US20140162943A1

US20140162943A1 - N-terminus conformationally constrained glp-1 receptor agonist compounds

Info

Publication number: US20140162943A1
Application number: US14/165,705
Authority: US
Inventors: Josue Alfaro-Lopez; Abhinandini Sharma; Soumitra S. Ghosh
Original assignee: Amylin Pharmaceuticals LLC; AstraZeneca Pharmaceuticals LP
Current assignee: Amylin Pharmaceuticals LLC; AstraZeneca Pharmaceuticals LP
Priority date: 2009-04-01
Filing date: 2014-01-28
Publication date: 2014-06-12
Also published as: US20120046222A1; WO2010120476A2; EP2413955A2; WO2010120476A3; US8642544B2; EP2413955A4

Abstract

The disclosure provides N-terminus conformationally constrained compounds, which may comprise peptide mimetics and/or amino acid substitutions, which may be used in peptides, such as GLP-1 receptor agonist compounds, to induce β-turn secondary structure at the N-terminus. The N-terminus conformationally constrained compounds may be used for research purposes; to produce GLP-1 receptor agonist compounds having improved GLP-1 receptor binding activity, enzymatic stability, or in vivo glucose lowering activity; and to develop GLP-1 receptor agonist compounds which have fewer amino acid residues. The disclosure also provides GLP-1 receptor agonist compounds, such as exendins, exendin analogs, GLP-1(7-37), GLP-1(7-37) analogs, comprising the N-terminus conformationally constrained compounds. The compounds are useful for treating various diseases, such as diabetes and obesity. The disclosure also provides methods for chemically synthesizing the N-terminus conformationally constrained compounds.

Description

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a divisional of U.S. application Ser. No. 13/260,702, filed on Sep. 27, 2011, which is a U.S. national stage of International Application No. PCT/US2010/28883, filed Mar. 26, 2010, which claims priority to U.S. Application No. 61/165,604 filed Apr. 1, 2009.

FIELD

Provided herein are N-terminus conformationally constrained GLP-1 receptor agonist compounds and therapeutic methods for their use.

BACKGROUND

Peptides and proteins play critical roles in the regulation of biological processes. Peptides, for example, play a regulatory role as hormones and inhibitors, and are also involved in immunological recognition. The significant biological role of peptides makes it important to understand their interactions with the receptors to which they bind.
The determination of the receptor-bound conformation of a peptide is invaluable for the rational design of peptide analogs. Marshall et al, Ann. Rep. Med. Chem., 13:227-238 (1978) disclose that peptides are characteristically highly flexible molecules, the structures of which are strongly influenced by the environment in which they reside. Thus, peptides are not generally useful for determining their receptor-bound conformation.
As no approach is available to predict which new ligand-receptor interactions will lead to antagonists and which will lead to agonists of greater or less potency, it is necessary to perform classical structure-function studies in a systematic way to provide information about the specific amino acid residues and functional groups in a peptide that are important to biological activity. Studies of this nature can utilize conformationally constrained peptide mimetics. For example, Hruby, Trends Pharmacol. Sci., 8:336-339 (1987) suggests that conformational constraints can provide information about the different requirements that a receptor has for a ligand to be an agonist or antagonist.
Generally, peptide mimetics can be defined as structures which serve as appropriate substitutes for peptides in interactions with receptors and enzymes. The development of rational approaches for discovering peptide mimetics is a major goal of medicinal chemistry. Such development has been attempted both by empirical screening approaches and by specific synthetic design. Specific design of peptide mimetics has utilized both peptide backbone modifications and chemical mimics of peptide secondary structures. The beta-turn has been implicated as an important site for molecular recognition in many biologically active peptides. Consequently, peptides containing conformationally constrained mimetics of beta-turns are particularly desirable.
There is a need in the art for new GLP-1 receptor agonist compounds that have good stability, resistance to degradation, and good glucagon-like peptide-1 (GLP-1) receptor binding activity and in vivo glucose lowering activity. To solve these needs, the disclosure herein provides, among other things, novel N-terminus conformationally constrained compounds, novel N-terminus conformationally constrained GLP-1 receptor agonist compounds containing modifications, such as peptide mimetics and/or amino acid substitutions, that provide a conformationally constrained N-terminus that results in improved GLP-1 receptor binding and in vivo blood glucose lowering activity.

SUMMARY

It was previously believed that the N-terminus of exendin-4 and exendin analogs was a random coil. It has now been unexpectedly discovered that the N-terminus shows a high beta-turn characteristic in a specific site, and therefore mimics the receptor bound conformation of this region of the peptides. The disclosure herein is based on this discovery.
Provided herein are N-terminus conformationally constrained compounds having the formula: Xaa₁Xaa₂Xaa₃-Z and Xaa₁Xaa₂Xaa₃Xaa₄-Z, where the substituents are defined herein. These N-terminus conformationally constrained compounds may induce a β-turn conformational constraint at the N-terminus when they are used in GLP-1 receptor agonist compounds. The N-terminus conformationally constrained compounds may be used for therapeutic purposes (e.g., treat diabetes); for research purposes; and to produce GLP-1 receptor agonist compounds having improved GLP-1 receptor binding activity, enzymatic stability, and improved in vivo glucose lowering activity. The disclosure provides pharmaceutical compositions comprising therapeutically effective amounts of the N-terminus conformationally constrained compounds. The disclosure also provides methods for synthesizing the N-terminus conformationally constrained compounds.
Provided herein are GLP-1 receptor agonist compounds, such as exendins, exendin analogs, GLP-1(7-37) (SEQ ID NO: 84), and GLP-1(7-37) analogs, comprising an N-terminus conformationally constrained compound having the formula Xaa₁Xaa₂Xaa₃- or Xaa₁Xaa₂Xaa₃Xaa₄-, where the substituents are defined herein. In one embodiment, the GLP-1 receptor agonist compounds comprise Xaa₁Xaa₂Xaa₃Xaa₄, where the substituents are defined herein, at positions 1-4 at the N-terminus. In one embodiment, the GLP-1 receptor agonist compounds comprise Xaa₁Xaa₂Xaa₃, where the substituents are defined herein, at positions 1-3 at the N-terminus. The disclosure provides pharmaceutical compositions comprising therapeutically effective amounts of these N-terminus conformationally-constrained GLP-1 receptor agonist compounds.
Provided herein are exendins and exendin analogs having the formula:

(SEQ ID NO: 1)

Xaa₁Xaa₂Xaa₃Xaa₄TFTSDLSKQXaa₁₄EEEAVRLFIEXaa₂₅LKN-Z;

(SEQ ID NO: 2)

Xaa₁Xaa₂Xaa₃Xaa₄TFTSDLSKQXaa₁₄EEEAVRLFIEXaa₂₅LK-R₁₀-Z;

(SEQ ID NO: 3)

Xaa₁Xaa₂Xaa₃Xaa₄TFTSDLSKQXaa₁₄EEEAVRLFIEXaa₂₅

LKNGGPSSGAPPPS-Z;

where Xaa₁₄, Xaa₂₅, R₁₀, and Z are defined herein; and at least one of Xaa₁, Xaa₂, Xaa₃, and Xaa₄are modifications, such as peptide mimetics and/or amino acid substitutions, that induce a conformational constraint at the N-terminus. The disclosure provides pharmaceutical compositions comprising therapeutically effective amounts of these exendin analogs.

Provided herein are GLP-1(7-37) (SEQ ID NO: 84) and GLP-1(7-37) analogs having the formula:
(SEQ ID NO: 4)

Xaa₁Xaa₂Xaa₃Xaa₄TFTSDVSSYXaa₁₄EGQAAKEFIAXaa₂₅

LVXaa₂₈GRXaa₃₁-Z;

where Xaa₁₄, Xaa₂₅, Xaa₂₈, Xaa₃₁, and Z are as defined herein; and at least one of Xaa₁, Xaa₂, Xaa₃, and Xaa₄are modifications, such as peptide mimetics and/or amino acid substitutions, that induce a conformational constraint at the N-terminus. The disclosure provides pharmaceutical compositions comprising therapeutically effective amounts of these GLP-1(7-37) analogs.
Provided herein are GLP-1 receptor agonist compounds, such as exendins, exendin analogs, GLP-1(7-37) (SEQ ID NO: 84), and GLP-1(7-37) analogs wherein position 1 comprises an imidazole ring (e.g., His) and position 3 is proline; where the GLP-1 receptor agonist compounds bind in a RIN cell membrane receptor binding assay with an affinity of less than 1 nM (or less than 0.1 nM).
The disclosure provides methods for treating diabetes; treating insulin resistance; treating postprandial hyperglycemia; lowering blood glucose levels; lowering HbA1c levels; stimulating insulin release; reducing gastric motility; delaying gastric emptying; reducing food intake; reducing appetite; reducing weight; treating overweight; and treating obesity in patients in need by administering therapeutically effective amounts of the N-terminus conformationally constrained compounds and/or the N-terminus conformationally constrained GLP-1 receptor agonist compounds described herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1: FIG. 1A is exendin-4 amide (SEQ ID NO: 86); FIG. 1B is dAla²,Pro³-exendin-4 amide, and FIG. 1C is dAla²,Pro³,Leu¹⁴,Phe²⁵-exendin-4 (1-28) amide. FIG. 1D is a graph showing the change in blood glucose in mice administered the compounds shown in FIGS. 1A-C based on the in vivo blood glucose assay described in Example 17. The compounds were injected IP at t=0 immediately following a baseline sample in 2-hour fasted NIH/Swiss mice. Blood glucose samples were taken at t=30, 60, 120, 180, and 240 minutes with a ONETOUCH® ULTRA® (LifeScan, Inc., Milpitas, Calif.).

FIG. 2: FIG. 2A is an exendin analog (SEQ ID NO: 87), described, e.g., in WO 2007/139941. FIGS. 2B-J show the exendin analog of FIG. 2A having a modification at Glu³. R₁is GTFTSDLSKQLEEEAVRLFIEWLKQGGPSKEIIS-NH₂(SEQ ID NO: 5). FIG. 2K shows the exendin analog (SEQ ID NO: 5) of FIG. 2A having modifications at Gly²Glu³. FIGS. 2L-2U show N-terminus conformationally constrained compounds. FIGS. 2B-J provide examples of exendin analogs containing the N-terminus conformationally constrained compounds shown in FIGS. 2L-2U.

FIGS. 3A-G are dAla²,Pro³-exendin-4 (FIG. 3A), Ala²,Pro³-exendin-4 (SEQ ID NO: 88) (FIG. 3B), Pro³,Ala⁴-exendin-4 (SEQ ID NO: 89) (FIG. 3C), Ala²,Pro³,Ala⁴-exendin-4 (SEQ ID NO: 90) (FIG. 3D), Pro³,dAla⁴-exendin-4 (FIG. 3E), Val²,Pro³-exendin-4 (SEQ ID NO: 91) (FIG. 3F), and NMeAla²,Pro³-exendin-4 (SEQ ID NO: 92) (FIG. 3G). The compound in FIGS. 1B and 3A are the same.

FIG. 4 is a graph showing the change in blood glucose in mice administered the compounds shown in FIGS. 3G, 2I, and 2B based on the in vivo blood glucose assay described in Example 17. The compounds were injected IP at t=0 immediately following a baseline sample in 2-hour fasted NIH/Swiss mice. Blood glucose samples were taken at t=30, 60, 120, 180, and 240 minutes with a ONETOUCH® ULTRA® (LifeScan, Inc., Milpitas, Calif.).

FIG. 5: FIG. 5A is Leu¹⁴,Phe²⁵-exendin-4(1-28) (SEQ ID NO: 93), described in WO 2007/139941. Leu¹⁴,Phe²⁵-exendin-4(1-28) (SEQ ID NO: 93) refers to amino acid residues 1-28 in exendin-4 (i.e., exendin-4(1-28) (SEQ ID NO: 99), where the amino acid residue at position 14 in exendin-4 is replaced with Leu (i.e., Leu¹⁴), and the amino acid residue at position 25 is replaced with Phe (i.e., Phe²⁵). FIGS. 5B-G show the exendin analog of FIG. 5A having a modification at Glu³or Gly²Glu³at the N-terminus. R₄is GTFTSDLSKQLEEEAVRLFIEFLKN-NH₂(SEQ ID NO: 6).

FIGS. 6A-E show the exendin analog of FIG. 5A having a modification at Gly²Glu³or Gly²Glu³Gly⁴at the N-terminus. The C-terminal amino acid in each compound shown in FIGS. 6A-E is amidated. FIGS. 6A through 6D disclose SEQ ID NOS 94 and 94-96, respectively.

FIG. 7 is a graph showing the change in blood glucose in mice administered the compounds shown in FIGS. 6A, 8B, 6E, 1C, 5B, and 5A based on the in vivo blood glucose assay described in Example 17. The compounds were injected IP at t=0 immediately following a baseline sample in 2-hour fasted NIH/Swiss mice. Blood glucose samples were taken at t=30, 60, 120, 180, and 240 minutes with a ONETOUCH® ULTRA® (LifeScan, Inc., Milpitas, Calif.).

FIG. 8: FIGS. 8A-D show the exendin analog in FIG. 5A having modifications at Gly²Glu³Gly⁴at the N-terminus, where the C-terminal amino acid is amidated. FIGS. 8E-H show the exendin analog in FIG. 2A having modifications at Gly²Glu³Gly⁴at the N-terminus. FIGS. 8A and 8E disclose SEQ ID NOS 97-98, respectively.

FIG. 9: FIGS. 9A-H show the exendin analog in FIG. 5A having a modification at Gly²Glu³or Gly²Glu³Gly⁴at the N-terminus. Each compound in FIGS. 9A-H is amidated at the C-terminal amino acid. FIGS. 9A through 9H disclose

SEQ ID NOS

6, 6, 6, 6, 100, 6, 6 and 6, respectively. FIG. 9I is a graph showing the change in blood glucose in mice administered the compounds shown in FIGS. 9B, 9C, 9D, 9F, 5A, and 1C based on the in vivo blood glucose assay described in Example 17. The compounds were injected IP at t=0 immediately following a baseline sample in 2-hour fasted NIH/Swiss mice. Blood glucose samples were taken at t=30, 60, 120, 180, and 240 minutes with a ONETOUCH® ULTRA® (LifeScan, Inc., Milpitas, Calif.).

FIGS. 10A-I show the exendin analog in FIG. 5A having a modification at His¹at the N-terminus. R₄is Xaa₂Xaa₃GTFTSDLSKQLEEEAVRLFIEFLKN-NH₂(SEQ ID NO: 7), where Xaa₂is Gly, dAla, or Aib; and Xaa₃is Glu or Pro. Alternatively, R₄is Xaa₂Xaa₃GTFTSDLSKQLEEEA VRLFIEWLKNGGPSSGAPPPS-NH₂(SEQ ID NO: 8), where Xaa₂is Gly, dAla, or Aib; and Xaa₃is Glu or Pro; provided that Xaa₃is not Glu when Xaa₂is Gly, or Xaa₂is not Gly when Xaa₃is Glu. Alternatively, R₄is Xaa₂Xaa₃GTFTSDLSKQLEEEAVRLFIEWLKQGGPSKEIIS-OH (SEQ ID NO: 9), where Xaa₂is Gly, dAla, or Aib; and Xaa₃is Glu or Pro.

FIGS. 11A-B show exendin-4 of FIG. 1A having a modification at His¹at the N-terminus. R₂is GEGTFTSDLSKQLEEEAVRLFIEWLKNGGPSSGAPPPS-NH₂(SEQ ID NO: 10).

FIG. 12 shows the compound of FIG. 10E with an amino acid substitution of Trp²⁵. The compound in FIG. 12 is amidated at the C-terminal amino acid residue (SEQ ID NO: 101).

FIG. 13 shows an exendin analog having a modification at His¹Gly²Glu³. The compound in FIG. 13 is amidated at the C-terminal amino acid residue (SEQ ID NO: 102).

FIG. 14: FIG. 14A is a generic structure (where Xaa₂is, e.g., Gly, dAla, or Aib; and the other substituents are defined herein) of an exendin analog of FIG. 1A, 2A, or 5A having modifications at His¹Gly²Glu³at the N-terminus, where R is a peptide, such as any one of the following: GTFTSDLSKQLEEEAVRLFIEFLKN-NH₂(SEQ ID NO: 6); GTFTSDLSKQLEEEAV-RLFIEWLKQGGPSKEIIS-OH (SEQ ID NO: 5); or GTFTSDLSKQLEEEAVRLFIEWLKNGGPSSGAPPPS-NH₂(SEQ ID NO: 11). Aib is α-methylalanine FIGS. 14B-C are examples of compounds from the structure in FIG. 14A that have modifications at His¹and Glu³. The compounds in FIGS. 14B-C are amidated at the C-terminal amino acid residue. FIGS. 14B through 14C disclose

SEQ ID NOS

6 and 6, respectively. FIG. 14D provides the generic structure (where Xaa₂is Gly, dAla, or Aib; and the other substituents are defined herein) of an N-terminus conformationally constrained compound. FIGS. 14E-R are exendin analogs comprising an N-terminus conformationally constrained compound. FIG. 14Q discloses SEQ ID NO: 38. R₁is GTFTSDLSKQLEEEAVRLF IEWLKQGGPSKEIIS-OH (SEQ ID NO: 5). R₄is GTFTSDLSKQLEEEAVRLFIEFLKN-NH₂(SEQ ID NO: 6). R₅is PGTFTSDLSKQLEEEAVRLFIEWLKNGGPSSGAPPPS-NH₂(SEQ ID NO: 12).

FIGS. 15A-D are exendin analogs that comprise isomers of nipecotic acid as the N-terminus conformationally constrained compound. R₁is FTSDLSKQLEEEAVRLFI EWLKQGGPSKEIIS-NH₂(SEQ ID NO: 13).

FIG. 16: FIGS. 16A-H are exendin analogs containing a modification at the N-terminus. R₁is FTSDLSKQLEEEAVRLFIEWLKQGGPSKEIIS-OH (SEQ ID NO: 13). R₂is FTSDLSKQLE EEAVRLFIEWLKNGGPSSGAPPPS-NH₂(SEQ ID NO: 14). R₃is FTSDVSSYLEGQAAKEFIAWLVKGRG-NH₂(SEQ ID NO: 15). FIGS. 16I-L are exendin analogs containing a modification at the N-terminus. The modification is designed to mimic amino acid residues His¹Gly²Glu³. R₄is GTFTSDLSKQLEE EAVRLFIEFLKN-NH₂(SEQ ID NO: 6).

FIG. 17: FIGS. 17A-F are exendin analogs containing a thiazolidine-proline peptide mimetics at Gly²Glu³. R₄is GTFTSDLSKQLEEEAVRLFIEFLKN-NH₂(SEQ ID NO: 6). FIGS. 17G-N are exendin analogs having a modification at His¹and containing a thiazolidine peptide mimetic at Gly²Glu³. R₁, R₂, and R₃are each independently hydrogen, methyl, or ethyl. In this embodiment, Xaa₃is Glu, Asp, Pro, or Gly. R₄is GTFTSDLSKQLEEEAVRLFIEFLKN-NH₂(SEQ ID NO: 6).

FIG. 18: FIG. 18A is a process for preparing (5,5)-Glu-Gly-OH, a dipeptide mimetic that can be used to induce a β-turn conformational constraint, for example, at the N-terminus in a GLP-1 receptor agonist compound. FIG. 18B is a generic structure of the compound that can be produced by the process shown in FIG. 18A. The skilled artisan can choose compounds with different stereochemistries during the reaction process to provide for various stereochemistries in the final product. * represents a chiral carbon atom.

FIG. 19: FIG. 19A is a process for preparing γ-lactam Glu-Gly-OH, a dipeptide mimetic that can be used to induce a β-turn conformational constraint, for example, at the N-terminus in a GLP-1 receptor agonist compound. FIG. 19B is a generic structure of the compound that can be produced by the process shown in FIG. 19A. The skilled artisan can choose compounds with different stereochemistries during the reaction process to provide for various stereochemistries in the final product. * represents a chiral carbon atom.

FIG. 20: FIG. 20A is a process for preparing (6,5)-Asp-Gly-OH, a dipeptide mimetic that can be used to induce a β-turn conformational constraint, for example, at the N-terminus in a GLP-1 receptor agonist compound. FIG. 20B is a generic structure of the compound that can be produced by the process shown in FIG. 20A. The skilled artisan can choose compounds with different stereochemistries during the reaction process to provide for various stereochemistries in the final product. * represents a chiral carbon atom.

FIG. 21: FIG. 21A is a process for preparing δ-lactam Asp-Gly-OH and Asp-Ala-OH, both of which are dipeptide mimetics that can be used to induce a β-turn conformational constraint, for example, at the N-terminus in a GLP-1 receptor agonist compound. FIG. 21B is a generic structure of the compound that can be produced by the process shown in FIG. 21A. The skilled artisan can choose compounds with different stereochemistries during the reaction process to provide for various stereochemistries in the final product. * represents a chiral carbon atom.

FIG. 22 is a process for preparing a peptide mimetic which can be used to induce a β-turn conformational constraint, for example, at the N-terminus in a GLP-1 receptor agonist compound. FIG. 22 discloses SEQ ID NOS 103-104, respectively, in order of appearance.

DETAILED DESCRIPTION

“GLP-1 receptor agonist compounds” refer to compounds that elicit a biological activity of an exendin reference peptide (e.g., exendin-4) or a GLP-1(7-37) (SEQ ID NO: 84) reference peptide when evaluated by art-known measures such as receptor binding studies or in vivo blood glucose assays as described, e.g., Examples 16 and 17, and by Hargrove et al, Regulatory Peptides, 141:113-119 (2007), the disclosure of which is incorporated by reference herein. GLP-1 receptor agonist compounds include, for example, native exendins, exendin analogs, native GLP-1, GLP-1 analogs, GLP-1(7-37) (SEQ ID NO: 84), and GLP-1(7-37) analogs.
The term “exendin” includes naturally occurring (or synthetic versions of naturally occurring) exendin peptides that are found in the salivary secretions of the Gila monster. Exendin-3 (HSDGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-NH₂) (SEQ ID NO: 16) is present in the salivary secretions of Heloderma horridum and exendin-4 (FIG. 1A) is present in the salivary secretions of Heloderma suspectum. Exendins include the amidated forms, the acid form, the pharmaceutically acceptable salt form, and any other physiologically active form of the molecule. In one embodiment, the term exendin can be used interchangeably with the term “exendin agonist.”
“Exendin analog” refers to peptides, peptides containing peptide mimetics, amino acid substitutions, and/or other modifications, peptides containing the N-terminus conformationally constrained compounds described herein, and/or other chemical moieties, or other compounds which elicit a biological activity similar to that of an exendin reference peptide (e.g., exendin-4), when evaluated by art-known measures such as receptor binding assays or in vivo blood glucose assays as described, e.g., Examples 16 and 17, and by Hargrove et al, Regulatory Peptides, 141:113-119 (2007), the disclosure of which is incorporated by reference herein. Preferably, the exendin analogs will bind in such receptor binding assays with an affinity of less than 1 μM; an affinity of less than 5 nM; an affinity of less than 1 nM, or an affinity of less than 0.1 nM. In one embodiment, the term “exendin analog” refers to a peptide that has an amino acid sequence with 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions, insertions, deletions, or a combination of two or more thereof, when compared to the amino acid sequence of exendin-4 shown in FIG. 1A. In other embodiment, the term “exendin analog” refers to a peptide that has at least 85%, at least 88%, at least 90%, at least 93%, at least 95%, or at least 98% sequence identity to the amino acid sequence of exendin-4 shown in FIG. 1A. Exendin analogs include the amidated forms, the acid form, the pharmaceutically acceptable salt form, and any other physiologically active form of the molecule. In one embodiment, the term exendin analog can be used interchangeably with the term “exendin agonist analog.”
“GLP-1(7-37) analogs” refers to peptides, peptides containing peptide mimetics and/or other modifications, peptides containing the N-terminus conformationally constrained compounds described herein, and/or other chemical moieties, or other compounds which elicit a biological activity similar to that of GLP-1(7-37) (SEQ ID NO: 84), when evaluated by art-known measures such as receptor binding assays or in vivo blood glucose assays as described, e.g., Examples 16 and 17, and by Hargrove et al, Regulatory Peptides, 141:113-119 (2007), the disclosure of which is incorporated by reference herein. In one embodiment, the term “GLP-1(7-37) analog” refers to a peptide that has an amino acid sequence with 1, 2, 3, 4, 5, 6, 7, or 8 amino acid substitutions, insertions, deletions, or a combination of two or more thereof, when compared to the amino acid sequence of GLP-1(7-37) (SEQ ID NO: 84). In one embodiment, the GLP-1(7-37) analog is GLP-1(7-36) (SEQ ID NO: 85). GLP-1(7-37) analogs include the amidated forms, the acid form, the pharmaceutically acceptable salt form, and any other physiologically active form of the molecule.
“N-Terminus conformationally constrained GLP-1 receptor agonist compounds” refers to compounds in which one, two, three, or four of the amino acid residues at positions 1-4 at the N-terminus of “GLP-1 receptor agonist compounds” (e.g., exendins, exendin analogs, GLP-1, GLP-1 analogs, GLP-1(7-37) (SEQ ID NO: 84), GLP-1(7-37) analogs) have been modified or substituted with amino acids (e.g., natural and/or non-natural amino acids), peptidomimetics, or beta-turn dipetidemimetics. This substitution(s) or modification(s) to the N-terminus of the parent GLP-1 receptor agonist compound changes the flexible random coil structure of this specific region into a more rigid secondary structure with beta-turn characteristics.
The glycine residues at positions 2 and 4 at the N-terminus of exendin-4 may indicate the presence of a β-turn in this region. In order to produce a conformationally constrained N-terminus, exendin analogs having a mimetic or other structural modification that restricted the conformational flexibility of the His¹side chain were synthesized. Restricting the flexibility of the His¹side chain was hypothesized to provide structural information about the possible bioactive conformation of GLP-1 receptor agonist compounds and thus enhance GLP-1 receptor binding, in vivo blood glucose lowering activity, and enzymatic stability.
An Ala scan of exendin-4 showed that the Glu³residue was important for biological activity. Additionally, Glu³or Asp³are present in many members of the super-family of glucagon-related peptides, which indicates the importance of an acidic side chain at that residue.
It was postulated that the negative charge of Glu³or Asp³interacted through an ionic bond with the positive charge of the His¹side chain to position the key imidazole ring of the His¹side chain in the right space for interaction with the GLP-1 receptor. It was thus proposed that a β-turn would be formed by the sequence Gly²Glu³Gly⁴Thr⁵(SEQ ID NO: 17) in the super-family of glucagon related peptides, such as exendin-4 and exendin analogs.
In order to maintain the negative charge of Glu³, thought to be essential for biological activity, 13-turn peptide mimetics were synthesized to mimic the amino acid residues Glu³Gly⁴. In one embodiment, the disclosure provides N-terminus conformationally constrained compounds of Formula (F):
Xaa₁Xaa₂Xaa₃-Z;
wherein:
Xaa₁is a compound of Formula (1), as described herein;
Xaa₂is Gly, Ala, dAla, or Aib;

Xaa₃is:

- wherein * indicates a chiral carbon atom; and R₂is hydrogen or a C_1-4alkyl (e.g., methyl, ethyl); and

Z is:

- (i) OH;
- (ii) NH₂;
- (iii) TFTSDLSKQXaa₁₄EEEAVRLFIEXaa₂₅LKN-Z₁(SEQ ID NO: 18);
- (iv) TFTSDLSKQXaa₁₄EEEAVRLFIEXaa₂₅LKNGGPSSGAPPPS-Z₁(SEQ ID NO: 19);
- (v) TFTSDLSKQXaa₁₄EEEAVRLFIEXaa₂₅LK-R₁₀-Z₁(SEQ ID NO: 20); or
- (vi) TFTSDVSSYXaa₁₄EGQAAKEFIAXaa₂₅LVXaa₂₈GRXaa₃₁-Z₁(SEQ ID NO: 21);
  - wherein:
  - Z₁is OH or NH₂;
  - Xaa₁₄is Leu or Met;
  - Xaa₂₅is Phe or Trp;
  - Xaa₂₈is Lys or Arg;
  - Xaa₃₁is Gly or absent; and
  - R₁₀is QGGPSKEIIS (SEQ ID NO: 22); QGGPSSGAPPPS (SEQ ID NO: 23); NG; NGG; NGGP (SEQ ID NO: 24); NGGPS (SEQ ID NO: 25); NGGPSS (SEQ ID NO: 26); NGGPSSG (SEQ ID NO: 27); NGGPSSGA (SEQ ID NO: 28); NGGPSSGAP (SEQ ID NO: 29); NGGPSSGAPP (SEQ ID NO: 30); NGGPSSGAPPP (SEQ ID NO: 31); NGGPSSGAPPS (SEQ ID NO: 32); NGGPSSGAPPSK (SEQ ID NO: 33); NGGPSSGAPPS(K)_2-6(SEQ ID NO: 34); NGGPSSGAPPPSK (SEQ ID NO: 35); or NK.

When Z is OH or NH₂, the compounds of Formula (F) are N-terminus conformationally constrained compounds. When Z is (iii), (iv), (v) or (vi), the compounds of Formula (F) are N-terminus conformationally constrained GLP-1 receptor agonist compounds.
Exemplary compounds of Formula (F) include the compounds in FIGS. 15A-D and 16A-H, each of which may be optionally amidated at the C-terminal amino acid residue. The reaction schemes for preparing the compounds are shown, e.g., in FIGS. 18-20.
Additional studies were undertaken to restrict the N-terminus conformation of GLP-1 receptor agonist compounds and it was unexpectedly discovered that the β-turn in GLP-1 receptor agonist compounds, such as exendin and exendin analogs, was provided by His¹Gly²Glu³Gly⁴(SEQ ID NO: 36). Thus, GLP-1 receptor agonist compounds were produced to constrain or mimic a β-turn defined by residues His¹Gly²Glu³Gly⁴(SEQ ID NO: 36) in exendin-4 and other GLP-1 receptor agonist compounds; or to constrain or mimic a β-turn defined by residues His¹Ala²Glu³Gly⁴(SEQ ID NO: 37) in GLP-1, GLP-1 analogs, GLP-1(7-37) (SEQ ID NO: 84), or GLP-1(7-37) analogs.
Provided herein are N-terminus conformationally constrained compounds of Formula (A): Xaa₁Xaa₂Xaa₃Xaa₄-Z.
In one embodiment, the disclosure provides the compound of Formula (A):
Xaa₁Xaa₂Xaa₃Xaa₄-Z;
wherein:

Xaa₁is a compound of Formula (1):

- wherein R₂₀and R₂₁are each independently a single bond or a carbon atom; R₂₃, R₂₄, R₂₅and R₂₆are each independently absent, hydrogen, hydroxyl, C_1-4alkyl, carboxyl, or C_1-4alkoxy; - - - - - - is a single bond or a double bond; and R₂₁is a chiral or achiral carbon atom;
Xaa₂is Gly, dAla, Aib, Ala, Val, NMeAla, a compound of Formula (3), as described herein; or a compound of Formula (4) and described herein; and Xaa₂is absent when Xaa₃is:

Xaa₃is Pro; a compound of Formula (2), as described herein; a compound of Formula (3), as described herein; a Compound of Formula (4), as described herein;

Xaa₄is Gly, dAla, or Aib; and
Z is OH or NH₂. In other embodiments, Xaa₃is Pro. In other embodiments, Xaa₂is dAla, Aib, Ala, Val, NMeAla, a compound of Formula (3), or a compound of Formula (4).

In other embodiments, the disclosure provides the compound of Formula (A):
Xaa₁Xaa₂Xaa₃Xaa₄-Z;
wherein:

Xaa₁is

Xaa₂is Gly, dAla, Aib, Ala, Val, NMeAla, a compound of Formula (3), as described herein; or a compound of Formula (4), as described herein; and Xaa₂is absent when Xaa₃is:

Xaa₃is Glu; Pro; a compound of Formula (2), as described herein; a compound of Formula (3), as described herein; a Compound of Formula (4), as described herein;

Xaa₄is Gly, dAla, or Aib; and
Z is OH or NH₂.

Also provided herein are N-terminus conformationally constrained GLP-1 receptor agonist compounds of Formula (B)-(E):

(B)

(SEQ ID NO: 1)

Xaa₁Xaa₂Xaa₃Xaa₄TFTSDLSKQXaa₁₄EEEAVRLFIEXaa₂₅LKN-

Z;

(C)

(SEQ ID NO: 2)

Xaa₁Xaa₂Xaa₃Xaa₄TFTSDLSKQXaa₁₄EEEAVRLFIEXaa₂₅LK-

R₁₀-Z;

(D)

(SEQ ID NO: 3)

Xaa₁Xaa₂Xaa₃Xaa₄TFTSDLSKQXaa₁₄EEEAVRLFIEXaa₂₅LKNGG

PSSGAPPPS-Z;

(E)

(SEQ ID NO: 4)

Xaa₁Xaa₂Xaa₃Xaa₄TFTSDVSSYXaa₁₄EGQAAKEFIAXaa₂₅LV

Xaa₂₈GRXaa₃₁-Z.

The substituents for the compounds of Formula (A), (B), (C), (D), and (E) are as follows:

Xaa₁is a compound of Formula (1), as described herein;
Xaa₂is Gly; dAla; Aib; Ala; Val; NMeAla; a compound of Formula (3), as described herein; or a compound of Formula (4), as described herein; and Xaa₂is absent when Xaa₃is:

Xaa₃is Pro; Glu; Asp; a compound of Formula (2), as described herein; a compound of Formula (3), as described herein; a Compound of Formula (4), as described herein;

Xaa₄is Gly, dAla, or Aib;
Xaa₁₄is Leu or Met;
Xaa₂₅is Phe or Trp;
Xaa₂₈is Lys or Arg;
Xaa₃₁is Gly or absent;
R₁₀is QGGPSKEIIS (SEQ ID NO: 22); QGGPSSGAPPPS (SEQ ID NO: 23); NG; NGG; NGGP (SEQ ID NO: 24); NGGPS (SEQ ID NO: 25); NGGPSS (SEQ ID NO: 26); NGGPSSG (SEQ ID NO: 27); NGGPSSGA (SEQ ID NO: 28); NGGPSSGAP (SEQ ID NO: 29); NGGPSSGAPP (SEQ ID NO: 30); NGGPSSGAPPP (SEQ ID NO: 31); NGGPSSGAPPS (SEQ ID NO: 32); NGGPSSGAPPSK (SEQ ID NO: 33); NGGPSSGAPPS(K)_2-6(SEQ ID NO: 34); NGGPSSGAPPPSK (SEQ ID NO: 35); or NK; and
Z is OH or NH₂.

The compounds of Formula (A)-(E) may optionally be in the form of a pharmaceutically acceptable salt.
The compound of Formula (1) is:
wherein R₂₀and R₂₁are each independently a single bond or a carbon atom; R₂₃, R₂₄, R₂₅and R₂₆are each independently absent, hydrogen, hydroxyl, C_1-4alkyl, carboxyl, amino, or C_1-4alkoxy; - - - - - - is a single bond or a double bond; and R₂₁is a chiral or achiral carbon atom.
In one embodiment for the compound of Formula (1), R₂₀and R₂₁are each independently a single bond or a carbon atom; R₂₃and R₂₄are each independently absent, hydrogen, hydroxy, a C_1-4alkyl, carboxyl, or a C_1-4alkoxy; R₂₅and R₂₆are each independently absent, hydrogen, hydroxy, a C_1-4alkyl, carboxyl, amino, or a C_1-4alkoxy; - - - - - - is a single bond or a double bond; and R₂₁is a chiral or achiral carbon atom.
In one embodiment for the compound of Formula (1), R₂₀and R₂₁are each independently a single bond or a carbon atom; R₂₃and R₂₄are each independently absent, hydrogen, hydroxy, methyl, ethyl, or carboxyl; R₂₅and R₂₆are each independently absent or hydrogen; - - - - - - is a single bond or a double bond; and R₂₁is a chiral or achiral carbon atom.
In one embodiment, the compound of Formula (1) is a compound of Formula (1a), (1b), (1c), (1d), (1e), (1f), (1g), (1h), (1j), (1k), (1m), or (1n):
In one embodiment, the compound of Formula (1) is a compound of Formula (1a), (1b), (1c), (1d), (1e), (1f), (1g), (1 h), (1j), (1k), or (1m).
In one embodiment, the compound of Formula (1) is a compound of Formula (1a), (1b), (1c), (1d), (1e), (1f), (1g), (1 h), or (1k).
In one embodiment, the compound of Formula (1) is a compound of Formula (1j) or (1m).
In one embodiment, the compound of Formula (1) is a compound of Formula (1n).
The compounds of Formula (2) and Formula (3) are:
wherein Y₁and Z₁are each independently a single bond, a carbon, or a sulfur; and W₁, W₂and W₃are each independently selected from hydrogen, C_1-6alkyl, C_1-6alkoxy, hydroxyl, and amino; and when one Y₁or Z₁is sulfur, the sulfur may be bonded to two oxygen atoms to form a sulfonyl group; and - - - - - - is
or
.
In one embodiment for the Compounds of Formula (2) and (3), Y₁and Z₁are each independently a single bond, a carbon, or a sulfur; and W₁, W₂and W₃are each independently selected from hydrogen, C_1-4alkyl, C_1-4alkoxy, and amino; and when one Y₁or Z₁is sulfur, the sulfur may be bonded to two oxygen atoms to form a sulfonyl group; and - - - - - - is
or
.
In one embodiment for the Compounds of Formula (2) and (3), Y₁and Z₁are each independently a single bond, a carbon, or a sulfur; and W₁, W₂and W₃are each independently selected from hydrogen, C_1-4alkyl, and C_1-4alkoxy; and when one Y₁or Z₁is sulfur, the sulfur may be bonded to two oxygen atoms to form a sulfonyl group; and - - - - - - is
or
.
In one embodiment for the Compounds of Formula (2) and (3), - - - - - - is
.
In one embodiment for the Compounds of Formula (2), Y₁and Z₁are each independently a single bond, a carbon, or a sulfur; and W₁, W₂and W₃are each independently selected from the group consisting of hydrogen, methyl, ethyl, and propyl; and - - - - - - is
.
In one embodiment for the Compounds of Formula (3), Y₁and Z₁are each independently a single bond or carbon; and W₁, W₂and W₃are each independently selected from the group consisting of hydrogen, methyl, ethyl, and propyl; and - - - - - - is
.
In one embodiment, the compound of Formula (2) is a compound of Formula (2Z):
wherein R₄₀, R₄₁, and R₄₂are each independently selected from the group consisting of hydrogen and C_1-4alkyl (preferably methyl or ethyl). Exemplary compounds of Formula (2Z) include compounds of Formula (2d), (2e), (2f), (2g), and (2h) described below.
In one embodiment, the compound of Formula (2) is a compound of Formula (2a), (2b), (2c), (2d), (2e), (2f), (2g), (2h), or (2j):
In one embodiment, the compound of Formula (2) is a compound of Formula (2d), (2e), (20, (2g), or (2h).
In one embodiment, the compound of Formula (3) is a compound of Formula (3a), (3b), or (3c):
The compound of Formula (4) is:
wherein R₃₀, R₃₁, and R₃₂are each independently hydrogen or a C_1-4alkyl; or R₃₀and R₃₁, together with the nitrogen¹and the carbon², form a 5-membered or 6-membered heterocyclic ring; or R₃₁and R₃₂, together with the carbon², form a 3-, 4-, or 5-membered carbocyclic ring.
In one embodiment for the compound of Formula (4), R₃₀, R₃₁, and R₃₂are each independently hydrogen, methyl, or ethyl; or R₃₀and R₃₁, together with the nitrogen¹and carbon², form a 5-membered or 6-membered heterocyclic ring; or R₃₁and R₃₂, together with the carbon², form a 3-, or 4-membered carbocyclic ring;
In one embodiment, the compound of Formula (4) is a compound of Formula (4a), (4b), (4c), (4d), or (4e):
In one embodiment for the Compounds of Formula (A)-(F), Xaa₂is dAla, Aib, Ala, Val, or NMeAla.
In one embodiment for the Compounds of Formula (A)-(F), Xaa₂is a compound of Formula (3).
In one embodiment for the Compounds of Formula (A)-(F), Xaa₂is a compound of Formula (4).
In one embodiment for the Compounds of Formula (A)-(F), Xaa₂is Gly.
In one embodiment for the Compounds of Formula (A)-(F), Xaa₂is dAla.
In one embodiment for the Compounds of Formula (A)-(F), Xaa₂is dAla or Aib.
In one embodiment for the Compounds of Formula (A)-(F), Xaa₃is Pro.
In one embodiment for the Compounds of Formula (A)-(F), Xaa₃is Glu.
In one embodiment for the Compounds of Formula (A)-(F), Xaa₃is a compound of Formula (2), as described herein.
In one embodiment for the Compounds of Formula (A)-(F), Xaa₃is a compound of Formula (3), as described herein.
In one embodiment for the Compounds of Formula (A)-(F), Xaa₂is absent and Xaa₃is
In one embodiment for the Compounds of Formula (A)-(F), Xaa₄is Gly or dAla.
In one embodiment for the Compounds of Formula (A)-(F), Z is NH₂.
For the Compounds of Formula (A)-(F): in one embodiment R₁₀is QGGPSKEIIS (SEQ ID NO: 22); in one embodiment R₁₀is NG; in one embodiment R₁₀is NGG; in one embodiment R₁₀is NGGP (SEQ ID NO: 24); in one embodiment R₁₀is NGGPS (SEQ ID NO: 25); in one embodiment R₁₀is NGGPSS (SEQ ID NO: 26); in one embodiment R₁₀is NGGPSSG (SEQ ID NO: 27); in one embodiment R₁₀is NGGPSSGA (SEQ ID NO: 28); in one embodiment R₁₀is NGGPSSGAP (SEQ ID NO: 29); in one embodiment R₁₀is NGGPSSGAPP (SEQ ID NO: 30); in one embodiment R₁₀is NGGPSSGAPPP (SEQ ID NO: 31); in one embodiment R₁₀is NGGPSSGAPPS (SEQ ID NO: 32); in one embodiment R₁₀is NGGPSSGAPPSK (SEQ ID NO: 33); in one embodiment R₁₀is NGGPSSGAPPS(K)_2-6(SEQ ID NO: 34); in one embodiment R₁₀is NGGPSSGAPPPSK (SEQ ID NO: 35); and in one embodiment R₁₀is NK; and in one embodiment R₁₀is QGGPSSGAPPPS (SEQ ID NO: 23).
For the Compounds of Formula (A)-(F): in one embodiment Xaa₁₄is Met and Xaa₂₅is Trp; in one embodiment Xaa₁₄is Leu and Xaa₂₅is Phe; in one embodiment Xaa₁₄is Met and Xaa₂₅is Phe; and in one embodiment Xaa₁₄is Leu and Xaa₂₅is Trp.
With respect to the compounds of Formula (D), Xaa₂, Xaa₃, Xaa₁₄, and Xaa₂₅cannot simultaneously be Gly, Glu, Met, and Trp, respectively, except when the compound of Formula (1) is a compound of Formula 1(j) or 1(m). Thus, when Xaa₂, Xaa₃, Xaa₁₄, and Xaa₂₅are Gly, Glu, Met, and Trp, respectively, the compound of Formula (D) may be one of the following:
In one embodiment, the N-terminus conformationally constrained GLP-1 receptor agonist compound may be Pro³-exendin-4 (SEQ ID NO: 40); Pro³,Leu¹⁴-exendin-4 (SEQ ID NO: 41); Pro³,Leu¹⁴,Phe²⁵-exendin-4 (SEQ ID NO: 42); Pro³-exendin-4(1-28) (SEQ ID NO: 43); Pro³,Leu¹⁴-exendin-4(1-28) (SEQ ID NO: 44); Pro³,Leu¹⁴,Phe²⁵-exendin-4(1-28) (SEQ ID NO: 45); Pro³-exendin-4(1-36) (SEQ ID NO: 46); Pro³,Leu¹⁴-exendin-4(1-36) (SEQ ID NO: 47); Pro³,Leu¹⁴,Phe²⁵-exendin-4(1-36) (SEQ ID NO: 48); or HGPGTFTSDLSKQLEEEAVRLFIEWLKQGGPSKEIIS (SEQ ID NO: 49), each of which may optionally be amidated and which may optionally be in the form of a pharmaceutically acceptable salt.
In one embodiment, the N-terminus conformationally constrained GLP-1 receptor agonist compound may be Pro³-exendin-3 (SEQ ID NO: 50); Pro³-exendin-4(1-29) (SEQ ID NO: 51); Pro³,Leu¹⁴,Phe²⁵-exendin-4(1-29) (SEQ ID NO: 52); Pro³-exendin-4(1-30) (SEQ ID NO: 53); Pro³,Leu¹⁴,Phe²⁵-exendin-4(1-30) (SEQ ID NO: 54); Pro³-exendin-4(1-31) (SEQ ID NO: 55); Pro³,Leu¹⁴,Phe²⁵-exendin-4(1-31) (SEQ ID NO: 56); Pro³-exendin-4(1-32) (SEQ ID NO: 57); Pro³,Leu¹⁴,Phe²⁵-exendin-4(1-32) (SEQ ID NO: 58); Pro³-exendin-4(1-33) (SEQ ID NO: 59); Pro³,Leu¹⁴,Phe²⁵-exendin-4(1-33) (SEQ ID NO: 60); Pro³-exendin-4(1-34) (SEQ ID NO: 61); Pro³,Leu¹⁴,Phe²⁵-exendin-4(1-34) (SEQ ID NO: 62); Pro³-exendin-4(1-35) (SEQ ID NO: 63); Pro³,Leu¹⁴,Phe²⁵-exendin-4(1-35) (SEQ ID NO: 64); Pro³-exendin-4(1-37) (SEQ ID NO: 65); Pro³,Leu¹⁴,Phe²⁵-exendin-4(1-37) (SEQ ID NO: 66); Pro³-exendin-4(1-38) (SEQ ID NO: 67); or Pro³,Leu¹⁴,Phe²⁵-exendin-4(1-38) (SEQ ID NO: 68), each of which may optionally be amidated and which may optionally be in the form of a pharmaceutically acceptable salt.
Examples of the compounds of Formula (A), (B), (C), (D), and (E) are shown in FIGS. 1B-C, 2A-U, 3A-G, 5B-G, 6A-E, 8A-H, 9A-H, 10A-I, 11A-B, 12, 13, 14A-Q, 15C-D, 16I-L, and 17A-N.
The N-terminus conformationally constrained compounds (e.g., compounds of Formula (A) and (F)) and the N-Terminus conformationally constrained GLP-1 receptor agonist compounds (e.g., compounds of Formula (B)-(F)) described herein (collectively referred to as “the compounds”) can optionally be covalently linked to one or more polymers to provide beneficial biological properties to the compounds. Such beneficial properties may include conferring another therapeutic property to the compounds; increasing the in vivo half life of the compounds; decreasing the rate of clearance of the compounds by the kidney; decreasing the immunogenicity of the compounds; decreasing the proteolysis rate of the compounds; or increasing the stability of the compounds. Exemplary polymers that can be covalently linked to the compounds include peptides, polyethylene glycols, albumin, fatty acids, dextran, polyamino acids, alkyl chains, immunoglobulins, signaling moieties, gelatin, polyvinyl pyrrolidone, polyvinyl alcohol, N-(2-hydroxypropyl)-methacrylamide, and the like. In one embodiment, two or more polymers (e.g., peptides, polyethylene glycols, albumin, fatty acids, dextran, polyamino acids, alkyl chains, immunoglobulins, gelatin, polyvinyl pyrrolidone, polyvinyl alcohol, N-(2-hydroxypropyl)-methacrylamide) are covalently attached together and linked to the N-terminus conformation constrained compounds described herein. For example, the two more polymers that are linked together may be polyethylene glycol(s) and fatty acid(s) or a peptide, polyethylene glycol(s), and fatty acids.
In one embodiment, the compounds are linked to another peptide to provide additional therapeutic benefits. Such peptides include amylin, amylin agonist analogs (e.g., amylin analogs that function as amylin agonists), PYY, PYY analogs, GIP, GIP analogs, leptin, metraleptin, leptin analogs, metraleptin analogs, and the like. Hybrid peptides comprising the compounds and another therapeutic peptide are described, for example, in WO 2005/077072 and WO 2007/022123, the disclosures of which are incorporated by reference herein.
In one embodiment, compounds are linked to one, two, or three polyethylene glycols. In one embodiment, the compounds are linked to one polyethylene glycol. The polyethylene glycol can have a molecular weight from about 200 daltons to about 80,000 daltons; from about 5,000 from about 10,000 daltons to about 60,000 daltons; from about 10,000 daltons to about 50,000 daltons; or from about 15,000 daltons to about 40,000 daltons. The polyethylene glycol may be linear or branched.
In one embodiment, compounds are linked to one or two polyethylene glycols, where the polyethylene glycol is further linked to a lipophilic moiety. In one embodiment, the polyethylene glycol may have a molecular weight from about 200 to about 7,000 daltons or from about 500 to about 5,000 daltons. The lipophilic moiety may be an alkyl group (e.g., C_1-20alkyl group; C_1-10alkyl group; C_1-6alkyl group; C_1-4alkyl group), a fatty acid (e.g., C_4-28fatty acid; C_4-20fatty acid; C_4-10fatty acid), cholesteryl, adamantyl, and the like. The alkyl group may be linear or branched, preferably linear. In one embodiment, the fatty acid is an acetylated fatty acid or an esterified fatty acid. The -(polyethylene glycol)-(lipophilic moiety) may be linked to the compound at a C-terminal amino acid residue, an N-terminal amino acid residue, an internal amino acid residue (e.g., an internal Lys amino acid residue), or a combination thereof (e.g., the compound is linked at the N-terminal and C-terminal amino acid residues via a lysine residue). Examplary peptides linked to such groups are shown in Example 20.
In one embodiment, the compounds are linked to a polyamino acid. Exemplary polyamino acids include poly-lysine, poly-aspartic acid, poly-serine, poly-glutamic acid, and the like. The polyamino acid may be in the D or L form, preferably the L form. The polyamino acids may comprise from 1 to 12 amino acid residues; from 2 to 10 amino acid residues; or from 2 to 6 amino acid residues.
In one embodiment, compounds are linked to a fatty acid. The fatty acid may be a C₄-C₂₈fatty acid chain, a C₈-C₂₄fatty acid chain, or a C₁₀-C₂₀fatty acid chain. In one embodiment, the fatty acid is an acetylated fatty acid. In one embodiment, the fatty acid is an esterified fatty acid.
In one embodiment, the compounds are linked to albumin. The albumin may be a recombinant albumin, serum albumin, or recombinant serum albumin. In another embodiment, the compounds are linked to an albumin-fatty acid (i.e., an albumin linked to a fatty acid).
In one embodiment, the compounds are linked to an immunoglobulin or an immunoglobulin Fc region. The immunoglobulin may be IgG, IgE, IgA, IgD, or IgM. In one embodiment, the compounds are linked to an IgG Fc region or an IgM Fc region. The immunoglobulin Fc region is (i) the heavy chain constant region 2(C_H2) of an immunoglobulin; (ii) the heavy chain constant region 3(C_H3) of an immunoglobulin; or (iii) both the heavy chain constant regions 2(C_H2) and 3(C_H3) of an immunoglobulin. The immunoglobulin Fc region may further comprise the hinge region at the heavy chain constant region. Other embodiments for the immunoglobulin Fc region that can be linked to exendin analog peptides are described in WO 2008/082274, the disclosure of which is incorporated by reference herein.
In one embodiment, the compounds are linked to one or more signalling moieties. Exemplary signalling moieties include, biotin, antigens, antibodies, receptors, enzymes, chemiluminescent groups, photoreactive groups, fluorescent groups, heavy metal-containing compounds (e.g., ferritin), and the like.
When the compounds described herein are covalently linked to one or more polymers, such as those described herein, any linking group known in the art can be used. The linking group may comprise any chemical group(s) suitable for linking the peptide to the polymer. Alternatively, compounds can be directly attached to the polymer without any linking group. Exemplary linking groups include amino acids, maleimido groups, dicarboxylic acid groups, succinimide groups, or a combination of two or more thereof. Methods for linking peptides to one or more polymers are known in the art and described, for example, in U.S. Pat. No. 6,329,336; U.S. Pat. No. 6,423,685; U.S. Pat. No. 6,924,264; WO 2005/077072, WO 2007/022123, WO 2007/053946; WO 2008/058461; and WO 2008/082274, the disclosures of which are incorporated by reference herein.
The compounds described herein may be prepared using biological, chemical, and/or recombinant DNA techniques that are known in the art. Exemplary methods are described in U.S. Pat. No. 6,872,700; WO 2007/139941; WO 2007/140284; WO 2008/082274; WO 2009/011544; and US Publication No. 2007/0238669, the disclosures of which are incorporated herein by reference. Other methods for preparing the compounds are set forth herein.
The compounds described herein may be prepared using standard solid-phase peptide synthesis techniques, such as an automated or semiautomated peptide synthesizer. Typically, using such techniques, an alpha-N-carbamoyl protected amino acid and an amino acid attached to the growing peptide chain on a resin are coupled at room temperature in an inert solvent (e.g., dimethylformamide, N-methylpyrrolidinone, methylene chloride, and the like) in the presence of coupling agents (e.g., dicyclohexylcarbodiimide, 1-hydroxybenzo-triazole, and the like) in the presence of a base (e.g., diisopropylethylamine, and the like). The alpha-N-carbamoyl protecting group is removed from the resulting peptide-resin using a reagent (e.g., trifluoroacetic acid, piperidine, and the like) and the coupling reaction repeated with the next desired N-protected amino acid to be added to the peptide chain. Suitable N-protecting groups are well known in the art, such as t-butyloxycarbonyl (tBoc) fluorenylmethoxycarbonyl (Fmoc), and the like. The solvents, amino acid derivatives and 4-methylbenzhydryl-amine resin used in the peptide synthesizer may be purchased from Applied Biosystems Inc. (Foster City, Calif.).
Solid phase peptide synthesis may be carried out with an automatic peptide synthesizer (Model 430A, Applied Biosystems Inc., Foster City, Calif.) using the NMP/HOBt (Option 1) system and tBoc or Fmoc chemistry (See Applied Biosystems User's Manual for the ABI 430A Peptide Synthesizer, Version 1.3B Jul. 1, 1988, section 6, pp. 49-70, Applied Biosystems, Inc., Foster City, Calif.) with capping. Boc-peptide-resins may be cleaved with HF (−5° C. to 0° C., 1 hour). The peptide may be extracted from the resin with alternating water and acetic acid, and the filtrates lyophilized. The Fmoc-peptide resins may be cleaved according to standard methods (e.g., Introduction to Cleavage Techniques, Applied Biosystems, Inc., 1990, pp. 6-12). Peptides may be also be assembled using an Advanced Chem Tech Synthesizer (Model MPS 350, Louisville, Ky.).
Peptides may be purified by RP-HPLC (preparative and analytical) using a Waters Delta Prep 3000 system. A C4, C8 or C18 preparative column (10μ, 2.2×25 cm; Vydac, Hesperia, Calif.) may be used to isolate peptides, and purity may be determined using a C4, C8 or C18 analytical column (5μ, 0.46×25 cm; Vydac). Solvents (A=0.1% TFA/water and B=0.1% TFA/CH₃CN) may be delivered to the analytical column at a flow rate of 1.0 ml/min and to the preparative column at 15 ml/min. Amino acid analyses may be performed on the Waters Pico Tag system and processed using the Maxima program. Peptides may be hydrolyzed by vapor-phase acid hydrolysis (115° C., 20-24 h). Hydrolysates may be derivatized and analyzed by standard methods (Cohen et al, The Pico Tag Method: A Manual of Advanced Techniques for Amino Acid Analysis, pp. 11-52, Millipore Corporation, Milford, Mass. (1989)). Fast atom bombardment analysis may be carried out by M-Scan, Incorporated (West Chester, Pa.). Mass calibration may be performed using cesium iodide or cesium iodide/glycerol. Plasma desorption ionization analysis using time of flight detection may be carried out on an Applied Biosystems Bio-Ion 20 mass spectrometer.
The compounds described herein may also be prepared using recombinant DNA techniques using methods known in the art, such as Sambrook et al., Molecular Cloning: A Laboratory Manual, 2d Ed., Cold Spring Harbor (1989). Non-peptide compounds may be prepared by art-known methods. For example, phosphate-containing amino acids and peptides containing such amino acids, may be prepared using methods known in the art, such as described in Bartlett et al, Biorg. Chem., 14:356-377 (1986).
The disclosure also provides pharmaceutical compositions comprising at least one of the N-terminus conformationally constrained compounds or the N-terminus conformationally constrained GLP-1 receptor agonist compounds described herein and a pharmaceutically acceptable carrier. The N-terminus conformationally constrained compounds or the N-terminus conformationally constrained GLP-1 receptor agonist compounds can be present in the pharmaceutical composition in a therapeutically effective amount and can be present in an amount to provide a minimum blood plasma level for therapeutic efficacy.
Pharmaceutical compositions containing the compounds described herein may be provided for peripheral administration, such as parenteral (e.g., subcutaneous, intravenous, intramuscular), topical, nasal, or oral administration. Suitable pharmaceutically acceptable carriers and their formulation are described in standard formulation treatises, such as Remington's Pharmaceutical Sciences by Martin; and Wang et al, Journal of Parenteral Science and Technology, Technical Report No. 10, Supp. 42:2 S (1988).
The compounds described herein can be provided in parenteral compositions for injection or infusion. They can, for example, be suspended in water; an inert oil, such as a vegetable oil (e.g., sesame, peanut, olive oil, and the like); or other pharmaceutically acceptable carrier. In one embodiment, the compounds are suspended in an aqueous carrier, for example, in an isotonic buffer solution at a pH of about 3.0 to 8.0, or about 3.0 to 5.0. The compositions may be sterilized by conventional sterilization techniques or may be sterile filtered. The compositions may contain pharmaceutically acceptable auxiliary substances as required to approximate physiological conditions, such as pH buffering agents. Useful buffers include for example, acetic acid buffers. A form of repository or “depot” slow release preparation may be used so that therapeutically effective amounts of the preparation are delivered into the bloodstream over many hours or days following subcutaneous injection, transdermal injection or other delivery method. The desired isotonicity may be accomplished using sodium chloride or other pharmaceutically acceptable agents such as dextrose, boric acid, sodium tartrate, propylene glycol, polyols (such as mannitol and sorbitol), or other inorganic or organic solutes. Sodium chloride is preferred particularly for buffers containing sodium ions.
The compounds can also be formulated as pharmaceutically acceptable salts (e.g., acid addition salts) and/or complexes thereof. Pharmaceutically acceptable salts are non-toxic salts at the concentration at which they are administered. Pharmaceutically acceptable salts include acid addition salts such as those containing sulfate, hydrochloride, phosphate, sulfamate, acetate, citrate, lactate, tartrate, methanesulfonate, ethanesulfonate, benzenesulfonate, p-toluenesulfonate, cyclohexylsulfamate and quinate. Pharmaceutically acceptable salts can be obtained from acids such as hydrochloric acid, sulfuric acid, phosphoric acid, sulfamic acid, acetic acid, citric acid, lactic acid, tartaric acid, malonic acid, methanesulfonic acid, ethanesulfonic acid, benzenesulfonic acid, p-toluenesulfonic acid, cyclohexylsulfamic acid, and quinic acid. Such salts may be prepared by, for example, reacting the free acid or base forms of the product with one or more equivalents of the appropriate base or acid in a solvent or medium in which the salt is insoluble, or in a solvent such as water which is then removed in vacuo or by freeze-drying or by exchanging the ions of an existing salt for another ion on a suitable ion exchange resin.
Carriers or excipients can also be used to facilitate administration of the compounds. Examples of carriers and excipients include calcium carbonate, calcium phosphate, various sugars such as lactose, glucose, or sucrose, or types of starch, cellulose derivatives, gelatin, vegetable oils, polyethylene glycols and physiologically compatible solvents.
If desired, solutions of the above compositions may be thickened with a thickening agent such as methyl cellulose. They may be prepared in emulsified form, either water in oil or oil in water. Any of a wide variety of pharmaceutically acceptable emulsifying agents may be employed including, for example, acacia powder, a non-ionic surfactant (such as a Tween), or an ionic surfactant (such as alkali polyether alcohol sulfates or sulfonates, e.g., a Triton).
Compositions may be prepared by mixing the ingredients following generally accepted procedures. For example, the selected components may be simply mixed in a blender or other standard device to produce a concentrated mixture which may then be adjusted to the final concentration and viscosity by the addition of water or thickening agent and possibly a buffer to control pH or an additional solute to control tonicity.
The therapeutically effective amount of the compounds described herein to treat the diseases described herein will typically be from about 0.01 μg to about 5 mg; about 0.1 μg to about 2.5 mg; about 1 μg to about 1 mg; about 1 μg to about 50 μg; or about 1 μg to about 25 μg. Alternatively, the therapeutically effective amount of the GLP-1 receptor agonist compounds may be from about 0.001 μg to about 100 μg based on the weight of a 70 kg patient; or from about 0.01 μg to about 50 μg based on the weight of a 70 kg patient. These therapeutically effective doses may be administered once/day, twice/day, thrice/day, once/week, biweekly, or once/month, depending on the formulation. The exact dose to be administered is determined, for example, by the formulation, such as an immediate release formulation or an extended release formulation. For transdermal, nasal or oral dosage forms, the dosage may be increased from about 5-fold to about 10-fold.
The compounds described herein and pharmaceutical compositions comprising the compounds are useful for treating diabetes. The diabetes can be Type I diabetes, Type II diabetes, or gestational diabetes. The methods for treating diabetes provide administering to a patient in need thereof a therapeutically effective amount of one or more of the N-terminus conformationally constrained compounds or the N-terminus conformationally constrained GLP-1 receptor agonist compounds described herein to treat diabetes in the patient.
The compounds described herein and pharmaceutical compositions comprising the compounds are useful for treating insulin resistance and stimulating insulin release. The methods for treating insulin resistance provide administering to a patient in need thereof a therapeutically effective amount of one or more of the N-terminus conformationally constrained compounds or the N-terminus conformationally constrained GLP-1 receptor agonist compounds described herein to treat insulin resistance in the patient. The methods for treating stimulating insulin release provide administering to a patient in need thereof a therapeutically effective amount of one or more of the N-terminus conformationally constrained compounds or the N-terminus conformationally constrained GLP-1 receptor agonist compounds described herein to stimulate insulin release in the patient.
The compounds described herein and pharmaceutical compositions comprising the compounds are useful for treating postprandial hyperglycemia. The methods for treating postprandial hyperglycemia provide administering to a patient in need thereof a therapeutically effective amount of one or more of the N-terminus conformationally constrained compounds or the N-terminus conformationally constrained GLP-1 receptor agonist compounds described herein to treat postprandial hyperglycemia in the patient.
The compounds described herein and pharmaceutical compositions comprising the compounds are useful for lowering blood glucose levels and lowering HbA1c levels. The methods for lowering blood glucose levels provide administering to a patient in need thereof a therapeutically effective amount of one or more of the N-terminus conformationally constrained compounds or the N-terminus conformationally constrained GLP-1 receptor agonist compounds described herein to lower blood glucose levels in the patient. In one embodiment, the blood glucose levels can be fasting blood glucose levels. The methods for lowering HbA1c levels provide administering to a patient in need thereof a therapeutically effective amount of one or more of the N-terminus conformationally constrained compounds or the N-terminus conformationally constrained GLP-1 receptor agonist compounds described herein to lower HbA1c levels in the patient. HbA1c levels are generally a long-term measure of a patient's blood glucose levels.
The compounds described herein and pharmaceutical compositions comprising the compounds are useful for reducing gastric motility and delaying gastric emptying. The methods for reducing gastric motility provide administering to a patient in need thereof a therapeutically effective amount of one or more of the N-terminus conformationally constrained compounds or the N-terminus conformationally constrained GLP-1 receptor agonist compounds described herein to reduce gastric motility in the patient. The methods for delaying gastric emptying provide administering to a patient in need thereof a therapeutically effective amount of one or more of the N-terminus conformationally constrained compounds or the N-terminus conformationally constrained GLP-1 receptor agonist compounds described herein to delay gastric emptying in the patient.
The compounds described herein and pharmaceutical compositions comprising the compounds are useful for reducing food intake, reducing appetite, increasing satiety, and reducing weight. The methods for reducing food intake provide administering to a patient in need thereof a therapeutically effective amount of one or more of the N-terminus conformationally constrained compounds or the N-terminus conformationally constrained GLP-1 receptor agonist compounds described herein to reduce food intake in the patient. The methods for reducing appetite provide or increasing satiety administering to a patient in need thereof a therapeutically effective amount of one or more of the N-terminus conformationally constrained compounds or the N-terminus conformationally constrained GLP-1 receptor agonist compounds described herein to reduce appetite in the patient. The methods for treating reducing weight provide administering to a patient in need thereof a therapeutically effective amount of one or more of the N-terminus conformationally constrained compounds or the N-terminus conformationally constrained GLP-1 receptor agonist compounds described herein to reduce weight in the patient. In the methods described herein, the patient may be in need of a reduced intake in food, of a reduced appetite, or of reduced weight. In other methods described herein, the patient may be desirous of having a reduced intake in food, of having a reduced appetite, or of having a reduced weight. The patient may be of any weight, and can be overweight or obese.
The compounds described herein and pharmaceutical compositions comprising the compounds are useful for treating overweight and obesity. The methods for treating overweight provide administering to a patient in need thereof a therapeutically effective amount of one or more of the N-terminus conformationally constrained compounds or the N-terminus conformationally constrained GLP-1 receptor agonist compounds described herein to treat overweight in the patient. The methods for treating obesity provide administering to a patient in need thereof a therapeutically effective amount of one or more of the N-terminus conformationally constrained compounds or the N-terminus conformationally constrained GLP-1 receptor agonist compounds described herein to treat obesity in the patient.
The disclosure also provides drug delivery devices having at least one therapeutically effective dose of the compounds described herein or the pharmaceutical composition containing the compounds described herein. The drug delivery devices can be single or multiple-use vials, single or multiple-use pharmaceutical pens, single or multiple-use cartridges, and the like. In one embodiment, the drug delivery devices contain the compounds or pharmaceutical compositions described herein in amounts capable of providing a patient with from about 7 to about 40 doses or enough doses to last about one week or about one month.

EXAMPLES

The following examples are for purposes of illustration only and are not intended to limit the scope of the claims.

Example 1

Preparation of Compound in FIG. 16A

A calculated 100 μmol of Fmoc-Ser(OtBu)-Wang resin was weighed into a reaction vessel in a Symphony peptide synthesizer, and the peptide elongation was carried out following standard Fmoc peptide synthesis protocol up to residue Thr⁵. The resulting peptide-resin intermediate (0.3 g, 0.0927 mmol) was swollen in dimethylformamide (DMF) and to the slurry was added compound 7 (0.110 g, 2.2 eq), followed by O-(benzotriazol-1-yl)-N,N,N′,N′ tetramethyluronium hexafluorophosphate (HBTU) (0.035 g, 2.2 eq), N-hydroxybenzotriazole (HOBt) (0.03 g, 2.2 eq) and methylmorpholine (NMM) (0.04 mL, 4.4 eq). After 3 hours, the resin was washed with DMF 6×, treated with 20% piperidine in DMF 2×25 min and washed with DMF 6×. The above cycle was repeated with Fmoc-Ala-OH and Fmoc-His(Trt)-OH followed by cleavage of the peptide from the resin with 10 ml TFA/H₂O/PhOH/TIPS (95:2:2:1) (TFA is trifluoroacetic acid; TIPS is triisopropylsilyl), precipitated by methyl-tert-Butyl ether and the obtained residue applied to a reverse-phase high performance liquid chromatography (HPLC) column (C₁₈, 20-50% CH₃CN in 0.1% TFA/H₂O over 30 min gradient) to afford the titled compound as a white powder (16.4 mg, 6%): Retention time in reverse phase-high performance liquid chromatography (RP-HPLC) (C₁₈, 5-75% CH₃CN in 0.1% TFA/H₂O over 15 min) is 9.78 min; Calculated mass for C₁₈₉H₂₉₄N₄₈O₆₀S (M+H)⁺ 4231.84, found by liquid chromatography/mass spectrometry (LC-MS) 1411.3 (M+3H)³⁺, 1059.6 (M+4H)⁴⁺, 2117.2 (M+2H)²⁺.

Example 2

Preparation of Compound in FIG. 16B

A calculated 100 μmol of Fmoc-Ser(OtBu)-Wang resin was weighed into a reaction vessel in a Symphony peptide synthesizer, and the peptide elongation was carried out following standard Fmoc peptide synthesis protocol up to residue Thr⁵. The resulting peptide-resin intermediate (0.3 g, 0.0927 mmol) was swollen in DMF and to the slurry was added compound 24 (0.122 g, 2.2 eq), followed by HBTU (0.035 g, 2.2 eq), HOBt (0.03 g, 2.2 eq) and NMM (0.04 mL, 4.4 eq). After 3 hours, the resin was washed with DMF 6×, treated with 20% piperidine in DMF 2×25 min and washed with DMF 6×. The above cycle was repeated with Fmoc-His(Trt)-OH followed by cleavage of the peptide from the resin with 10 ml TFA/H₂O/PhOH/TIPS (95:2:2:1), precipitated by methyl-tert-Butyl ether. The obtained crude peptide was dissolved in 1.5 mL of MeOH:ACN (1:1) (ACN is acetonitrile), followed by addition of 2M LiOH (0.6 mL) and the reaction stirred at RT for 6 h. The crude was dissolved and applied to a reverse-phase HPLC column (C₁₈, 20-50% CH₃CN in 0.1% TFA/H₂O over 30 min gradient) to afford the titled compound as a white powder (15 mg, 6%): Retention time in RP-HPLC (C₁₈, 5-75% CH₃CN in 0.1% TFA/H₂O over 15 min) is 9.82 min; Calculated mass for C₁₈₈H₂₉₄N₄₈O₆₀(M+H)⁺4186.72, found by LC-MS 1396.7 (M+3H)³⁺, 1048.6 (M+4H)⁴⁺, 2114.2 (M+2H)²⁺.

Example 3

Preparation of Compound in FIG. 16C

A calculated 100 μmol of Fmoc-Ser(OtBu)-Wang resin was weighed into a reaction vessel in a Symphony peptide synthesizer, and the peptide elongation was carried out following standard Fmoc peptide synthesis protocol up to residue Thr⁵. The resulting peptide-resin intermediate (0.3 g, 0.0927 mmol) was swollen in DMF and to the slurry was added compound 18 (0.131 g, 2.2 eq), followed by HBTU (0.035 g, 2.2 eq), HOBt (0.03 g, 2.2 eq) and NMM (0.04 mL, 4.4 eq). After 3 hours, the resin was washed with DMF 6×, treated with 20% piperidine in DMF 2×25 min and washed with DMF 6×. The above cycle was repeated with Fmoc-His(Trt)-OH followed by cleavage of the peptide from the resin with 10 ml TFA/H₂O/PhOH/TIPS (95:2:2:1), precipitated by methyl-tert-Butyl ether. The obtained crude peptide was dissolved in 1.5 mL of MeOH:ACN (1:1), followed by addition of 2M LiOH (0.6 mL) and the reaction stirred at RT for 6 h. The crude was dissolved and applied to a reverse-phase HPLC column (C₁₈, 20-50% CH₃CN in 0.1% TFA/H₂O over 30 min gradient) to afford the titled compound as a white powder (19.7 mg, 8%): Retention time in RP-HPLC (C₁₈, 5-75% CH₃CN in 0.1% TFA/H₂O over 15 min) is 8.58 min; Calculated mass for C₁₈₉H₂₉₄N₄₈O₆₀5 (M+H)⁺4230.80, found by LC-MS 1411. (M+3H)³⁺, 1059.6 (M+4H)⁴⁺, 2117.2 (M+2H)²⁺.

Example 4

Preparation of Compound in FIG. 16E

The synthesis of this compound was accomplished following the same experimental procedure as described for Example 3. The only difference was the sequence of the peptide-resin intermediate, starting with Rink-amide resin. The crude was dissolved and applied to a reverse-phase HPLC column (C₁₈, 20-50% CH₃CN in 0.1% TFA/H₂O over 30 min gradient) to afford the titled compound as a white powder (15.7 mg, 7%): Retention time in RP-HPLC (C₁₈, 5-75% CH₃CN in 0.1% TFA/H₂O over 15 min) is 8.25 min; Calculated mass for C₁₈₈H₂₈₆N₄₈O₆₀S (M+H)⁺4238.74, found by LC-MS 1414.8 (M+3H)³⁺, 1061.6 (M+4H)⁴⁺, 2121.2 (M+2H)²⁺.

Example 5

Preparation of Compound in FIG. 16D

A calculated 100 μmol of Fmoc-Ser(OtBu)-Wang resin was weighed into a reaction vessel in a Symphony peptide synthesizer, and the peptide elongation was carried out following standard Fmoc peptide synthesis protocol up to residue Thr⁵. The resulting peptide-resin intermediate (0.3 g, 0.0927 mmol) was swollen in DMF and to the slurry was added compound 11 (0.100 g, 2.2 eq), followed by HBTU (0.035 g, 2.2 eq), HOBt (0.03 g, 2.2 eq) and NMM (0.04 mL, 4.4 eq). After 3 h, the resin was washed with DMF 6×, treated with 20% piperidine in DMF 2×25 min and washed with DMF 6×. The above cycle was repeated with Fmoc-Ala-OH and Fmoc-His(Trt)-OH followed by cleavage of the peptide from the resin with 10 ml TFA/H₂O/PhOH/TIPS (95:2:2:1), precipitated by methyl-tert-Butyl ether and the obtained residue applied to a reverse-phase HPLC column (C₁₈, 20-50% CH₃CN in 0.1% TFA/H₂O over 30 min gradient) to afford the titled compound as a white powder (20.3 mg, 10%): Retention time in RP-HPLC (C₁₈, 5-75% CH₃CN in 0.1% TFA/H₂O over 15 min) is 9.74 min; Calculated mass for C₁₈₈H₂₉₄N₄₈O₆₀(M+H)⁺4186.72, found by LC-MS 1396.7 (M+3H)³⁺, 1048.6 (M+4H)⁴⁺, 2114.2 (M+2H)²⁺.

Example 6

Preparation of Compound in FIG. 16F

A calculated 100 μmol of Fmoc-Ser(OtBu)-Wang resin was weighed into a reaction vessel in a Symphony peptide synthesizer, and the peptide elongation was carried out following standard Fmoc peptide synthesis protocol up to residue Thr⁵. The resulting peptide-resin intermediate (0.3 g, 0.0927 mmol) was swollen in DMF and to the slurry was added compound 21 (0.125 g, 2.2 eq), followed by HBTU (0.035 g, 2.2 eq), HOBt (0.03 g, 2.2 eq) and NMM (0.04 mL, 4.4 eq). After 3 h, the resin was washed with DMF 6×, treated with 20% piperidine in DMF 2×25 min and washed with DMF 6×. The above cycle was repeated with Fmoc-His(Trt)-OH followed by cleavage of the peptide from the resin with 10 ml TFA/H₂O/PhOH/TIPS (95:2:2:1), precipitated by methyl-tert-Butyl ether. The obtained crude peptide was dissolved in 1.5 mL of MeOH:ACN (1:1), followed by addition of 2M LiOH (0.6 mL) and the reaction stirred at RT for 6 h. The crude was dissolved and applied to a reverse-phase HPLC column (C₁₈, 20-50% CH₃CN in 0.1% TFA/H₂O over 30 min gradient) to afford the titled compound as a white powder (8 mg, 4%): Retention time in RP-HPLC (C₁₈, 5-75% CH₃CN in 0.1% TFA/H₂O over 15 min) is 7.93 min; Calculated mass for C₁₈₉H₂₉₆N₄₈O₆₀(M+H)⁺4200.75, found by LC-MS 1401.8 (M+3H)³⁺, 1051.6 (M+4H)⁴⁺, 2102.2 (M+2H)²⁺.

Example 7

Preparation of Compound in FIG. 16G

The synthesis of this compound was accomplished following the same experimental procedure as described for Example 3. The only difference was the sequence of the peptide-resin intermediate. The crude was dissolved and applied to a reverse-phase HPLC column (C₁₈, 20-50% CH₃CN in 0.1% TFA/H₂O over 30 min gradient) to afford the titled compound as a white powder (27.4 mg, 18%): Retention time in RP-HPLC (C₁₈, 5-75% CH₃CN in 0.1% TFA/H₂O over 15 min) is 8.25 min; Calculated mass for C₁₅₃H₂₂₈N₄₀O₄₇S (M+H)⁺3411.83, found by LC-MS 1138.6 (M+3H)³⁺, 854.5 (M+4H)⁴⁺, 1707.9 (M+2H)²⁺.

Example 8

Preparation of (5,5)-Glu-Gly-OH as shown in FIG. 18

Preparation of Boc-Asp(OBn)-OMe: Boc-Asp(OBn)-OH 2.4 g (1 equiv, 7.45 mmol) was dissolved in 17 mL of dry DMF in a 100 mL round-bottom flask. Finely ground K₂CO₃(1.5 g, 11 mmol) was added to the solution to form a suspension. The mixture was cooled to 0° C. in an ice-bath over five minutes. MeI (1 mL, 15 mmol) was then added to the mixture over 20 seconds under a positive flow of nitrogen. A yellow color developed within 30 min. The resulting mixture was stirred for 3 hours. The ice-bath was removed and 25 mL of water were added and the mixture left standing at room temperature 2 hours. The mixture was then treated with 30 mL of water and extracted with 3×25 mL AcOEt. The organics were washed with saturated NaHCO ₃1×, brine 3×, dried over Na₂SO₄filtered and concentrated to yield a yellow oil. This material was passed through a short silica column (eluting w/ AcOEt), after concentration of the pure fractions 2.42 g of Boc-Asp(OBn)-OMe as a yellow oil was obtained (98% yield). ¹H NMR (DMSO d6, 500 mHz): δ 7.36 (m, 5H), 5.10 (s, 2H), 4.40 (q, 1H), 3.6 (s, 3H), 2.80 (dd, 1H), 2.72 (dd, 1H), 1.38 (s, 9H). LCMS (C₁₈, 2-98% CH₃CN in 0.1% TFA/H₂O over 6 min); Calculated mass for C₁₇H₂₃NO₆(M+H)⁺338.23, found by LC-MS 338.2.
Preparation of Compound 1 in FIG. 18: Boc-Asp(OBn)-OMe (26.4 g, 78.3 mmol) was dissolved in a 500 mL round-bottom flask with 110 mL of dry THF and the mixture cooled at negative 42° C. To this mixture was added lithium bis(trimethylsilyl)amide (LiHMDS) (173 mL, 1 M solution, 2.2 eq). This solution was stirred under a gentle argon flow for 45 min followed by slow addition (via syringe) of tert-butyl bromo acetate (14.5 mL, 1.25 eq). Thin layer chromatography (TLC) (Hex:AcOEt, 8/2) showed after 4 h>95% of expected product. The reaction was quenched by addition of saturated NH₄Cl (70 mL). The mixture was evaporated and the residue re-dissolved with 100 mL of dichloromethane (DCM). The emulsion formed was separated by standing overnight. The organics were collected and washed with saturated NH₄Cl 2×, brine 1×, dried over Na₂SO₄, filtered and concentrated to give Compound 1 as a red orange oil. Flash chromatography (Hex:AcOEt, 7:3) gave 27.4 g of a yellow oil (78% yield). ¹H NMR (DMSO d6, 500 mHz): δ 7.35 (m, 5H), 5.08 (s, 2H), 4.57 (dd, 1H), 4.05 (m, 1H), 3.52 (s, 3H), 1.39 (s, 9H), 1.34 (s, 9H). LCMS (C₁₈, 2-98% CH₃CN in 0.1% TFA/H₂O over 6 min); Calculated mass for C₂₃H₃₃NO₈(M+H)⁺452.23, found by LC-MS 452.2.
Preparation of Compound 2 in FIG. 18. Compound 2 is tert-butyl-(1S,2R)-2-[(carboxy)-3-tert-butoxycarbonyl)-1-metoxhycarbonyl)]propylcarbamate. Compound 1 (5.8 g, 12.8 mmol) was dissolved in 30 mL of a 6:4 mixture of MeOH/THF. Argon was bubbled in the catalyst for 5 minutes, followed by incorporation of two hydrogen balloons attached via syringe to the reaction flask. After 4 hours, LCMS shows complete transformation. The crude mixture was filtrated through celite and concentrated to yield 5.25 g of Compound 2 as an orange oil which was used without further purification in the next step. Calculated mass for C₁₆H₂₇NO₈(M+H)⁺362.23, found by LC-MS 362.2.
Preparation of Compound 3 in FIG. 18. Compound 3 is tert-butyl-(1S,2R)-2-[(benzylthio)-carbonyl)-3-tert-butoxycarbonyl)-1-metoxhycarbonyl)]propylcarbamate. Compound 2 (2.1 g, 5.8 mmol) was dissolved with DCM (7 mL). To this clear mixture was added ethylthiol (0.4 g, 1.1 eq) and 4-dimethylaminopyridine (DMAP) (0.07 g, 0.1 eq). To the clear solution was added dicyclohexyl carbodiimide (DCC) (1.3 g, 1.1 eq) as a solid and the mixture was stirred at room temperature for 5 hours. The mixture was diluted with DCM (40 mL) and washed with 1N HCl (2×), dried over Na₂SO₄, filtered and concentrated to give 2.15 g of Compound 3 as a yellow oil, which was used without further purification in the next step. Calculated mass for C₁₈H₃₁NO₇S (M+H)⁺406.13, found by LC-MS 406.2.
Preparation of Compound 4 in FIG. 18. Compound 4 is tert-butyl-(1S,2R)-3-(tert-butoxcarbonyl)-1-(methoxycarbonyl)-2-formylpropylcarbamate. To Compound 3 (5.9 g, 14.5 mmol) and Pd—C 10% wt (0.35 g) were added acetone (36 mL) and the mixture cooled down to about 4-8° C. To this mixture was added drop wise, under positive flow of Argon, triethylsilyl (TES) (11.5 mL, 5 eq). After addition of TES the mixture was kept at 10-15° C. After 3.5 hours LCMS showed no more starting material. The mixture was filtrated through celite, and the solution concentrated to give 6.6 g of Compound 4 as a green oil which was used without further purification in the next step. Calculated mass for C₁₆H₂₇NO₇(M+H)⁺346.13, found by LC-MS 346.2.
Preparation of Compound 5 in FIG. 18. Compound 5 is methyl-[3S,4R,8R]-1-Aza-3-tert-butoxycarbonyl-4-(tert-butoxycarbonyl)methyl)-2-oxo-6-thiabicyclic[3.3.0]-octane-8-carboxylate. To Compound 4 (1.3 g, 3.76 mmol), L-Cys-OH—HCl (0.98 g, 1.5 eq) and 4° A molecular sieves (2g) was added dry pyridine (10 mL) and the mixture stirred, under argon, at room temperature for 4 hours in high pressure vessel. After this time, 3.5 mL more of pyridine were added and the reaction was stirred at 50° C. for 5 days. After this time, the crude mixture was filtered through celite, the solution was re-dissolved in AcOEt and washed with 2N HCl 2×, dried over Na₂SO₄, filtered and concentrated to yield 1.2 g of Compound 5 as a yellowish semisolid, which was used without further purification in the next step. Calculated mass for C₁₉H₃₀N₂O₇S (M+H)⁺ 431.13, found by LC-MS 431.2.
Preparation of Compound 6 in FIG. 18. Compound 6 is [3S,4R,8R]-1-Aza-3-amino-4-(tert-butoxycarbonyl)methyl)-2-oxo-6-thiabicyclic[3.3.0]-octane-8-carboxylate. Crude Compound 5 was treated with 10 mL of a 50% TFA-DCM mixture at 0° C. The mixture was let to warm up at 10° C. and stirred for 2.5 hours. The mixture is then concentrated and the residue re-dissolved with 5 mL of a 2M LiOH solution and stirred at room temperature for 2.5 hours. LCMS analysis showed complete transformation to Compound 6. The reaction was concentrated and the residue passed through a short column packed with ion-exchange resin (H⁺, Dowex), eluting with 1:1 MeOH/H₂O. The residue was concentrated and then lyophilized to give 1.8 g of crude Compound 6 as a yellow semisolid, which was used without further purification in the next step. Calculated mass for C₁₃H₂₀N₂O₅S (M+H)⁺317.13, found by LC-MS 317.2.
Preparation of Compound 7 in FIG. 18. Compound 7 is the (5,5)-Glu-Gly bicylic dipeptide mimetic. Compound 7 is also referred to as [3S,4R,8R]-1-Aza-3-fluorenylmethyl-carbonyl-4-(tert-butoxycarbonyl)methyl)-2-oxo-6-thiabicyclic[3.3.0]-octane-8-carboxylate. Crude Compound 6 (1.2 g, 3.8 mmol) was dissolved in 17 mL of dry DCM. To this solution was added FmocOSu (1.8 g, 1.4 eq) followed by N,N-diisopropylethylamine (DIEA) (1.3 mL, 2 eq). The reaction was stirred at room temperature for 3 hours. To the mixture was added 50 mL of DCM and washed with 2M HCl 2×, brine 1×, dried over Na₂SO₄, filtered and concentrated to give 1.42 g of a yellow oil. Purification by flash chromatography using an increasing polarity solvent gradient (Hex:AcOEt 1:1 to DCM:MeOH 9:1) gave 0.150 g of pure Compound 7. Calculated mass for C₂₈H₃₀N₂O₇S (M+H)⁺539.13, found by LC-MS 539.2.

Example 9

Preparation of Compound 11 in FIG. 19

Compound 9 is 1-Aza-3-amino-tert-butoxylcarbonyl-4-(tert-butoxycarbonyl)methyl)-2-oxo-1-methoxyacetyl. Compound 4 was prepared as described above. A solution of Compound 4 (preparation described in Example 8) (4.1 g, 12 mmol) and the HCl salt of NH₂-Gly-OMe (1.66 g, 1.1 eq) in 20 mL of DMF were stirred at room temperature. To this mixture was added NaBH(OAc)₃(5.0 g, 2 eq) dissolved in 18 mL of DMF. After 1 hour LCMS shows complete transformation to the desired secondary amine 8. Calculated mass for C₁₉H₃₄N₂O₈(M+H)⁺419.1, found by LC-MS 419.2. To this crude mixture was added AcOEt (80 mL), washed with sat. NaHCO ₃3×, water 1×, brine 1×, dried over Na₂SO₄, filtered and concentrated to give 3.9 g of a crude yellow oil. LCMS of this material showed that the five-member ring lactam (Compound 9) was formed during work-up. The residue was concentrated to give 3.9 g of crude Compound 9 as a yellow oil, which was used without further purification in the next step. Calculated mass for C₁₈H₃₀N₂O₇(M+H)⁺ 387.1, found by LC-MS 387.2.
Preparation of Compound 10 in FIG. 19. Compound 10 is 1-amino-4-(tert-butoxy-carbonyl)methyl)-2-oxo-1-methoxyacetyl. Crude Compound 9 (3.8 g) was cooled at 10° C. in an ice-water bath. To this stirred mixture was added in a drop wise manner 15 mL of a 50% solution of TFA in DCM. The mixture was stirred at that temperature for 2 h. LCMS showed a selective N-Boc deprotection. The mixture was concentrated to yield crude compound 10 as clear semisolid, which was used without further purification in the next step. Calculated mass for C₁₃H₂₂N₂O₅(M+H)⁺287.1, found by LC-MS 287.2.
Preparation of Compound 11 in FIG. 19. Compound 11 is a γ-lactam-Glu-Gly bicyclic dipeptide mimetic which is 1-Aza-3-aminofluorenylmethylcarbonyl-4-(tert-butoxycarbonyl)methyl)-2-oxo-1-acetylcarboxylate. Crude Compound 10 (2.8 g, 9.8 mmol) was dissolved in 8 mL of a 1:1 mixture of dioxane/MeOH at room temperature. To this mixture was added 15 mL of a 2M LiOH (2.8 eq) and the mixture stirred at room temperature for 3 hours. The mixture was then concentrated and passed trough a short column of Dowex H⁺ ion-exchange resin. The pooled fractions containing M+1=273 by LCMS were collected and lyophilized. This crude material was then dissolved in DCM (40 mL) followed by addition of DIEA (3.5 mL, 2 eq) and FmocOSu (3.9 g, 1.2 eq). The mixture was stirred at room temperature for 3 hours. LCMS analysis shows no more starting material, thus to the reaction was added AcOEt (60 mL), washed with sat. NaHCO ₃3×, water 1×, brine 1×, dried over Na₂SO₄, filtered and concentrated to give 2.5 g of a crude Compound 11. The mixture was purified by flash chromatography using AcOEt:Hex (1:1). Collection of the pure fractions identified by LCMS gave 100 mg of pure Compound 11. Calculated mass for C₂₇H₃₀N₂O₇(M+H)⁺495.1, found by LC-MS 495.2.

Example 10

Preparation of Compound 18 in FIG. 20

Compound 1 (i.e., tert-butyl-1(1S,2R)-2-[(benzyloxy)carbonyl)-3-tert-butoxycarbonyl)-1-metoxhycarbonyl)]propylcarbamate) was prepared as described in Example 8 above.
Preparation of Compound 12 in FIG. 20. Compound 12 is tert-Butyl-(1S,2R)-2-[(benzylcarboxylate)-3-carboxy)-1-metoxhycarbonyl)]propylcarbamate. To Compound 1 (13.9 g, 30.8 mmol) was added 45 mL of a 2M solution of HCl in diethyl ether. The clear yellow solution was stirred at room temperature for 18 hours. The mixture was triturated with cold ether which gave a yellow foam. Drying of this solid gave 8.32 g (84% yield) of the hydrochloride salt intermediate which was used without further purification in the next step. This HCl salt crude (8.3 g, 25 mmol) was dissolved in THF (80 mL). To this clear solution was added TEA (7.6 mL, 2.2 eq) followed by Boc₂O (6.5 g, 1.2 eq). The mixture was stirred for 18 hours at room temperature. The mixture was then concentrated, re-dissolved in AcOEt (150 mL), washed with 1N HCl 2×, sat. NaCl 1×, dried over Na₂SO₄, filtered, concentrated and dried under high vacuum to yield Compound 12 as a brownish oil (11.1 g, 100% crude yield), which was used without further purification in the next step. Calculated mass for C₁₉H₂₅NO₈(M+H)⁺396.1, found by LC-MS 396.2.
Preparation of Compound 13 in FIG. 20. Compound 13 is tert-Butyl-(1S,2R)-2-[(benzylcarboxylate)-3-ethylthiocarboxylate)-1-metoxhycarbonyl)]propylcarbamate. DCC (5.9 g, 1.1 eq) was added to a solution of crude Compound 12 (10.4 g, 26.2 mmol), EtSH (2.15 mL, 1.1 eq) and 4-dimethylaminopyridine (DMAP) (0.32 g, 0.1 eq) dissolved in DCM (28 mL). After 6 hours the mixture was concentrated, re-dissolved in AcOEt (150 mL) washed with 1N HCl 2×, sat. NaCl 1×, dried over Na₂SO₄, filtered, concentrated and dried under high vacuum to yield Compound 13 as a brown oil (7.6 g), which was used without further purification in the next step. Calculated mass for C₂₁H₂₉NO₇S (M+H)⁺440.1, found by LC-MS 440.2.
Preparation of Compound 14 in FIG. 20. Compound 14 is (1S,2R)-2-[(benzyl-carboxylate)-3-ethylthiocarboxylate)-1-metoxhycarbonyl)]propylamine hydrochloride salt. Crude Compound 13 (7.6 g, 17.2 mmol) was dissolved in ethyl ether (8 mL), followed by addition of a 2N solution of HCl in diethyl ether (40 mL). The clear mixture was stirred at room temperature for 18 hours. The mixture was concentrated to give Compound 14 as an orange oil (7.5 g) which was used without further purification in the next step. Calculated mass for C₁₆H₂₁NO₅S (M+H)⁺338.1, found by LC-MS 338.2.
Preparation of Compound 15 in FIG. 20. Compound 15 is (1S,2R)-2-[(benzyl-carboxylate)-3-ethylthiocarboxylate)-1-metoxhycarbonyl)]propyl-L-N-Boc-Ala. Crude Compound 14 (6.6 g, 17.7 mmol), Boc-L-Ala-OH (3.7 g, 1.1 eq) and 2-(7-aza-1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluoride (HATU) (12.1 g, 1.8 eq) were dissolved in ACN (150 mL). To this solution was added DIEA (6.1 mL, 2 eq) and the mixture was stirred at room temperature for 6 hours. The mixture was concentrated, re-dissolved in AcOEt (150 mL), washed with sat NH₄Cl 2×, sat. NaCl 1×, dried over Na₂SO₄, filtered, concentrated and dried under high vacuum to yield compound 15 as a brown oil (16.5 g of crude). Purification by flash chromatography using Hex; AcOEt (6:4) gave pure compound 15 (3.55 g, 40% yield). Calculated mass for C₂₄H₃₄N₂O₈S (M+H)⁺511.1, found by LC-MS 511.2.
Preparation of Compound 16 in FIG. 20. Compound 16 is (1S,2R)-2-[(benzyl-carboxylate)-3-formyl)-1-metoxhycarbonyl)]propyl-L-N-Boc-Ala. Triethylsilane (5.6 mL, 5 eq) was slowly added, under argon, to a solution of Compound 15 (3.5 g, 6.9 mmol) and Pd—C (0.6 g) dissolved in acetone (16 mL) in an ice bath (˜4° C.). After addition of TES the reaction was stirred at 10-20° C. After 4 hours, thin layer chromatography (TLC) showed complete transformation. The reaction was filtered and concentrated to give an off-green oil. Purification by flash chromatography using Hex; AcOEt (6:4) gave pure Compound 16 (2.9 g, 93% yield). Calculated mass for C₂₂H₃₀N₂O₈(M+H)⁺452.1, found by LC-MS 452.2.
Preparation of Compound 17 in FIG. 20. Compound 17 is (3R,6S,7R)-7-(benzyloxy-carbonyl)-6-amino-(L-Boc-Ala)-hexahydro-5-oxo-2H-thiazolo[3,2-a]pyridine-3-carboxylic acid. In a high pressure vial crude Compound 16 (2 g, 4.55 mmol) was dissolved in dry pyridine (12 mL). To this mixture was added L-Cys-OH (0.88 g, 1.6 eq) followed by addition of 2.7 g of activated 4° A molecular sieves (powder). The mixture was stirred vigorously at room temperature for 4 hours. Dry-pyridine (10 mL) was added to the mixture, the vial was capped and the mixture stirred at 50° C. for 4 days. The mixture was then filtered through a bed of celite and washed with tetrahydrofuran (THF) and MeOH. The mixture was concentrated to give Compound 17 as yellow foam (1.6 g) which was used without further purification in the next step. Calculated mass for C₂₄H₃₁N₃O₈S (M+H)⁺522.1, found by LC-MS 522.2.
Preparation of Compound 18 in FIG. 20. Compound 18 is (3R,6S,7R)-7-(benzyloxy-carbonyl)-6-amino-(L-Ala)-hexahydro-5-oxo-2H-thiazolo[3,2-a]pyridine-3-carboxylic acid. In a round bottom flask crude Compound 17 (1.6 g, 3.1 mmol) was dissolved in dioxane (15 mL) followed by addition of 4M HCl. The mixture was stirred at room temperature and after 3 hours LCMS analysis showed complete conversion to the free amine. The mixture was concentrated, dissolved with DCM, MeOH, concentrated again and then dried under high vacuum to give the hydrochloride salt as a brownish semisolid (1.5 g) which was used without further purification in the next step. Calculated mass for C₂₄H₃₁N₃O₈S (M+H)⁺522.1, found by LC-MS 522.2.
This HCl salt was dissolved in DCM (14 mL) followed by addition of DIEA (2 mL, 3.5 eq) and FmocOSu (1.2 g, 1.1 eq) in portions as solid. The mixture was stirred for 3 hours showing complete transformation by LCMS. The mixture was concentrated, dissolved in AcOEt (60 mL), washed with 2N HCl 2×, brine, dried over Na₂SO₄, filtered and concentrated to give 2.1 g of a brownish oil. Purification by flash chromatography using Hex; AcOEt (5:95) followed by 100% MeOH gave pure compound 18 (0.821 g, 44% yield). Calculated mass for C₃₄H₃₃N₃O₈S (M+H)⁺644.1, found by LC-MS 644.2.

Example 11

Preparation of Compounds in FIG. 21

Preparation of compound 16 has been described in Example 10.
Preparation of Compound 19 in FIG. 21. Compound 19 is (1S,2R)-[2-(benzylcarboxylate)-3-formyl-4-(tert-butoxy-N-2-methyl acetyl)-1-(methoxycarbonyl)]butyl-L-N-Boc-Ala. Crude compound 16 (1.01 g, 2.24 mmol) and L-Ala-OtBu. HCl (0.449 g, 1.1 eq) were dissolved in DMF (4.5 mL) at room temperature. NaBH(OAc)₃(0.95 g, 2 eq) was dissolved separately in DMF (4.5 mL) at room temperature, the two solutions were mixed and stirred at room temperature for about 2 hours. The reaction was washed with saturated NaHCO₃solution 2×, water 1×, sat. NaCl 1×, dried over Na₂SO₄, filtered, concentrated to yield 0.92 g of Compound 19 as a colorless oil, which was used without further purification in the next step. Calculated mass for C₂₉H₄₅N₃O₉(M+H)⁺579.68, found by LC-MS 579.6.
Preparation of Compound 20 in FIG. 21. Compound 20 is (1S,2R)-tert-butyl-[4-(benzylcarboxylate)-3-(N-L-Boc-Ala)-2-oxopiperidine)-1-methyl-1-yl]acetate. Crude Compound 19 (0.550 g, 0.95 mmol) was dissolved in DMF (3 mL), DIEA (0.445 mL, 2 eq) was heated using microwaves at 140° C. for 20 min. The reaction was checked and then washed with 0.5M HCl 2×, water 1×, sat. NaCl 1×, dried over Na₂SO₄, filtered, concentrated to give 0.50 g of Compound 20 as a light brownish oil, which was used without further purification in the next step. Calculated mass for C₂₈H₄₁N₃O₈(M+H)⁺547.64, found by LC-MS 547.6.
Preparation of Compound 21 in FIG. 21. Compound 21 is (1S,2R)-[4-(benzyl-carboxylate)-3-(N-L-Fmoc-Ala)-2-oxopiperidine)-1-methyl-1-yl]acetic acid. Crude Compound 20 (0.501 g, 0.91 mmol) was dissolved in 5 mL of 20% TFA in DCM and stirred for about an hour at room temperature. The reaction was concentrated and then dried under high vacuum to give the corresponding TFA salt as brownish oil (0.47 g) which was used without further purification in the next step. Calculated mass for C₁₉H₂₅N₃O₆(M+H)⁺505.64, found by LC-MS 505.6
This TFA salt (0.47 g, 0.91 mmol) was dissolved in aqueous 10% Na₂CO₃solution (12 mL) and DMF (4 mL), and cooled down to 0° C. A solution of FmocOSu (0.472 g, 1.5 eq) in DMF (8 mL) was added dropwise to the cold aqueous solution. After 5 minutes the ice bath was removed and the reaction was stirred overnight, quenched by adding 40 mL water followed by washings with AcOEt. The aqueous layer was acidified using 2N HCl (pH=2) and washed with AcOEt 3×, NaCl 1×, dried over Na₂SO₄, filtered and concentrated. Purification was done by washing the crude compound with Hex:AcOEt (6:4) 2× at room temperature to give Compound 21 as light colored oil (0.290 g, 52% yield). Calculated mass for C₃₄H₃₅N₃O₈(M+H)⁺613.24, found by LC-MS 613.2.
Preparation of Compound 22 in FIG. 21. Compound 22 is (1S,2R)-[2-(benzylcarboxylate)-3-(formyl)-4-(tert-butoxy-N-acetyl)-1-(methoxycarbonyl)]butyl-L-N-Boc-Ala. Crude compound 16 (1.01 g, 2.24 mmol) and AcOH. NH₂-Gly-OtBu (0.478 g, 1.1 eq) were dissolved in dry DMF (4.5 mL) at room temperature. NaBH(OAc)₃(0.95 g, 2 eq) was dissolved separately in dry DMF (4.5 mL) at room temperature, the two solutions were mixed and stirred at room temperature for about 2 hours. The reaction was washed with saturated NaHCO₃solution 2×, water 1×, sat. NaCl 1×, dried over Na₂SO₄, filtered and concentrated to yield 1.09 g of Compound 22 as a colorless oil, which was used without further purification in the next step. Calculated mass for C₂₈H₄₃N₃O₉(M+H)⁺565.68, found by LC-MS 565.3.
Preparation of Compound 23 in FIG. 21. Compound 23 is (1S,2R)-tert-butyl-[4-(benzylcarboxylate)-3-(N-L-Boc-Ala)-2-oxopiperidine)-1-yl]acetate. Crude compound 22 (0.550 g, 0.95 mmol) was dissolved in DMF (3 mL), DIEA (0.445 mL, 2 eq) and then heated using microwaves at 140° C. for 20 min (Biotage™, Initiator8). The reaction was checked by LCMS. The reaction crude was washed with 0.5M HCl 2×, water 1×, sat. NaCl 1×, dried over Na₂SO₄, filtered and concentrated to yield 0.41 g of Compound 23 as a light brownish oil, which was used without further purification in the next step. Calculated mass for C₂₇H₃₉N₃O₈(M+H)⁺533.64, found by LC-MS 533.6.
Preparation of Compound 24 in FIG. 21. Compound 24 is (1S,2R)-[4-(benzylcarboxylate)-3-(N-L-Fmoc-Ala)-2-oxopiperidine)-1-yl]acetic acid. Crude Compound 23 (0.41 g, 0.76 mmol) was dissolved in 5 mL of 20% TFA in DCM; reaction was stirred for about an hour at room temperature. The reaction was concentrated and then dried under high vacuum to give the corresponding TFA salt as a brownish oil (0.45 g) which was used without further purification in the next step. Calculated mass for C₁₈H₂₃N₃O₆(M+H)⁺491.3, found by LC-MS 491.4
This TFA salt (0.45 g, 0.92 mmol) was dissolved in aqueous 10% Na₂CO₃solution (12 mL) and DMF (4 mL), and cooled down to 0° C. A solution of FmocOSu (0.472 g, 1.5 eq) in DMF (8 mL) was added dropwise to the cold aqueous solution. After 5 min, the ice bath was removed and the reaction was stirred overnight, then quenched by adding about 40 mL water, followed by washings with AcOEt. The aqueous layer was acidified using 2N HCl (pH=2) and washed with AcOEt 3×, NaCl 1×, dried over Na₂SO₄, filtered, concentrated. Purification was done by washing the crude compound with Hex:AcOEt (6:4) 2× at room temperature giving Compound 24 as light colored oil (0.15 g, 27% yield). Calculated mass for C₃₃H₃₃N₃O₈(M+H)⁺599.6, found by LC-MS 599.4.

Example 12

Preparation of Compounds in FIGS. 1A, 2A, and 5A

The compound in FIG. 1A may be prepared following the methods described, e.g., in U.S. Pat. No. 6,872,700, the disclosure of which is incorporated by reference. The compounds in FIGS. 2A and 5A may be prepared following the methods described, e.g., in WO 2007/139941, the disclosure of which is incorporated by reference.

Example 13

Preparation of Compounds in FIGS. 1B-C, 2B-U, 3A-G, 5B-G, 6A-E, 10A-I, 11A-C, 12, 13A-B, 14B-C, 14E-R

For each compound in FIGS. 1B, 1C, 2B, 2C, 2D, 2E, 2F, 2G, 2H, 2I, 2J, 2K, 3A, 3B, 3C, 3D, 3E, 3F, 3G, 5B, 5C, 5D, 6A, 6B, 6C, 6D, and 6E, a calculated 100 μmol of Fmoc-Ser(OtBu)-Wang resin or Fmoc Rink amide resin was weighed into a reaction vessel in a Symphony peptide synthesizer, and peptide elongation was carried out following standard Fmoc peptide synthesis protocol. If an unnatural amino acid was used at position 2 this was incorporated manually in a polypropylene syringe. Cleavage of the peptide from the resin was done with 10 mL TFA/H₂O/PhOH/TIPS (95:2:2:1), then precipitated by methyl-tert-Butyl ether. The crude was dissolved and applied to a reverse-phase HPLC column (C₁₈, 20-50% CH₃CN in 0.1% TFA/H₂O over 30 min gradient) to afford the titled compounds as white powders. All characterized by LC-MS. The compounds in FIGS. 2L-U, 5E-G, 10A-I, 13B, 14B-C and 14E-K will be prepared following the methods described in this example.
With respect to the compounds in FIGS. 14L-R, a calculated 100 μmol of Fmoc-Rink-amide resin was weighed into a reaction vessel in a Symphony peptide synthesizer, and peptide elongation was carried out following standard Fmoc peptide synthesis protocol. If an unnatural amino acid was used at position 2 this was incorporated manually in a polypropylene syringe. Only for the compounds in FIGS. 14N-O, this procedure was carried out under the following microwave-assisted conditions: A microwave vial was loaded with the corresponding peptide-resin intermediate (0.3 g, 0.14 mmol), Fmoc-AA-OH (4 eq), PyBrop (0.32 g, 4.8 eq), 2,6-lutidine (0.25 mL, 15 eq), dichloroethane (DCE) (2-3 mL) and DMF (about 0.3 mL). The vial was capped and heated with microwaves (Biotage™, Initiator8) at 100° C. for 11 min. The resin was filtered, washed with DCE and MeOH. Cleavage of the peptide from the resin was done with 10 mL TFA/H₂O/PhOH/TIPS (95:2:2:1), then precipitated by methyl-tert-Butyl ether. The crude was dissolved and applied to a reverse-phase HPLC column (C₁₈, 20-50% CH₃CN in 0.1% TFA/H₂O over 30 min gradient) to afford the titled compounds as white powders. All characterized by LC-MS.

Example 14

Preparation of Compounds in FIGS. 8A-H

For the compounds in FIGS. 8B, 8C, 8D, 8E, 8F, 8G, and 8H, a calculated 100 mol of Fmoc-Ser(OtBu)-Wang or Fmoc-Rink-amide resin was weighed into a reaction vessel in a Symphony peptide synthesizer, and the peptide elongation was carried out following standard Fmoc peptide synthesis protocol. The corresponding L- or D-Cys(Trt)-OH was introduced at positions 2 and 4. Cleavage of the peptide from the resin with 10 mL TFA/H₂O/PhOH/TIPS (95:2:2:1) then precipitated by methyl-tert-Butyl ether. The crude peptides were dried overnight under high vacuum.
Disulfide cylclization: Clear-OX resin (0.39 g, 3× molar excess) was swollen on DCM for 45 min at room temperature, then washed with DCM 2×, DMF 3×, MeOH 3×, deionized water 3× and finally H₂O:ACN (1:1) 3×. The corresponding crude peptide (0.1 g) was dissolved in degassed 1:1 v/v solution of 0.1M NH₄OAc buffer (pH=6.5)/ACN. The peptide solution was then added to the pre-swollen Clear-OX resin and the slurry was shacked at room temperature. After 2-3 hours the cyclization was complete. The resin was washed with a small amount of ACN/H₂O (1:1) solution, the filtrate concentrated to remove volatiles and then lyophilized. The crude was dissolved and applied to a reverse-phase HPLC column (C₁₈, 20-50% CH₃CN in 0.1% TFA/H₂O over 30 min gradient) to afford the titled compounds as white powders. All characterized by LC-MS. The compound in FIG. 8A will be prepared following the methods described in this example.

Example 15

Preparation of Compounds in FIGS. 9A-H

For each compound in FIGS. 9A, 9B, 9C, 9D, 9E, 9G, and 9H, a calculated 100 μmol of Fmoc-Rink amide resin was weighed into a reaction vessel in a Symphony peptide synthesizer, and the peptide elongation was carried out following standard Fmoc peptide synthesis protocol. The amino acid at position 2 of all these compounds was introduced by special coupling conditions. A microwave vial was loaded with the corresponding peptide-resin intermediate (0.3 g, 0.14 mmol), Fmoc-AA-OH (4 eq), PyBrop (0.32 g, 4.8 eq), 2,6-lutidine (0.25 mL, 15 eq), dichloroethane (DCE) (2-3 mL) and DMF (−0.3 mL). The vial was capped and heated with microwaves (Biotage™, Initiator8) at 100° C. for 11 min. The resin was filtered, washed with DCE and MeOH. For some compounds the above process was repeated twice. The resin is then treated in a cycle of deprotection, coupling with Fmoc-His(Trt)-OH, HBTU and HOBt, deprotection. Cleavage of the peptide from the resin was done with 10 mL TFA/H₂O/PhOH/TIPS (95:2:2:1), then precipitated by methyl-tert-Butyl ether. The crude was dissolved and applied to a reverse-phase HPLC column (C₁₈, 20-50% CH₃CN in 0.1% TFA/H₂O over 30 min gradient) to afford the titled compounds as white powders. All characterized by LC-MS. The compound in FIG. 9F will be prepared following the methods described in this example.

Example 16

In Vitro Assay

The compounds shown in Table 1 below were analyzed in an in vitro functional assay at the GLP-1 receptor. The assay was conducted as follows: Membrane fractions were prepared from confluent cultures of RIN m5f cells. Compounds were serially diluted with an assay buffer, and then added to a 96-well assay plate containing RIN m5f cell membranes in an ATP/GTP mixture. Cyclase activities were determined by measuring the production of cAMP induced through GLP-1 receptor activation. Quantification of cAMP production was achieved through a competitive chemiluminescence assay with a biotinylated-cAMP probe using Perkin Elmer Fusion™-Alpha Microplate Analyzer (AlphaScreen™ technology). The compound EC₅₀values were obtained through fitting the concentration-response curves to a four-parameter logistic equation within GraphPad PRISM® software. The results of the assay are presented in Table 1 below.

Example 17

In Vivo Assay

Some compounds shown in Table 1 below were analyzed in an in vivo basal glucose lowering assay using the following procedure: A subcutaneous injection of either 200 μl phosphate-buffered saline (PBS) vehicle or test article was given immediately following baseline glucose (t=0) to NIH/Swiss female mice. Tail blood glucose samples were measured at t=2 and 4 hours post dose using a OneTouch® Ultra® (LifeScan, Inc., a Johnson & Johnson Company, Milpitas, Calif.). Body weight was measured daily. Significant test sample effects were identified by ANOVA (p<0.05), followed by Dunnett's post test using GraphPad Prism version 4.00 for Windows (GraphPad Software, San Diego Calif.). The results are presented in Table 1 below.

TABLE 1

Compound in	Cyclase GLP-1 Receptor	In Vivo
FIG.	EC₅₀(nM)	Glucose-Lowering Assay

1A	0.01	not tested
1B	0.004	not tested
1C	0.029	not tested
2A	0.008	greater than 3 hours
2B	0.0089	more than 4 hours
2C	6	not tested
2D	0.04	not tested
2E	2.15	not tested
2F	0.027	more than 4 hours
2G	0.89	not tested
2H	0.124	not tested
2I	0.054	more than 4 hours
2J	7.2	not tested
2K	0.29	similar to GLP-1
3A	0.007	more than 4 hours
3B	0.007	more than 2 hours
3C	0.022	not tested
3D	1.03	not tested
3E	0.06	more than 2 hours
3F	1.089	not tested
3G	0.065	similar to GLP-1
5A	1.83	more than 4 hours
5B	0.53	more than 4 hours
5C	1.056	not tested
5D	10.5	not tested
6A	0.144	similar to GLP-1
6B	0.52	not tested
6C	151.9	not tested
6D	0.282	not tested
6E	0.60	more than 4 hours
8B	1.26	similar to GLP-1
8C	968.6	not tested
8D	106.2	not tested
9A	0.21	not tested
9B	0.03	more than 4 hours
9C	0.05	more than 4 hours
9D	0.01	more than 4 hours
9E	133	not tested
9F	0.07	more than 4 hours
9G	1.9	not tested
9H	0.1	not tested
11A	0.155	not tested
11B	0.007	not tested
11C	0.011	not tested
12	0.028	not tested
13	0.274	not tested
14J	0.054	not tested
14K	3.5	not tested
14L	0.05	not tested
14M	0.2 (partial agonist)	not tested
14N	0.004	not tested
14O	0.24	not tested
14P	0.25	not tested
14Q	0.02	not tested
14R	0.41 (partial agonist)	not tested
15A	0.57	inactive
15B	1000	not tested
15C	2.46	inactive
15D	1.7	inactive
16A	0.52	inactive
16B	0.18	similar to GLP-1
16C	1.1	inactive
16D	1	inactive
16E	1	inactive
16F	0.61	about 30 minutes
16G	56	not tested
16H	1000	not tested
17A	105	not tested
17B	668	not tested
17C	10,000	not tested

Example 18

Preparation of Compounds in FIGS. 17A-F

A calculated 100 μmol of Rink amide resin was weighed into a reaction vessel in a Symphony peptide synthesizer, and the peptide elongation was carried out following standard Fmoc peptide synthesis protocol up to residue Pro³. The resulting peptide-resin intermediate (0.3 g, 0.0927 mmol) was swollen in DCM with around 5% DMF and to the slurry was added Fmoc-Cys(Trt)-OH (0.351 g, 4 eq), followed by Bromo-tris-pyrrolidino-phosphonium hexafluorophosphate (PyBrop) (0.34 g, 4.8 eq) and 2,6-lutidine (0.27 mL, 15 eq). The vial was capped and heated in a microwave apparatus (Biotage Initiator8) at 100° C. for 11 min. The resin was filtered and washed with DMF 6×, treated with 20% piperidine in DMF 2×25 min and washed with DMF 6×, DCM 4× followed by treatment with 2% TFA/1.5% TIS in DCM 4×10 min. then washed with DCM 4×, DMF 2× and TMOF 3×. In a polypropylene syringe the resin is swollen in TMOF, followed by addition of compound 25 (0.2 g, 3 eq) and dry pyridine (0.06 mL, 5 eq). The resin was shaken at room temperature for 16 hours, followed by washings with TMOF 3×, DCM 3×, MeOH 3× and dried under high vacuum. Cleavage of the peptide from the resin was carried out with 10 ml TFA/H₂O/PhOH/TIPS (95:2:2:1) (TFA is trifluoroacetic acid; TIPS is triisopropylsilyl), precipitated by methyl-tert-Butyl ether. The residue was dissolved in 0.8 ml of MeOH and 0.8 ml of ACN. To this solution 0.1 ml of DEA were added and the mixture stirred at room temperature overnight. The resulting residue was applied to a reverse-phase high performance liquid chromatography (HPLC) column (C₁₈, 20-50% CH₃CN in 0.1% TFA/H₂O over 30 min gradient) to afford compound 17A as a white powder (5.3 mg, 3%): Retention time in reverse phase-high performance liquid chromatography (RP-HPLC) (C₁₈, 5-75% CH₃CN in 0.1% TFA/H₂O over 15 min) is 8.71 min; Calculated mass for C₁₄₆H₂₂₈N₃₈O₄₃S (M+H)⁺3235.74, found by liquid chromatography/mass spectrometry (LC-MS) 1079.6 (M+3H)³⁺, 1619.9 (M+2H)²⁺.
The synthesis of compounds 17B and 17C was performed similar as reported above for compound 17A, with the difference of using Fmoc-D-Cys(Trt)-OH and Fmoc-Pen-OH, respectively. After purification compound 17B was obtained as a white powder (8 mg, 7%): Retention time in reverse phase-high performance liquid chromatography (RP-HPLC) (C₁₈, 5-75% CH₃CN in 0.1% TFA/H₂O over 15 min) is 8.55 min; Calculated mass for C₁₄₆H₂₂₈N₃₈O₄₃S (M+H)⁺3235.74, found by liquid chromatography/mass spectrometry (LC-MS) 1079.6 (M+3H)^3α, 1619.9 (M+2H)²⁺. After purification compound 17C was obtained as a white powder (1.5 mg, 3%): Retention time in reverse phase-high performance liquid chromatography (RP-HPLC) (C₁₈, 5-75% CH₃CN in 0.1% TFA/H₂O over 15 min) is 8.55 min; Calculated mass for C₁₄₈H₂₃₂N₃₈O₄₃S (M+H)⁺3263.79, found by liquid chromatography/mass spectrometry (LC-MS) 1089.6 (M+3H)³⁺, 1632.9 (M+2H)²⁺.

Example 19

Preparation of Compound 25

In a round bottom flask Fmoc-His(Boc)-OH (4 g, 1 eq) was dissolved in DCM (50 mL). To this solution was added DCC (1.9 g, 1.1 eq), EtSH (0.7 mL, 1.1 eq) and DMAP (0.102 g, 0.1 eq). The mixture was stirred at room temperature for 6 h. The reaction mixture was filtered and the solution concentrated to yield 4.9 g of an off-white solid. The crude product was purified by flash chromatography using Hex:AcOEt (1:1). After concentration of the pure fractions the corresponding thioester was obtained as a clear oil (3.6 g, 84%). The thioester (0.69 g, 1 eq) was dissolved in THF (11 mL) and stirred under Argon atmosphere for few minutes. To this solution was added Pd—C (0.180 g) and the mixture stirred under Argon for 10 min followed by dropwise addition of TES (0.75 mL, 3.5 eq) and the mixture stirred at room temperature. After 4 h one more equivalent of TES was added, one hour later the crude mixture was filtered through a bed of silica/celite and washed with THF. The filtrate was concentrated to give a dark brown oil, which was purified by flash chromatography in a short (20 mL) silica gel column using Hex:AcOEt (2:8). After concentration of the pure fractions compound 25 was obtained as a clear semisolid (0.26 g, 50%).

Example 20

Modified Exendin Peptides

N-Terminus conformationally constrained GLP-1 receptor agonist compounds described herein were covalently linked to one or more polyethylene glycol and/or fatty acids, as described herein. In particular, the following twelve compounds 23A-L were prepared:
Compound 23A: 4-imidazopropionyl-dAla-PGTFTSDLSK¹²QMEEEAVRLFIE-WLKNGGPSSGAPPPS-NH₂, wherein K¹²was modified with —C(═O)—CH₂(OCH₂CH₂)₂NH—C(═O)CH₂—(OCH₂CH₂)₂NH—C(═O)—(CH₂)₆—CH₃(SEQ ID NO:105).
Compound 23B: 4-imidazopropionyl-dAla-PGTFTSDLSK¹²QMEEEAVRLFIE-WLKNGGPSSGAPPPS-NH₂, wherein K¹²was modified with —C(═O)—CH₂(OCH₂CH₂)₂NH—C(═O)CH₂(OCH₂CH₂)₂NH—C(═O)—CH(NH₂)—(CH₂)₇—CH₃(SEQ ID NO:106).
Compound 23C: 4-imidazopropionyl-dAla-PGTFTSDLSK¹²QMEEEAVRLFIE-WLKNGGPSSGAPPPS-NH₂, wherein K¹²was modified with —C(═O)—CH₂(OCH₂CH₂)₂NH—C(═O)CH₂(OCH₂CH₂)₂NH—C(═O)—CH[NH—C(═O)(CH₂)₆CH₃]-(CH₂)₇—CH₃(SEQ ID NO:107).
Compound 23D: 4-imidazopropionyl-dAla-PGTFTSDLSK¹²QMEEEAVRLFIE-WLKNGGPSSGAPPPS-NH₂, wherein K¹²was modified with —C(═O)—CH₂(OCH₂CH₂)₂NH—C(═O)CH₂(OCH₂CH₂)₂NH—C(═O)—CH₂CH₂—CH[C(OH)(═O)]—NH—C(═O)—(CH₂)₁₆-[C(OH)(═O)] (SEQ ID NO:108).
Compound 23E: 4-imidazopropionyl-dAla-PGTFTSDLSKQMEEEAVRLFIEW-LK²⁷NGGPSSGAPPPS-NH₂, wherein K²⁷was modified with —C(═O)—CH₂(OCH₂CH₂)₂NH—C(═O)CH₂(OCH₂CH₂)₂NH—C(═O)—(CH₂)₆—CH₃(SEQ ID NO:109).
Compound 23F: 4-imidazopropionyl-dAla-PGTFTSDLSKQMEEEAVRLFIEW-LK²⁷NGGPSSGAPPPS-NH₂, wherein K²⁷was modified with —C(═O)—CH₂(OCH₂CH₂)₂NH—C(═O)CH₂(OCH₂CH₂)₂NH—C(═O)—CH(NH₂)—(CH₂)₇—CH₃(SEQ ID NO:110).
Compound 23G: 4-imidazopropionyl-dAla-PGTFTSDLSKQMEEEAVRLFIEW-LK²⁷NGGPSSGAPPPS-NH₂, wherein K²⁷was modified with —C(═O)—CH₂(OCH₂CH₂)₂NH—C(═O)CH₂(OCH₂CH₂)₂NH—C(═O)—CH[NH—C(═O)(CH₂)₆CH₃]-(CH₂)₇—CH₃(SEQ ID NO:111).
Compound 23H: 4-imidazopropionyl-dAla-PGTFTSDLSKQMEEEAVRLFIEW-LK²⁷NGGPSSGAPPPS-NH₂, wherein K²⁷was modified with —C(═O)—CH₂(OCH₂CH₂)₂NH—C(═O)CH₂(OCH₂CH₂)₂NH—C(═O)—CH₂CH₂—CH[C(OH)(═O)]—NH—C(═O)—(CH₂)₁₆-[C(OH)(═O)](SEQ ID NO:112).
Compound 23I: 4-imidazopropionyl-dAla-PGTFTSDLSKQMEEEAVRLFIEWLKN-GGPSSGAPPPS³⁹, wherein S³⁹was modified with -Lys(NH₂)—C(═O)—CH₂(OCH₂CH₂)₂NH—C(═O)CH₂(OCH₂CH₂)₂NH—C(═O)—(CH₂)₆—CH₃(SEQ ID NO:113).
Compound 23J: 4-imidazopropionyl-dAla-PGTFTSDLSKQMEEEAVRLFIEWLKN-GGPSSGAPPPS³⁹, wherein S³⁹was modified with -Lys(NH₂)—C(═O)—CH₂(OCH₂CH₂)₂NH—C(═O)CH₂(OCH₂CH₂)₂NH—C(═O)—CH(NH₂)—(CH₂)₇—CH₃(SEQ ID NO:114).
Compound 23K: 4-imidazopropionyl-dAla-PGTFTSDLSKQMEEEAVRLFIEWLKN-GGPSSGAPPPS³⁹, wherein S³⁹was modified with -Lys(NH₂)—C(═O)—CH₂(OCH₂CH₂)₂NH—C(═O)CH₂(OCH₂CH₂)₂NH—C(═O)—CH[NH—C(═O)(CH₂)₆CH₃]-(CH₂)₇—CH₃(SEQ ID NO:115).
Compound 23L: 4-imidazopropionyl-dAla-PGTFTSDLSKQMEEEAVRLFIEWLKN-GGPSSGAPPPS³⁹, wherein S³⁹was modified with —C(═O)—CH₂(OCH₂CH₂)₂NH—C(═O)CH₂(OCH₂CH₂)₂NH—C(═O)—CH₂CH₂—CH[C(OH)(═O)]—NH—C(═O)—(CH₂)₁₆-[C(OH)(═O)] (SEQ ID NO:116).
For each of Compound Nos. 23A-L above, a calculated 100 μmol of Fmoc-Rink-amide resin was weighed into a reaction vessel in a Symphony peptide synthesizer, and peptide elongation was carried out following standard Fmoc peptide synthesis protocol. The dAla at position 2 was incorporated manually in a polypropylene syringe. The orthogonal protection (alloc group) of the side chain group (Lys¹², Lys²⁷or Lys⁴⁰) was performed as follows: The peptide-resin was swollen in DCM and dimethylamino borane-complex (6 eq) followed after about 3 minutes by tetrakis(triphenylphosphine)palladium(0) (0.1 eq). The resin was shaken for 15 minutes, washed with DCM 3× and the process repeated. Then, the resin was washed with DCM 3×, 10% DIEA in DCM 2×, DCM 3× and MeOH 2×. At this point, the peptide-resin gave a positive chloranil test. The resulting peptide-resin intermediate (0.3 g, 0.0927 mmol) was swollen in DMF and to the slurry was added the corresponding polyethylene glycol (2.2 eq), followed by HBTU (0.035 g, 2.2 eq), HOBt (0.03 g, 2.2 eq) and NMM (0.04 mL, 4.4 eq). After 3 hours, the resin was washed with DMF 6×, treated with 20% piperidine in DMF 2×25 min and washed with DMF 6×. The above cycle was repeated with a second Fmoc-(polyethylene glycol)-OH in most cases. The coupling of the corresponding fatty acid chain was done using two different microwave-assisted methods: the corresponding acyl chloride (5 eq) and NMM (7 eq) were added to the resin swollen in a 1:1 DMF:DCM mixture, and then heated using microwaves at 75° C. for 20 min (Biotage™, Initiator8); or the corresponding carboxylic or dicarboxylic acid (5 eq), HOAt (5 eq) and DIC (5 eq) were added to the slurry resin on DMF, and then heated using microwaves at 75° C. for 15 min (Biotage™, Initiator8). Cleavage of the peptide from the resin was done with 10 mL TFA/H₂O/PhOH/TIPS (95:2:2:1), then precipitated by methyl-tert-Butyl ether. The crude was dissolved and applied to a reverse-phase HPLC column (C₁₈, 20-50% CH₃CN in 0.1% TFA/H₂O over 30 min gradient) to afford the titled compounds as white powders. All were characterized by LC-MS.
Each of these compounds was analyzed in an in vitro functional assay at the GLP-1 receptor following the methods described in Example 16. The results are in Table 2:

	TABLE 2

		Cyclase GLP-1 Receptor
	Compound	EC₅₀(nM)

	23A	0.027
	23B	0.027
	23C	0.06
	23D	0.22
	23E	0.3
	23F	0.1
	23G	0.7
	23H	0.08
	23I	0.01
	23J	0.01
	23K	0.02
	23L	0.185

Example 21

Modified Exendin Peptides

N-Terminus conformationally constrained GLP-1 receptor agonist compounds described herein were covalently linked to one or more biotin, as described herein. In particular, the following compounds 24A-L were prepared:

Compound 24A:

(SEQ ID NO: 1117)

His-dAla-PGTFTSDLSKQMEEEAVRLFIEWL-Lys(biotin)-

NGGPSSGAPPS-Lys[(NH₂)(biotin)].

Compound 24B:

(SEQ ID NO: 69)

4-imidazopropionyl-GEGTFTSDLSKQMEEEAVRLFIEWL-

Lys(biotin)-NGGPSSGAPPS-Lys[(NH₂)(biotin)].

Compound 24C:

(SEQ ID NO: 70)

4-imidazopropionyl-GEGTFTSDLSKQMEEEAVRLFIEWLKN-

Lys[(NH₂)(biotin)].

Compound 24D:

(SEQ ID NO: 71)

4-imidazopropionyl-APGTFTSDLSKQMEEEAVRLFIEWLKN-

Lys[(NH₂)(biotin)].

Compound 24E:

(SEQ ID NO: 118)

His-dAla-PGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS-

Lys(NH2)-(O-CH₂-CH₂)₂-KAKAEAEAKAKAEAEA-biotin.

Compound 24F:

(SEQ ID NO: 119)

His-dAla-PGTFTSDLSKQMEEEAVRLFIEWLE[-(O-CH₂-CH₂)₂-

(biotin)]-NGGPSSGAPPPS-NH₂.

Compound 24G:

(SEQ ID NO: 120)

His-dAla-PGTFTSDLSEK[-(O-CH₂-CH₂)₂-(biotin)]-

QMEEEAVRLFIEWLKNGGPSSGAPPPS-NH₂.

For each of Compound Nos. 24A-G above, a calculated 100 μmol of Fmoc-Rink-amide resin was weighed into a reaction vessel in a Symphony peptide synthesizer, and peptide elongation was carried out following standard Fmoc peptide synthesis protocol. If an unnatural amino acid was used at position 2 this was incorporated manually in a polypropylene syringe. The orthogonal protection (alloc group) of the side chain group (Lys²⁷, Lys²⁸or Lys⁴⁰) was performed as follows: The peptide-resin was swollen in DCM and dimethylamino borane-complex (6 eq) followed after about 3 minutes by tetrakis(triphenylphosphine)palladium(0) (0.1 eq). The resin was shaken for 15 min, washed with DCM 3× and the process repeated. Then, the resin was washed with DCM 3×, 10% DIEA in DCM 2×, DCM 3× and MeOH 2×. At this point, the peptide-resin gave a positive chloranil test. The biotin moiety (one or two) was coupled to the free amino group using the standard solid-phase coupling conditions described above (HBTU, HOBt, NMM). Cleavage of the peptide from the resin was done with 10 mL TFA/H₂O/PhOH/TIPS (95:2:2:1), then precipitated by methyl-tert-Butyl ether. The crude was dissolved and applied to a reverse-phase HPLC column (C₁₈, 20-50% CH₃CN in 0.1% TFA/H₂O over 30 min gradient) to afford the titled compounds as white powders. All were characterized by LC-MS.
Each of these compounds was analyzed in an in vitro functional assay at the GLP-1 receptor following the methods described in Example 16. The results are in Table 3:

	TABLE 3

		Cyclase GLP-1 Receptor
	Compound	EC₅₀(nM)

	24A	0.04
	24B	0.03
	24C	0.07
	24D	0.77
	24E	0.06
	24F	0.06
	24G	0.01

Example 22

Compounds of FIG. 10

With respect to FIG. 10, the following compounds 10J-10N were made:

10J:
(SEQ ID NO: 72)
4-imidazopropionyl-GPGTFTSDLSKQLEEEAVRLFIEWLKNGGPSSGAPPPS-NH₂

10K:
(SEQ ID NO: 121)
4-imidazopropionyl-dAla-PGTFTSDLSKQLEEEAVRLFIEWLKNGGPSSGAPPPS-NH₂.

10L:
(SEQ ID NO: 73)
4-imidazopropionyl-Aib-PGTFTSDLSKQLEEEAVRLFIEWLKNGGPSSGAPPPS-NH₂

10M:
(SEQ ID NO: 74)
4-imidazopropionyl-GEGTFTSDLSKQLEEEAVRLFIEWLKQGGPSKEIIS-OH

10N:
(SEQ ID NO: 122)
4-imidazopropionyl-dAla-PGTFTSDLSKQLEEEAVRLFIEWLKQGGPSKEIIS-OH.

For each of Compounds 10J-N, a calculated 100 μmol of Fmoc-Rink-amide resin was weighed into a reaction vessel in a Symphony peptide synthesizer, and peptide elongation was carried out following standard Fmoc peptide synthesis protocol. If an unnatural amino acid was used at position 2 this was incorporated manually in a polypropylene syringe. Only for the compound in FIG. 10L, this procedure was carried out under the following microwave-assisted conditions: a microwave vial was loaded with the corresponding peptide-resin intermediate (0.3 g, 0.14 mmol), Fmoc-AA-OH (4 eq), PyBrop (0.32 g, 4.8 eq), 2,6-lutidine (0.25 mL, 15 eq), dichloroethane (DCE) (2-3 mL) and DMF (˜0.3 mL). The vial was capped and heated with microwaves (Biotage™, Initiator8) at 100° C. for 11 min. The resin was filtered, washed with DCE and MeOH. Cleavage of the peptides from the resins was done with 10 mL TFA/H₂O/PhOH/TIPS (95:2:2:1), then precipitated by methyl-tert-Butyl ether. The crude was dissolved and applied to a reverse-phase HPLC column (C₁₈, 20-50% CH₃CN in 0.1% TFA/H₂O over 30 min gradient) to afford the titled compounds as white powders. All characterized by LC-MS.
Compounds 10J-L were analyzed in an in vitro functional assay at the GLP-1 receptor following the methods described in Example 16. The results are in Table 4:

	TABLE 4

		Cyclase GLP-1 Receptor
	Compound	EC₅₀(nM)

	10J	0.1
	10K	0.03
	10L	0.005
	10M	0.007
	10N	0.01

All publications and patent applications are incorporated herein by reference. Although the foregoing has been described in detail for purposes of clarity of understanding, it will be apparent to one of ordinary skill in the art that changes and modifications may be made without departing from the spirit or scope of the disclosure or appended claims.

Claims

1-130. (canceled)

131. A polypeptide comprising the amino acid sequence as set forth in Formula (C):

(SEQ ID NO: 2) Xaa₁Xaa₂Xaa₃Xaa₄TFTSDLSKQXaa₁₄EEEAVRLFIEXaa₂₅LK-R₁₀-Z;

wherein:

Xaa₁is His; or a compound of Formula (1);

Xaa₂is Gly, dAla, Aib, Ala, Val, NMeAla, a compound of Formula (3), or a compound of Formula (4); and Xaa₂is absent when Xaa₃is:

Xaa₃is Pro; a compound of Formula (2); a compound of Formula (3); a Compound of Formula (4);

Xaa₄is Gly, dAla, or Aib;

Xaa₁₄is Leu or Met;

Xaa₂₅is Phe or Trp;

R₁₀is QGGPSKEIIS (SEQ ID NO:22); NG; NGG; NGGP (SEQ ID NO:24); NGGPS (SEQ ID NO:25); NGGPSS (SEQ ID NO:26); NGGPSSG (SEQ ID NO:27); NGGPSSGA (SEQ ID NO:28); NGGPSSGAP (SEQ ID NO:29); NGGPSSGAPP (SEQ ID NO:30); NGGPSSGAPPP (SEQ ID NO:31); QGGPSSGAPPPS (SEQ ID NO:32); NGGPSSGAPPS (SEQ ID NO:33); NGGPSSGAPPSK (SEQ ID NO:34); NGGPSSGAPPS(K)_2-5(SEQ ID NO:35); NGGPSSGAPPPSK (SEQ ID NO:36); or NK; and

Z is OH or NH₂;

wherein the compound of Formula (1) is:

wherein R₂₀and R₂₁are each independently a single bond or a carbon atom; R₂₃, R₂₄, R₂₅and R₂₆are each independently absent, hydrogen, hydroxyl, C_1-6alkyl, carboxyl, amino, or C_1-6alkoxy; - - - - - - is a single bond or a double bond; and R₂₁is a chiral or achiral carbon atom;

wherein the compound of Formula (2) is:

wherein Y₁and Z₁are each independently a single bond, a carbon, or a sulfur; and W₁, W₂and W₃are each independently selected from hydrogen, C_1-6alkyl, C_1-6alkoxy, hydroxyl, and amino; and when one Y₁or Z₁is sulfur, the sulfur may be bonded to two oxygen atoms to form a sulfonyl group; and - - - - - - is

or

;

wherein the compound of Formula (3) is:

or

; and

wherein the compound of Formula (4) is:

wherein R₃₀, R₃₁, and R₃₂are each independently hydrogen or a C_1-6alkyl; or R₃₀and R₃₁, together with the nitrogen¹and the carbon², form a 5-membered or 6-membered heterocyclic ring; or R₃₁and R₃₂, together with the carbon², form a 3-, 4-, or 5-membered carbocyclic ring.

132. The polypeptide of claim 131, wherein Xaa₃is Pro.

133. The polypeptide of claim 132, wherein the polypeptide comprises the sequence set forth as: Pro³-exendin-4 (SEQ ID NO:40); Pro³,Leu¹⁴-exendin-4 (SEQ ID NO:41); Pro³,Leu¹⁴,Phe²⁵-exendin-4 (SEQ ID NO:42); Pro³-exendin-4(1-28) (SEQ ID NO:43); Pro³,Leu¹⁴-exendin-4(1-28) (SEQ ID NO:44); Pro³,Leu¹⁴,Phe²⁵-exendin-4(1-28) (SEQ ID NO:45); Pro³-exendin-4(1-29) (SEQ ID NO:51); Pro³,Leu¹⁴-exendin-4(1-29) (SEQ ID NO:75); Pro³,Leu¹⁴,Phe²⁵-exendin-4(1-29) (SEQ ID NO:52); Pro³-exendin-4(1-30) (SEQ ID NO:53); Pro³,Leu¹⁴-exendin-4(1-30) (SEQ ID NO:76); Pro³,Leu¹⁴,Phe²⁵-exendin-4(1-30) (SEQ ID NO:54); Pro³-exendin-4(1-31) (SEQ ID NO:55); Pro³,Leu¹⁴-exendin-4(1-31) (SEQ ID NO:77); Pro³,Leu¹⁴,Phe²⁵-exendin-4(1-31) (SEQ ID NO:56); Pro³-exendin-4(1-32) (SEQ ID NO:57); Pro³,Leu¹⁴-exendin-4(1-32) (SEQ ID NO:78); Pro³,Leu¹⁴,Phe²⁵-exendin-4(1-32) (SEQ ID NO:58); Pro³-exendin-4(1-33) (SEQ ID NO:59); Pro³,Leu¹⁴-exendin-4(1-33) (SEQ ID NO:79); Pro³,Leu¹⁴,Phe²⁵-exendin-4(1-33) (SEQ ID NO:60); Pro³-exendin-4(1-34) (SEQ ID NO:61); Pro³,Leu¹⁴-exendin-4(1-34) (SEQ ID NO:80); Pro³,Leu¹⁴,Phe²⁵-exendin-4(1-34) (SEQ ID NO:62); Pro³-exendin-4(1-35) (SEQ ID NO:63); Pro³,Leu¹⁴-exendin-4(1-35) (SEQ ID NO:81); Pro³,Leu¹⁴,Phe²⁵-exendin-4(1-35) (SEQ ID NO:64); Pro³-exendin-4(1-36) (SEQ ID NO:46); Pro³,Leu¹⁴-exendin-4(1-36) (SEQ ID NO:47); Pro³,Leu¹⁴,Phe²⁵-exendin-4(1-36) (SEQ ID NO:48); Pro³-exendin-4(1-37) (SEQ ID NO:65); Pro³,Leu¹⁴-exendin-4(1-37) (SEQ ID NO:82); Pro³,Leu¹⁴,Phe²⁵-exendin-4(1-37) (SEQ ID NO:66); Pro³-exendin-4(1-38) (SEQ ID NO:67); Pro³,Leu¹⁴-exendin-4(1-38) (SEQ ID NO:83); Pro³,Leu¹⁴,Phe²⁵-exendin-4(1-38) (SEQ ID NO:68); Pro³-exendin-3 (SEQ ID NO:50); or HGPGTFTSDLSKQLEEEAVRLFIEWLKQGGPSKEIIS (SEQ ID NO:49).

134. A method for treating diabetes, treating insulin resistance, treating postprandial hyperglycemia, lowering blood glucose levels, lowering HbA1c levels, stimulating insulin release, reducing gastric motility, delaying gastric emptying, reducing food intake, reducing appetite, reducing weight, treating overweight, or treating obesity in a patient in need thereof, the method comprising:

administering to the patient a therapeutically effective amount of the peptide of claim 131 to treat diabetes, treat insulin resistance, treat postprandial hyperglycemia, lower blood glucose levels, lower HbA1c levels, stimulate insulin release, reduce gastric motility, delay gastric emptying, reduce food intake, reduce appetite, reduce weight, treat overweight, or treat obesity in the patient.