 -C 5 heterocycloalkyl), or 2 to 4 carbon atoms (C 2 -C 4Heterocycloalkyl having a (heterocycloalkyl) is exemplified, but not limited thereto. In some embodiments, the heterocycloalkyl is a 3- to 6-membered heterocycloalkyl. In some embodiments, the heterocycloalkyl is a 5- to 6-membered heterocycloalkyl. Unless otherwise specified herein, the heterocycloalkyl radical may be of a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which ring system may include a fused ring system (when fused to an aryl or heteroaryl ring, the heterocycloalkyl is bonded through a non-aromatic ring atom) or a bridged ring system, and the nitrogen, carbon, or sulfur atoms in the heterocycloalkyl radical may optionally be oxidized, and the nitrogen atom may optionally be quaternized. Examples of such heterocycloalkyl radicals include aziridinyl, azetidinyl, dioxolanyl, thienyl[1,3]dithianyl, decahydroisoquinolyl, imidazolinyl, imidazolidinyl, isothiazolidinyl, isoxazolidinyl, morpholinyl, octahydroindolyl, octahydroisoindolyl, 2-oxopiperazinyl, 2-oxopiperidinyl, 2-oxopyrrolidinyl, oxazolidinyl, piperidinyl, piperazinyl, 4-piperidonyl, pyrrolidinyl, pyrazolidinyl, quinuclidinyl, thiazolidinyl, tetrahydrofuryl, trithianyl, tetrahydropyranyl, thiomorpholinyl, thiamorpholinyl, 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, 2-oxo-1,3-dioxol-4-yl, but are not limited thereto. The term "heterocycloalkyl" includes all cyclic forms of carbohydrates, including but not limited to monosaccharides, disaccharides, and oligosaccharides. Unless otherwise specified, the heterocycloalkyl has 2 to 10 carbons in the ring.When referring to the number of carbon atoms in a heterocycloalkyl, it is understood that the number of carbon atoms in the heterocycloalkyl is not the same as the total number of atoms (including heteroatoms) (the skeletal atoms of the heterocycloalkyl ring) that make up the heterocycloalkyl. Examples of partially saturated heterocycloalkyls include dihydropyrrolyl and tetrahydropyridine. Unless otherwise defined herein, heterocycloalkyl is optionally substituted, for example, by oxo, halogen, amino, nitrile, nitro, hydroxyl, alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, and the like. In some embodiments, heterocycloalkyl is optionally substituted by oxo, halogen, methyl, ethyl, -CN, -CF. 3 , -OH, -OMe, -NH 2 , or -NO 2 and is optionally substituted thereby. In some embodiments, heterocycloalkyl is optionally substituted by oxo, halogen, methyl, ethyl, -CN, -CF 3 , -OH, or -OMe. In some embodiments, heterocycloalkyl is optionally substituted by halogen.

[0255] "Heteroalkyl" refers to an alkyl group in which one or more skeletal atoms of the alkyl are atoms other than carbon, for example, oxygen, nitrogen (e.g., -NH-, -N(alkyl)-), sulfur, or a combination thereof. Heteroalkyl is attached to the remainder of the molecule at a carbon atom of the heteroalkyl. In one aspect, heteroalkyl is C 1 -C 6heteroalkyl, where heteroalkyl is composed of 1 to 6 carbon atoms and one or more atoms other than carbon, such as oxygen, nitrogen (e.g., -NH-, -N(alkyl)-), sulfur, or combinations thereof, and heteroalkyl is attached to the remainder of the molecule at a carbon atom of the heteroalkyl. Unless otherwise defined herein, heteroalkyl is optionally substituted, for example, by oxo, halogen, amino, nitrile, nitro, hydroxyl, alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, etc. In some embodiments, heteroalkyl is optionally substituted by oxo, halogen, methyl, ethyl, -CN, -CF 3 , -OH, -OMe, -NH 2 , or -NO 2 . In some embodiments, heteroalkyl is optionally substituted by oxo, halogen, methyl, ethyl, -CN, -CF 3 , OH, or -OMe. In some embodiments, heteroalkyl is optionally substituted by halogen.

[0256] "Heteroaryl" refers to a 5- to 14-membered ring system radical containing a hydrogen atom, 1 to 13 carbon atoms, 1 to 6 heteroatoms selected from nitrogen, oxygen, phosphorus, and sulfur, and at least one aromatic ring. The heteroaryl radical may be a monocyclic, bicyclic, tricyclic, or tetracyclic ring system, which may include a fused ring system (when fused to a cycloalkyl or heterocycloalkyl ring, the heteroaryl is bonded through an aromatic ring atom) or a bridged ring system, and the nitrogen, carbon, or sulfur atoms in the heteroaryl radical may optionally be oxidized, and the nitrogen atom may optionally be quaternized. In some embodiments, the heteroaryl is a 5- to 10-membered heteroaryl. In some embodiments, the heteroaryl is a 5- to 6-membered heteroaryl. In some embodiments, the heteroaryl is a 5-membered heteroaryl. In some embodiments, the heteroaryl is a 6-membered heteroaryl.Examples include, but are not limited to, azepinyl, acridinyl, benzimidazolyl, benzothiazolyl, benzindolyl, benzodioxolyl, benzofuranyl, benzoxazolyl, benzothiazolyl, benzothiadiazolyl, benzo[b][1,4]dioxepinyl, 1,4-benzodioxanyl, benzonaphthofuranyl, benzoxazolyl, benzodioxolyl, benzodioxinyl, benzopyranyl, benzopyranonyl, benzofuranyl, benzofuranonyl, benzothienyl, benzothiophenyl, benzotriazolyl, benzo[4,6]imidazo[1,2-a]pyridinyl, carbazolyl, cinnolinyl, dibenzofuranyl, dibenzothiophenyl, furanyl, furanonyl, isothiazolyl, imidazolyl, indazolyl, indolyl, indazolyl, isoindolyl, indolinyl, isoindolinyl, isoquinolyl, indolizinyl, isoxazolyl, naphthyridinyl, oxadiazolyl, 2-oxoazepinyl, oxazolyl, oxiranyl, 1-oxidopyridinyl, 1-oxidopyrimidinyl, 1-oxidopyrazinyl, 1-oxidopyridazinyl, 1-phenyl-1H-pyrrolyl, phenazinyl, phenothiazinyl, phenoxazinyl, phthalazinyl, pteridinyl, purinyl, pyrrolyl, pyrazolyl, pyridinyl, pyrazinyl, pyrimidinyl, pyridazinyl, quinazolinyl, quinoxalinyl, quinolinyl, quinuclidinyl, isoquinolinyl, tetrahydroquinolinyl, thiazolyl, thiadiazolyl, triazolyl, tetrazolyl, triazinyl, thiophenyl (i.e., thienyl). Unless otherwise defined herein, heteroaryl is optionally substituted, for example, by halogen, amino, nitrile, nitro, hydroxyl, alkyl, alkenyl, alkynyl, haloalkyl, alkoxy, aryl, cycloalkyl, heterocycloalkyl, heteroaryl, etc. In some embodiments, heteroaryl is halogen, methyl, ethyl, -CN, -CF 3 , -OH, -OMe, -NH 2 , or -NO 2 and is optionally substituted thereby. In some embodiments, heteroaryl is halogen, methyl, ethyl, -CN, -CF 3is optionally substituted by -OH, or -OMe. In some embodiments, the heteroaryl is optionally substituted by halogen.

[0257] The term "percent identity" refers to a comparison between two nucleic acid or amino acid sequences. Such comparisons are performed using any number of alignment methods known in the art, including but not limited to global (e.g., the Needleman-Wunsch algorithm) or local alignments (e.g., Smith-Waterman, Sellers, or other algorithms). Percent identity often represents the proportion of positions in a section of adjacent positions where the two sequences match, and the two sequences are aligned to maximize the matching positions and minimize the displacement of non-matching positions. In some examples, an alignment is performed with no displacement between the two sequences. In some instances, as a result of the alignment, the displacement is less than 5%, less than 3%, or less than 1%. Additional methods of sequence comparison or alignment are also consistent with the present disclosure.

[0258] As used herein, the term "identity" may refer to that which can be determined by aligning sequences for optimal comparison purposes in calculating "identity" or "percent identity" between two or more amino acid sequences (e.g., gaps may be introduced into the sequence of the first sequence). Then, the amino acids at corresponding positions may be compared, and the percent identity between the two sequences may be a function of the number of identical positions shared by the sequences (i.e., % identity = number of identical positions / total number of positions × 100). For example, a position in the first sequence may be occupied by the same amino acid as the corresponding position in the second sequence, and the molecules are identical at that position. The percent identity between two sequences may be a function of the number of identical positions shared by the sequences, and the number and length of the gaps are considered, which need to be introduced for the optimal alignment of the two sequences. In some embodiments, the length of the sequences aligned for comparison purposes may 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. A BLAST search may be used to determine the identity between two sequences. The identity may be between the entire lengths of the two sequences or between fractions of the entire lengths of the two sequences. The two sequences may be peptide sequences, amino acid sequences, or fragments thereof. The actual comparison of two sequences can be achieved using well-known methods, such as mathematical algorithms. Non-limiting examples of such mathematical algorithms may be described in Karlin, S. and Altschul, S., Proc. Natl. Acad. Sci. USA, 90-5873-5877 (1993). Such algorithms can be incorporated into the programs NBLAST and XBLAST (version 2.0) as described in Altschul, S. et al., Nucleic Acids Res., 25:3389-3402 (1997). When using the BLAST and Gapped BLAST programs, the relevant parameters of each program (e.g., NBLAST) can be used.For example, the parameters for array comparison can be set or changed to score = 100, word length = 12 (e.g., W = 5 or W = 20). Other examples include the algorithms of Myers and Miller, CABIOS (1989), ADVANCE, ADAM, BLAT, and FASTA. In another embodiment, the percent identity between two amino acid sequences can be achieved, for example, using the GAP program (Accelrys, Cambridge, UK) in the GCG software package.

[0259] "Pharmaceutically acceptable" means approved or approvable by a regulatory agency of the Federal or State government or listed in the U.S. Pharmacopeia or other generally recognized pharmacopeias for use in animals including humans.

[0260] "Pharmaceutically acceptable salt" refers to a salt of a compound that is pharmaceutically acceptable and has the desired pharmacological activity of the parent compound.

[0261] "Pharmaceutically acceptable excipient, carrier, or adjuvant" refers to an excipient, carrier, or adjuvant that can be administered to a subject together with at least one antibody of the present disclosure, which do not destroy its pharmacological activity and are non-toxic when administered in a dosage sufficient to deliver a therapeutic amount of the compound.

[0262] "Pharmaceutically acceptable vehicle" refers to a diluent, adjuvant, excipient, or carrier with which at least one antibody of the present disclosure is administered together.

[0263] The terms "treating", "treatment", or "to treat", or "alleviating" or "to alleviate", etc., may refer to 1) therapeutic means for curing, delaying, reducing the symptoms of, and / or halting the progression of a diagnosed medical condition or disorder, and / or 2) prophylactic or disease-preventing means for preventing and / or delaying the onset of a target medical condition or disorder. "Treatment" represents a clinical intervention in an attempt to modify the natural course of the individual or cell being treated and can be done for prophylaxis or during the course of a clinical pathology. Desirable treatment effects include prevention of the occurrence or recurrence of a disease, alleviation of symptoms, reduction of any direct or indirect pathological consequences of the disease, prevention of metastasis, reduction in the rate of disease progression, improvement or alleviation of the medical condition, and remission or improved prognosis. Thus, those in need of treatment can include those already suffering from a disorder, those predisposed to suffering from a disorder, and those in whom a disorder should be prevented.

[0264] "Amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs or mimetics that function in a manner similar to naturally occurring amino acids. Naturally occurring amino acids include those encoded by the genetic code, as well as amino acids that are later modified, such as hydroxyproline, gamma-carboxyglutamate, O-phosphoserine. Amino acid analogs refer to compounds having the same basic chemical structure as naturally occurring amino acids, such as homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium, which have, for example, a hydrogen, carboxyl group, amino group, and an alpha carbon bonded to an R group. Such analogs can have a modified R group (e.g., norleucine) or a modified peptide backbone, but can retain the same basic chemical structure as naturally occurring amino acids. Amino acid mimetics refer to chemical compounds having a structure that is different from the general chemical structure of amino acids but functions in a manner similar to naturally occurring amino acids.

[0265] "Disorder" or "disease" refers to a condition that benefits from treatment by a substance / molecule disclosed in this specification (e.g., a peptide conjugate as disclosed in this specification) or a method. This includes chronic and acute disorders or diseases, including conditions that cause a mammal to suffer from the disorder of interest.

[0266] "Mammal" for treatment purposes refers to humans, rodents (e.g., mice and rats), and monkeys; domestic and farm animals; and any animal classified as a mammal, including animals for zoos, sports, research, or pets, such as dogs, cats, cows, horses, sheep, pigs, goats, rabbits, etc. In some embodiments, the mammal is selected from humans, rodents, or monkeys.

[0267] "Unmodified peptide" refers to either an unmodified sequence (wild-type peptide) or a modified sequence without staples.

Examples

[0268] Peptides were synthesized by standard solid-phase peptide synthesis (SPPS) techniques and purified via HPLC as described.

[0269] Unless otherwise specified, all reagents were purchased from 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 of HPLC grade. Reactions were monitored by LC-MS or thin-layer chromatography (TLC) on Merck 50×100 mm silica gel 60 aluminum sheets stained with an aqueous solution of KMnO4.

[0270] Flash chromatography purification was performed on a CombiFlash® Rf (Teledyne Isco) with a silica gel-packed column (40 μm, RediSep® Rf from Teledyne Isco). The purified final compound eluted as a single and symmetric peak (thereby confirming a purity of over 95%). Preparative chromatography was carried out on a Shimadzu HPLC equipped with a Phenomenex Luna column (C18, 100 Å pore size, 10 μm particle size, 250×10.0 mm, flow rate: 4 mL / min), or on an Agilent 1200 HPLC equipped with a Phenomenex Luna column (C18, 100 Å pore size, 5 μm particle size, 150×21.2 mm, flow rate: 20 mL / min).

[0271] 1 H and 13 C NMR spectra were recorded on a Bruker 400 system with respect to d 6 -DMSO, CDCl 3 3, or CD 3 3OD. Chemical shifts are provided in parts per million (ppm) using tetramethylsilane as an internal standard. Abbreviations are used 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 provided in Hertz (Hz). Low-resolution mass spectra were recorded on a Waters Acquity UPLC equipped with a hemomenex Luna Omega C18 column (C18, 100 Å pore size, 1.6 μm particle size, 50×2.1 mm, flow rate: 0.4 mL / min). Solvents: A - H 2 2O + 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.

[0272] High-resolution mass spectrum (HRMS) was recorded on an Agilent 1200 Series Accurate Mass Time-of-Flight (TOF) equipped with an Aeris Widepore column (XB-C8, 3.6 μm particle size, 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.

[0273] General protocol A for the loading of chlorotrityl chloride resin 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 of DMF and stirring for 30 minutes. The resin was then washed with DMF (3 times), DCM (3 times), and treated with CH 3 OH / DCM / DIEA (8:1:1) for 10 minutes to cap the unreacted trityl chloride sites, dried under vacuum, and stored in a desiccator.

[0274] General protocol B for the deprotection of Fmoc protecting group Piperidine dissolved in DMF (20%) was added to the resin. The mixture was shaken and drained for 5 minutes. Fresh 20% piperidine was added and this time the mixture was shaken for 15 minutes. The ninhydrin and / or TNBS tests were positive. The resin was then washed with DMF (3 times), DCM (3 times).

[0275] General protocol C for the deprotection of ivDde protecting group After washing with DMF and DCM, the resin was treated with 2% hydrazine dissolved in DMF (5 mL, twice for 15 minutes). The ninhydrin and / or TNBS tests were positive. Subsequently, the resin was washed with DMF (3 times) and DCM (3 times).

[0276] General protocol D for peptide coupling The resin was treated with the specified carboxylic acid derivative (3 eq) using the coupling reagents HATU (3.3 eq) and DIEA (3.3 eq) in DMF (5 mL), or the treatment was repeated until the ninhydrin and / or TNBS tests became negative. Subsequently, the resin was washed with DMF (3 times) and DCM (3 times).

[0277] General protocol E for bromoacetylation on resin Subsequently, the resin was treated with bromoacetic anhydride anhydride (2.4 eq) and DIEA (2.6 eq) in 200 mL of DCM for 30 minutes.

[0278] General protocol F for cleavage of peptides from chlorotrityl resin The resin was treated with DCM (3 times), and the product was cleaved from the resin using 5 mL of 10% TFA in DCM containing 10% H 2 O and 10% triisopropylsilane for 1 hour.

Example

[0279] Synthesis of L1

[0280]

Chemical formula

[0281] A solution of 1,4-diaminobutane (80 μL, 0.795 mmol, 1 eq) in DCM (10 mL) at 0 °C was added with DIEA (276 μL, 1.59 mmol, 2 eq), followed by bromoacetic anhydride (413 g, 1.59 mmol, 2 eq) dissolved in 1 mL of DCM. Subsequently, the reaction mixture was stirred at 0 °C for 30 minutes and at room temperature for 1.5 hours, and the solvent was removed. Purification on silica gel by flash column chromatography gave 1L as a white solid (162 mg, 0.49 mmol, 61%). MS (ES + ) m / z 331.0 ([M+H] + ). 1 H NMR (400 MHz, methanol-d 4 ) δ 3.94 (s, 4H), 3.40 - 3.30 (m, 4H), 1.68 (p, J = 3.5 Hz, 4H).

Example

[0282] Synthesis of L1B

[0283]

Chemical formula

[0284] A solution of 1,2-ethylenediamine (30 μL, 0.448 mmol, 1 eq) in DCM (5 mL) at 0 °C was added with DIEA (172 μL, 0.985 mmol, 2.2 eq), followed by bromoacetic anhydride (233 mg, 0.897 mmol, 2 eq) dissolved in 1 mL of DCM. Subsequently, the reaction mixture was stirred at 0 °C for 30 minutes and at room temperature for 1.5 hours, and the solvent was removed. Purification on silica gel by flash column chromatography gave L1B as a white solid (43.9 mg, 0.145 mmol, 32%). MS (ES + ) m / z 302.55 ([M+H] + ), 304.54 ([M+H] + ). 1 H NMR (400 MHz, methanol-d 4 ) δ 2.49 (s, 4H), 2.06 (s, 4H).

Example

[0285] Synthesis of L1C

[0286] [Chemical formula]

[0287] A solution of 1,3-diaminopropane (30 μL, 0.359 mmol, 1 eq) dissolved in DCM (5 mL) at 0 °C was added with DIEA (138 μL, 0.789 mmol, 2.2 eq), followed by bromoacetic anhydride (186 mg, 0.718 mmol, 2 eq) dissolved in 1 mL of DCM. Subsequently, the reaction mixture was stirred at 0 °C for 30 minutes and at room temperature for 1.5 hours, and the solvent was removed. Purification on silica gel by flash column chromatography gave L1C as a white solid (60.8 mg, 0.19 mmol, 53%). MS (ES + ) m / z 316.32 ([M+H] + ), 318.6 ([M+H] + ). 1 H NMR (400 MHz, methanol-d 4 ) δ 3.86 (s, 4H), 3.27 (t, J = 6.8 Hz, 4H), 1.74 (p, J = 6.8 Hz, 2H). [Example]

[0288] Synthesis of L1D

[0289] [Chemical formula]

[0290] A solution of 1,7-diaminohexane (65 μLmg, 0.499 mmol, 1 eq) in DCM (15 mL) at 0 °C was added to DIEA (208 μL, 1.197 mmol, 2.4 eq), 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 and at room temperature for 1.5 hours, and the solvent was removed. Purification on silica gel by flash column chromatography gave L1D as a white solid (120 mg, 0.322 mmol, 64%). MS (ES + ) m / z 372.71 ([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).

Example

[0291] Synthesis of L1E

[0292]

Chem.

[0293] A solution of 1,1-diaminoundecane (48 μLmg, 0.257 mmol, 1 eq) in DCM (10 mL) at 0 °C was added to DIEA (108 μL, 0.616 mmol, 2.4 eq), followed by 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 and at room temperature for 1.5 hours, and the solvent was removed. Purification on silica gel by flash column chromatography gave L1E as a white solid (62.3 mg, 0.145 mmol, 56%). MS (ES + ) m / z 428.33 ([M+H] + ). 11H NMR (400 MHz, chloroform-d) δ 6.53 (s, 2H), 3.91 (s, 4H), 3.30 (q, J = 6.8 Hz, 4H), 1.57 (q, J = 7.2 Hz, 4H), 1.42 - 1.20 (m, 14H).

Example

[0294] Synthesis of L1F

[0295]

Chem.

[0296] A solution of cadaverine (48 mg, 0.257 mmol, 1 eq) dissolved in DCM (20 mL) at 0 °C was added with DIEA (284 μL, 1.63 mmol, 2.4 eq), followed by bromoacetic anhydride (353 mg, 1.36 mmol, 2 eq) dissolved in 1 mL of DCM. Subsequently, the reaction mixture was stirred at 0 °C for 30 minutes and at room temperature for 1.5 hours, and the solvent was removed. Purification on silica gel by flash column chromatography gave L1F as a white solid (156 mg, 0.453 mmol, 66%). MS (ES + ) m / z 344.65 ([M+H] + ), 346.64 ([M+H] + ). 1 1H NMR (400 MHz, methanol-d 4 ) 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).

Example

[0297] Synthesis of L1G

[0298]

Chem.

[0299] Intermediate L1Ga A solution of tert-butylbis(2-aminoethyl)carbamate (167 mg, 0.82 mmol, 1 eq) in DCM (20 mL) was added with DIEA (342 μL, 11.96 mmol, 2.4 eq), followed by bromoacetic anhydride (426 mg, 1.64 mmol, 2 eq) dissolved in 1 mL of DCM. Subsequently, the reaction mixture was stirred at 0 °C for 30 minutes and at room temperature for 1.5 hours, and the solvent was removed. Purification on silica gel by flash column chromatography gave L1Ga as a white solid (289 mg, 0.65 mmol, 79%). MS(ES + ) m / z 445.71([M+H] + ), 447.7([M+H] + ). 1 H NMR(400 MHz, methanol-d 4 ) δ 3.85(s, 4H), 3.39(s, 9H), 1.50(s, 10H).

[0300] L1G 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-evaporation with hexane) to give compound L1G as an oil. This product was used directly in the previous step. MS(ES + ) m / z 345.2([M+H] + ).

Example

[0301] Synthesis of L3

[0302]

Chem.

[0303] Intermediate L3a Myristic acid (184 mg, 0.805 mmol, 1 eq) was dissolved in 4 mL of DMF. HATU (321 mg, 0.845 mmol, 1.1 eq) and DIEA (154 μL, 0.885 mmol, 1.1 eq), followed by Boc-NH-PEG 2-COOH (200 mg, 0.805 mmol, 1 eq) was added. Subsequently, the reaction mixture was stirred for 1.5 h and the solvent was removed. The product was dissolved in EtOAc. The organic layer was washed successively with 1 M HCl, saturated NaHCO 3 , and brine, dried over Na 2 SO 4 , filtered, and concentrated. Purification on silica gel by flash column chromatography gave the desired compound L3a as a white solid (254 mg, 0.55 mmol, 69%). 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). t R = 2.21 min (Agilent). MS (ES + ) m / z 459.6 ([M + H] + ).

[0304] Intermediate L3b A solution of compound L3a (242 mg, 0.527 mmol, 1 eq) dissolved in DCM (2 mL) was treated with TFA (2 mL) for 30 min. The mixture was concentrated and co-evaporated with hexane. BocNH-PEG 2 -CO 2 H (146 mg, 0.527 mol, 1 eq) dissolved in DMF (5 mL) was added with HATU (224 mg, 0.59 mmol, 1.1 eq). The deprotected compound L3a dissolved in DMF and DIEA (183 μL, 1.05 mmol, 2 eq) were 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 successively with 1 M HCl, saturated NaHCO 3 , and brine, dried over Na 2 SO 4 , filtered, and concentrated. Purification on silica gel by flash column chromatography gave the desired compound L3b as an oil (129 mg, 0.209 mmol, 40%).1 1H NMR (400 MHz, chloroform-d) δ 6.76 (s, 1H), 6.19 (s, 1H), 5.29 (s, 1H), 3.76 (t, J = 5.8 Hz, 2H), 3.69 - 3.62 (m, 8H), 3.57 (dt, J = 12.3, 5.0 Hz, 6H), 3.48 (dt, J = 10.4, 5.5 Hz, 4H), 3.33 (s, 2H), 2.51 (t, J = 5.8 Hz, 2H), 2.20 (t, J = 7.0 Hz, 2H), 1.90 - 1.75 (m, 4H), 1.64 (p, J = 7.3 Hz, 2H), 1.46 (s, 9H), 1.33 - 1.22 (m, 17H).

[0305] Intermediate L3c A solution of compound L3b (129 mg, 0.209 mmol, 1 eq) dissolved 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 eq) was added to a solution of Boc-Orn(Boc)-OH (69 mg, 0.209 mmol, 1 eq) dissolved in DMF (5 mL). The deprotected compound L3b dissolved in DMF and DIEA (73 μL, 0.419 mmol, 2 eq) were added to the reaction mixture. The reaction mixture was stirred at room temperature for 2 hours. The product was diluted with EtOAc. The organic layer was washed successively with 1M HCl, saturated NaHCO 3 , and brine, dried over Na 2 SO 4 , filtered, and concentrated. Purification by flash column chromatography on silica gel gave the desired compound L3c as an oil (137 mg, 0.164 mmol, 78%). t R = 4.07 min (Agilent). MS (ES + ) m / z 832.9 ([M + H] + ). 11H NMR (400 MHz, chloroform-d) δ 7.12 (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, 5.5 Hz, 7H), 3.53 - 3.36 (m, 6H), 3.29 - 3.00 (m, 2H), 2.51 (t, J = 5.8 Hz, 2H), 2.20 (t, J = 7.7 Hz, 2H), 2.00 - 1.74 (m, 6H), 1.71 - 1.51 (m, 5H), 1.45 (s, 18H), 1.35 - 1.22 (m, 21H).

[0306] L3 A solution of compound L3c (137 mg, 0.165 mmol, 1 eq) in DCM (2 mL) was treated with TFA (2 mL) for 30 minutes. The mixture was concentrated, co-evaporated with hexane, dissolved in 10 mL of DCM and cooled to 0 °C. DIEA (115 μL, 0.66 mmol, 4 eq) was added, followed by bromoacetic anhydride (85.8 g, 0.33 mmol, 2 eq) dissolved in 1 mL of DCM. The reaction mixture was then stirred at 0 °C for 30 minutes and at room temperature for 1.5 hours, and the solvent was removed. Purification on silica gel by flash column chromatography gave L3 as a white solid (56 mg, 0.064 mmol, 39%). t R = 3.4 min (Agilent). MS (ES + ) m / z 872.4 ([M + H] + ), 874.3 ([M + H] + ).

Example

[0307] Synthesis of L4

[0308]

Chem.

[0309] Intermediate L4a A solution of Boc-Orn(Boc)-OH (595 mg, 1.79 mmol, 1 eq) in DMF (5 mL) was added to a solution of HATU (750 mg, 1.79 mmol, 1.1 eq), DIEA (343 μL, 1.97 mmol, 1.1 eq), and amine-PEG 3 -N 3 (391 mg, 1.79 mmol, 1 eq) dissolved in 1 mL of DMF. The reaction mixture was stirred at room temperature for 16 h. The product was diluted with EtOAc. The organic layer was washed successively with 1 M HCl, saturated NaHCO 3 , brine, dried over Na 2 SO 4 , filtered, and concentrated. Purification on silica gel by flash column chromatography gave the desired compound L4a as an oil (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).

[0310] Intermediate L4b To a solution of compound L4a (548 mg, 1.02 mmol, 1 eq) dissolved in anhydrous MeOH (10 mL) under argon was added Pd / C (10.9 mg, 0.102 mmol, 0.1 eq), and the argon was replaced with H 2 . The reaction mixture was stirred at room temperature for 6 h, filtered through celite, and evaporated to give compound L4b as an oil (516 mg, 1.02 mmol, quantitative). This product was used without further purification.

[0311] Intermediate L4c A solution of octadecanedioic acid monoter - butyl ester (370 mg, 1.02 mmol, 1 eq) in DMF (5 mL) was added with HATU (387 mg, 1.02 mmol, 1.1 eq), DIEA (186 μL, 1.07 mmol, 2 eq), and compound L4b (516 mg, 1.02 mmol, 1 eq) dissolved in 1 mL of DMF. The reaction mixture was stirred at room temperature for 3 hours. The product was diluted with EtOAc. The organic layer was successively washed with 1 M HCl, saturated NaHCO 3 , brine, dried over Na 2 SO 4 , filtered, and concentrated. Purification on silica gel by flash column chromatography gave the desired compound L4c as an oil (697 mg, 0.81 mmol, 79%). 1 H NMR (400 MHz, chloroform - d) δ 6.94 (s, 1H), 6.42 (s, 1H), 4.81 (s, 1H), 4.20 (s, 1H), 3.65 (d, J = 6.7 Hz, 8H), 3.59 (dt, J = 9.7, 5.1 Hz, 4H), 3.51 - 3.35 (m, 4H), 3.31 - 3.18 (m, 1H), 3.17 - 3.06 (m, 1H), 2.20 (q, J = 8.0 Hz, 4H), 1.87 (s, 4H), 1.71 - 1.53 (m, 6H), 1.45 (s, 26H), 1.26 (s, 24H).

[0312] L4 A solution of L4c (422 mg, 0.49 mmol, 1 eq) in DCM (2 mL) was treated with TFA (2 mL) for 30 minutes. The mixture was concentrated, co - evaporated with hexane, dissolved in 20 mL of DCM, and cooled to 0 °C. DIEA (327 μL, 1.96 mmol, 4 eq) was added, followed by bromoacetic anhydride (254 mg, 0.98 mmol, 2 eq) dissolved in 1 mL of DCM. Then the reaction mixture was stirred at 0 °C for 30 minutes and at room temperature for 1.5 hours, and the solvent was removed. Purification on silica gel by flash column chromatography gave L4 as a white solid (53 mg, 0.063 mmol, 12%). MS (ES + ) m / z 845.08 ([M + H] + ), 847.07 ([M + H]+ ) 1 1H NMR (400 MHz, methanol-d 4 ) 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).

Example

[0313] Synthesis of L4A

[0314]

Chem.

[0315] Intermediate L4Aa To a solution of tert-butyl bis(2-aminoethyl)carbamate (500 mg, 2.45 mmol, 1 eq) and DIEA (1.02 mL, 5.88 mmol, 2 eq) in DCM (20 mL) at 0 °C was added dropwise bromoacetic anhydride (1.31 g, 5.04 mmol, 2.05 eq in 1 mL of DCM). The reaction mixture was stirred at 0 °C for 30 minutes and at room temperature for 6 hours and then evaporated under vacuum. Purification by flash chromatography gave the product as an oil (883 mg, 81%). 1 1H NMR (400 MHz, methanol-d 4 ) δ 1.50 (s, 9H), 3.39 (s, 8H), 3.85 (s, 4H). t R = 1.04 min. MS (ES + ) m / z 445.71 / 447.70 ([M+H] + ).

[0316] Intermediate L4Ab A solution of compound L4Aa (1 eq) in DCM / TFA (1:1, v / v) was stirred at room temperature for 30 minutes and then concentrated under vacuum (co-evaporated with heptane). Compound L4Ab was used directly in the next step without purification. tR = 0.58 min. MS(ES + ) m / z 345.65 / 347.67 ([M+H] + )。

[0317] Intermediate L4Ac To a solution of mono-tert-butyl succinate (1.05 eq) in DMF was added HATU (1.05 eq). The reaction mixture was stirred at room temperature for 5 minutes. Compound L4Ab and DIEA (4 eq) were dissolved in DMF (1 mL) and added to the reaction mixture. The reaction was stirred at room temperature overnight and diluted with AcOEt. The organic phase was washed with 1N HCl, saturated NaHCO 3 solution, dried over MgSO4, and evaporated. Purification by flash chromatography was performed to obtain the product as an oil. t R = 1.07 min. MS(ES + ) m / z 501.52 / 503.80 ([M+H] + )。

[0318] Intermediate L4Ad A solution of compound L4Ac (1 eq) in DCM / TFA (1:1, v / v) was stirred at room temperature for 30 minutes and concentrated under vacuum (co-evaporated with heptane). Compound L4Ad was used directly in the next step without purification. t R = 0.57 min. MS(ES + ) m / z 445.71 / 447.73 ([M+H] + )。

[0319] Intermediate L4Ae Octadecanedioic acid mono-tert-butyl ester acid (200 mg, 0.54 mmol, 1 eq) was dissolved in 5 mL of DMF. HATU (225 mg, 0.59 mmol, 1.1 eq) and DIEA (103 μL, 0.59 mmol, 1.1 eq), followed by Boc-NH-PEG 3 -NH 2 (157.8 g, 0.54 mmol, 1 eq) was added. Then, the reaction mixture was stirred for 3 hours and the solvent was removed. The product was dissolved in EtOAc. The organic layer was washed with saturated NaHCO 3, washed successively with 1 M HCl and brine, and dried over Na 2 SO 4 , filtered, and concentrated. Purification on silica gel by flash column chromatography gave the desired product L4Ae as a white solid (281 mg, 0.43 mmol, 81%). MS (ES + ) m / z 645.5 ([M+H] + ). 1 H NMR (400 MHz, chloroform-d) δ 3.76 - 3.61 (m, 8H), 3.63 - 3.54 (m, 4H), 3.48 (q, J = 5.1 Hz, 2H), 3.34 (s, 2H), 2.20 (dt, J = 9.8, 7.6 Hz, 4H), 1.67 - 1.55 (m, 4H), 1.49 - 1.44 (m, 17H), 1.30 (s, 6H), 1.30 - 1.24 (m, 19H).

[0320] L4A 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.

Example

[0321] Synthesis of L5

[0322]

Chemical Formula

[0323] General protocols A, B, D (mono-tert-butyl octadecanedioate), C, D (Fmoc-PEG 2 -propionic acid), B, D (Fmoc-PEG 2 -propionic acid), B, D (Fmoc-Orn(Fmoc)-OH), B, E, F.

[0324] The crude product was purified by semi-preparative HPLC with mass detection to obtain the product L5 as a white solid (73 mg, 0.065 mmol, 11%). 1 H NMR (400 MHz, methanol-d 4 ) δ 4.36 (td, J = 8.9, 5.1 Hz, 2H), 3.89 (q, J = 11.4 Hz, 2H), 3.82 (s, 2H), 3.74 (t, J = 6.2 Hz, 2H), 3.60 (s, 4H), 3.54 (t, J = 5.5 Hz, 2H), 3.37 (q, J = 5.2 Hz, 2H), 3.29 - 3.11 (m, 5H), 2.44 (t, J = 6.2 Hz, 2H), 2.26 (dt, J = 12.3, 7.5 Hz, 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).

Example

[0325] Synthesis of L5A

[0326]

Chemical formula

[0327] Intermediate L5Aa 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 N 2 under -40 °C and stirred for 2 hours. The completion of the reaction was confirmed by LCMS. The crude product in the solution was used directly in the next step without purification. 1 H NMR (400 MHz, DMSO-d 6 ) δ 7.88 (d, J = 7.6 Hz, 4H), 7.68 (d, J = 7.6 Hz, 4H), 7.43 - 7.24 (m, 10H), 4.30 (d, J = 6.4 Hz, 4H), 4.21 (d, J = 6.4 Hz, 2H), 3.06 (d, J = 5.6 Hz, 4H), 2.57 (d, J = 7.6 Hz, 4H). MS (ES + ) m / z 548.2 ([M + H] + ).

[0328] Intermediate L5Ab To a solution of compound L5Aa (106 g, 194 mmol) in DCM (400 mL), DMAP (4.74 g, 38.8 mmol) and tetrahydrofuran-2,5-dione (67.9 g, 678 mmol) were added, and the mixture was stirred at 25 °C for 14 hours. The completion of the reaction was confirmed by LCMS. 1N HCl was added to the reaction mixture until pH = 5 - 6, and the mixture was 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 Na 2 SO 4 and concentrated under vacuum. Using DCM / MeOH (80:0 - 5:1) as the eluent, the crude product was purified by column chromatography on silica gel to obtain compound L5Ab (57.6 g, 45% yield) as a white solid powder. 1 1H NMR (400 MHz, DMSO-d 6 ) δ 12.09 (s, 1H), 7.87 (d, J = 7.5 Hz, 4H), 7.66 (d, J = 7.0 Hz, 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] + ).

[0329] L5A General protocols A, B, D (octadecanedioic acid mono-tert-butyl ester), C, D (Fmoc-PEG 2 -propionic acid), B, D (Fmoc-PEG 2 -propionic acid), B, D (compound L5Ab), B, E, F.

[0330] The crude product was purified by HPLC to obtain product L5A as a white solid (5.2 g, 11% yield). MS (ES + ) m / z 1188.5 ([M + H] + ).

Example

[0331] Synthesis of L6

[0332] [Chemical formula]

[0333] Intermediate L6a Palmitic acid (235 mg, 0.919 mmol, 1.05 eq) was dissolved in 4 mL of DMF. HATU (349 mg, 0.919 mmol, 1.1 eq) and DIEA (167 μL, 0.963 mmol, 1.05 eq), followed by Boc-NH-PEG 2 -NH 2 (200 mg, 0.875 mmol, 1 eq) was added. The reaction mixture was then stirred for 2 hours and the solvent was removed. The product was dissolved in EtOAc. The organic layer was washed successively with 1 M HCl, saturated NaHCO 3 , HCl, and brine, dried over Na 2 SO 4 and filtered and concentrated to give the desired compound L6a as a white solid (412 mg, 0.84 mmol, 97%). 1 1H 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).

[0334] Intermediate L6b A solution of compound L6a (412 mg, 0.84 mmol, 1 eq) in DCM (2 mL) was treated with TFA (2 mL) for 30 minutes. The mixture was concentrated and co-evaporated with hexane. BocNH-PEG 2 -CO 2A solution of H (258 mg, 0.931 mmol, 1.1 eq) dissolved in DMF (5 mL) was added to HATU (353 mg, 0.931 mmol, 1.1 eq). The deprotected compound L6a dissolved in DMF and DIEA (294 μL, 1.69 mmol, 2 eq) were added to the reaction mixture. The reaction mixture was stirred at room temperature for 2 hours. The product was diluted with EtOAc. The organic layer was washed successively with 1M HCl, saturated NaHCO 3 , HCl, and brine, dried over Na 2 SO 4 , filtered, and concentrated. Purification on silica gel by flash column chromatography gave the desired compound L6b as an oil (329 mg, 0.51 mmol, 60%). 1 H NMR (400 MHz, chloroform-d) δ 6.79 (s, 1H), 6.28 (s, 1H), 5.28 (s, 1H), 3.68 (t, J = 5.8 Hz, 2H), 3.61 - 3.44 (m, 14H), 3.38 (p, J = 5.6 Hz, 4H), 3.24 (q, J = 5.5 Hz, 2H), 2.42 (t, J = 5.8 Hz, 2H), 2.11 (t, J = 7.9 Hz, 2H), 1.55 (p, J = 7.2 Hz, 2H), 1.38 (s, 9H), 1.32 - 1.10 (m, 24H), 0.81 (t, J = 6.7 Hz, 3H).

[0335] Intermediate L6c A solution of compound L6b (329 mg, 0.51 mmol, 1 eq) dissolved in DCM (2 mL) was treated with TFA (2 mL) for 30 minutes. The mixture was concentrated and co-evaporated with hexane. A solution of Boc-Orn(Boc)-OH (186 mg, 0.56 mmol, 1.1 eq) dissolved in DMF (5 mL) was added to HATU (213 mg, 0.56 mmol, 1.1 eq). The deprotected compound L6b dissolved in DMF and DIEA (177 μL, 1.02 mmol, 2 eq) were added to the reaction mixture. The reaction mixture was stirred at room temperature for 2 hours. The product was diluted with EtOAc. The organic layer was washed successively with saturated NaHCO 3 , 1M HCl, and brine, dried over Na 2 SO 4It was dried, filtered, and concentrated. Purification on silica gel by flash column chromatography gave the desired compound L6c as an oil (326 mg, 0.37 mmol, 94%). 1 H NMR (400 MHz, chloroform-d) δ 7.18 (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 - 3.29 (m, 8H), 3.19 - 2.86 (m, 2H), 2.42 (t, J = 5.9 Hz, 2H), 2.10 (d, J = 7.3 Hz, 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 = 6.7 Hz, 3H).

[0336] L6 A solution of compound L6c (100 mg, 0.116 mmol, 1 eq) in DCM (2 mL) was treated with TFA (2 mL) for 30 minutes. The mixture was concentrated, co-evaporated with hexane, dissolved in 10 mL of DCM, and cooled to 0 °C. DIEA (80.8 μL, 0.46 mmol, 4 eq) was added, followed by bromoacetic anhydride (61.9 mg, 0.238 mmol, 2.05 eq) dissolved in 1 mL of DCM. The reaction mixture was then stirred at 0 °C for 30 minutes and at room temperature for 1.5 hours, and the solvent was removed. Purification on silica gel by flash column chromatography gave L6 as a white solid (50.1 mg, 0.055 mmol, 40%). 1 H NMR (400 MHz, methanol-d 4)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 - 3.36 (m, 6H), 3.25 (t, J = 13.9, 6.8 Hz, 2H), 2.49 (t, J = 6.2 Hz, 2H), 2.21 (t, J = 7.5 Hz, 2H), 1.91 - 1.79 (m, 1H), 1.75 - 1.53 (m, 5H), 1.42 - 1.25 (m, 24H), 0.92 (t, J = 6.7 Hz, 3H).

Example

[0337] Synthesis of L7

[0338]

Chem.

[0339] Intermediate L7a Stearic acid (261 mg, 0.919 mmol, 1.05 eq) was dissolved in 4 mL of DMF. HATU (349 mg, 0.919 mmol, 1.1 eq) and DIEA (167 μL, 0.963 mmol, 1.05 eq), followed by Boc-NH-PEG 2 -NH 2 (200 mg, 0.875 mmol, 1 eq) were added. The reaction mixture was then stirred for 2 hours and the solvent was removed. The product was dissolved in EtOAc. The organic layer was washed successively with 1M HCl, saturated NaHCO 3 , and brine, dried over Na 2 SO 4 and filtered and concentrated to give the desired compound L7a as a white solid (430 mg, 0.83 mmol, 95%). 11H NMR (400 MHz, chloroform-d) δ 3.69 - 3.59 (m, 4H), 3.56 (t, J = 5.1 Hz, 4H), 3.46 (q, J = 5.2 Hz, 2H), 3.40 - 3.23 (m, 2H), 2.18 (t, J = 7.6 Hz, 2H), 1.62 (t, J = 7.3 Hz, 2H), 1.45 (s, 9H), 1.35 - 1.19 (m, 30H), 0.88 (t, J = 6.7 Hz, 4H).

[0340] Intermediate L7b A solution of compound L7a (426 mg, 0.87 mmol, 1 eq) in DCM (2 mL) was treated with TFA (2 mL) for 30 minutes. The mixture was concentrated and co-evaporated with hexane. BocNH-PEG 2 -CO 2 H (266 mg, 0.96 mmol, 1.1 eq) was added to a solution of HATU (366 mg, 0.96 mmol, 1.1 eq) in DMF (5 mL). The deprotected compound L7a dissolved in DMF and DIEA (304 μL, 1.75 mmol, 2 eq) were added to the reaction mixture. The reaction mixture was stirred at room temperature for 2 hours. The product was diluted with EtOAc. The organic layer was washed successively with 1M HCl, saturated NaHCO 3 , and brine, dried over Na 2 SO 4 , filtered, and concentrated. Purification by flash column chromatography on silica gel gave the desired compound L7b as an oil (360 mg, 0.53 mmol, 61%). 1 1H NMR (400 MHz, chloroform-d) δ 6.75 (s, 1H), 6.18 (s, 1H), 5.26 (s, 1H), 3.75 (t, J = 5.8 Hz, 2H), 3.69 - 3.52 (m, 14H), 3.47 (p, J = 5.4 Hz, 4H), 3.33 (q, J = 5.5 Hz, 2H), 2.50 (t, J = 5.8 Hz, 2H), 2.19 (t, J = 7.5 Hz, 2H), 2.07 (s, 1H), 1.63 (p, J = 7.3 Hz, 2H), 1.46 (s, 9H), 1.37 - 1.19 (m, 29H), 0.89 (t, J = 6.7 Hz, 3H).

[0341] Intermediate L7c A solution of compound L7b (360 mg, 0.53 mmol, 1 eq) dissolved 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 eq) was added to a solution of Boc-Orn(Boc)-OH (195 mg, 0.58 mmol, 1.1 eq) dissolved in DMF (5 mL). The deprotected compound L7b dissolved in DMF and DIEA (186 μL, 1.07 mmol, 2 eq) were added to the reaction mixture. The reaction mixture was stirred at room temperature for 2 hours. The product was diluted with EtOAc. The organic layer was washed successively with 1M HCl, saturated NaHCO 3 , and brine, dried over Na 2 SO 4 , filtered, and concentrated. Purification on silica gel by flash column chromatography afforded the desired compound L7c as an oil (373 mg, 0.42 mmol, 78%). 1 H NMR (400 MHz, chloroform-d) δ 7.14 (s, 1H), 6.84 (s, 1H), 6.35 (s, 1H), 5.53 (d, J = 8.2 Hz, 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 = 5.8 Hz, 2H), 2.17 (t, J = 7.8 Hz, 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 = 6.9 Hz, 3H).

[0342] L7 A solution of compound L7c (100 mg, 0.112 mmol, 1 eq) in DCM (2 mL) was treated with TFA (2 mL) for 30 minutes. The mixture was concentrated and co-evaporated with hexane, dissolved in 10 mL of DCM, and cooled to 0 °C. DIEA (78 μL, 0.44 mmol, 4 eq) was added, followed by bromoacetic anhydride (62 mg, 0.24 mmol, 2.05 eq) dissolved in 1 mL of DCM. The reaction mixture was then stirred at 0 °C for 30 minutes and at room temperature for 1.5 hours, and the solvent was removed. The product was dissolved in EtOAc. The organic layer was washed successively with 1 M HCl, saturated NaHCO 3 , and brine, dried over Na 2 SO 4 , filtered, and concentrated. Purification by flash column chromatography on silica gel gave L7 as a white solid (95 mg, 0.10 mmol, 91%). MS (ES + ) m / z 931.31 ([M+H] + ), 933.25 ([M+H] + ). 1 H NMR (400 MHz, methanol-d 4 ) δ 4.39 (dd, J = 8.5, 5.4 Hz, 1H), 3.91 (q, J = 11.3 Hz, 2H), 3.84 (s, 2H), 3.76 (t, J = 6.2 Hz, 2H), 3.63 (d, J = 7.0 Hz, 8H), 3.57 (t, J = 5.5 Hz, 6H), 3.42 - 3.35 (m, 6H), 3.31 - 3.13 (m, 4H), 2.49 (t, J = 6.2 Hz, 2H), 2.20 (t, J = 7.4 Hz, 2H), 1.91 - 1.79 (m, 1H), 1.75 - 1.56 (m, 6H), 1.39 - 1.26 (m, 26H), 0.92 (t, J = 6.3 Hz, 3H).

Example

[0343] Synthesis of L8

[0344]

Chem.

[0345] General protocols A, B, D (mono-tert-butyl hexadecanedioate), C, D (Fmoc-PEG 2 -propionic acid), B, D (Fmoc-PEG 2 -propionic acid), B, D (Fmoc-Orn(Fmoc)-OH), B, E, F.

[0346] The crude product was purified by preparative HPLC with mass detection to give the product L8 as a white solid (42.6 mg, 0.038 mmol, 22%). 1 1H NMR (400 MHz, methanol-d 4 ) 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 - 3.37 (m, 4H), 3.31 - 3.16 (m, 4H), 2.48 (dt, J = 15.7, 6.2 Hz, 4H), 2.28 (dt, J = 12.6, 7.5 Hz, 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).

Example

[0347] Synthesis of L9

[0348]

Chem.

[0349] General protocols A, B, D (mono-tert-butyl heptadecanedioate), C, D (Fmoc-PEG 2 -propionic acid), B, D (Fmoc-PEG 2 -propionic acid), B, D (Fmoc-Orn(Fmoc)-OH), B, E, F.

[0350] The crude product was purified by preparative HPLC with mass detection to give the product L9 as a white solid (49 mg, 0.089 mmol, 9%).1 1H NMR (400 MHz, methanol-d 4 ) δ 4.45 - 4.33 (m, 2H), 3.92 (t, J = 10.9 Hz, 2H), 3.85 (d, J = 1.1 Hz, 2H), 3.77 (q, J = 6.0 Hz, 4H), 3.63 (s, 8H), 3.57 (t, J = 5.6 Hz, 4H), 3.40 (t, J = 5.5 Hz, 4H), 3.25 (dq, J = 22.7, 6.7 Hz, 4H), 2.48 (dt, J = 15.6, 6.2 Hz, 4H), 2.29 (dt, J = 13.2, 7.4 Hz, 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).

Example

[0351] Synthesis of L12

[0352]

Chem.

[0353] General protocols A, B, D (octadecanedioic acid), C, D (Fmoc-PEG 2 -propionic acid), B, D (Fmoc-Orn(Fmoc)-OH), B, E, F.

[0354] The crude product was purified by preparative HPLC with mass detection to obtain the product L12 as a white solid (51.7 mg, 0.054 mmol, 3%). 1 1H NMR (400 MHz, methanol-d 4) 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 = 5.1 Hz, 2H), 3.30 - 3.12 (m, 6H), 2.47 (t, J = 6.1 Hz, 2H), 2.29 (dt, J = 12.1, 7.4 Hz, 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).

Example

[0355] Synthesis of L14

[0356]

Chem.

[0357] Intermediate L14a To a solution of hexadecanedioic acid monoter - butyl ester (102 mg, 0.3 mmol, 1 eq) dissolved in DMF (5 mL), HATU (125 mg, 0.33 mmol, 1.1 eq), DIEA (51 μL, 0.33 mmol, 1.1 eq), and compound L4b (151.9 mg, 0.30 mmol, 1 eq) dissolved in 1 mL of DMF were added. The reaction mixture was stirred at room temperature for 3 hours. The product was diluted with EtOAc. The organic layer was washed successively with 1M HCl, saturated NaHCO 3 , brine, and dried over Na 2 SO 4 . It was filtered and concentrated. Purification on silica gel by flash column chromatography gave the desired compound L14a as an oil (147 mg, 0.176 mmol, 59%). 11H NMR (400 MHz, chloroform-d) δ 6.87 (s, 1H), 6.40 (s, 1H), 5.32 (s, 2H), 4.79 (s, 1H), 4.20 (s, 1H), 3.66 (d, J = 7.0 Hz, 8H), 3.60 (dt, J = 10.0, 5.1 Hz, 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, 6.0 Hz, 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).

[0358] L14 A solution of compound L14a (40 mg, 0.048 mmol, 1 eq) in DCM (2 mL) was treated with TFA (2 mL) for 30 minutes. The mixture was concentrated, co-evaporated with hexane, dissolved in 20 mL of DCM, and cooled to 0 °C. DIEA (34 μL, 0.1924 mmol, 4 eq) was added, followed by bromoacetic anhydride (23.63 mg, 0.098 mmol, 2.05 eq) dissolved in 1 mL of DCM. The reaction mixture was then stirred at 0 °C for 30 minutes and at room temperature for 1.5 hours, and the solvent was removed. Purification on silica gel by flash column chromatography gave L14 as a white solid (18.3 mg, 0.022 mmol, 46%). MS (ES + ) m / z 817.1 ([M + H] + ), 819.09 ([M + H] + ). 1 1H NMR (400 MHz, methanol-d 4 ) δ 4.38 (dd, J = 8.4, 5.5 Hz, 1H), 3.92 (q, J = 11.2, 10.6 Hz, 2H), 3.84 (s, 2H), 3.69 - 3.61 (m, 8H), 3.56 (td, J = 5.5, 2.6 Hz, 4H), 3.44 - 3.36 (m, 4H), 3.30 - 3.14 (m, 2H), 2.29 (t, J = 7.4 Hz, 2H), 2.21 (t, J = 7.5 Hz, 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).

Example

[0359] Synthesis of L15

[0360]

Chemical formula

[0361] Intermediate L15a To a solution of 20-(tert-butoxy)-20-oxoicosanoic acid (360 mg, 0.90 mmol, 1.05 eq) in DMF (5 mL) were added HATU (343 mg, 0.90 mmol, 1.05 eq) dissolved in 1 mL of DMF, DIEA (300 μL, 1.71 mmol, 2 eq), and compound L4b (435 mg, 0.858 mmol, 1 eq). The reaction mixture was stirred at room temperature for 3 hours. The product was diluted with EtOAc. The organic layer was washed successively with 1 M HCl, saturated NaHCO 3 , brine, dried over Na 2 SO 4 , filtered, and concentrated. Purification on silica gel by flash column chromatography gave the desired compound L15a as an oil (555 mg, 0.625 mmol, 72%). 1 H NMR (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).

[0362] L15 A solution of compound L15a (100 mg, 0.112 mmol, 1 eq) in DCM (2 mL) was treated with TFA (2 mL) for 30 minutes. The mixture was concentrated, co-evaporated with hexane, dissolved in 20 mL of DCM, and cooled to 0 °C. DIEA (79 μL, 0.45 mmol, 4 eq) was added, followed by bromoacetic anhydride (60 mg, 0.231 mmol, 2.05 eq) dissolved in 1 mL of DCM. The reaction mixture was then stirred at 0 °C for 30 minutes and at room temperature for 1.5 hours, and the solvent was removed. Purification on silica gel by flash column chromatography gave L15 as a white solid (17.5 mg, 0.02 mmol, 18%). MS (ES + ) m / z 873.21 ([M+H] + ), 875.20 ([M+H] + ) 1 H NMR (400 MHz, methanol-d 4 ) δ 4.38 (dd, J = 8.4, 5.5 Hz, 1H), 3.91 (q, J = 11.4 Hz, 2H), 3.84 (s, 2H), 3.72 - 3.61 (m, 8H), 3.56 (td, J = 5.5, 2.7 Hz, 4H), 3.44 - 3.35 (m, 5H), 3.30 - 3.17 (m, 2H), 2.29 (t, J = 7.4 Hz, 2H), 2.21 (t, J = 7.5 Hz, 2H), 1.92 - 1.77 (m, 1H), 1.75 - 1.53 (m, 7H), 1.40 - 1.27 (m, 27H).

Example

[0363] Synthesis of L16

[0364]

Chem.

[0365] Intermediate L16a To a solution of Boc-Orn(Boc)-OH (400 mg, 1.2 mmol, 1 eq) in DMF (10 mL) were added HATU (504 mg, 1.32 mmol 1.1 eq), DIEA (230 μL, 1.32 mmol, 1.1 eq), and amine-PEG 2 -N3 (210 mg, 1.20 mmol, 1 eq) was added as a solution in 1 mL of DMF. The reaction mixture was stirred at room temperature for 4 hours. The product was diluted with EtOAc. The organic layer was washed successively with 1 M HCl, saturated NaHCO 3 , brine, and dried over Na 2 SO 4 . It was filtered and concentrated. Purification on silica gel by flash column chromatography gave the desired compound L16a as an oil (471 mg, 0.96 mmol, 80%). 1 H NMR (400 MHz, methanol-d 4 ) 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).

[0366] Intermediate L16b To a solution of compound L16a (471 mg, 0.9 mmol, 1 eq) dissolved in anhydrous MeOH (10 mL) under argon, Pd / C (10.2 mg, 0.09 mmol, 0.1 eq) was added, and the argon was replaced with H 2 . The reaction mixture was stirred at room temperature for 6 hours, filtered through celite, and evaporated to give compound L16b as an oil (295.5 mg, 0.64 mmol, 71%). This product was used without further purification. MS (ES + ) m / z 462.51 ([M + H] + ).

[0367] Intermediate L16c A solution of octadecanedioic acid monoter - butyl ester (281 mg, 0.76 mmol, 1 eq) in DMF (5 mL) was added to HATU (288 mg, 0.76 mmol, 1 eq), DIEA (132 μL, 0.76 mmol, 1 eq), and compound L16b (351 mg, 0.76 mmol, 1 eq) 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 successively washed with 1 M HCl, saturated NaHCO 3 , brine, dried over Na 2 SO 4 , filtered, and concentrated. Purification on silica gel by flash column chromatography gave the desired compound L16c as an oil (351 mg, 0.43 mmol, 57%). 1 H NMR (400 MHz, methanol - d 4 ) δ 3.61 (s, 4H), 3.54 (td, J = 5.6, 2.3 Hz, 4H), 3.40 - 3.34 (m, 4H), 3.04 (t, J = 6.6 Hz, 2H), 2.20 (td, J = 7.6, 5.9 Hz, 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).

[0368] L16 A solution of compound L16c (31 mg, 0.038 mmol, 1 eq) in DCM (2 mL) was treated with TFA (2 mL) for 30 min. The mixture was concentrated, co - evaporated with hexane, dissolved in 20 mL of DCM, and cooled to 0 °C. DIEA (27 μL, 0.152 mmol, 4 eq) was added, followed by bromoacetic anhydride (21 mg, 0.078 mmol, 2.05 eq) dissolved in 1 mL of DCM. The reaction mixture was then stirred at 0 °C for 30 min and at room temperature for 1.5 h, and the solvent was removed. Purification on silica gel by flash column chromatography gave L16 as a white solid (12.6 mg, 0.015 mmol, 41%). MS (ES + ) m / z 801.13 ([M + H] + ), 803.12 ([M + H] + ). 11H NMR (400 MHz, methanol-d 4 ) δ 4.37 (dd, J = 8.5, 5.4 Hz, 1H), 3.91 (q, J = 11.3 Hz, 2H), 3.84 (s, 2H), 3.63 (s, 4H), 3.57 (td, J = 5.6, 2.6 Hz, 4H), 3.43 - 3.36 (m, 4H), 3.31 - 3.17 (m, 1H), 2.29 (t, J = 7.4 Hz, 2H), 2.21 (t, J = 7.5 Hz, 2H), 1.90 - 1.79 (m, 1H), 1.76 - 1.54 (m, 7H), 1.41 - 1.30 (m, 26H).

Example

[0369] Synthesis of L17

[0370]

Chem.

[0371] Intermediate L17a A solution of Boc-Orn(Boc)-OH (400 mg, 1.2 mmol, 1 eq) in DMF (10 mL) was added to a solution of HATU (504 mg, 1.32 mmol, 1.1 eq), DIEA (230 μL, 1.32 mmol, 1.1 eq), and amine-PEG 2 -N 3 (316 mg, 1.20 mmol, 1 eq) dissolved in 1 mL of DMF. The reaction mixture was stirred at room temperature for 4 h. The product was diluted with EtOAc. The organic layer was washed successively with 1 M HCl, saturated NaHCO 3 , brine, dried over Na 2 SO 4 , filtered, and concentrated. Purification on silica gel by flash column chromatography gave the desired compound L17a as an oil (454 mg, 0.78 mmol, 66%). 1 1H NMR (400 MHz, methanol-d 4)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).

[0372] Intermediate L17b To a solution of compound L17a (454 mg, 0.9 mmol, 1 eq) dissolved in anhydrous MeOH (10 mL) under argon, Pd / C (8.3 mg, 0.078 mmol, 0.1 eq) was added, and argon was replaced with H 2 The reaction mixture was stirred at room temperature for 6 hours, filtered through celite, and evaporated to obtain compound L17b as an oil (192 mg, 0.35 mmol, 45%). This product was used without further purification.

[0373] Intermediate L17c To a solution of monoter - butyl octadecanedioate (225 mg, 0.61 mmol, 1 eq) dissolved in DMF (5 mL), HATU (231 mg, 0.61 mmol, 1 eq), DIEA (106 μL, 0.61 mmol, 1 eq), and compound L17b (335 mg, 0.61 mmol, 1 eq) dissolved in 1 mL of DMF were added. The reaction mixture was stirred at room temperature for 2 hours. The product was diluted with EtOAc. The organic layer was washed successively with 1M HCl, saturated NaHCO 3 , brine, dried over Na 2 SO 4 filtered, and concentrated. Purification on silica gel by flash column chromatography gave the desired compound L17c as an oil (178 mg, 0.20 mmol, 32%). 11H NMR (400 MHz, chloroform-d) δ 5.32 (s, 2H), 3.74 - 3.63 (m, 11H), 3.59 (dt, J = 10.9, 5.0 Hz, 4H), 3.52 - 3.43 (m, 4H), 3.27 - 3.08 (m, 2H), 2.22 (d, J = 7.6 Hz, 4H), 1.69 - 1.52 (m, 6H), 1.51 - 1.42 (m, 27H), 1.27 (s, 26H).

[0374] L17 A solution of compound L17c (45.6 mg, 0.05 mmol, 1 eq) in DCM (2 mL) was treated with TFA (2 mL) for 30 minutes. The mixture was concentrated, co-evaporated with hexane, dissolved in 20 mL of DCM, and cooled to 0 °C. DIEA (36 μL, 0.202 mmol, 4 eq) was added, followed by bromoacetic anhydride (27 mg, 0.103 mmol, 2.05 eq) dissolved in 1 mL of DCM. The reaction mixture was then stirred at 0 °C for 30 minutes and at room temperature for 1.5 hours, and the solvent was removed. Purification on silica gel by flash column chromatography gave L17 as a white solid (14.9 mg, 0.017 mmol, 33%). MS (ES + ) m / z 889.18 ([M + H] + ), 891.17 ([M + H] + ) 1 1H NMR (400 MHz, methanol-d 4 ) δ 4.38 (dd, J = 8.3, 5.5 Hz, 1H), 3.92 (q, J = 11.3 Hz, 2H), 3.84 (s, 2H), 3.67 - 3.60 (m, 7H), 3.56 (td, J = 5.5, 3.5 Hz, 4H), 3.45 - 3.35 (m, 5H), 3.32 - 3.15 (m, 3H), 2.29 (t, J = 7.4 Hz, 2H), 2.21 (t, J = 7.5 Hz, 2H), 1.90 - 1.76 (m, 1H), 1.74 - 1.57 (m, 7H), 1.41 - 1.26 (m, 25H).

Example

[0375] Synthesis of L18

[0376]

Chem.

[0377] General protocols A, B, D (octadecanedioic acid mono-), C, D (Fmoc-PEG 2 -propionic acid), B (Fmoc-PEG 2 -propionic acid), B, (Fmoc-PEG 2 -propionic acid), B, D (Fmoc-Orn(Fmoc)-OH), B, E, F.

[0378] 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] + ).

[0379] General procedure for stapling / conjugation of bromoacetyl peptides The peptide was dissolved at a concentration of 2 mM in 1:3 (v / v) MeCN / 30 mM NH 4 HCO 3 buffer (pH 8.5) using 1.5 eq of bromoacetyl staple. The pH of the reaction mixture was readjusted with ammonium hydroxide to correct for the pH drop caused by the peptide TFA counterion. In the case of particularly insoluble peptides, more MeCN was added. The reaction was stirred at room temperature for 2 - 4 h, then acetic acid was added dropwise to acidify to pH 5. The resulting solution was lyophilized and purified by reverse-phase HPLC.

[0380] General solid-phase protocol for lactam stapling Peptide-resin bearing amine side-chain orthogonal protection (Dde / Mmt) at each stapling position was swollen in DMF for 1 hour. Treatment with a 2% hydrazine solution in DMF (twice for 15 minutes) removed the Dde protecting group from the first side chain. The TNBS test was positive. The linker building blocks specified below were coupled as described, and the TNBS test was negative. The solvent was exchanged to DCM, and treatment with 1% TFA in DCM containing 5% TIPS (five times for 2 minutes) removed the Mmt group from the second side chain. The resin was washed with DCM, 10% DIEA in DMF, and DMF, and the TNBS test was positive. The linker was cyclized and the staple PEG-fatty acid moiety (where applicable) was extended as described below. The fully stapled peptide was cleaved from the resin using 95% TFA, 2.5% TIPS, 2.5% H 2 O for 3 hours. The peptide cleavage mixture was evaporated to an oil, triturated, washed with diethyl ether, and purified by reverse-phase HPLC. The Dde / Alloc protection scheme can also be used in this approach, but this requires the addition of allyl alcohol as a scavenger to the Dde deprotection cocktail to prevent the concurrent reduction of the Alloc allyl moiety.

[0381] Synthesis of the K(Fmoc) linker

[0382]

Chemical Structure

[0383] Intermediate Ka

[0384] 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 mixture was stirred at room temperature for 3.5 h. The solvent was evaporated, and the residue was dissolved in MeOH and purified by flash column chromatography on silica gel (hexane / EtOAc) to give the product as a white solid (0.802 g, 56%). 1 1H NMR (400 MHz, chloroform-d) δ 7.78 (d, J = 7.4 Hz, 2H), 7.62 (d, J = 7.4 Hz, 2H), 7.42 (t, J = 7.4 Hz, 2H), 7.33 (t, J = 7.4 Hz, 2H), 5.66 (t, J = 5.7 Hz, 1H), 4.35 (d, J = 7.3 Hz, 2H), 4.23 (t, J = 7.3 Hz, 1H), 4.10 (s, 2H), 4.02 (s, 2H), 3.56 (q, J = 5.7 Hz, 2H), 2.55 (t, J = 5.7 Hz, 2H), 1.49 (s, 18H).

[0385] K(Fmoc) linker Compound Ka was treated with 20 mL of 1:1 TFA / DCM for 2 h. The solvent was evaporated, and the residue was triturated and washed with diethyl ether to give the K(Fmoc) linker as a white solid (0.371 g, 58%). MS(ES + ) m / z 427.15 ([M+H] + ).

[0386] Synthesis of A(Fmoc) linker

[0387]

Chemical Structure

[0388] A solution of 5-aminoisophthalic acid (1.00 g, 5.5 mmol) dissolved in 10 mL of dioxane was treated with Na 2 CO 3(1.46 g, 5.5 mmol) was added to the degassed solution dissolved in 15 mL of water. Then, the solution was cooled on ice, and a solution of Fmoc chloride (1.42 g, 5.5 mmol) dissolved in 10 mL of dioxane was added dropwise with stirring over 15 minutes. Then, the reaction mixture was stirred for 1 hour and then at room temperature for 24 hours. Dioxane was removed under vacuum, and the remaining aqueous solution was acidified with 1 M HCl. Then, the resulting solid precipitate was washed with diethyl ether (4 times with 10 mL), redissolved in EtOAc, filtered, washed with brine, and dried over Na 2 SO 4 to obtain the A(Fmoc) linker as a white solid (119 mg, 5%). 1 H NMR (500 MHz, DMSO-d 6 ) δ 13.24 (s, 2H), 10.12 (s, 1H), 8.33 (d, J = 1.5 Hz, 2H), 8.12 (t, J = 1.5 Hz, 1H), 7.91 (d, J = 7.6 Hz, 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).

[0389] General protocol G for simple lactam staples of the "A1" and "K1" series In linker coupling, an appropriate diacidic building block (2 eq) was attached using HATU (4 eq) and DIEA (4 eq) in DMF (once for 2 hours). The cyclization step was achieved using HATU (1 eq) and DIEA (2 eq) in DMF (once for 2 hours).

[0390] General protocol H for trifunctional lactam staples of "K" PEG fatty acids In the linker coupling, the coupling of the symmetric anhydride within the molecule of the building block K(Fmoc) linker (2 eq) was carried out at room temperature for 10 minutes using DIC (2 eq) and the catalyst DMAP in dry DCM. The peptide-resin solvent was exchanged with DCM, then the anhydride was added and stirred overnight. The resin was drained and washed with DCM and DMF. The linker was cyclized overnight by treatment with DIC (1 eq) and HOBt or HOAt (1 eq) in DMF, and the TNBS test was negative. The remaining uncyclized linker was capped by treatment with 10% acetic anhydride in DMF (30 minutes). The Fmoc group of the linker was deprotected by treatment with 20% piperidine in DMF (10 minutes twice). TNBS was positive. 20% piperidine in DMF was used for the deprotection cycle (5 + 10 minutes, room temperature), and the subsequent staple PEG and fatty acid building blocks were successively attached to the linker-free amine via the standard coupling chemistry: building block (3 eq), HATU (3 eq), and DIEA (6 eq) in DMF (1 hour at room temperature).

[0391] General Protocol I for the trifunctional lactam staple of "A" PEG fatty acid In linker coupling, the building block A (Fmoc) linker (2 eq) was attached using HATU (4 eq) and DIEA (4 eq) in DMF (once for 2 h). The cyclization step was achieved using HATU (1 eq) and DIEA (2 eq) in DMF (once for 2 h). The remaining uncyclized linkers were capped by treatment with 10% acetic anhydride in DMF (30 min). The linker Fmoc groups were deprotected by treatment with 20% piperidine in DMF (twice for 10 min). It was not possible to observe a positive TNBS test for the aniline nitrogen. Fmoc-β-Ala-OH (3 eq) was coupled as a symmetrical anhydride using HATU (3 eq) and DIEA (6 eq) in DMF (four times for 1 h at room temperature) or using DIC / DMAP in DCM (2 h at room temperature). 20% piperidine in DMF was used for the deprotection cycle (5 + 10 min at room temperature), and the subsequent staple PEG and fatty acid building blocks were successively attached to the linker-free amine via standard coupling chemistry: building block (3 eq), HATU (3 eq), and DIEA (6 eq) in DMF (1 h at room temperature).

[0392] In some embodiments, the peptide conjugates described herein include the staples of Table 2.

[0393] [Table 2-1]

[0394] [Table 2-2]

[0395] [Table 2-3]

[0396] [Table 2-4]

[0397]

Chem.

[0398]

Chem.

[0399] In some embodiments, the peptide conjugates described herein are as shown in Table 3.

[0400]

Table 3-1

[0401]

Table 3-2

[0402]

Table 3-3

[0403] Example A: In vitro GLP-2 Receptor Activation Reporter Assay (Receptor-Mediated cAMP Synthesis) The activity and potency of the peptides in GLP-2R activation were determined using a stable HEK293 cell line that overexpresses a luciferase reporter induced by a cAMP response element (CRE) and human GLP-2R in the presence of 10% FBS. GLP2-2G (teduglutide) was used as a positive control.

[0404] HEK293-GLP-2R-CRE cells were seeded at a density of 5000 cells / well in a 384-well plate and cultured in DMEM containing 10% FBS at 37 °C and 5% CO2 for 18 hours. Cells were treated with the peptide for 16 hours in a dose-dependent manner, and receptor activation was determined 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 from three independent experiments. The results are shown in Table 4 below.

[0405] [Table 4]

[0406] Example B: Pharmacokinetics (half-life) of the peptide in mice To determine the in vivo half-life of the GLP-2 agonist, a pharmacokinetics (PK) study was conducted on male CD1 mice (n = 4 per group) by intravenous or subcutaneous injection of the peptide at 10 nmol / kg. Plasma levels of the peptide at various time points (5 minutes, 30 minutes, 1 hour, 3 hours, 7 hours, 24 hours) were determined using an in vitro GLP-2R-mediated cell-based reporter assay. The estimated terminal half-life after intravenous or subcutaneous administration is shown in Table 5 below.

[0407] Briefly, female CD-1 mice from Charles River Laboratory (4 mice per group) were fasted overnight, and 100 μL of each peptide was dissolved in phosphate-buffered saline and administered intravenously (iv) or subcutaneously (sc). Immediately after bleeding at the 30-minute time point, the mice were given food. Blood was drawn and transferred to heparin tubes, and centrifuged at 3000 g for 15 minutes. As a result, plasma floated on the surface, which was stored at -80 °C for peptide concentration determination. The peptide concentration in plasma at each time point was determined by in vitro cell-based activity assays. HEK293-GLP-2R-CRE cells were treated with plasma samples at various time points (5-point dose response, dilution starting from 1:10 to 1:100 in each plasma sample), incubated in DMEM containing 10% FBS at 37 °C, 5% CO2 for 16 hours, and then the firefly luciferase activity was measured. At the same time, using the same peptide, standard curves and parameters in Bottom, Top, EC50, and Hill slope were obtained. Using the relative light units (RLU) in each plasma sample, the peptide concentration (nmol / L) in plasma was calculated using the parameters derived from the standard curve (RLU = Bottom + (Top - Bottom) / (1 + 10(logEC50 - Conc)Hill slope)). By using WinNonLin Phoenix software (Pharsight Corp, St. Louis, MO) to obtain the peptide concentration in plasma and plot it against time points, the in vivo half-life of each peptide was obtained.

[0408]

Table 5

[0409] Example C: In vitro Potency of Long-Acting GLP2R Agonists against Human GLP2R In this example, the potency of long-acting GLP2 against GLP2R was evaluated. Due to the increase in GLP2R activity, an increase in free cAMP was observed due to a decrease in the ratio of 665 / 615.

[0410] GLP2-2G (teduglutide) was used as a positive control. As shown in Figure 1A, as the teduglutide concentration increased, the 665 / 615 ratio decreased, indicating an increase in GLP2R activity. The concentration variations of GLP2-2G-10Nle-1K-EX4-K5, GLP2-2G-1-EX4-L5A, and GLP2-2G-10Nle-1-EX4-L5A resulted in activity values similar to those of teduglutide. From this data, IC 50 values were calculated as shown in Table 6. As shown in Figure 1B, when 10% fetal bovine serum was added to this assay, the teduglutide curve became steeper than that of the long-acting GP2R agonist curve. As listed in Table 6, the obtained IC 50 values were higher for all three long-acting GLP2R agonists.

[0411]

Table 6

[0412] Example D: In vitro Potency of Long-Acting GLP2R Agonists against Mouse GLP2R The potency of long-acting GLP2R agonists against mouse GLP2R was determined. An increase in free cAMP was observed due to an increase in GLP2R activity, as indicated by a decrease in the 665 / 615 ratio.

[0413] As shown in Figure 2, the ratio of fluorescence at 665 to fluorescence at 615 was plotted against the molecular concentration, and this data was used to calculate the IC 50 values, which are listed in Table 7. Teduglutide (GLP2-2G) and albuglutide (synthetic GLP-2 analog) were used as positive controls. The IC 50 values of the long-acting GLP2R agonists were found to be in a similar range to those of teduglutide and albuglutide, indicating that the long-acting GLP2R agonists exhibit relatively high potency against mouse GLP2R.

[0414]

Table 7

[0415] Example E: In vitro Potency of a Long-Acting GLP2R Agonist against Cynomolgus GLP2R The potency of a long-acting GLP2R agonist against cynomolgus GLP2R was determined. To determine the potency of the long-acting GLP2R agonist against cynomolgus, as shown in Figure 3, a ratio of 615 to 665 was plotted against concentration nM. The EC50 was calculated using the data in Figure 3 and this value is listed in Table 8. The EC50 value of the long-acting GLP2R agonist was in the range of 0.119 nM to 0.156 nM, whereby it was recognized that the long-acting GLP2R agonist showed relatively high potency against cynomolgus GLP2R.

[0416] [Table 8]

[0417] Example F: The long-acting GLP2R agonist was highly selective for GLP2R over other G-protein coupled receptors. In this example, the effect of a stable GLP2R agonist on other G-protein coupled receptors (GPCRs) was evaluated.

[0418] Due to the increase in GLP2R activity, an increase in free cAMP was observed due to the decrease at a ratio of 665 / 615.

[0419] As shown in Figure 4A, neither GLP2 nor the stabilized molecule of the test subject (GLP2-2G-10Nle-1K-EX4-K5 or GLP2-2G-10Nle-1-L5A) caused any significant change in the activity value of GLP-1R compared to the changes caused by various concentrations of semaglutide, i.e., a GLP-1R agonist. IC 50When the values were calculated and listed in Table 9, these values were extremely higher than those of semaglutide which is a positive control. From this, it was recognized that an extremely high concentration of GLP2 and a long-acting GLP2R agonist are required before GLP-1R is activated.

[0420]

Table 9

[0421] As shown in Fig. 4B, for the long-acting GLP2R agonist, within the concentration range between 10 -2 ~10 2 nm, no change occurred in the GCGR activity value. However, when the glucagon concentration was increased, the activity value of GCGR was affected. The IC 50 of glucagon is 0.04, and it is recognized that this value affects the activity value of GCPR at a low concentration. However, as listed in Table 9, the IC 50 of GLP2 and the long-acting GLP2R agonist exceeded 500.

[0422] As shown in Fig. 4C, for the long-acting GLP2R agonist, within the concentration range between 10 -2 ~10 2 nm, no change occurred in the GCGR activity value. However, when the GIP concentration was increased, the activity value of GIPR was affected. The IC 50 value was calculated as listed in Table 9. The IC 50 of GIP is 0.04, and it is recognized that this value was relatively effective in affecting the activity value of GIPR. However, the IC 50 value of GLP2 and the long-acting GLP2R agonist exceeded 500.

[0423] Furthermore, GLP2-2G-1-EX4-L5A and GLP2-2G-10Nle-1K-EX4-K5 were profiled by DiscoverRx against the gpcrMAX panel. 168 GPCR targets were examined by primary screens of agonists and antagonists. This assay was performed using the PathHunter β-arrestin enzyme fragment complementarity (EFC) technology. In agonist mode, no target with activity exceeding 30% was identified except for GLP2. In antagonist mode, no target with inhibition exceeding 35% was identified.

[0424] Example G: Stability of long-acting GLP2R agonists at various temperatures In this example, the stability of GLP2R agonists stable over a long period at various temperatures was evaluated.

[0425] As shown in Figure 5A, GLP2-2G-1-EX4-L5A (GLP2-L5A) and GLP2-2G-10Nle-1K-EX4-L5A (GLP2-K5) are stable at 4°C for 4 days. As shown in Figure 5B, at 25°C, 3% oxidation was observed in GLP2-2G-1-EX4-L5A, while GLP2-2G-10Nle-1K-EX4-K5 was moderately intact for 4 days. At 37°C, 11% oxidation was observed in GLP2-2G-1-EX4-L5A, and a 4% increase was observed in the +12Da impurity on the second day. As shown in Figure 5C, the ratio of intact peptide over 4 days in GLP2-2G-10Nle-1K-EX4-K5 was higher than that in GLP2-2G-1-EX4-L5A. At 70°C (forced degradation), many racemized products were present in both peptides. As shown in Figure 5D, at 70°C for 4 days, the ratio of intact peptide was less than 50% in both GLP2-2G-1-EX4-L5A and GLP2-2G-10Nle-1K-EX4-K5.

[0426] Example H: Stability of long-acting GLP2R agonists in various solutions As listed in Table 10, the stability of the compounds in various solutions at 0 hours was measured. In both GLP2-2G-1-EX4-L5A and GLP2-2G-10Nle-1-EX4-L5A, the target compounds were not detected at 24 hours. In GLP2-2G-10Nle-1K-EX4-K5, good protection was observed in the glutathione group at 24 hours. Overall, it was recognized from this that the most stable peptide was GLP2-2G-10Nle-1-EX4-L5A, and the peptide with the least stability was GLP2-2G-1-EX4-L5A.

[0427]

Table 10

[0428] Example E: Long-Term Stability of Thioether Peptides in Liquid and Solid Forms In this example, the long-term stability of thioether peptides was tested.

[0429] The stability of the thioether peptide was measured against wet oxidation. After 10 days, Met oxidation was observed in GLP2-2G-1-EX4-L5A, and 16% decomposition was observed. GLP2-2G-10Nle-1-EX4-L5A was more stable against wet oxidation. Thus, it was recognized that the thioether bridge was stable against oxidation for at least 10 days.

[0430] The powder was stored at 4 °C as the HCl salt. After 4 months, no signs of Met oxidation were observed for GLP2-2G-1-EX4-L5A (GLP2-L5A). Similarly, after 7 months, no signs of Met oxidation were observed for GLP2-2G-1-EX4-L5A (GLP2-L5A).

[0431] Example F: Stability of Long-Acting GLP2R Agonists at Various pH Values The stability of the peptide was measured over a range of pH values and temperatures.

[0432] At pH 3.3 and room temperature, GLP2-2G-1-EX4-L5A (GLP2-L5A) was 100% stable over 4 days. As shown in Figure 6A, GLP2-2G-10Nle-1K-EX4-K5 was also stable, with 95% of the peptide remaining intact up to 5 days. As shown in Figure 6B, GLP2-2G-10Nle-1K-EX4-K5 and GLP2-2G-1-EX4-L5A (GLP2-L5A) were less stable at pH 3.3 and 37 °C compared to room temperature. At 37 °C, GLP2-2G-10Nle-1K-EX4-K5 and GLP2-2G-1-EX4-L5A (GLP2-L5A) underwent major hydrolysis at pH 3.4 (-18 Da and -775 Da). In addition, GLP2-2G-1-EX4-L5A (GLP2-L5A) was not soluble at pH 4.6.

[0433] As shown in Figure 6C, both GLP2-2G-10Nle-1K-EX4-K5 and GLP2-2G-1-EX4-L5A (GLP2-L5A) were 100% stable for 4 days at pH 7.5 and room temperature. As shown in Figure 6D, at 37 °C, GLP2-2G-10Nle-1K-EX4-K5 underwent 1-% degradation and L5A underwent 1% degradation. As shown in Figure 6E, both GLP2-2G-10Nle-1K-EX4-K5 and GLP2-2G-1-EX4-L5A (GLP2-L5A) were 100% stable for 4 days at pH 8.9 and room temperature. As shown in Figure 6F, at 37 °C and pH 8.9, GLP2-2G-10Nle-1K-EX4-K5 underwent 1-% degradation and GLP2-2G-1-EX4-L5A (GLP2-L5A) underwent 1% degradation.

[0434] Example G: Stability of long-acting GLP2R agonists in hepatocytes The hepatocyte stability of the long-acting GLP2R agonist was measured over time. As shown in Figure 7A, for GLP2-2G-1-EX4-L5A, the values in both mice and MC slightly exceeded 100% after 120 minutes. However, as shown in Figures 7B - 7C, for both GLP2-2G-10Nle-1-EX4-L5A and GLP2-2G-10Nle-1K-EX4-K5, the mouse values slightly exceeded 100%, but the MC values decreased to approximately 60% after 120 minutes.

[0435] As listed in Table 11, the biological half-life (T 1 / 2 ) and intrinsic clearance (CLint) values were calculated for each peptide from the data. The half-life in both hepatocytes and the liver of GLP2-2G-1-EX4-L5A was the highest, and the CLint was the lowest. The half-life in both hepatocytes and the liver of GLP2-2G-10Nle-1-EX4-L5A was the lowest, and the CLint value was the highest.

[0436]

Table 11

[0437] Example H: GLP2-2G-1-EX4-L5A exhibited a long in vivo half-life in mice. As shown in Figure 8, for male C57BL / 6 mice, 1.5 mg / kg of GLP2-2G-1-EX4-L5A was dissolved in PBS (pH 7.5, clear solution) and administered, and the plasma concentration of the agonist was tracked for 96 hours. An LC-MS assay with a lower limit of quantification of 20 ng / mL was used to analyze the plasma concentration. As shown in Table 12, using these values, other pharmacokinetic properties of this compound in mice administered the drug by intravenous injection and subcutaneous injection were also calculated. A long in vivo half-life of around 8.4 hours was observed, and this value was similar to the 8-hour half-life of semaglutide in rodents.

[0438]

Table 12

[0439] Example I: GLP2-2G-1-EX4-L5A exhibited a long in vivo half-life in cynomolgus monkeys. As shown in Figure 9, for male cynomolgus monkeys, 1.0 mg / kg of GLP2-2G-1-EX4-L5A was dissolved in PBS (pH 7.5, clear solution) and administered, and the plasma concentration of the agonist was tracked for 504 hours. As listed in Table 13, the pharmacokinetic properties of GLP2-2G-1-EX4-L5A in drug delivery by intravenous injection and subcutaneous injection were analyzed. An LC-MS assay with a lower limit of quantification of 10 ng / mL was used to analyze the plasma concentration. A long in vivo half-life was observed around 70 hours, and this value was longer than the 50-hour half-life of semaglutide in monkeys. It was recognized that this long in vivo half-life might potentially enable a transition to once-weekly dosing for humans.

[0440] [Table 13]

[0441] Example J: GLP2-2G-10Nle-1-EX4-L5A exhibited a long in vivo half-life in mice. For male C57BL / 6 mice, GLP2-2G-10Nle-1-EX4-L5A at a concentration of 1.5 mg / kg was dissolved in PBS (pH 7.5) and administered subcutaneously (SC) or intravenously (IV). As shown in Figure 10, the plasma concentration of the agonist was tracked for 96 hours after drug administration. An LC-MS assay with a lower limit of quantification of 5 ng / mL was used to analyze the plasma concentration. The pharmacokinetic properties including the half-life in mice were calculated from this data, and the values are listed in Table 14. When this drug was administered to mice, a long in vivo half-life of around 8 hours was observed.

[0442] [Table 14]

[0443] Example K: GLP2-2G-10Nle-1-EX4-L5A exhibited a long half-life in cynomolgus monkeys. For male cynomolgus monkeys, GLP2-2G-10Nle-1-EX4-L5A at a concentration of 1.0 mg / kg was dissolved in PBS (pH 7.5) and administered subcutaneously (SC) or intravenously (IV). As shown in Figure 11, the plasma concentration of the agonist was followed for 504 hours after drug administration. An LC-MS assay with a lower limit of quantification of 5 ng / mL was used to analyze the plasma concentration. Pharmacokinetic properties including the half-life in monkeys were calculated from this data and the values are listed in Table 15. When this drug was administered to monkeys, a long in vivo half-life of around 57 hours was observed.

[0444]

Table 15

[0445] Example L: GLP2-2G-10Nle-1K-EX4-K5 exhibited a long in vivo half-life in mice. For male C57BL / 6 mice, GLP2-2G-10Nle-1K-EX4-K5 at a concentration of 1.5 mg / kg was dissolved in PBS (pH 7.5) and administered subcutaneously (SC) or intravenously (IV). As shown in Figure 12, the plasma concentration of the agonist was followed for 72 hours after drug administration. An LC-MS assay with a lower limit of quantification of 5 ng / mL was used to analyze the plasma concentration. Pharmacokinetic properties including the half-life in mice were calculated from this data and the values are listed in Table 16. When this drug was administered to mice, a long in vivo half-life of around 7 hours was observed.

[0446]

Table 16

[0447] Example M: GLP2-2G-10Nle-1K-EX4-K5 exhibited a long half-life in cynomolgus monkeys. For male cynomolgus monkeys, GLP2-2G-10Nle-1K-EX4-K5 at a concentration of 1.0 mg / kg was dissolved in PBS (pH 7.5) and administered subcutaneously (SC) or intravenously (IV). As shown in Figure 13, the plasma concentration of the agonist was followed for 504 hours after drug administration. An LC-MS assay with a lower limit of quantification of 5 ng / mL was used to analyze the plasma concentration. As listed in Table 17, the pharmacokinetic properties including the half-life in monkeys were calculated from this data. When this drug was administered to monkeys, a long in vivo half-life of around 36 hours was observed.

[0448]

Table 17

[0449] Example N: GLP2-L5A produced an enteric effect in mice. As listed below, 13-week-old female CD1 mice were divided into five treatment groups: 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). There were five mice in each group. The relevant doses were administered subcutaneously to the mice once a day (QD) or twice a day (BID) using DPBS as a vehicle with a volume of 5 mL / kg, and then the body weight was monitored daily.

[0450] On the 10th day after administration, GI tract measurements were collected. These measurements included taking a terminal bleed, dissecting the small intestine, measuring the length and weight of the small intestine, and recording the length and weight of the empty large intestine.

[0451] As shown in Figure 14A, no significant increase was observed in the small intestine weight of treated mice (Groups C - F) administered with any long - acting GLP2R agonist at least at 0.1 mg / kg compared to untreated mice (Group A). As shown in Figure 14B, the small intestine length increased in all mice administered with the long - acting GLP2R agonist (agBonist) compared to untreated mice (Group A), and significantly increased in mice administered with 0.1 mg / kg of GLP2 - 2G - 5 - L5A (Group E). As shown in Figure 14C, no significant change in body weight was observed over 10 days in the treatment groups.

[0452] Example O: GLP2 - 2G - 10Nle - 1 - EX4 - L5A was effective in treating a mouse model of acute colitis. In this example, the enteric - acting effect of GLP2 - 2G - 10Nle - 1 - Ex4 - L5A in mice was evaluated.

[0453] Male C57B6 mice, 7 - 8 weeks old, were grouped into six 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). There were six mice in each treatment group except Group A, which had four mice. FPBS was used as the vehicle at a dose of 5 mL / kg, and the mice were administered subcutaneously once a day (QD) or twice a day (BID). Body weight was monitored daily and 10 days after dosing, and GI tract measurements were collected. These measurements included taking terminal bleeding, dissecting the small intestine, measuring the length and weight of the small intestine, and recording the length and weight of the empty colon.

[0454] As shown in FIGS. 15C to 15D, in all treatment groups (B to F), the length and weight of the small intestine were significantly increased compared to the control group. Even at a low dose of GLP2-2G-10Nle-1K-EX4-K5 (group C) such as 0.03 mg / kg, a significant effect on both measurements of the length and weight of the small intestine was observed. Furthermore, in the group D with a dose of 0.1 mg / kg of GLP2-2G-10Nle-1-EX4-K5, an effect equivalent to the administration of 0.5 mg / kg of teduglutide (group B) was observed.

[0455] In the mice administered with high doses of GLP2-2G-10Nle-1-EX4-K5 (groups E to F), the length of the colon was significantly increased compared to the untreated control (group A). In all treatment groups, the weight of the colon was significantly increased compared to the untreated control. However, the maximum increase was observed in the mice of groups E and F administered with high doses of GLP2-2G-10Nle-1-EX4-K5.

[0456] Example P: GLP2-2G-1-EX4-L5A was effective in the treatment of a mouse model of acute colitis. Acute colitis was induced in mice by a single administration of 3% dextran sulfate sodium (DSS) over a 5-day period. The mice were divided into four treatment groups as listed: A (control mice not administered DSS), B (mice administered DSS and subcutaneously injected with PBS), C (mice administered DSS and subcutaneously administered 1 mg / kg of GLP2-2G-1-EX4-L5), and D (mice administered DSS and intraperitoneally administered 20 mg / kg of cyclosporine).

[0457] As shown in Fig. 16A, body weight was measured over a 12-day period. In mice that received treatment for acute colitis (Groups C and D), body weight did not decrease as much as in untreated induced colitis mice (Group B). In addition, in mice of Group C administered with GLP2-2G-1-EX4-L5A, as shown in Figs. 16B to 16C, the weights of the colon and small intestine were significantly increased compared to untreated mice induced with acute colitis. In mice administered with GLP2-2G-1-EX4-L5A, the lengths of both the colon and small intestine were significantly increased compared to untreated controls induced with acute colitis (figure not shown). Treatment with GLP2-2G-1-EX4-L5A also improved the histopathology of the colon and small intestine in the DSS-induced colitis model. As shown in Fig. 16D, the histopathology of mice administered both DSS and L5A showed a colon crypt depth similar to that of mice not receiving DSS treatment, while mice administered only DSS showed a significant decrease in crypt length. As shown in Fig. 16E, in the jejunum of the small intestine, the length of the jejunal villi was longer in mice of Group C than in mice of Group A or Group B. A significant increase in the length of the jejunal villi was observed when comparing mice treated with GLP2-2G-1-EX4-L5A and untreated mice. In addition, mice of Group C did not show the villus deformation and abscesses presented by mice of Group B.

[0458] Example Q: GLP2-2G-10Nle-1K-EX4-K5 was effective in the treatment of a mouse model of acute colitis. As listed below, 8-week-old male C57BL / 6 mice were divided into seven treatment groups: A (control mice not administered DSS), B (mice administered DSS and subcutaneously injected with PBS), C (mice administered DSS and subcutaneously administered 0.1 mg / kg of GLP2-2G-10Nle-1K-EX4-K5), D (mice administered DSS and subcutaneously administered 0.3 mg / kg of GLP2-2G-10Nle-1K-EX4-K5), E (mice administered DSS and subcutaneously administered 1 mg / kg of GLP2-2G-10Nle-1K-EX4-K5), F (mice administered DSS and subcutaneously administered 0.5 mg / kg of teduglutide), and G (mice administered DSS and intraperitoneally administered 20 mg / kg of cyclosporine). There were six mice in each group except group A, which had four mice. As shown in Figure 17A, acute colitis was induced in the mice by a single administration of 3% dextran sulfate sodium (DSS) over a 5-day period. An appropriate amount was administered to the animals once a day for 11 days for each treatment group. Body weight was monitored daily. If the body weight decreased by more than 20%, the animal was sacrificed. On days 10 - 11, samples were collected at 0, 1, 3, 7, and 24 hours after administration for pharmacokinetic analysis. On day 11, the animals were sacrificed and necropsied. Terminal bleeding was collected in heparinized collection tubes and processed into plasma. The small intestine and colon were collected and their weights and lengths were measured. GI tissue was collected for histological examination.

[0459] As shown in Figure 17A, when examining the body weight over time, it was observed that in the mice of group G administered cyclosporine, the rate of body weight decrease during DSS treatment was higher than that of the mice in other treatment groups, but the body weight increased after the end of DSS treatment. In the mice administered DSS without treatment (group D), the rate of body weight decrease after the end of DSS treatment was the highest compared to all other treatment groups. In the mice administered GLP2-2G-10Nle-1K-EX4-K5 (groups C - E) or teduglutide (group F), no significant change in body weight was observed during this time, together with the mice not administered DSS (group A).

[0460] Treatment with GLP2-2G-10Nle-1K-EX4-K5 prevented weight loss and restored colonic shortening in mice. As shown in Figure 17B, colonic length was significantly increased in mice administered high doses of GLP2-2G-10Nle-1K-EX4-K5 or teduglutide (Groups D - F) compared to the untreated group (Group B). There was no change in colonic weight. As shown in Figures 17C - 17D, both the length and weight of the small intestine were significantly increased in the groups administered teduglutide or GLP2-2G-10Nle-1K-EX4-K5 (Groups C - F) compared to untreated mice. The lowest dose of GLP2-2G-10Nle-1K-EX4-K5 at 0.1 mg / ml (Group C) showed an effect equivalent to that of teduglutide at 0.5 mg / kg BID.

[0461] Treatment with GLP2-2G-10Nle-1K-EX4-K5 also affected the histological characteristics of the intestinal tract. As seen in Figure 17E, a significant increase in villus height was observed in all teduglutide and GLP2-2G-10Nle-1K-EX4-K5 treatment groups (Groups C - F) compared to untreated mice (Groups A - B). Administration of a low dose of GLP2-2G-10Nle-1K-EX4-K5 at 0.1 mg / kg once daily showed an effect equivalent to that of administering teduglutide at 0.5 mg / kg twice daily. Furthermore, as shown in Figure 17F, no increase in proliferation was observed in any of the treatment groups by Ki67 staining. Thus, no evidence of abnormal proliferation associated with treatment with GLP2-2G-10Nle-1K-EX4-K5 was found.

[0462] The pharmacokinetic properties of treatment groups C - F are plotted in Figure 17G. Teduglutide could not be detected 3 hours after treatment. However, GLP2-2G-10Nle-1K-EX4-K5 was detectable up to 24 hours after treatment for all administrations.

[0463] Example R: Long-acting GLP2R agonists were effective in the treatment of acute colitis. Mice were divided into nine treatment groups as follows: A (non-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 contained six 8-week-old male C57BL / 6 mice. Mice were administered 3% DSS for 7 days and simultaneously appropriate treatment for 8 days to induce acute colitis. All animals except group I were administered a vehicle of 5 ml / kg volume and DPBS subcutaneously, and in group I the vehicle was olive oil. Pharmacokinetic samples were collected from groups C - H at 0, 1, 3, 7, and 24 hours post-administration on days 6 - 7. Measurements were taken on day 9. These measurements included collecting terminal bleeding, dissecting the small intestine, measuring the length and weight of the small intestine, and recording the length and weight of the empty large intestine.

[0464] All long-acting GLP-2 agonists showed dose-related protection against weight loss compared to untreated animals. This protection was significant at a dose of 0.1 mg / kg. As shown in Figure 18A, the total body weight of animals treated with any dose of GLP2-2G-10Nle-1-Ex4-L5A (groups C, D) was greater than that of untreated animals (group B). As shown in Figure 18B, the total body weight of animals treated with any dose of GLP2-2G-1-EX4-L5A (groups E, F) was greater than that of untreated animals (group B). As shown in Figure 18C, the total body weight of animals treated with any dose of GLP2-2G-10Nle-1K-Ex4-K5A (groups G, H) was greater than that of untreated animals (group B). Furthermore, 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.

[0465] As shown in Figure 18D, all three long-acting GLP-2 agonists significantly increased the length of the colon in an acute DSS-induced colitis model at 0.1 mg / kg. Furthermore, as shown in Figure 18E, a non-significant increasing trend in the weight of the colon was observed when comparing animals administered long-acting GLP2 agonists with untreated DSS animals. Furthermore, as shown in Figures 18F - 18G, all three long-acting GLP2 agonists exhibited dose-related trophic effects on both the weight and length of the small intestine.

[0466] As shown in Figure 18H, treatment with a GLP2R agonist also increased the size of the gallbladder. In addition, as shown in Figure 18I, the fecal occult blood volume was measured using the Hemoccult II test. Various treatment parameters of 1L5A, such as both doses of 0.03 mg / kg and 0.1 mg / kg, decreased the Hemoccult value compared to untreated DSS model mice.

[0467] The levels of the long-acting GLP2 agonist were measured over time. As shown in Fig. 18J, at a dose of 0.03 mg / kg, the concentration increased until 7 hours after administration and was still detectable even 24 hours after administration. As shown in Fig. 18K, at a dose of 0.1 mg / kg, the concentration increased until 7 hours after administration and was still detectable even 24 hours after administration. At both doses of 0.03 mg / kg and 0.1 mg / kg, the levels in GLP2-2G-1-EX4-L5A were the highest, followed by GLP2-2G-10Nle-1-Ex4-L5A and GLP2-2G-10Nle-1K-Ex4-K5 in that order. As shown in Figs. 18L to 18N, in all three drugs tested in the subjects, administration at higher doses resulted in the drug being present at higher concentrations at all test time points.

[0468] A significant decrease in the mRNA value in inflammatory cytokines was observed in the colon tissue.

[0469] Example S: GLP2-2G-1-EX4-L5A was effective in the treatment of chronic colitis. 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, followed by 7 days of recovery for 3 cycles. During the last DSS induction cycle, animals were treated daily for 7 days. The therapeutic agents were administered once daily (QD) or twice daily (BID), subcutaneously (S) or intraperitoneally (IP). Mice were treated according to the treatment groups listed: A (non-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)). Body weight was monitored three times a week. Pharmacokinetic sampling was performed 3 - 4 days before necropsy. On day 33, necropsy was performed and measurements were taken. These measurements included collecting terminal bleeding, dissecting the small intestine, measuring the length and weight of the small intestine, and recording the length and weight of the empty colon.

[0470] GLP2-2G-1-EX4-L5A was effective in treating weight loss in a mouse model of chronic colitis. As shown in Figure 19A, mice treated with GLP2-2G-1-EX4-L5A or teduglutide (groups C, D, F) did not show the same percentage of weight loss or total body weight loss as that seen in untreated mice (group B). The protection against weight loss was dose-dependent, with increased protection at higher doses. In addition, these effects were equivalent to the administration of teduglutide 0.3 mg / kg, QD.

[0471] As shown in Figure 19B, in GLP2-2G-1-EX4-L5A, the colon length recovered dose-dependently compared to untreated mice. Furthermore, when comparing treatment with 0.3 mg / kg of GLP2-2G-1-EX4-L5A to an equivalent amount of teduglutide, the effect of GLP2-2G-1-EX4-L5A was less variable than that of teduglutide treatment. As seen in Figure 19C, the colon weight was also affected by treatment with GLP2-2G-1-EX4-L5A and teduglutide. The colon weight of mice treated with low-dose GLP2-2G-1-EX4-L5A was similar to that of mice treated with teduglutide.

[0472] As shown in Figure 19D, at higher doses of GLP2-2G-1-EX4-L5A, a significant increase in small intestine weight was observed compared to untreated mice. This effect was equivalent to that produced by teduglutide.

[0473] Example T: GLP2-2G-10Nle-1-EX4-L5A was effective in treating a mouse model of chronic colitis. 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, followed by 3 cycles of 7-day recovery. During the last DSS induction cycle, the animals were treated daily for 7 days. The therapeutic agents were administered subcutaneously (S) or intraperitoneally (IP) once daily (QD) or twice daily (BID). Mice were treated according to the treatment groups listed: A (non-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)).

[0474] Body weight was monitored three times a week. Pharmacokinetic sampling was performed for 3 - 4 days before necropsy. On day 33, necropsy was performed and measurements were taken. These measurements included collecting terminal bleeding, dissecting the small intestine, measuring the length and weight of the small intestine, and recording the length and weight of the empty large intestine.

[0475] A moderate effect on weight loss was observed with low - dose GLP2 - 2G - 10Nle - 1 - Ex4 - L5A. Treatment at doses of 0.03 mg / kg and 0.3 mg / kg was protective against weight loss compared to mice that did not receive treatment (figure not shown).

[0476] As shown in Figures 20A - 20B, GLP2 - 2G - 10Nle - 1 - Ex4 - L5A increased both the length and weight of the colon in a chronic DSS - induced colitis model. The colon length was significantly increased in animals treated with GLP2 - 2G - 10Nle - 1 - Ex4 - L5A at doses of 0.1 mg / kg or higher, as well as in animals treated with teduglutide (groups D - G), compared to untreated animals (group B). The colon weight increased in animals treated with GLP2 - 2G - 10Nle - 1 - Ex4 - L5A at doses of 0.3 mg / kg or higher, as well as in animals treated with teduglutide, compared to untreated animals.

[0477] GLP2 - 2G - 10Nle - 1 - Ex4 - L5A also had a significant effect on the weight and length of the small intestine. As shown in Figure 20C, in addition to treatment with GLP2 - 2G - 10Nle - 1 - Ex4 - L5A at doses of 0.1 mg / kg or higher, treatment with teduglutide significantly increased the length of the small intestine compared to untreated animals. Treatment with GLP2 - 2G - 10Nle - 1 - Ex4 - L5A at doses of 0.3 mg / kg or higher significantly increased the weight of the small intestine compared to untreated mice (not shown).

[0478] Example U: GLP - 2 - 2G - 5 - L5A Treatment in the NASH Model Five-week-old C57BL / 6 mice were fed a choline-deficient diet (CDAA, Dyets#518753) or an AA-supplemented control diet (CSAA, Dyets#518754) for a total of 19 weeks. The mice were divided into three treatment groups as listed, with 8 mice in each group: CSAA control diet, treated with vehicle only; CDAA diet, treated with vehicle (MCT, PO; saline, SC); and CDAA diet, treated with 1 mg / kg of GLP2-2G-5-EX4-L5A subcutaneously. After 15 weeks, the mice were treated with vehicle or compound for 4 weeks. Body weight was monitored weekly during the dietary induction phase and three times a week during the treatment phase. After 19 weeks, the animals were sacrificed and terminal blood and liver samples were collected for serum panels, tissue architecture, and gene expression.

[0479] Long-term treatment with GLP2-2G-5-EX4-L5A improved markers of liver function. As shown in FIGS. 21A-21B, in mice fed a choline-deficient diet, both serum ALT and serum AST were significantly decreased compared to untreated mice fed the same diet. Total serum bilirubin was also decreased in mice treated with GLP2-2G-5-EX4-L5A compared to untreated mice fed the same diet. The gallbladder was enlarged in 7 / 8 of the mice treated with GLP-2. As shown in FIG. 21C, as a result of treatment with GLP2-2G-5-EX4-L5A, the liver fibrosis score was decreased by 20%. Collagen accumulation / fibrosis was observed with picrosirius red, and severity was graded using the following scale: 0 = absent, 1 = minimal, 2 = mild, 3 = moderate, 4 = marked, 5 = severe. No significant effect on body weight was observed with this treatment, indicating that the treatment was tolerated.

[0480] The effects of this treatment on fatty liver and inflammation were also analyzed. Steatosis was analyzed by the percentage of hepatocyte vacuolization by crisp, round, and non-stained lipid vacuoles, and grades were assigned based on the scale listed below: 0 indicates less than 5%, 1 indicates 5 - 33%, 2 indicates 33 - 66%, and 3 indicates more than 66%. As shown in Figure 21D, treatment with GLP2-2G-5-EX4-L5A did not significantly affect the grade of hepatic steatosis. Lobular inflammation was analyzed by the assessment of inflammatory foci in the infiltration of neutrophils, lymphocytes, and macrophages. Lobular inflammation was scored using the following scale: 0 indicates no foci, 1 indicates 2 foci / 200x field, 2 indicates 2 - 4 foci / 200x field, and 3 indicates more than 4 foci / 200x field. As shown in Figure 21E, treatment with GLP-2-2G-5-L5A decreased the level of lobular inflammation compared to untreated animals fed a CDAA diet. In this example, it was observed that treatment with GLP2-2G-5-EX4-L5A improved markers of liver injury and prevented the exacerbation of hepatic fibrosis in the CDAA-NASH model.

[0481] Example V: Long-acting GLP2 agonists treat a mouse model of environmental enteric dysfunction (EED). Using a weaned malnourished model, the ability of GLP2-2G-10Nle-1K-EX4-K5 to treat environmental enteric dysfunction (EED) was evaluated. All dams were fed an isocaloric Northeast Brazil (Regional Basic Diet - RBD), and when their offspring reached 10 days of age, they were moderately deficient in protein, fat, and minerals. At weaning (3 weeks of age), the offspring were either fed a standard control diet (CD) or continued on the RBD. At 4 weeks of age, the weaned animals were administered a drug or placebo (formulated at 0.1 mg / kg in PBS (vehicle)) subcutaneously once a day for 2 - 3 weeks. Body weight and food consumption were measured twice a week. Feces were collected at weaning, 6 weeks of age, and 8 weeks of age for calorimetry and microbiome analysis. Oral FITC-dextran was used as a measure of barrier function. At 6 weeks of age, the mice were sacrificed and jejunal tissue was collected for morphological examination, immunohistochemical examination, and chamber analysis of oral mucosal resistance and permeability. As shown in Figures 22A - 22B, both male and female RBD mice that switched to CD after weaning and were treated with either teduglutide or GLP2-2G-10Nle-1K-EX4-K5 showed a tendency for weight gain. However, as shown in Figures 22C - 22D, male and female mice that switched to RBD after weaning showed a tendency for weight loss when administered teduglutide or a long-acting GLP2 agonist.

[0482] Treatment with both teduglutide and the long-acting GLP2 agonist had a significant effect on the wet weight and length of the intestine. As shown in Figure 22E, in male C57BL / 6J mice treated with either teduglutide or GLP2-2G-10Nle-1K-EX4-K5, a significant increase in the wet weight of the small intestine per body weight was observed compared to untreated males. As shown in Figure 22F, in female C57BL / 6J mice treated with either teduglutide or GLP2-2G-10Nle-1K-EX4-K5, a significant increase in the wet weight of the small intestine per body weight was observed compared to untreated females. Furthermore, as a result of treatment with GLP2-2G-10Nle-1K-EX4-K5, a significant increase was also observed compared to treatment with teduglutide. In male BALB / c mice treated with either teduglutide or GLP2-2G-10Nle-1K-EX4-K5, a significant increase in the wet weight of the small intestine per body weight was observed compared to untreated males. In female BALB / c mice, a significant increase in the wet weight of the small intestine per body weight was observed only in animals treated with GLP2-2G-10Nle-1K-EX4-K5 compared to untreated animals. In both animals switched to a C57BL / 6J diet after weaning and animals switched to a BALB / c diet after weaning, as a result of treatment with GLP2 or teduglutide, a significant increase in the length of the small intestine was observed compared to untreated animals.

[0483] Treatment with GLP2-2G-10Nle-1K-EX4-K5 also had an enterotropic effect on the animals. In male C57BL / 6J mice treated with either teduglutide or GLP2-2G-10Nle-1K-EX4-K5, the villus height was significantly longer than that of untreated males. In female C57BL / 6J mice treated with GLP2-2G-10Nle-1K-EX4-K5, the villus length was also significantly longer than that of untreated females. Regarding the crypt depth in treated and untreated C57BL / 6J animals, in males treated with either teduglutide or GLP2-2G-10Nle-1K-EX4-K5, the crypt depth was longer than that of untreated males.

[0484] Intestinal permeability was also measured in these mice, and intestinal permeability increased as the relative fluorescence of FITC-dextran increased. In male CD mice treated with either teduglutide or GLP2-2G-10Nle-1K-EX4-K5, a tendency for decreased permeability was observed in the treated mice compared to the untreated mice. In female CD mice treated with either teduglutide or GLP2-2G-10Nle-1K-EX4-K5, a tendency for decreased permeability was observed compared to untreated female CD mice. Treatment with either teduglutide or GLP2-2G-10Nle-1K-EX4-K5 had no effect on permeability in female RBD or male RBD compared to untreated mice.

[0485] In untreated CD mice, the level of intestinal permeability was found to be higher than that in untreated RBD mice. The levels of permeability in CD and RBD mice were overall similar when treated with teduglutide. In female mice, the level of permeability was slightly higher when treated with GLP2-2G-10Nle-1K-EX4-K5 compared to male mice fed the same diet.

Claims

1. a) A peptide that regulates the GLP-2 receptor, comprising an amino acid sequence selected from the group consisting of SEQ ID NOs: 1-9 and 21-29, or an amino acid sequence having one amino acid addition, deletion, or substitution as compared to a sequence selected from the group consisting of SEQ ID NOs: 1-9 and 21-29, and b) A staple that binds to the peptide at a first cysteine and a second cysteine, wherein the first cysteine has the i-position in the peptide, the second cysteine has the (i + 7)-position in the peptide, and the staple is 【Chemical 1】 wherein in the formula, n is from 1 to 4, m is from 6 to 20, each "S" is the sulfur atom of the first cysteine or the second cysteine, a staple and a peptide conjugate comprising the same.

2. The staple of the peptide conjugate according to claim 1, wherein the staple is [Chemical Formula 2] as described.

3. The staple of the peptide conjugate according to claim 1, wherein the staple is [Chemical Formula 3] as described.

4. a) A peptide that regulates the GLP-2 receptor, comprising any one of SEQ ID NOs: 1-3 and 21-23, and b) A staple that binds to the peptide at a first cysteine and a second cysteine, having the following structure 【Chemical Formula 4】 and each "S" is the sulfur atom of the first cysteine or the second cysteine, a staple and a peptide conjugate comprising the same.

5. a) A peptide that regulates the GLP-2 receptor, comprising SEQ ID NO: 1, and b) A staple that binds to the peptide at a first cysteine and a second cysteine, wherein the staple has the following structure [Chemical Formula 5] and each "S" is the sulfur atom of the first cysteine or the second cysteine, a staple and a peptide conjugate comprising the same.

6. a) A peptide that regulates the GLP-2 receptor, comprising SEQ ID NO: 2 or 22, and b) A staple that binds to the peptide at a first cysteine and a second cysteine, having the following structure [Chemical Formula 6] and each "S" is the sulfur atom of the first cysteine or the second cysteine, a staple and a peptide conjugate comprising the same.

7. a) A peptide that regulates the GLP-2 receptor, comprising SEQ ID NO: 30 or 10, wherein the amino acid at the 10th position is L-norleucine, and b) A staple that binds to the peptide at a first lysine and a second lysine, having the following structure 【Chemical Formula 7】 having, each "NH" being an amine of said first lysine or said second lysine, a staple, and a peptide conjugate comprising.

8. A pharmaceutical composition comprising the peptide conjugate according to any one of claims 1 to 7 and a pharmaceutically acceptable excipient.

9. Use of the peptide conjugate according to any one of claims 1 to 7 in the manufacture of a medicament for use in the treatment of a disease or disorder in a subject in need thereof.

10. Use according to claim 9, wherein said disease or disorder is diabetes or obesity, or a medical condition associated with diabetes or obesity.

11. Use according to claim 9, wherein said disease or disorder is non-alcoholic fatty liver disease (NAFLD), non-alcoholic steatohepatitis (NASH), or a cardiovascular disease.

12. Use according to claim 9, wherein said disease or disorder is a gastrointestinal (GI) disorder selected from short bowel syndrome (SBS), irritable bowel syndrome (IBS), or inflammatory bowel disease (IBD).

13. Use according to claim 12, wherein said inflammatory bowel disease (IBD) is Crohn's disease or ulcerative colitis.

14. Use according to claim 9, wherein said disease or disorder is psoriasis.

15. Use according to claim 9, wherein said disease or disorder is Alzheimer's disease, Parkinson's disease, or Huntington's disease.

16. Use of the peptide conjugate according to any one of claims 1 to 7 in the manufacture of a medicament for use in the treatment of radiation-induced GI mucositis, chemotherapy-induced diarrhea (CID), or total parenteral nutrition (TPN)-induced intestinal atrophy.

Images